Author
FLUORESCENCE RESONANCE ENERGY TRANSFER-BASED
BIOSENSORS FOR MONITORING PROSTATE SPECIFIC ANTIGEN by
Chunyao Jenny Mu
B.S., Biomedical Engineering, The Johns Hopkins University, 2000
Submitted to the Department of Mechanical Engineering in Partial Fulfillment of the Requirements for the Degree of
Master of Science in Mechanical Engineering at the
MASSACHUSETTS INS
OF TECHNOLOGY
E
Massachusetts Institute of Technology
June 2005
JUN 16RAR
© 2005 Massachusetts Institute of Technology
All rights reserved
(n a tent of Mechanical Engineering
May 12, 2005
Certified by
Bruce R. Zetter
Charles Nowiszewski Professor of Cancer Biology
Children's Hospital
Thesis Supervisor
Certified by
H. Frederick Bowman
Senior Academic Administrator in HST
Thesis Reader
Accepted by
Lalit Anand
Chairman, Departmental Committee on Graduate Students

Fluorescence Resonance Energy Transfer-Based Biosensors for Monitoring Prostate
Specific Antigen by
Chunyao Jenny Mu
Submitted to the Department of Mechanical Engineering on May 12, 2005 in
Partial Fulfillment of the Requirements for the Degree of
Master of Science in Mechanical Engineering
Prostate cancer has become the most commonly diagnosed cancer in men in the United States.
Clinical diagnostic procedures currently include prostate-specific antigen (PSA) screening, digital rectal exam, and prostatic needle biopsy. However, these methods lack the sensitivity to detect small lesions that occur in the early stages of cancer and metastasis. I propose a molecular imaging modality that provides a biochemical characterization of localized regions of prostate tissue. Using fluorescence resonance energy transfer (FRET), several peptide substrates have been designed to respond to varying concentrations of PSA with a concomitant increase in fluorescence. In the near-infrared wavelength range, these fluorescent substrates can be imaged through thin sections of tissue to allow surface volume imaging of biochemical function, and thus, to provide additional insight into prostate cancer localization and progression.
The goal of this study was to develop novel fluorescent substrates for prostate-specific antigen to serve as indicators of prostate cancer progression. PSA is a biomolecular marker that has gained widespread clinical use in prostate cancer detection. Produced primarily by prostate epithelium, PSA is an androgen-regulated serine protease that acts to cleave semenogelins.
Several peptide substrates for PSA have been identified and optimized for specific and efficient hydrolysis. Two of these substrates, QFYSSN and SSIYSQTEEQ, were modified with fluorescent dye and quencher molecules to suppress fluorescence in the inactivated form. Light absorbed by the fluorescent molecule is dissipated via nonradiative interaction with the quencher molecule. Disruption of dye and quencher interaction, as in substrate proteolysis, results in an increase in fluorescence. I report several promising substrates that generate significant increases in fluorescence upon cleavage by PSA in purified systems as well as with human prostate cancer cell lines. Selected FRET substrates can distinguish between PSA- producing and non-PSA-producing human prostate cancer cells.
Thesis Supervisor: Bruce R. Zetter, Ph.D.
Title: Charles Nowiszewski Professor of Cancer Biology, Children's Hospital
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This work could not have been possible without the encouragement and support of my research advisors, Bruce Zetter and Robert Langer. Their pioneering spirit served as an inspiration to me throughout this project. I am indebted to them for all the opportunities that they have afforded me in my graduate education. One such opportunity was the chance to share this journey with
David LaVan whose creativity first launched us into uncertain waters with a sense of anticipation for great things to come. David has navigated through the peaks and troughs with me, and for that, I am grateful. I would also like to express my appreciation to all the members of the Zetter and Langer labs, in particular, Jacqueline Banyard, Vanja Vuletic, Lloyd
Hutchinson, Yadong Wang, and Ying Chau, for their contributions towards my education as a biologist and chemist. However, my transformation from an engineer to an interdisciplinary jack-of-all-trades could not have been complete without Richard Cook and Derek Hudson.
Their expertise in peptide chemistry was the beacon of light that finally guided me to shore.
My livelihood in the lab was sustained in great part by a number of entertaining officemates. Humor and intellectual discourse were provided by David Nguyen, Paul George, and Steven Charles. My home in the HST Lounge was made much more pleasant by their presence. Outside of the lab, I have been blessed with friends whom I consider to be a part of my extended family. I hold these individuals close to my heart as they have seen me through the most challenging times of my life. This thesis is owed in part to Chethan Gangireddy whose friendship has lasted the test of time and who continues to be my trusted confidant. I am grateful to my friends, Aaron Aguirre, Melissa Barbagelata, Joaquin Blaya, Todd Coleman,
Blanca Himes, Priya Natarajan, Thanh-Nga Tran, Roxanna Webber, and Peter Wu for sustaining my spirit with laughter and companionship along the way. To all my friends near and far, including Catherine Lee, Serena Leung, Isaac Weingrod, Supreet Rangi-Bauer, Jiyoung Dang, and First Piluek, thank you for sharing your lives with me and for helping me take my life a little less seriously.
The source of my strength has always been my family. I am indebted to them for providing the love and support that has guided me throughout my life. There are no words to express my love and appreciation for my parents who have worked tirelessly for their children's futures. I hope that they see this modest work as a testament to their perseverance and sacrifice as much as it was mine. I am also grateful to my grandparents, aunt, uncle, and cousin, for their support and encouragement. To my brother and sister, thank you for always being there. Last but not least, I would like to express my love and gratitude to David DiPaola for being my biggest fan and cheering me onwards to the finish line.
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PSA Prostate specific antigen
TRUS Transrectal ultrasound
LLD Lower limit of detection
DRE Digital rectal exam
PET Positron emission tomography
CT Computed tomography
MRI Magnetic resonance imaging
CCD Charge coupled device
FRET Fluorescence resonance energy transfer
PAP Prostatic acid phosphatase
BPH Benign prostatic hyperplasia
TGF Transforming growth factor
A 4ACR x-methylacyl-coenzyme A racemase
AFM Anterior fibromuscular stroma
IG FBP-3 Insulin-like growth factor binding protein-3 hK2 Human glandular kallikrein 2
ACT a1-antichymotrypsin a2M a2-macroglobulin tPSA Total prostate specific antigen
SgI, II Semenogelin I, II
TMR Tetramethylrhodamine
I MSO Dimethylsulfoxide
TFA Trifluoroacetic acid
YFP Yellow fluorescent protein
PPAR Peroxisome proliferatoractivated receptors
GTP Guanosine triphosphate
PCI fPSA
FAM
DMF
PBS
GFP
CFP
LCFA
GDP
CCK
IRMA
TUUS
BPH
TNM
SPECT
RF
Fluc
TZ
PZ tPA
FGF
IGF
PCa
PIN siRNA
HUVEC
Immunoradiometric assay
Transurethral ultrasound
Benign prostatic hyperplasia
Tumor/node/metastasis system
Single photon emission computed tomography
Radio frequency
Firefly luciferase gene
Transition zone
Peripheral zone
Tissue plasminogen activator
Fibroblast growth factor
Insulin-like growth factor
Prostate cancer
Prostatic intraepithelial neoplasia
Small interfering RNA
Human umbilical vein endothelial cell
Protein C inhibitor
Free prostate specific antigen
Fluorescein
Dimethylformamide
Phosphate-buffered saline
Green fluorescent protein
Cyan fluorescent protein
Long chain fatty acid
Guanosine diphosphate
Cholecystokinin
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cAMP Cyclic 3',5' adenosine monophosphate
EGFR Epidermal growth factor receptor
ODN Oligonucleotide
DABCYL 4-(4'-dimethylaminophenylazo) benzoic acid
MMP Matrix metalloproteinase
DNP Dinitrophenol
PTP Protein tyrosine phosphatase
PMT Photomultiplier tube
FT14 (FAM)-QFYSSNK(TMR)
FT19 (FAM)-SSIYSQTEEQK(TMR)
MS Mass spectrometry
GPCR
CHO
ATP
EDANS
PCR
Trp
LF
NIRF
DIPEA
FT15
MALDI-TOF
G-protein coupled receptor
Chinese hamster ovary cell
Adenosine triphosphate
5-(2'-aminoethyl) aminonaphthalene-1-sulfonic acid
Polymerase chain reaction
Tryptophan
Bacillus anthracis lethal factor
Near-infrared fluorescence
N,N-diisopropylethylamine
(FAM)-6GQFYSSN6GK(TMR)
Matrix assisted laser desorption/ ionization-time of flight technique
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Figure 1-1 Gleason histologic grade .......................................................................................... 15
Figure 2-1 Genitourinary anatomy with emphasis on prostate gland ................................. 23
Figure 2-2 View s of the prostate.................................................................................................. 25
Figure 2-3 Immunohistochemical staining for AMACR........................................................ 28
Figure 2-4 Jablonski diagram of light absorption and emission............................................35
Figure 2-5 Jablonski diagram demonstrating the FRET mechanism .................................... 39
Figure 2-6 Absorption and emission spectra for energy transfer from D to A....................39
Figure 2-7 Relevant angles in defining the orientation factor................................................40
Figure 3-1 Chromophore absorption-concentration curves...................................................51
Figure 3-2 Fluorescence curve for dye dilution series ............................................................ 52
Figure 4-1 Initial reaction velocity of FAM-TMR substrates with PSA ................................. 59
Figure 4-2 Comparison of FAM to reference fluorescence signals in PSA kinetics.......60
Figure 4-3 Comparison of FAM to TMR fluorescence signals in PSA kinetics .......... 61
Figure 5-1 Western blot of LNCaP conditioned media and lysate.........................................66
Figure 5-2 Western blot of PC-3 conditioned media and lysate in wild-type and transfected cells.........................................................................................................
Figure 5-3 FT14 in cell culture ....................................................................................................
66
Figure 5-4
Figure 5-5
FT15 in cell culture ....................................................................................................
FT19 in cell culture ....................................................................................................
66
67
67
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Table 3-1
Table 3-2
Table 3-3
Summary of linear fit to absorption-concentration curves..................................51
Extinction coefficients for dye molecules...............................................................52
Manufacturer reported fluorescence parameters................................................. 55
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1 Introduction
1.1 Current Practices in Prostate Cancer Assessment........................................................11
1.2 Molecular Imaging........................................................................ ... ........ 16
1.3 Scope of the Thesis..........................................................................................
.
..... 21
2 Background
2.1 Neoplastic Processes in the Prostate.............................................................................23
2.2 Prostate Specific A ntigen ........................................................................................
29
2.3 Theory of FRET...................................................................................................34
2.4 Applications of FRET....................................................................................40
2.5 Motivation for this Study..........................................................................
.... 46
3 Fluorophore Characterization
3.1 Experimental Methods.................................................................................
3.2 R esults............................................................................--
... 47
...... ..... -------------------................... 49
3.3 D iscu ssion .................................................................. ...... -.. ----. -------------------..................... 53
3.4 C onclusions...................................................................-------- .... .
.
---------------....................
55
4 Substrate Evaluation with Purified Human PSA
4.1 Experimental Methods..........................................................
4.2 R esu lts...................................................................
..... ... .... 57
............. ------. .
-------------.......................
58
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4.3 D iscussion ..............................................................................................................................
4.4 Conclusions............................................................................................................................62
5 Substrate Evaluation with Human Prostate Cancer Cells
5.1 Experim ental M ethods ....................................................................................................
5.2 Results.....................................................................................................................................65
5.3 D iscussion ..............................................................................................................................
5.4 Conclusion .............................................................................................................................
6 Summary of Conclusions
7 Future Direction
References .................................................................................................................................................
61
65
69
71
68
68
72
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1
In the United States, prostate cancer is the second leading cause of cancer death in men. Since the discovery of prostate-specific antigen (PSA) as a potentially useful serum marker for prostate cancer, this form of neoplastic disease has become the most commonly diagnosed cancer in the male population. In 2005, it is estimated that 232,090 new cases will be identified with 30,350 deaths resulting from cancer-related complications. The main risk factors associated with prostate cancer are age, ethnicity, and family history. Men over the age of 65 account for more than 70% of all prostate cancer cases. Moreover, African-American men have the highest incidence rates in the world. In 5-10% of prostate cancer cases, there may be a strong genetic predisposition to the disease
1
.
As with most degenerative disorders, early detection in prostate cancer can significantly improve a patient's prognosis. The advent of PSA screening in the 1980s has revolutionized prostate cancer diagnosis by allowing physicians to monitor the levels of a specific protein as an early indicator of prostate dysfunction. Elevated serum PSA levels can be detected even before neoplastic growth has reached a palpable mass.
Tumors that are indistinguishable from normal prostate tissue using ultrasound can be detected through PSA screening. Consequently, annual PSA tests are recommended for men beginning at the age of 50 as a simple yet effective method of early detection.
The utility of any biomolecular marker for disease, such as PSA, depends strongly on the sensitivity with which it can be detected and the level of correlation to disease progression.
PSA has gained wide clinical acceptance in the diagnosis of prostate cancer due to highsensitivity assays that can detect serum PSA at the level of 0.02 ng/ml and clinical studies that have established its correlation with cancer incidence. Moreover, the complexity of prostate
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cancer management has required the use of serum PSA levels as a clinical measure of treatment efficacy. As a result, in vitro assays for PSA have proven indispensable in the early detection and management of neoplasms. However, these assays only measure the total level of PSA secreted into the bloodstream and cannot provide tissue-specific characterization of biochemical activity. This thesis outlines a method of PSA screening in vivo that may allow physicians to examine local levels of PSA activity within the prostate. To accomplish this, peptide substrates specific for PSA were synthesized and modified to increase in fluorescence upon proteolysis by the target molecule. These substrates may be injected into the prostate and allowed to diffuse freely within the entire tissue volume. Subsequent fluorescence intensities can be used as a measure of local PSA levels, providing a whole-organ image of localized disease. The work described in this thesis pertains to the design and development of these novel enzyme substrates and their utility in identifying human prostate cancer cells.
Early detection of prostate cancer requires specific and sensitive methods of discrimination between normal and neoplastic tissue. Beyond patient presentation of associated clinical symptoms, the most common and direct means of cancer diagnosis is through morphological and histological examination of the affected tissue. Tumors develop as a result of unfettered cell division, creating an abnormal mass of cells. These neoplasms may be either benign or malignant based on morphological considerations, but such classifications are ultimately subjective. Benign tumors are generally well-differentiated and grow as cohesive expansile masses that remain self-contained in a fibrous capsule. They grow slowly over a period of years and do not metastasize to distant sites. In contrast, malignant tumors progressively invade, infiltrate, and destroy surrounding tissue. These neoplastic cells have the ability to metastasize to other tissues and organs within the body. Tumor classification based on degree of cell differentiation can be misleading, because malignant neoplasms range from well-differentiated to undifferentiated. Moreover, malignant tumors may resemble benign growths in the early stages when invasion has not occurred and the neoplasm is experiencing slow growth
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surrounded by a fibrous capsule. Consequently, the use of invasion as the main criterion for distinguishing between malignant and benign neoplasms can also lead to an inaccurate diagnosis.
Clinical assessment of prostate cancer begins with PSA screening in men over the age of
50 or younger, depending on family history. Prostate cancer is typically asymptomatic in the early stages; patients present with symptoms in advanced stages of the disease when the cancer has metastasized to the bone and/or soft tissues
2
. PSA screening involves the withdrawal of a sample of blood from the patient. Patient serum is then tested using a variety of commercially available PSA assays. The Hybritech Tandem-R test (Beckman Coulter) has become widely used in hospitals and clinics because of its reliability and sensitivity. This immunoradiometric assay (IRMA) is approved by the Federal Drug Administration (FDA) and has a linear range of
2-100 ng/ml with a lower limit of detection (LLD) of 0.1-0.2 ng/m
3
. The Hybritech Tandem-R assay was designed as a two-site IRMA in which two monoclonal antibodies to different epitopes on the PSA molecule are used to label the target antigen. One set of monoclonal antibodies is coated onto plastic beads to facilitate the separation of bound PSA from other molecules in the patient serum.
1251 isotope is conjugated to the other monoclonal antibody to form a radioisotope complex whose y radiation can be counted as a measure of bound PSA.
Recently, nonradioisotopic assays have been developed that rely, instead, on fluorescent, chemiluminescent, or absorption measurements in PSA detection. An elevated level of PSA is based on a serum value greater than 4 ng/ml. Contra-indications for elevated PSA may include inflammation, infection, ejaculation, infarction, massage, biopsy and benign prostatic hyperplasia (BPH).
Abnormal PSA levels signal the possibility of prostate dysfunction and are addressed by digital rectal exam (DRE). An urologist performs this exam by digital palpation of the prostate gland via the rectum. The patient is instructed to exert pressure such as that experienced during defecation to assist in the exam. The urologist then palpates the prostate through the wall of the rectum, checking for signs of tenderness, swelling, or atypical nodules. During the exam, the physician can also determine any abnormalities in prostate size or shape. Although gland enlargement, in the form of BPH, is common in older patients, it can also signal the
-
12 -

presence of malignant growth. DRE is performed routinely in conjunction with PSA screening to establish morphological and biochemical baselines for each individual patient. Significant deviations from normal values raise the suspicion of prostate cancer. However, neither DRE nor PSA screening can confirm the diagnosis of prostate cancer. Prostatic biopsy is currently the only reliable means of cancer determination.
Subsequent diagnosis by prostatic needle biopsy is typically facilitated by transrectal ultrasound (TRUS) imaging. Ultrasound provides gross morphological images of prostate tissue on the millimeter scale. Current TRUS systems yield an average voxel resolution of 200 pim 3 , whereas newly developed transurethral ultrasound (TUUS) devices can generate an average voxel resolution of 1 pm 3 . TUUS imaging benefits from the central location of the urethra with respect to the prostate gland. TUUS only needs to image through half the tissue required of TRUS to delineate gland boundaries. Consequently, higher transducer frequencies
(10 MHz) can be used to improve image resolution
4
. Studies have suggested that tumors arising in the peripheral posterior region of the prostate are more aggressive and more common than those that occur in the internal portion of the gland
5
. In such situations, TRUS imaging is preferable due to the positional proximity of the imaging probe to the region of interest. Greyscale TRUS imaging currently represents the standard of care in prostate cancer detection.
Ultrasound images are evaluated for hypoechoic lesions, for which there is a 17-57% chance of being malignant. Unfortunately, malignant lesions can also appear isoechoic on grey-scale
TRUS, confounding the identification of at least 27% of prostate carcinomas. Although TRUS imaging can be a highly sensitive modality, its specificity for malignant lesions is low, typically within the range of 20-60%6. In the case of hypoechoic lesions, TRUS imaging is used to guide site-specific needle biopsies that remove tissue cores for pathological examination. For isoechoic images, systematic sampling of the prostate is achieved through the sextant biopsy technique originally outlined by Hodge et al. in 1989. The sextant biopsy targets the mid-lobe of each side of the prostate at the apex, middle, and base. However, there has been much debate over the incidence of false-negative biopsies and the number of tissue cores required for optimal diagnostic yield. Clinical studies have suggested that the sextant technique represents a significant undersampling of the prostate volume. Sampling more than twice as many biopsy
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-

cores, particularly in patients with glands greater than 50 g by ultrasound, has demonstrated an increase in the overall cancer detection rate
7
. The debate has also continued over the value of transitional zone biopsies and its contribution to the detection of clinically insignificant disease.
Nearly 24% of prostate cancers originate in the transition zone but usually have a favorable prognosis, as indicated in part, by their low Gleason grade 8
.
The Gleason grading system was introduced in the 1970s and has become the most commonly used means of classifying prostate cancer under histologic examination. Biopsy specimens are stained and evaluated for the architectural pattern of the glands of the prostate tumor. A well-differentiated tumor resembles normal prostate tissue in its glandular structure and presumably, its biological function. Consequently, these tumors are classified as minimally aggressive and receive a low Gleason grade. Poorly differentiated tumors are identified by a complete loss of glandular architecture with no resemblance to their native tissue. These tumors are awarded a high Gleason grade to reflect the extreme aggressiveness and malignancy of the cancer. Figure 1-1 is an adaptation of the conceptual diagram drawn by D.F. Gleason in his 1977 article outlining the histologic grading scheme that bears his name. It illustrates the continuum in gland structure that typifies each Gleason grade. Gleason grades 1-3 represent well-differentiated tissue structure in that normal and distinct gland units are still visible.
However, the progression of disease from grade 1 to grade 3 reveals prominent glandular invasion of the stroma. A significant transition occurs between Gleason grades 3 and 4, as expression of complete gland units is completely lost. The histologic distinction between these two grades, and thus, between well-differentiated and poorly differentiated tissue, is perhaps one of the most important decisions in grading. Gleason grade 5 is represented by undifferentiated tumors that exhibit no attempts to form separate, individual gland units and often resemble layers of confluent cells. Pathologists examine the major and minor histologic patterns present in the biopsy specimen and assign a separate Gleason grade to each section. A composite Gleason score is then derived from the sum of these two grades. High Gleason scores tend to correlate with worse pathologic stages, from which a poorer prognosis is predicted for a Gleason grade of 4 or greater or a Gleason score of 7 or greater. However,
Gleason scores alone are insufficient to predict pathologic stage. A combination of clinical
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staging, based on the tumor, node, and metastasis (TNM) system, serum PSA level, and Gleason score from prostate biopsy is used to more accurately determine pathologic stage. Several studies have contributed towards the construction of tables based on these parameters to predict the probability of specific pathologic states
9
. Accurate determination of the extent of prostate cancer remains essential in formulating a patient's course of treatment and prognosis.
1 2 3 4 5
Figure 1-1 Gleason histologic grade Courtesy of UPMC Cancer Centers, Pittsburgh, PA
(www.upmccancercenters.com/cancer/prostate/pfv/gradingsystems.html), 2000
The modem practice of prostate cancer diagnosis includes PSA screening, digital rectal exam (DRE), transrectal ultrasound (TRUS), and prostatic needle biopsy. DRE, TRUS, and biopsy fall within the traditional realm of palpation, tissue imaging, and histology as the dominant methods of diagnosis. However, the introduction of PSA as a serological marker for cancer has changed the way disease progression is assessed. As researchers gain a greater understanding of the pathogenesis and molecular basis of prostate cancer, key biochemical pathways are emerging, which may serve as targets for diagnosis and treatment. Biomolecular and genetic markers for prostate cancer may become important clinical correlates of disease that will supplement current methods of diagnosis. They will enable clinicians to evaluate local disease activity with greater specificity and sensitivity than current methods.
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Molecular imaging is an emergent technique that integrates clinical imaging modalities with our ever expansive understanding of molecular and cellular pathogenesis. Massoud and
Gambhir defined molecular imaging as the visual representation, characterization, and quantification of biological processes at the cellular and subcellular levels within intact living organisms
0
. Clinical imaging techniques are currently used to detect nonspecific physiological and metabolic changes at the macroscopic level to distinguish pathological from normal tissue.
The aim of molecular imaging is to provide a visual context for cellular and molecular pathways as an extension of the anatomic and physiologic information yielded by traditional imaging techniques. Rapid growth in this field of biomedical research has been fueled by advances in molecular and cell biology techniques and the use of transgenic animal models to study the molecular basis of disease. The validation of conventional molecular assays in vitro has also driven translational research to make them applicable to medical imaging techniques.
Molecular imaging has the potential to advance integrative biology, promote early detection and characterization of disease progression, and allow for greater ease in the evaluation of medical treatments. This new paradigm in medical imaging has the ability to visualize specific molecular events in intact living organisms and to track dynamic processes in real time.
Applications that may benefit from molecular imaging include phenotypic screening with respect to physiological abnormalities that arise from genetic mutations and biodistribution or pharmacokinetic studies. In both cases, biopsy is unnecessary for quantitative evaluation of tissue specimens, and temporal changes in expression can be monitored in the same living subject. Pathologic states that arise from a complex array of physiological interactions can thus be evaluated in vivo with greater authenticity.
Current implementations of molecular imaging involve traditional imaging modalities combined with unique contrast agents that are designed to target specific molecular and cellular processes. Standard clinical imaging techniques include positron emission tomography (PET), single photon emission computed tomography (SPECT), optical bioluminescence and fluorescence imaging, magnetic resonance imaging (MRI), computed tomography (CT), and
16 -

ultrasound. Contrast agents, or molecular probes, are designed for each technique to maximize the signal-to-background ratio for a particular tissue or biochemical pathway. A comparison of these imaging modalities can be made based on spatial and temporal resolution, depth of penetration, energy required in image generation, availability of molecular probes, and the probe detection threshold. PET is a highly sensitive imaging technique that utilizes positronemitting isotopes to label species of interest. The positron collides with an electron to produce two y-rays whose paths are nearly 1800 apart from each other. These high-energy 7-rays are detected by scintigraphic instrumentation to form a composite image of tracer distribution. PET exhibits spatial resolution at 1-2 mm with no limits in penetration depth. Its principal uses are in monitoring metabolic changes, receptor-ligand interactions, and enzyme targeting with sensitivity in the picomolar range. Distinct morphological features can be distinguished with tracer uptake in several million cells. However, isotope production for PET imaging relies on the use of a cyclotron or generator and results in high operational costs. Furthermore, these tracer molecules have short half-lives at approximately 110 minutes. Molecular imaging strategies using PET must target biochemical processes that occur rapidly within the limit of the isotope half-life. SPECT is another form of radionuclide imaging that detects y-emitting isotopes using a gamma camera that rotates around the subject. SPECT has spatial and temporal resolutions comparable to those of PET, since the signal source and detection schemes are nearly the same in both imaging applications. Due to the isotropic emission of 7-rays in this application, SPECT detection requires the use of a lead collimator to facilitate image reconstruction. However, the use of a collimator results in low detection efficiency and severely limits sensitivity to 10-0-10-1 mole/L. SPECT does allow for simultaneous imaging of multiple tracers, since different isotopes emit 7-rays of different energies. Using PET, tracer detection can only be performed in series. The two y-rays produced under positron emission have nearly identical energies and thus, cannot be modulated to distinguish multiple molecular events occurring simultaneously. Applications of PET and SPECT in molecular imaging involve receptor-ligand interactions and reporter-gene expression in which radiolabeled tracers are selectively internalized by certain cell populations.
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In order to achieve the highest spatial resolution for molecular imaging applications, researchers have explored the use of magnetic resonance imaging (MRI) in conjunction with tailored molecular probes. The underlying principle of MRI is that unpaired nuclear spins, or magnetic dipoles, align themselves when exposed to a magnetic field. A temporary radiofrequency (RF) pulse then perturbs the nuclear spin alignment. The time required for the magnetic dipole to return to the baseline orientation is then measured using the same RF coil that generated the disruption pulse. This relaxation time is the quantifiable parameter represented in the MRI image. MRI scanners incorporate both a strong magnet to generate the magnetic field as well as a RF coil to detect changes in magnetic dipole alignment. MRI is sensitive to soft-tissue differences and provides a spatial resolution of 25-100 ptm and no limit to the penetration depth. However, temporal resolution is on the order of minutes to hours for one complete scan. MRI on the molecular level can be accommodated by the use of paramagnetic metal cations or superparamagnetic nanoparticles that are modified for targeting specificity. With the use of contrast agents such as chelated gadolinium or dysprosium, physiological/molecular characterization and anatomical information can be extracted simultaneously. The distinct disadvantage to MRI is that it is several orders of magnitude less sensitive than radionuclide and optical techniques. Signal-to-noise ratio can be improved with the use of high-powered magnets that generate fields as high as 14 T. However, the cost of such a scanner becomes prohibitive. One recent application in molecular imaging involves receptorbased MRI imaging, where transferrin-monocrystalline iron oxide nanoparticle probes were used to identify cell populations that overexpressed engineered transferrin receptors (TfR) in vivo,,.
Computed tomography is another widely-used clinical imaging system. CT images are constructed from the differential absorption of X-rays as they pass through component tissues in the body. Small animal CT scanners rely on high-resolution phosphor screen/CCD detectors to improve image quality. Typical spatial resolution is on the order of 50-200 Pm with no limit in penetration depth. However, CT has been used principally for morphological characterization. It is not as sensitive as MRI in detecting soft-tissue contrast, necessitating the administration of iodinated contrast media to delineate anatomical features. Limited molecular
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applications have been explored for CT due to the inherent obstacles in adapting the technology. Recent research has focused on developing CT-based molecular probes that are tagged with X-ray absorbing species to provide contrast enhancement. Aside from issues with cell uptake and tissue accumulation of these probes, CT imaging exposes the subject to a significant radiation dose that precludes repeated scans of the same subject. Adverse health risks and biological interactions have made CT a less attractive target for developing molecular imaging techniques. However, in cases where tissue uptake is non-limiting, such as in bone and tumor, CT imaging with contrast media can provide a highly detailed anatomical view.
Ultrasonography has emerged as the most prevalent clinical imaging modality used today. Advantages in using ultrasound include its low cost, availability, and safety. Highfrequency sound waves are emitted from a transducer placed against the skin. Reflected sound waves from internal tissue structures are processed based on their backscatter and attenuation properties using a number of algorithms to produce a contrast image. Ultrasound applications have spatial resolution in the range of 50-500 ptm and a penetration depth on the order of millimeters to centimeters. Molecular-level imaging with ultrasound has not progressed as rapidly as applications in MRI and optical techniques, because of its traditional role as a means for morphological characterization. However, echo contrast agents have been produced that enable molecular imaging of cell-surface receptors. Acoustic nanoparticles are modified with ligand molecules specific to the target receptor. These molecular probes increase the regional echogenicity of the cell surface once receptor-ligand binding is achieved.
Optical imaging is another class of imaging modalities that is being explored for use with fluorescent and bioluminescent probes to detect biochemical processes in vivo. For many years, optical imaging techniques have been the mainstay for applications in molecular and cell biology. Light photons serve as the signal source and are converted into an electrical charge pattern through a charge coupled device (CCD) detector. CCD chips have high sensitivity to light and can be cooled to reduce thermal noise for an improved signal-to-noise ratio.
Bioluminescence imaging relies on the emission of photons by either endogenous or exogenous molecular species. It differs from fluorescence imaging in that no excitation source is required to trigger light emission. One common application of bioluminescence is in the use of Firefly
-19 -

luciferase, an enzyme that oxidizes D-luciferin into a luminescent molecule, as a reporter for gene expression. The Firefly luciferase gene (Fluc) can be expressed in selected cell populations
by transfection. Bioluminescence activation is then achieved through systemic administration of the reporter probe, D-luciferin, and its oxidation by intracellular luciferase. In this manner, only cells expressing Fluc will exhibit luminescence upon substrate administration. This reporter gene/probe strategy may be particularly useful in cell trafficking studies for tracking metastases and monitoring cell proliferation and gene expression as a function of local environmental stresses or protein-protein interactions. A similar approach can also be used in fluorescence imaging. Fluorescent molecules are conjugated to proteins or ligands of interest.
Selective cellular uptake via receptor-ligand interactions or activation via the target biochemical reaction results in a concentrated localization of fluorescent markers. Spatial and temporal resolutions for both bioluminescence and fluorescence imaging are comparable. However, bioluminescence detection exhibits higher sensitivity due to minimal background luminescence.
Fluorescence imaging in intact living subjects suffers from tissue autofluorescence, which produces a much higher background signal. Optical imaging coupled with novel molecular probes has emerged as one of the more developed forms of molecular imaging. This phenomenon is due in large part to the ease with which fluorescence and luminescence-based in
vitro assays have been adapted to the needs of in vivo imaging. However, the major drawback in all forms of optical imaging is the limited penetration depth of light. Bioluminescence imaging has sufficient detection capabilities to a depth of 1-2 cm, whereas fluorescence imaging can only detect molecular probes at a depth of less than 1 cm. Light is easily absorbed and scattered through tissue sections, but judicious selection of the operating wavelengths can increase optical transmission. The near-infrared wavelength range exhibits optimal transmission properties due to low interaction with the main absorbing species in tissue, hemoglobin and water.
The promise of molecular imaging is in its ability to bridge the divide between clinical imaging techniques and molecular and cell biology tools. Both research areas have experienced tremendous growth and innovation within recent years and are poised to forge a new paradigm in medicine. Clinical medicine has embraced the concept of in vitro diagnostics, in which
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biological discoveries in the laboratory are being translated into clinically relevant assays. The next logical step is to bring the same strategy and approach to the development of in vivo diagnostics and monitoring. For many disease processes, pathogenesis, progression, and effects of treatment are poorly understood at the systemic level, the point at which most clinicians interact with their patients. Molecular imaging also bears tremendous scientific implications.
Biochemical pathways that were elucidated in isolated cell culture systems could be visualized at the ultimate scale with complete physiological input. It is anticipated that this new technique will raise new and interesting questions in biomedicine as well as increase the number of targets available for medical treatments. All of the imaging modalities described in this section have come into the standard repertoire of medical care. However, only a few of these lend themselves readily to adaptation for molecular imaging applications. Because the ultimate goal is microscale imaging of biomolecular events in live human subjects, many obstacles and health concerns must be addressed to achieve successful implementation. The work described herein tackles the design and optimization of molecular probes for use with a novel optical imaging platform that overcomes some of the traditional barriers to in vivo monitoring with light.
The objective of this thesis was to develop novel molecular imaging probes for the detection and characterization of prostate cancer in vivo. Prostate cancer is a well studied disease in clinical medicine with the important distinction that it expresses a widely accepted biomolecular marker in its initial stages of progression. We have developed a novel imaging strategy that combines tools from a number of research disciplines. As with all current molecular imaging approaches, there needs to be reliable imaging hardware and a specific contrast agent that enhances the signal-to-background ratio for the target molecular event. This thesis describes work relevant to the optimization of molecular probes used to identify and quantify PSA concentration as a marker for prostate cancer. The molecular probes were designed for use in fluorescence imaging, utilizing fluorescence resonance energy transfer
(FRET) to improve signal-to-background ratio and thus detection sensitivity. Key parameters in
-21 -

fluorescence spectroscopy are discussed and characterized with respect to fluorophores used in probe design. Experiments with purified human PSA and human prostate cancer cell lines are described and used to determine features of an optimal FRET probe.
-22 -

The prostate is a walnut-sized gland situated below the bladder (Figure 2-1) in the genitourinary region of the male anatomy. The structure of the prostate is comprised of 30-50 glands arranged in acini. The acini formation aids in channeling glandular secretions into the prostatic urethra. These secretions are milky and white, forming the bulk of semen, and help to maintain sperm function and motility by establishing an alkaline environment. The normal prostate has typical dimensions of 3-4 cm at the base, 4-6 cm along the cephalocaudad axis, and
2-3 cm in the anteroposterior domain
12
. The main arterial blood supply enters the prostate as branches of the internal iliac artery with drainage into the dorsal venous complex.
Bladder
Pubic bone
Urethra
Penis
1- Seminal vesicle k Prostate gland
-
-Rectum rg|||||y scroturn
Figure 2-1 Genitourinary anatomy with emphasis on prostate gland Courtesy of The Prostate Centre
(www.library.utoronto.ca/medicine/prostate/index.html), 2000
-23-

McNeal identified four histologically and biologically distinct regions in the prostate: the transition zone, peripheral zone, central zone, and the anterior fibromuscular stroma
13
. Figure
2-2 illustrates the four prostatic regions as described by McNeal. Using anatomic structures within the prostate gland as reference points, he also developed measurement parameters for cancers that develop in the transition zone (TZ) and the peripheral zone (PZ)14. The anterior fibromuscular stroma is composed of nonglandular tissue and extends from the bladder neck at the superior aspect to join the urethra at the prostate apex. It is comprised mostly of large bundles of smooth muscle cells. In the glandular regions of the prostate, the ducts and acini are arranged to function as distensible secretory reservoirs
15 . The main ducts of the prostate begin at the urethra and end near the capsule, except in the transition zone where the ducts terminate at the anterior fibromuscular stroma. Acini and ducts are usually uniformly spaced throughout the glandular zones, exhibiting similar calibers. In the epithelial compartment of the prostate, the secretory cells are separated from the basement membrane and stroma by a layer of basal cells. These cells produce a number of proteins found in seminal plasma, such as PSA and prostatic acid phosphatase (PAP). The central zone demonstrates differences in its secretory proteins. Studies suggest that this region is responsible for the production of enzymes, such as pepsinogen II and tissue plasminogen activator (tPA). Substrates for these enzymes are purportedly produced by secretory cells in the peripheral zone. Each zone within the prostate exhibit generalized morphology with various modifications in architecture and function that render it specific to its particular biological role.
-24-

OCC
Figure 2-2 Views of the prostate demonstrating the central zone (CZ), peripheral zone (PZ), transition zone (TZ), and anterior fibromuscular stroma (fm) (McNeal, 1988)
Common prostate pathologies include benign prostatic hyperplasia (BPH), prostate intraepithelial neoplasia (PIN), and cancer. BPH describes an age-related enlargement of the prostate, resulting from multifocal proliferation of glandular tissue into a benign tumor. This characteristic process in BPH produces multinodular, rather than diffuse, hyperplasia in both prostate stroma and epithelium. Both autopsy and radical prostatectomy studies have shown primary hyperplasia to be caused by budding and branching of acini and ductal structures lined with epithelial cells1
6
. Nodules often originate in the transition zone, where the predominant tissue involvement is with glands and stroma in this region. McNeal observed that nodules in periurethral tissue were few in number, small in size, and predominantly fibroblastic
7
. Because transition zone and periurethral tissues differentiate during prostatic embryogenesis, McNeal proposed that BPH may result from a reawakening of inductive interactions between incompletely developed prostatic glands and nonprostatic stroma. Cellular changes that occur in BPH include an increase in fibroblasts and nonmuscle myosin heavy chain which may be indicative of early-stage smooth muscle cell proliferation
8
. There is still much debate in the
-25-

literature as to whether smooth muscle content is increased or decreased in BPH, as nodules are often heterogeneous with tissue composition changing over time. Studies show evidence that there are a number of peptide growth factors, such as fibroblast growth factor (FGF), insulinlike growth factor (IGF), and transforming growth factor (TGF), that may stimulate tissue proliferation.
Prostatic intraepithelial neoplasia (PIN) represents the most significant risk factor for prostate cancer, as it is considered a precursor lesion to carcinoma. All men eventually develop
PIN as they age, but only a subset of this population develops prostate cancer within their lifespan. Findings associated with PIN include cellular proliferation within pre-existing ducts and glands with nuclear and nucleolar enlargement and expansion of pre-existing glands similar to PCa, but the basement membrane and basal cell layer are usually grossly intact with some fragmentation
19
. PIN is classified as either high grade (HGPIN) or low grade (LGPIN).
Pathologists rarely make note of LGPIN and focus on characterizing HGPIN lesions with histological findings and often immunohistochemical studies. HGPIN can exhibit four main architectural patterns: tufting, micropapillary, cribriform, and flat. These patterns describe the arrangement of acinar epithelial cells and have not shown any clinical significance with regards to prognosis. HGPIN can often be identified with proliferative changes that create a hyperchromatic appearance under histological examination. In situ measurements of telomere lengths in fixed and sectioned prostate specimens have shown that telomeres are shorter in luminal cells of HGPIN lesions than in basal cells of the same specimen
20
. Telomere shortening is often a manifestation of chromosomal instability and may play a role in human tumorigenesis. The changes found in luminal epithelial cells may predispose them to further neoplastic transformation.
PIN exhibits many of the same morphologic and molecular genetic changes that are observed in prostate cancer. These two lesions often colocalize within the same prostate zone, with a higher frequency of HGPIN in prostates that contain cancer. A recent study has demonstrated an immunohistochemical target for differentiating between normal prostate, PIN, and PCa 2 1 . Figure 2-3 illustrates a histological section from prostate that contains all three states stained for a-methylacyl-coenzyme A racemase (AMACR). AMACR is upregulated in prostate
-26
-

cancer and highlights the presence, and not necessarily the aggressiveness, of the cancer. The glandular structure of the cancerous lesion seen in Figure 2-3 is abnormally round to oval as compared to the normal tissue section. Adenocarcinoma encompasses over 95% of diagnosed prostate cancers (Smith's General Urology, 2004); other types of prostate cancer include transitional cell carcinomas and neuroendocrine, or small cell, carcinomas or sarcomas.
Hyperchromasia, increased nuclear-to-cytoplasm ratio, and prominent nucleoli are the dominant cytologic characteristics in prostate cancer. In addition, the distinguishing feature between PCa and PIN is that there is an absence of the basal cell layer in PCa, as seen via keratin immunohistochemical staining. Nearly 60-70% of prostate adenocarcinomas arise in the peripheral zone, followed by 10-20% incidence in the transition zone and 5-10% incidence in the central zone (Smith's General Urology, 2004). Besides the known epidemiological risk factors for prostate cancer, studies suggest that certain susceptibility genes related to infectious activation, such as RNASEL, an endoribonuclease component of an interferon inducible RNA degradation pathway activated upon viral infection, exposure to dietary and inflammatory oxidants and electrophiles can also predispose an individual to prostate cancer
22
. There is renewed interest among the prostate cancer community in the role of inflammation in the pathogenesis of PCa.
Despite confounding factors in the epidemiological study of inflammation associated with PCa, correlations between an increase in prostate cancer risk and symptomatic prostatitis and sexually transmitted infections have been demonstrated (Nelson et al., 2004). Abnormal androgen metabolism can also predispose an individual to prostate cancer, as androgens, primarily testosterone and its metabolite, dihydrotestosterone, are often required for the growth, maintenance and function of prostate cancer cells. Bostwick and colleagues have recently published a comprehensive review of prostate cancer risk factors, so this section will not reiterate the findings but instead, refers the reader to the article for further insight
23
.
The primary site of prostate cancer metastasis is bone. Nearly 70% of patients with advanced breast or prostate cancer have metastatic bone lesions
24
. Bone metastases derived from prostate cancer are more likely to be osteoblastic lesions, whereas bone metastases derived from breast cancer are more often osteolytic lesions. Proposed theories regarding metastatic preference to bone suggest that high blood flow in red marrow predisposes this region to
-27-

metastatic lesion formation. Studies also indicate that prostate cancer cells secrete or display molecules on their cell surfaces that make them greatly adhesive to bone marrow, followed by growth stimulation with the numerous growth factors that reside in bone. This combination of favorable conditions may explain the prevalence of bone metastases found in advanced prostate cancer.
V
P N
LI
Figure 2-3 Immunohistochemical staining for alpha-methylacyl-coenzyme A racemase (AMACR) in normal prostate tissue (ni), prostatic intraepithelial neoplasia (PIN), and prostate cancer (CaP) (De Marzo
et al., 2003)
-28-

PSA is a biomolecular marker that has gained wide clinical acceptance in prostate cancer screening and monitoring , 2
6
. Clinical studies have determined that a cutoff value of 4.0 ng/ml for PSA levels aids in the detection of suspicious lesions 2
7
,2
8
.
Use of a lower cutoff value of 2.6 ng/ml has recently been reported to increase the detection rate for small, organ-confined tumors without becoming oversensitive to 'clinically insignificant' disease29. Produced primarily by prostate epithelium, PSA is an androgen-regulated serine protease that acts to cleave semenogelins in the seminal coagulum. One study has hypothesized that elevated serum PSA in pathologic states, primarily metastatic prostate cancer, is mediated by the biochemical breakdown of glandular and capillary basement membrane and stroma
3
O. The disruption of extracellular matrix components as well as the endothelial cell layer allows PSA to effectively
'leak' into the microvasculature, primarily as a result of prostate cancer. The most recent studies suggest that the physiological functions of PSA have both beneficial and deleterious effects on cancer progression. PSA is an insulin-like growth factor binding protein-3 (IGFBP-3) protease
3 1 . IGFBP-3 acts as a carrier protein for insulin-like growth factors (IGFs) in human seminal plasma. Once IGFBP-3 is cleaved by PSA, IGF-I loses significant affinity for IGFBP-3 and is able to stimulate increased mitogenic activity and growth in prostate cancer cells 32 . PSA also acts to cleave extracellular matrix glycoproteins, such as fibronectin and laminin, to promote cancer cell invasion in metastatic disease33. Despite indications that PSA enables prostate cancer cells to proliferate and metastasize, PSA expression in xenograft prostate tumors derived from the PC-3 cell line failed to increase tumor size as compared to wild type tumors that do not express PSA-'. The PSA protein secreted by transfected PC-3 cells was not enzymatically active, further supporting the idea that proteolytic action by PSA is an important function in cancer pathogenesis. Prostate cancer metastases occur predominantly in bone with primary osteoblastic lesions, although there is increasing evidence that suggests an interplay between osteoblastic and osteoclastic activity 3
5
. PSA appears to play a role in prostate cancer cell adhesion to bone marrow endothelial cells, which is presumably the first step towards invasion and metastasis. Addition of active, exogenous PSA increased cell-cell adhesion,
-29-

whereas antibodies to PSA and downregulation of PSA mRNA expression using small interfering RNA (siRNA) attenuated cell-cell interactions 3 6
.
Osteoblastic lesions can also promote prostate cancer cell proliferation and PSA expression through an androgenindependent pathway, further feeding the metastatic potential
7
. There is also evidence that
PSA promotes antiangiogenic activity by converting Lys-plasminogen into angiostatin-like fragments through proteolysis. These fragments, when purified, inhibit proliferation and tubular formation in human umbilical vein endothelial cells (HUVECs)
38.
Interestingly, recombinant PSA that has been enzymatically inactivated through deletion of the first amino acid, inhibits angiogenesis in vivo to a similar degree as active recombinant and native PSA 3
9
.
PSA is expressed in mammalian cells as an inactive pro-form, containing 244 amino acidso
41
. Once the pro-PSA is secreted into the lumen of the prostate, proteases in the seminal fluid cleave seven amino acids from the N-terminus to yield the enzymatically-active form of
PSA. Activation by trypsin, human glandular kallikrein (hK2), and prostin has been demonstrated and releases a pro-sequence of Ala-Pro-Leu-Ile-Leu-Ser-Arg
42 43
. PSA was first discovered and characterized as having a molecular weight of 33-34 kDa and an isoelectric point of 6.94. It can become complexed with several proteins in human serum, primarily ciiantichymotrypsin (ACT), a2-macroglobulin (a2M), and protein C inhibitor (PCI). Although PSA and ACT are found in human serum, prostatic fluid, and seminal plasma, PSA-ACT complex was found only in human serum by Western blot
45
. Qian et al. further discovered that the addition of exogenous ACT to prostatic fluid yielded quantities of PSA-ACT complex that varied with the amount of ACT added. However, the addition of exogenous PSA failed to produce the complex. This indicated that PSA is biologically active in prostatic fluid where
ACT exists in an inactive form. Espafia et al. found varying levels of all complexes (PSA-ACT,
PSA-a2M, and PSA-PCI) in prostatic fluid, seminal plasma, and seminal vesicle fluid using a much more sensitive sandwich ELISA4.
The primary interest in elucidating the different molecular forms of PSA is the potential application towards improving clinical diagnostics for prostate cancer. Prostate pathologies fall under several classifications, which include prostate cancer (PCa), prostate intraepithelial neoplasia (PIN), and benign prostatic hyperplasia (BPH). Significant efforts have been made
-30-

towards identifying diagnostic tools that can distinguish between benign and malignant lesions with greater specificity than total PSA. Stenman et al. reported that PSA-ACT is the predominant form of serum PSA in patients with prostate cancer; patients with BPH had a significantly lower level of PSA-ACT 47 . The ratio between PSA-ACT complex and total PSA has been used to further discriminate between these two pathologic states. In men with high serum levels of total PSA (>10 ng/ml), this ratio, at a cutoff of 0.62, becomes a highly sensitive and specific test for prostate cancer
48
. In the intermediate tPSA range of 4.1-20 ng/ml, a ratio of PSA-
ACT to prostate volume, known as ACT density, is the most reliable predictor of cancer as compared to tPSA alone
49
. Other molecular forms of PSA, primarily free PSA (fPSA), when expressed as a ratio against total PSA, have also proven valuable in positively identifying PCa and eliminating unnecessary prostate biopsies in patients with BPH 0 5
, 2,
3 , even in men with tPSA levels less than 4 ng/ml
54
. Lilja first characterized the enzymatic activity of endogenous
PSA as a serine proteinase with direct action against semenogelin and semenogelin-related proteins
55 56
. When purified from semen, approximately one-third of the PSA was found to be enzymatically inactive
57 . The remaining PSA formed stable complexes with both ACT and a2M, as documented in other reports. Complexation between PSA and ACT was initiated by chymotrypsin-like cleavage of ACT, although at a much slower rate than that observed between chymotrypsin and ACT. PSA and ACT complex formation can be reversed through prolonged incubation at 37*C in vitro to yield free active PSA
58
. Because PSA immunoreactivity is lost when it binds to a2M, presumably due to epitope shielding by the bound (2M, Leinonen et al. identified PSA-a2M complexes by determining the undetected fraction of PSA in a total mixture.
The undetected fraction, presumed to be PSA complexed with a2M, is 66% of the total PSA content. This result led to the conclusion that a2M is the major inhibitor to PSA in serum.
The enzymatic activity of PSA is highly dependent on its molecular form and complex state. Uncomplexed PSA, also known as free PSA, can act freely to cleave specific substrates.
Subsequently, fPSA has become the standard measure of enzymatically active PSA. Clinical measurements of serum PSA level are typically performed using the Tandem-R assay
(Hybritech/Beckman-Coulter, Brea, CA) and reflect the amount of total PSA, which includes
fPSA and PSA-ACT complex (PSA-a2M is undetectable using these assays). To evaluate the
-
31
-

levels of fPSA in the various fluid compartments accessible to the prostate, Denmeade et al. examined extracellular fluid and serum from human patients (normal and prostate cancer) as well as from tumor xenografts. Within the patient group, the researchers found that total PSA in the extracellular fluid, as measured by the Tandem-R and Tandem-MP assays, did not vary significantly between normal and diseased individuals. However, 89% of the total PSA in patients with prostate cancer was enzymatically active versus 78% for the normal individuals.
In addition, total serum PSA levels were extremely low in both groups with no enzymatically active fractions
9
. Tumor xenografts in nude mice demonstrated that only 18% of PSA secreted
by LNCaP cells are enzymatically active, as compared to 66% from PC-82 cells. In all cases, whether human primary tumors or xenograft, no active PSA was found in the serum. The absence of active PSA in human serum presents an unique opportunity to deliver proteolytically-activated fluorescent contrast agents, such as the ones described in this work, intravenously without fear of extra-glandular activation. Moreover, PSA enzymatic activity appears to be a better metric for differentiating between normal and disease states in patients than total protein level. PSA substrate specificity has been characterized as chymotrypsin-like towards its main physiological substrate, semenogelin I. Cleavage sites were shown to be predominantly after tyrosine or leucine residues
60
. Serine, glutamine, and aspartic acid have also been found at the P-1 position
1
. Malm et al. confirmed reports that divalent cations, particularly zinc, act to inhibit PSA proteolytic activity towards semenogelin-I as well as PSA complexation with ACT. However, Robert et al. demonstrated evidence that the enzyme inhibitory effect of zinc may be attributed to zinc binding to semenogelin-I and not PSA. In recent research, PSA, SgI, and SgII have all demonstrated binding affinity for zinc. Zincinhibited PSA can recover its enzymatic activity with the addition of SgI and SgII, to effect an indirect regulatory mechanism for PSA proteolytic activity 62
.
Historically, many of the synthetic substrates used to evaluate PSA enzymatic activity were chymotrypsin substrates. More specific substrates were necessary for targeted drug delivery applications, so Denmeade et al. evaluated several peptide sequences corresponding to the cleavage map for semenogelin-I and semenogelin-IlI to identify HSSKLQ as the best substrate with high specificity for PSA and low degradation in serum 6 3 .
Yang et al. used single-
-32
-

position minilibraries to determine an optimal hexapeptide substrate for PSA. Several studies had demonstrated increased substrate specificity for tyrosine at position P-1. Based on this data,
PSA was then found to have preference for serine at position Pi and phenylalanine at position P-
2, for a proposed optimal substrate of QFYSSNM. These synthetic substrates were optimized from base peptide sequences chosen arbitrarily from the semenogelin-I cleavage map, and thus, reflect the best substrates from a limited pool of candidates. To truly pan the SgI cleavage map for PSA substrate preferences and rules which govern amino acid arrangements, Coombs and coworkers employed an iterative optimization technique to systematically evaluate substrate efficiency as a function of single-position changes in amino acid residue. As a means of corroborating peptide sequence results from iterative optimization, substrate phage display was implemented and found to converge on SS(Y/F)-S(G/S)
65
.
The most labile substrate found through phage display, GAGLRLSSYY-SGAG, was cleaved by PSA at a kcat:Km value of
3100/Ms, nearly 100-times greater than the kcat:Km value reported for QFYSSN by Yang and coworkers. However, chymotrypsin was able to cleave this same substrate at an efficiency that was 24-times greater than PSA, as measured by kct:Km value. Rehault and coworkers managed to identify SSIYSQTEEQ as the best PSA substrate to date with a kcat:Km value of 60,000/Ms
66
.
These reports, taken together, illustrate the major barrier to developing efficient and specific substrates, an incomplete knowledge of PSA enzymatic mechanisms and interaction with its native substrate. Moreover, even the most optimal substrates presented in the literature demonstrate weak catalytic efficiency, at a rate that is nearly an order of magnitude less than chymotrypsin. Several studies have conjectured that there may be extended site interactions between PSA and its protein substrates which enhance its enzyme activity and which cannot be recapitulated by short peptide substrates. Interestingly, novel PSA-binding peptides have been found, through cyclic phage display, which increase PSA enzyme activity towards a chymotrypsin substrate
67
. Many of these substrates were originally developed for use in clinical assays for PSA. There has also been significant interest in using these substrates as cleavable
69
, ,
70
,
7
1
,
7
2 . Prodrugs have the advantages of reduced systemic toxicity and selective activation in the tumor for site-specific drug action. In this study, PSA-specific substrates are modified with fluorophore pairs that
-33 -

exhibit fluorescence resonance energy transfer to serve as reporter molecules for selective tumor detection.
2.3
Fluorescence describes the physical phenomenon by which a photon is absorbed and re-emitted as a lower energy photon. This process occurs on the scale of 10 ns, with many fluorescent molecules, or fluorophores, exhibiting subnanosecond lifetimes. Fluorophores are generally aromatic compounds that contain one or more benzene rings. A Jablonski diagram is typically used to illustrate the physical phenomenon of light absorption and emission (Figure 2-3). When a fluorescent molecule absorbs light, the energy of excitation shifts the molecule into a higher electronic state. There are several modes of decay that the excited molecule can undergo in its return to the ground state. These processes, known as Stokes' losses, include nonradiative dissipation, phosphorescence via intersystem crossing, fluorescence, and quenching or energy transfer. Each of the individual processes outlined in the Jablonski diagram occurs at different time scales. Light absorption is usually complete within 10-5 s followed by internal conversion
(10-12
s), in which excited state electrons relax to the lowest vibrational level in Si. From its excited state, the molecule can emit a photon (i.e., fluorescence) during its relaxation to the ground state, or not (i.e., quenching or nonradiative relaxation). Quenching can occur through collision between the excited fluorophore and other molecules in solution and through formation of nonfluorescent complexes with quencher molecules. Another pathway for molecules in the excited state is intersystem crossing in which electrons in the excited singlet state undergo spin conversion to a lower energy excited triplet state. Because the transition from a triplet excited state to a singlet ground state is much less probable than a singlet/singlet relaxation, the time scale for phosphorescence, 10-102 s, is many orders of magnitude greater than fluorescence. Competing processes, such as nonradiative decay and quenching, are so
highly effective in converting excited triplet state molecules to ground state that phosphorescence is rarely observed except under highly favorable conditions.
-34-

S
2
S,
I V
Intersystem
Crossing
Internal conversion
Quenching
Nonradiative decay
T, hvA
21
So
-r hvA
V V
V
Fluorescence hvF Phosphorescence hvp
,C.
V
*
Figure 2-4 Jablonski diagram of light absorption and emission
A fluorophore is characterized by two main parameters, quantum yield and fluorescence lifetime. Quantum yield refers to the fraction of absorbed light that is reemitted as a photon and is expressed as
F
Q F + knr where Q is the quantum yield, F is the emissive rate of the fluorophore, and knr is the rate of nonradiative processes. The quantum yield approaches unity when knr<< IF but can never reach unity due to Stokes' losses. Fluorescence lifetime, 'r, is the amount of time that a fluorophore spends in its excited state. It is defined by
F + knr
Both of these parameters are highly dependent on species interaction with the solvent3 and with neighboring molecules that may serve as a quencher. Calorimetric approaches have been used to determine that the absolute fluorescence quantum yields for two standard fluorophores, rhodamine 6G cation and fluorescein dianion, in water are 0.90 ± 0.02 and 0.92 ± 0.02, respectively
7
4. Studies that incorporate trends in fluorescence lifetime and quantum yield as a
35 -

function of solvent polarity can often hint at the underlying mechanisms for nonradiative processes.
One particular nonradiative process, fluorescence resonance energy transfer (FRET), was first described by Forster in the late 1940s
75
. FRET involves nonradiative energy transfer from a donor to an acceptor molecule. The requirement for resonance energy transfer is that the emission spectrum for the donor fluorophore overlaps with the absorption spectrum for the acceptor fluorophore. This also dictates that the fluorophore with higher energy absorption is designated the donor, with the acceptor having lower energy absorption. The energy transfer occurs as a dipole-dipole interaction between donor and acceptor, and not as a photonic exchange between these molecules. The Jablonski diagram in Figure 2-5 illustrates the various decay processes after initial light absorption, highlighting the FRET mechanism. The donor fluorophore is excited at a suitable wavelength to produce intrinsic fluorescence as well as energy transfer to the acceptor fluorophore, which stimulates acceptor fluorescence, also known as sensitized emission. Figure 2-6 provides an illustration of the effective spectral changes that are expected in a donor-acceptor FRET mechanism. The rate of energy transfer can be shown as
kT (r) =1
1_ II R
0
6
Td r where r is the distance between donor and acceptor, td, is the lifetime of the donor independent of the acceptor, and Ro is the Forster distance. The Forster distance represents the physical length scale between donor and acceptor at which energy transfer is 50% efficient and is expressed as
R6 = (
8
.
7 8
5 x 10-)K 2 n d where K
2 is an orientation factor,
4d is the quantum yield for the donor in the absence of the acceptor, J is the normalized spectral overlap integral, and n is the refractive index for the medium. I is calculated from
-36 -

J
= fF (2 C A ( 2))_4
JFD(A)dA d 2 where FD is the dimensionless fluorescence intensity for the donor at the particular wavelength,
k, and &A is the extinction coefficient for the acceptor, typically expressed as M-1 cm-
1
. The major source of uncertainty in any calculation of the Forster distance rests with the value of
12, defined as
K
2
= (COS OT-3cosOD
COS 9A where OT is the angle between the emission transition dipole of the donor and the absorption transition dipole of the acceptor, and OD and OA are the angles between these transition dipoles and the vector joining the two fluorophores (Figure 2-7). The orientation factor,
12 value, can range from 0, when the transition dipoles are perpendicular to each other and the vector between them, to 4, when the three vectors are collinear. Since
12 varies with the sixth power of the Forster distance, Ro, a deviation in the orientation factor from 1 to 4 produces a 26% change in the value for Ro. Consequently, K2 is often estimated at 2/3 to account for donor and acceptor randomization through rotational diffusion, as direct measurement of the orientation factor is impossible at this time. A range of values for
12 can be established by observing the fluorescence anisotropies exhibited by the FRET pair. However, the use of 2/3 for the orientation factor allows for no more than 35% error in calculating Ro.
The efficiency in energy transfer can be expressed in several forms. One method is to consider the efficiency as a generalized 'quantum yield' for energy transfer
76
,
E =
Energy transferred from D to A
Energy absorbed by donor and to introduce the quantities, FD and
FDA, as the steady-state fluorescence intensities for the donor alone and the donor in the presence of acceptor, respectively, both normalized with respect to the concentration of the donor. The efficiency of energy transfer can then be expressed as
-37-

E=1-
(A1
F, or as a ratio of the rate processes
E= kT to yield
1+
RO
Hence, given a known donor-acceptor pair, FRET energy efficiency is a function of the distance between the donor and acceptor only.
-38
-

i I I kD I I I kT
.E*IkF
V1 V
V
V
V D
A ---...
.
.
kFA
A
Figure 2-5 Jablonski diagram demonstrating the FRET mechanism where kD is the rate of dissipation, kF is the rate of donor fluorescence, kT is the rate of energy transfer, and kFA is the rate of acceptor fluorescence (adapted from Szollosi et al., 1998)
T1
.M
A
C
Wavcengt (X)
Figure 2-6 Absorption and emission spectra for energy transfer from D to A where Da is the donor absorption, De is the donor emission, Aa is the acceptor absorption, and Ae is the acceptor emission; note that in the presence of an acceptor molecule, FRET requires spectral overlap between De and Aa to induce a decrease in donor fluorescence and an increase in acceptor emission (Szollosi et al., 1998)
-39
-

9D
'.T OA
Figure 2-7 Relevant angles in defining the orientation factor
Fluorescence has emerged as a highly sensitive modality for imaging and assaying biological activity. The distance-dependent behavior in FRET emission has resulted in its application as a
'spectroscopic ruler' in many biochemical studies7. Effective energy transfer occurs over
Forster distances between 10 A and 100 A, which makes this technique highly suited to probing macromolecular interactions related to biological phenomena. FRET has been particularly useful in studies focused on conformational changes associated with biochemical events such as ligand-receptor binding
78
, DNA hybridization
7 80
, protein folding, membrane protein dynamics, and proteolytic activity. Many interesting problems in biology and clinical medicine center around the identification of endogenous ligands, interaction between extracellular proteins and cell-surface or nuclear receptors, and the intracellular signaling cascade that ensues.
Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated through ligand binding to control the transcription of genes involved in fatty acid oxidation and cell differentiation. PPARs have also been implicated in disease processes, such as diabetes, obesity, and cancer. Consequently, the elucidation of endogenous ligands for one member of the PPAR family, PPARa, and its binding interaction is important in the development of therapeutics targeted at regulating PPARa transcriptional activity. Long chain fatty acids (LCFAs) and LCFA-CoAs were proposed as natural ligands for PPARa and
-40-

demonstrated to bind with PPARa at an intermolecular distance of -25
A using FRET between the aromatic amino acid residues of PPARa and the ligands 81 . The close binding interaction between the ligands, cis-parinaroyl-CoA and trans-parinaric acid, and PPARa suggested specific binding and helped to confirm that LCFA-CoAs and unsaturated LCFAs exhibit high binding affinity for PPARa. When there is sufficient data on both agonist and receptor, structure-based drug design is possible with additional insight provided by the FRET technique.
Type A cholecystokinin (CCK) receptor is a G-protein coupled receptor that responds to a linear peptide hormone by regulating nutritional homeostasis, pancreatic exocrine secretion, and satiety. The structure of the receptor and amino acid sequence had been elucidated previously, so Harikumar and coworkers expressed several mutant CCK receptors containing monoreactive cysteine residues in Chinese hamster ovary (CHO) cell lines. The receptor constructs were prepared such that each mutant had a single reactive cysteine residue within one of the four regions of interest, the transmembrane helix and the three extracellular loops.
These modified cysteine residues were then tagged with Alexa568 as the acceptor molecule, while the hormone peptide, CCK, was labeled at the N-terminus with Alexa488 to provide a
FRET pairing. The study investigated the intermolecular distance between the peptide agonist and each of the four receptor domains to shed light on the conformational changes that occur when the receptor binds ligand
2
. The results concurred with a computational model of protein interaction that had been derived independently.
G-protein-coupled receptors are particularly interesting because they are involved in many important signaling cascades. Activated receptors will catalyze the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). The Py subunit then dissociates from the GTP-bound a subunit to effect downstream signaling. Janetopoulos and coworkers
8 3 introduced Ga2-cyan fluorescent protein (CFP) and Go-yellow fluorescent protein
(YFP) fusion proteins in Dictyostelium discoideum to observe the subunit interactions during G- protein activation in living cells. When treated with chemoattractant cyclic adenosine 3',5' monophosphate (cAMP), D. discoideum coordinates a physiological response to migrate, partially through GPCRs. cAMP triggered a decrease in the FRET-associated fluorescence signal in a dose-dependent manner, confirming the purported dissociation between Ga2 and
-41
-

Go subunits after G-protein activation. This study represented the first direct observation of G- protein activation in living cells. Genetically encoded fluorescent proteins, such as GFP, CFP, and YFP, have allowed unparalleled access to live-cell FRET imaging. This technique preserves cell membrane integrity and allows for nondestructive evaluation of biochemical signaling.
Methods which rely on antibodies directed towards proteins that become altered through a signal cascade, such as growth factor-induced phosphorylation
84 , require cell fixation before imaging and are impractical for observing dynamic processes that have length scales much smaller than the size of an antibody (i.e., DNA hybridization). Direct transfection of a gene construct expressing a fluorescent protein into the cell lines of interest gives researchers greater control over their experimental models. Tsien and many others in the field have been working for the last decade to expand the arsenal of engineered fluorescent proteins to span the visible range (emission wavelengths from 440 nm to 648 nm)
85
. CFP and YFP, mutants of GFP, remain the best FRET pairing among fluorescent proteins. Ting and coworkers used this pairing to demonstrate a general strategy for constructing genetically encoded indicator proteins for several tyrosine kinases
86
. Each indicator protein contained a substrate peptide joined to a phosphotyrosine binding protein via a peptide linker. This construct was then sandwiched between CFP and YFP. Each phosphorylation substrate was selected based on specificity for tyrosine kinases, Src, epidermal growth factor receptor (EGFR), or Abl. Once the kinase phosphorylates the appropriate substrate in the presence of adenosine triphosphate
(ATP), a conformational change is precipitated by high affinity between the newly phosphorylated tyrosine and the phosphotyrosine binding protein. The conformational change brings CFP and YFP into closer proximity, introducing an increase in sensitized fluorescence emission via FRET at 527 nm when CFP is excited at 433 nm. Addition of a phosphatase reverses the FRET change. Purified indicator proteins, in this study, experienced a 25-35% increase in fluorescence emission ratio between YFP and CFP when treated in vitro with Src,
EGFR, and Abl. This same response was observed over an approximate three minute period when mouse fibroblasts, expressing the indicator protein for EGFR, were stimulated with extracellular EGF. The wave of FRET change within a single fibroblast was seen to propagate from the plasma membrane into the cytosol. Removal of EGF or addition of EGFR inhibitor
-42
-

could return the emission ratio to its original unperturbed level. Under the same method for intracellular observation, Ting and coworkers found evidence that there is elevated Abl activity in the ruffles of PDGF-stimulated cells to indicate an as-yet-undetermined role of Abl in cell morphology. These observations demonstrate the power of FRET-based techniques to provide time and spatial resolution in characterizing dynamic processes.
Similar FRET-based approaches have been used to study DNA interactions. Donor and acceptor fluorophores can be introduced into oligonucleotides (ODNs) by the use of an aminohexyl linker on the 5' end or an aminohexylamino linker at the 3' end. Nucleic acid hybridization in solution can be monitored using this technique
87 as well as intracellularly through microinjection of oligonucleotides. A 28-mer phosphodiester oligonucleotide was functionalized at the 3' end with rhodamine (+PD-R) while the complementary, antisense phosphorothioate ODN was labeled with fluorescein at the 5' end (-PT-F)
88
. This FRET pair allowed researchers to observe the specific quenching of fluorescein emission after the addition of the rhodamine-labeled sense strand. Pre-formed -PT-F/+PD-R hybrids at 1:1 molar ratio were microinjected into mouse 3T3 cells, and fluorescence was observed over a time course.
The -PT-F/+PD-R hybrids dissociated intracellularly in the cytosol, possibly as a result of disruption or degradation by proteins or cytoplasmic nucleases. Transient hybridization between -PT-F and +PD-R was demonstrated in the nucleus following time-lagged serial injections of the two ODN constructs. Another approach to FRET measurements has been the use of fluorophore-quencher pairs. In this construct, the excited fluorophore transfers nonradiative energy to the quencher as in traditional FRET applications, but the quencher is a nonfluorescent chromophore that converts the transferred energy into heat.
Tyagi and Kramer first introduced the use of a fluorophore-quencher pairing to monitor
DNA hybridization through the concept of 'molecular beacons' in 199689. The use of a hairpin loop of DNA synthesized with complementary arm sequences allows for controlled positioning of the fluorophore, 5-(2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), and the quencher, 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) at the 5' and 3' ends, respectively. The chromophores were attached to the ends of the DNA strand via linkers. In its native state, prior to the addition of a complementary strand, the molecular beacon is self-
-43
-

annealed along its arm sequence to hold the fluorophore and quencher in close proximity to each other. Once its target is introduced into solution, the energy of hybridization overcomes the bonds holding the stem together to open the molecular beacon and separate the fluorophore from the quencher. Tyagi and Kramer observed a rise in EDANS fluorescence after hybridization (-25-fold in solution with target oligodeoxyribonucleotide at temperatures below the melting point of the stem structure). The use of RNA targets instead of DNA targets could also induce this same conformational change as evidenced by a rise in EDANS fluorescence.
Disruption of this RNA:DNA hybrid by addition of ribonuclease H effectively returned the
EDANS fluorescence signal back to low levels. This is a particularly attractive technique as the molecular beacon can be reversibly activated (i.e., fluorescence signal exists only in the presence of target and can be exposed to target repeatedly without loss of signal). Molecular beacons can be used to monitor polymerase chain reaction (PCR) in real time.
Another form of FRET-based sensing that has shown accelerated progress over recent years is in the detection of proteolysis. Similar to DNA in its finite alphabet and ease of synthesis, peptides have become the favored template for developing FRET probes for enzyme activity. A typical probe is designed with donor and acceptor fluorophores at either end of a peptide substrate. In its pre-digested form, the peptide conformation defines the distance between donor and acceptor (or fluorophore and quencher, in some cases) and allows efficient energy transfer between the two chromophores. After the probe is cleaved by the target enzyme, the FRET pair is no longer associated with each other via the peptide and loses enhanced acceptor emission related to energy transfer, or fluorescence suppression by the quencher. Disease-associated enzymes have been the target for many FRET probes in efforts to develop specific and sensitive clinical diagnostic assays. Matrix metalloproteinases (MMPs) have been implicated in cancer progression, as their main action is to break down major protein components of the extracellular matrix. One strategy for a MMP-specific FRET substrate has been to modify known substrates for MMP to include a tryptophan (Trp) residue at the P2' subsite as the fluorescent moiety and attach dinitrophenol (DNP) to the N-terminus as the quenching group
0
. Subsequent studies revealed that MMP proteolysis favors Trp in the P2' subsite, allowing these FRET substrates to be highly sensitive to MMP activity.
-44-

A similar design strategy was used in a detection scheme for Bacillus anthracis lethal factor (LF) protease
91
. A consensus substrate, composed of the shared sequence elements between all of the natural substrates for LF protease, was modified with several fluorophorequencher pairs at the N- and C-termini to determine the optimal pairing. The substrate with the largest signal:background ratio (16) at 100% cleavage was the consensus sequence appended with the fluorophore, coumarin, at the N-terminus and a quencher, QSY-35, attached to a distal lysine c-amino side chain. The addition of two glycine residues between the C-terminus of the consensus sequence and the resin was an attempt to space the lysyl side chain away for the resin and provide greater accessibility for labeling reactions. Interestingly, the same consensus sequence appended with QSY-35 at the N-terminus and coumarin on the lysine side chain yielded a signal:background ratio of only 10 at 100% cleavage. This observation indicates that there may be secondary interactions after proteolysis between coumarin and internal residues that may suppress its intrinsic fluorescence even in the absence of the quencher. Much of the literature on FRET applications in protease detection centers on double-labeled peptide substrates such as those described above.
Another strategy in constructing a FRET probe that is sensitive to biochemical modifications is to modulate the spectral overlap integral between the donor and acceptor fluorophores. Takakusa and coworkers demonstrated a novel method of FRET 'switching' by conversion of fluorescein from a lactone to a quinoid form after dephosphorylation by a protein tyrosine phosphatase (PTP)
92
. The fluorescein molecule acted as an acceptor to a coumarin dye.
These two fluorophores were held together via a rigid cyclohexane linker to prevent dye-to-dye contact and any changes in fluorescent properties that may ensue from dye complex formation.
Prior to PTP-induced hydrolysis, fluorescein is in the lactone form with minor absorption in the
UV region. Coumarin exhibits strongest fluorescence emission around 450 nm and no appreciable spectral overlap with fluorescein absorption while in the lactone form. In this state, there is no energy transfer between coumarin and fluorescein. However, once PTP removes the phosphate protecting groups, the fluorescein structure changes into the quinoid form. In this form, fluorescein has strong absorption in the emission region for courmarin. The increase in spectral overlap between coumarin emission and fluorescein absorption produces FRET-based
-45 -

sensitized emission at 515 nm and a decrease in donor fluorescence at 450 nm. These PTPsensitive FRET probes employ spectral overlap switching to shift emission wavelengths and represent a novel approach to biochemically-triggered indicator molecules.
2.5
Self-reporting constructs that provide real-time monitoring of biochemical phenomena are invaluable as in vitro tools for understanding the dynamic processes that determine cellular function and interaction with extracellular species. They are also gaining new applications in clinical diagnostics as well as in vivo detection. Weissleder and coworkers have established a new era in molecular imaging with activatable near-infrared fluorescent (NIRF) probes
93
.
Similar to the FRET probes used to determine MMP enzymatic activity in vitro, these NIRF conjugates have demonstrated specificity for their enzyme targets during in vivo whole animal imaging. The use of near-infrared fluorophores maximizes on the ability of light in this wavelength range to transmit through tissue with reduced absorption. NIRF conjugates specific for MMP-2 were developed and used to image MMP-expressing tumors in nude mice
94
. Bremer and coworkers were also able to demonstrate in these same mice that administration of MMP inhibitor effectively reduced MMP activity as evidenced by reduced activation of MMP-specific
NIRF conjugates and low fluorescence signal in the tumor.
In this study, the primary interest is in the development of sensitive and specific fluorescent indicator molecules for prostate cancer detection. PSA is a well-characterized biomarker for prostate cancer that is already used in clinical screening. While many studies have compared and optimized natural as well as synthetic substrates for PSA in the development of clinical diagnostic assays, this study examines the issues surrounding the design and synthesis of a FRET probe for PSA, characterizes the proteolytic dynamics with respect to substrate modifications, and proposes an optimal configuration and synthesis route for the probe. This thesis represents the first step towards developing prostate cancer specific imaging probes for in vivo detection.
-46
-

The use of FRET substrates required knowledge of the fluorophore and dye molecules used in the design of the molecular probes. Due to cost restrictions, design iterations were performed to select for the FRET substrate that exhibited the greatest signal-to-background response to
PSA. First-generation substrates were modified with fluorescein and tetramethylrhodamine
(TMR) as the donor-acceptor pair. Second-generation substrates were then designed using the best peptides from the first iteration and modifying them with dye-quencher pairs. These dyequencher pairs included near-infrared fluorescent molecules such as Cy5.5 and Alexa Fluor® 680 paired with a dark quencher molecule such as Cy7Q and BHQ2. In order to quantify the amount of FRET substrate in a particular solution, I performed a set of characterization studies to determine the extinction coefficients and linear range in fluorescence for the fluorophores used in substrate design.
5-(and-6)-carboxyfluorescein, succinimidyl ester (5(6)-FAM, SE; MW = 473.39), 5-(and-6)carboxytetramethylrhodamine, succinimidyl ester (5(6)-TAMRA, SE; MW = 527.53), and Alexa
Fluor® 680 carboxylic acid, succinimidyl ester (MW ~ 1150) were purchased from Molecular
Probes (Eugene, OR). Cy5.5 was purchased from Amersham Biosciences (Piscataway, NJ). The dark quencher, BHQ2, as analyzed in this assay, was conjugated onto a peptide (KHSSKLQK-
BHQ2) synthesized by Derek Hudson at Biosearch Technologies, Inc. (Novato, CA). The BHQ2-
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labeled peptide was delivered still on the resin. 6.7 mg resin was weighed out and cleaved with
1 ml solution containing 95% trifluoroacetic acid (TFA) and 5% water (v/v) for 20 hours. The cleavage solution was then filtered through a glass pipet fitted with glass wool to separate resin from the product. The solution was placed on a Schlenk line and evaporated under either argon or nitrogen. Cleavage products were reconstituted in 200 d dimethylformamide (DMF) and purified by reversed-phase high-pressure liquid chromatography (RP-HPLC) as three separate injections. I used a two-phase solvent system where solvent A contained 0.1% TFA in water and solvent B contained 90% acetonitrile and 10% solution of 0.09% TFA in water. The elution gradient was comprised of 10% B to 100% B in 20 minutes. The major peak was identified at
30.7 minutes by absorption at 215 nm and collected as the peptide product (P022-BHQ2) for lyophilization. The absorption assay for P022-BHQ2 was performed separately from the other dye molecules but with the same method.
To minimize the amount of DMSO required in each dye solution, I dissolved 1 mg of the most hydrophobic molecule, fluorescein, at its lower limit of solubility (5 mg/ml) in DMSO. The solution was then made up to a 10 mM solution using phosphate-buffered saline (PBS). 10 mM solutions of TMR, Cy5.5, and Alexa Fluor® 680 (AF680) were then prepared such that they all contained the same volumes of DMSO (37.9 pl) and PBS (362.1 pl) as the fluorescein stock solution. The dry weight of each dye molecule was determined either by microbalance for fluorescein and TMR or as reported by the manufacturer in the cases of Cy5.5 and AF680.
Dilution series were then prepared in PBS at a total volume of 100 pl per well in a Costar® 96- well UV ELISA plate (Corning Life Sciences, Coming, NY) at concentrations of 1 mM, 0.5 mM,
0.2 mM, 0.1 mM, 0.05 mM, 0.02 mM, 0.01 mM, and 0.001 mM. All samples were assayed in duplicate. A control sample of the solvent mixture (37.9 pl DMSO and 62.1 pl PBS) was prepared to determine the baseline absorption characteristics. A Spectramax UV-Visible plate reader (Molecular Devices, Sunnyvale, CA) was used to perform all absorption measurements.
Fluorescence measurements were made using a Spectramax Gemini spectrofluorometer under low photomultiplier tube (PMT) gain setting for all species assayed.
-48-

Optical density, or absorbance, is defined as
A = OD = logo -
T where T is the transmittance, evaluated as the ratio of the spectral transmitted radiant flux to the incident flux. The Beer-Lambert law expresses the relationship between absorption and concentration as
A =s -b-c where c is the molar extinction coefficient (L mol-1 cm-1), b is the path length of the sample (cm), and c is the sample concentration (mol L-1). In determining the concentration of pure FRET peptide in a given solution, I used
A(Ad..) =a /Idm'-Ca + [E(Ad.'Cd
A(Ax)= a(A .ca ]+ C] where
Xd max is the maximum absorption wavelength for the donor fluorophore,
Xa max is the maximum absorption wavelength for the acceptor fluorophore, e is the extinction coefficient (L mol-1), and c denotes the species concentration (mol L-1). Given the values of ea and d from standard fluorophore dilution curves, measurement of solution absorption at
Xd max and
Xa max will yield a complete set of equations for which the concentrations of the acceptor and donor fluorophores can be calculated. In the ideal case of a pure solution of FRET peptide, there is a
1:1 molar ratio between donor and acceptor molecules. Consequently, the concentrations of each fluorophore should be equivalent.
The UV-visible plate reader measures the sample absorbance as light passes vertically through the sample well. Consequently, the path length through each sample is dependent on the sample volume. The path length through the sample volume was determined by comparing
-49 -

the absorptions of the control well with a reference cuvette containing PBS. The control well contained 100 d PBS and represented the same volume as all the samples. The following formula was used to evaluate the path length:
PL =
OD,(1000nm) - OD,(900nm)
ODf (1000nm) ODr (900 nm) where ODw represents absorbance in the sample well, ODrej is the absorbance in the reference cuvette, and PL is in centimeters. For this assay, the path length was determined to be 0.264 cm.
This value was assumed for all sample wells, since the total volume and preparation were identical to those of the control well used in path length determination.
Extinction coefficients for each dye molecule were evaluated from the Beer-Lambert relation in which the absorption is directly proportional to the species concentration at a specified wavelength. Figure 3-1 illustrates the absorption data obtained from each dilution series and collected at the maximum absorption wavelength for each dye. Linear regression was performed on each data set to determine the slope. The y-intercept was set to be the absorption value for the control well at the specified wavelength for the particular dye. The slope of the absorption-concentration curve represents the product of path length and extinction coefficient, from which the desired parameter can be deduced. Linear fit parameters are outlined in Table 3-1, and Table 3-2 lists the extinction coefficient values for each dye molecule for several absorption wavelengths.
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-

4
3.5
3
2.5
.0
2
1.5
1
0.5
0'
0
-
0.1
-00
0.2 0.3 0.4 0.5 0.6 0.7
Concentration (mM)
-
0.8 0.9
-
* Fluorescein
0 Cy5.5
Linear (TMR)
Linear (PO22-BHQ2)
0 TMR
A P022-BHQ2
***,*V*'**Linear (Fluorescein) -
-
A AF680
Linear (Cy5.5)
Linear (AF680)
.0-.0A
1
Figure 3-1 Chromophore absorption-concentration curves
Chromophore absorptions were sampled at 495 nm (fluorescein), 555 nm (TMR), 675 nm (Cy5.5 and AF680), and 570 nm (P022-BHQ2)
Dye
Fluorescein
TMR
Cy5.5
Alexa Fluor® 680
P022-BHQ2
Table 3-1 Summary of linear fit to absorption-concentration curves
1.456
1.393
0.817
2.780
3.466
Linear Regression
2
VleWavelength
0.9999
0.9942
0.9923
0.9603
0.9856
495
555
675
675
570
(nm)
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495 nm
555 nm
570 nm
670 nm
675 nm.
Fluorescein
5,507
6.4
Table 3-2 Extinction coefficients for dye molecules
S
Extinction Coefficients (M-
1 cm-
1
)
TMR
1,260
5,270
C 5.5 AF680
2,373
3,058
3,090
10,443
10,520
P022-BHQ2
13,114
3,043
Fluorescence measurements were made to determine the linear portion in the intensity curve prior to the onset of the inner filter effect. It is also interesting to examine the relative brightness of each dye species for the same concentration. Figure 3-2 demonstrates the fluorescenceconcentration relationship for the four fluorescent molecules considered in the absorption assay.
Under the same excitation and emission wavelengths, Cy5.5 and AF680 exhibit very different fluorescence intensities.
45000
< Cy5.5 (670/700)
40000 - U AF680 (670/700)
35000 A TMR (550/580)
30000 .
0 FAM (490/520)
25000 -
20000 -
15000 -
10000
5000 -
0
0 0.1 0.2 0.3 0.4 0.5 0.6
Concentration (mM)
0.7 0.8 0.9
Figure 3-2 Fluorescence curve for dye dilution series
Excitation and emission wavelengths (nm) are indicated after each dye in the legend; RFU is an arbitrary measure of fluorescence units
1
-52-

Absorption measurements reveal much lower extinction coefficients than those reported in literature provided by the respective manufacturers. All extinction coefficients were measured to be less than 11,000 M-1 cm-' which are an order of magnitude less than the listed values.
Solvent effects do not explain this discrepancy in all cases, since extinction coefficients for Alexa
Fluor® 680 and Cy5.5 were determined in aqueous buffer (i.e., PBS for Cy5.5). Inconsistencies in weighing out the dye may affect the determination of extinction coefficients. To evaluate the extent of the error required to achieve the manufacturer listed extinction coefficient, I considered the most plausible scenario. Since the dilution series was made from a single stock solution, the relative concentrations should be accurate. The source of error would be in the starting concentration. Because the slope of the absorption-concentration curve is inversely proportional to the scaling in concentration, the actual sample concentrations would need to be
10-fold lower than the presumed values illustrated in Figure 3-1 for the extinction coefficients to be comparable to those reported in Table 3-3. Where I presumed to have prepared a 10 mM stock solution, there must have been an error in handling or product purity to only have produced a 1 mM solution to enable the measured extinction coefficient to be on the same order of magnitude as the listed values. Such an error is possible although not likely, particularly with the use of a pre-weighed sample purchased directly from the manufacturer in the cases of
Cy5.5 and AF680. However, for the purposes of substrate evaluation which is the reason behind the need for extinction coefficients, it is sufficient that relative concentrations can be derived from the values at hand; absolute concentrations are not necessary nor is the difficulty in obtaining such values warranted.
The fluorescence measurements shown in Figure 3-2 indicate the portion of the concentration scale that can be relied upon to provide a linear correlation between fluorescence intensity and concentration. I chose to plot concentration instead of optical density, as many texts do, because the substrates that are considered later in the thesis are modified with two different dye molecules. Consequently, a single value of optical density would not be of value in determining the quantity of a dual-labeled substrate, as is usually implied when researchers
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-

choose to use optical density as a measure of concentration. The linear range can be extended upwards to 0.1 mM with all dye molecules under consideration. Lakowicz
95 suggested the use of dilute solutions with GD 0.05 in all fluorescence applications to avoid the inner filter effect, exhibited as the concave nature of the fluorescence curve in Figure 3-2 for higher concentrations. However, the observed linear range for AF680 extends to an OD value of 0.37, which is nearly eight times the suggested cut-off value. It looks to be advantageous that the extinction coefficients are lower than expected. It allows us to work with a wider range of concentrations without the concern that most of the incident light would have been absorbed by the sample surface.
As an estimate of the relative quantum yield of particular dye molecules, one can examine the fluorescence intensity curves (Figure 3-2). Because weighing errors may have occurred with fluorescein and TMR, AF680 and Cy5.5 curves should represent fairly accurate concentrations. Thus, comparisons of fluorescence at the same concentration should be valid.
Within the linear range, AF680 exhibits more than eight times greater fluorescence intensity than Cy5.5 for the same excitation and emission wavelengths. Since the manufacturer listed the quantum yield for Cy5.5 as 0.23, the quantum yield for AF680 can be determined from the following formula:
I ODR n2
IR OD nR where Q is the quantum yield, I is the integrated intensity, OD is the optical density, and n is the refractive index. The subscript R refers to the reference fluorophore of known quantum yield. It is assumed that the two samples are excited at the same wavelength to preclude the need to correct for the difference in excitation intensity. Without knowing I for either dye molecules, we can use the fluorescence intensity at the maximum emission wavelength for each fluorophore at comparable OD values. In this case, Cy5.5 at 0.2 mM (OD=0.145) and AF680 at
0.05 mM (OD=0.191) have similar OD values. AF680 fluorescence intensity at 0.05 mM is more than twice that of Cy5.5 at 0.2 mM. Consequently, for similar OD values and identical refractive
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-

indices, the quantum yield for AF680 appears to be larger than that for Cy5.5. Low quantum yields are expected for these near-infrared fluorophores, as the nonradiative decay rates at these wavelengths are much higher due to thermal losses. In particular, Cy5.5 was designed with aryl sulfonate groups to make the fluorophore more water soluble. These chemical groups facilitate increased solvent interactions and may introduce higher rates of nonradiative dissipation, resulting in an even lower quantum yield.
Table 3-3 Manufacturer reported fluorescence parameters (NR = not reported)
Fluoresceina
TMRb
Alexa Fluor* 680c
EC (M-1 cm-') Quantum Yield Fluorescence Lifetime (ns)
74,000 NR
95,000
183,000
NR
NR
Cy5.5d 190,000 0.23
Measured at pH 9. EC and QY decreases dramatically in solvents with pH < 7.
NR
NR
1.2
1.0
c Measured in aqueous buffer (pH 7.5) at 20'C. Data provided by Molecular Probes, Inc. and attributed to
Pierre-Alain Muller, Max Planck Institute for Biophysical Chemistry, G6ttingen.
Data referenced by Amersham Biosciences and attributed to Mujumdar, S.R., Mujumdar, R.B., Grant,
C.M. & Waggoner, A.S. (1996) Bioconjugate Chem. 7, 356-362.
3.4 Conclusions
The extinction coefficients for fluorescein, tetramethylrhodamine, Alexa Fluor® 680, and Cy5.5, were determined to be less than 10% of the manufacturer reported values at their respective maximal absorption wavelengths. The discrepancies in EC values may be attributed to the use of different solvent systems in measuring dye absorptions. Extinction coefficients for fluorophores dissolved in the minimum amount of organic solvent to achieve solubility for the most hydrophobic dye are listed in Table 3-2. These values will be used to determine the concentrations of dual-labeled FRET substrates in subsequent experiments. An evaluation of the relative brightness of the four fluorophores under consideration yielded the relationship: fluorescein > TMR > AF680 > Cy5.5. Under the same excitation and emission wavelengths,
AF680 exhibited a higher quantum yield than Cy5.5.
-55 -

Human PSA has proteolytic activity after post-processing in seminal fluid. In this assay, purified PSA from human seminal fluid was used to determine the enzyme kinetics of PSA with the FRET substrates. These substrates utilize the same FRET pairing to establish a valid comparison between samples. Fluorescein and TMR were chosen as the donor and acceptor molecules, respectively, because of their low cost, historical use in FRET applications, ease in peptide conjugation, and relatively high quantum yields. The Forster distance for the fluorescein-TMR FRET pair was calculated to be -60 A96, allowing for sufficient energy transfer within the peptide sequences under consideration. The affinity portion of the molecular probe, the bare peptide, was chosen among several candidates reported in the literature. Denmeade et al.
97 reported HSSKLQ as a specific and efficient peptide substrate for PSA, but there was concern that the internal lysine would pose a problem in its specificity, as trypsin readily cleaves at lysines and arginines. Consequently, two other peptide substrates were chosen from the literature, QFYSSN and SSIYSQTEEQ. Previous experiments with FRET versions of the shorter peptides, HSSKLQ and QFYSSN, demonstrated that access to the cleavage site was being blocked by the bulky fluorophores. Subsequently, glycine residues were used to act as spacers between the donor and acceptor molecules attached at either end of the peptide substrate. This later design change resulted in the 6GQFYSSN6G peptide substrate.
SSIYSQTEEQ was chosen for the length of its native peptide sequence without the addition of spacer residues. The three substrates evaluated in this assay were QFYSSN, 6GQFYSSN6G, and
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-

SSIYSQTEEQ. The important measures of substrate performance included the rate of fluorescence activation and the signal-to-background ratio.
Fluorescein-TMR FRET conjugates were prepared using peptides that were synthesized at the
MIT Biopolymers Laboratory using an ABI Model 433A peptide synthesizer. All peptides were synthesized on Wang resin using FMOC chemistry and lyophilized prior to delivery. 5-(and-6)carboxyfluorescein, succinimidyl ester (5(6)-FAM, SE; MW = 473.39) and 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (5(6)-TAMRA, SE; MW = 527.53) were purchased from Molecular Probes (Eugene, OR). 10 mg FAM-SE in 1 ml of anhydrous DMF was added to
30 mg of peptide on resin in a 3-ml Wheaton V-vial. 40 pd of N,N-diisopropylethylamine
(DIPEA) was added dropwise into the resin solution while being stirred with a triangular stir bar. The reaction was allowed to proceed overnight at room temperature with sufficient protection from light. After approximately 18 hours, the reaction solution was filtered through a Whatman membrane under vacuum to eliminate excess free dye. Several washes with excess
DMF and methanol were performed. The dry resin was then mixed with 2 ml cleavage cocktail containing 95% TFA and 5% water by volume and allowed to stir for more than 2 hours in the dark at room temperature. The solution was then filtered through glass wool placed in a glass pipet and collected without the resin. The product was evaporated under argon to yield a yellow film. The yellow product was reconstituted in 200 pl DMF and purified via RP-HPLC as described in Chapter 3 using the same solvent system and elution gradient. The major peak was collected and lyophilized. A small fraction of this product was sent to the MIT
Biopolymers Laboratory for MALDI-TOF mass spectrometry (MS) to verify the species molecular weight. The remainder of the FAM-labeled peptide was mixed with 2 mg TMR-SE in
250 pl anhydrous DMF and 25 pl DIPEA in a Wheaton V-vial. The reaction was stirred overnight using a triangular stir bar. The reaction solution was purified using RP-HPLC as described above. The major peak was then collected and lyophilized. A small fraction of this
-57-

product was also sent to the MIT Biopolymers Laboratory for MALDI-TOF analysis. All FAM-
TMR FRET conjugate peptides were verified by MS to be of the correct molecular weight.
The PSA assay was performed using enzymatically-active human PSA from Calbiochem
(EMD Biosciences, San Diego, CA). The enzyme solution was prepared at a concentration of
100 ng/pil in PBS. The substrate samples were prepared in a Costar® 96-well UV-ELISA microplate (Corning Life Sciences, Coming, NY) from a 10 mM stock solution in DMSO and diluted according to the concentrations outlined in Chapter 3. The path length was also determined in the same manner as described previously. All wells contained a pre-enzyme volume of 100 pl. 200 ng of PSA were added to each sample well to initiate the kinetic study.
For ease of reference, FAM-QFYSSNK(TMR) will be known as FT14, FAM-
6GQFYSSN6GK(TMR) will be known as FT15, and FAM-SSIYSQTEEQK(TMR) will be known as FT19. All FRET substrate assays were performed in triplicate with one well serving as the control well for each substrate concentration (i.e., substrate only with no enzyme addition). A commercial fluorogenic PSA substrate (HSSKLQ-AFC from Calbiochem) was used to validate
PSA enzyme activity. 1 mg HSSKLQ-AFC was dissolved in 109.5 ptl DMSO to make an 8 mM solution. 5 pl of this solution was then added to 95 pil PBS to make a 0.4 mM substrate solution for use in the PSA assay. This assay was performed in duplicate. The commercial substrate assay was performed first to ensure that the PSA was enzymatically active prior to testing with the FRET substrates. Fluorescence readings were performed on a Spectramax Gemini spectrofluorometer (Molecular Devices, Sunnyvale, CA) using low PMT gain settings.
Absorbance readings were measured on a Spectramax UV-Visible plate reader (Molecular
Devices, Sunnyvale, CA).
The FAM-TMR substrates demonstrate interesting kinetics with PSA. The interaction between
FAM and TMR as donor and acceptor fluorophores, respectively, allowed several measurements to be made. Data not shown here indicates that the TMR fluorescence signal changes very little over time. Consequently, the changes in FAM fluorescence become the
-58
-

dominant signal. The first gauge of substrate efficiency in PSA proteolysis is the reaction velocity as measured by the increase in FAM fluorescence over time. As in Michaelis-Menten enzyme kinetics, the initial reaction velocity is the relevant parameter. In Figure 4-1, the initial reaction velocity is shown as a comparison between all three substrates grouped by the substrate concentration. Although velocity is typically expressed as the rate at which a specified quantity of substrate is cleaved, or peptide fragment is released, there are two main reasons for the use of fluorescence intensity as a direct substitution or correlate to the amount of cleaved peptide. One reason is that the FAM fluorescence signals for all three substrates are within the linear range discussed in Chapter 3. Consequently, a direct extrapolation from
Figure 3-2 can reveal the concentration of FAM released by PSA cleavage. The second reason is that all three substrates are modified with FAM and TMR in the same manner. The variations in the control sample fluorescence (i.e., native fluorescence of inactivated probe) do not affect the rates of reaction as reflected in fluorescence signal increase. These should not differ greatly from the actual reaction kinetics.
90
80 -
70 -
S50
60
-
S40
30 -
20
10 -f
IFT19 i!
if
~ iiiiiiiM
1 0.5 0.2 0.1
Concentration (mM)
0.05 0.02
Figure 4-1 Initial reaction velocity of FAM-TMR substrates with PSA
Initial reaction velocity is measured as the change in FAM fluorescence over the first 5-6 minutes after PSA addition
-59 -

In defining signal-to-background ratio, I considered both the idea of comparing FAM fluorescence between enzyme activated and inactivated samples and also the fluorescence ratio between FAM and TMR. FAM fluorescence comparison between the sample and reference wells provides a logical means of relating the signal as it would be detected in a sensor. This interpretation would only require one excitation source and one filter for detection. In contrast, the use of a FAM:TMR ratio as is traditionally calculated in FRET applications involving these two fluorophores, requires two sets of excitation sources and detectors. This would result in a cumbersome detection package at the level of molecular applications. However, this strategy does have the advantage that the reference fluorophore (TMR) is localized together with the indicator fluorophore (FAM) at all times. The method of using a reference sample that is not exposed to the target enzyme is difficult to employ in a physiological setting, although it is a much more intuitive approach. Both strategies are considered in this chapter.
1.6
1.4 -
1.2 -
0.8
0.6
0.4
0.2
-
-FT14
E- FT15
-FT19
0 20 40 60
Time (min)
80 100 120 140
Figure 4-2 Comparison of FAM to reference fluorescence signals in PSA kinetics
0.5 mM solution of each substrate was prepared in 100 p1 PBS with the addition of
200 ng of purified human PSA
-60-

S0.3
0.6
0.5 -
.;.....-:+ --- ;- FT14
- -FT15
--
A
FT19
0.4
-
0.2
0.1
--- --
0 200 400 600 800
Time (min)
1000 1200 1400 1600
Figure 4-3 Comparison of FAM to TMR fluorescence signals in PSA kinetics
1 mM solution of each substrate was prepared in 100 pl PBS with the addition of
200 ng of purified human PSA
4.3 Discussion
The two criteria, which were considered in the evaluation of substrate performance against
PSA, included initial reaction velocity and the signal-to-background ratio. These criteria were chosen to address the main concerns in the design of an optical imaging system, mainly time and contrast. Ideally, a molecular probe for PSA would experience a rapid change in fluorescence such that there would be sufficient contrast to distinguish the region of high PSA concentration from its neighboring PSA-deficient zone. Fast PSA kinetics is also advantageous in that fewer enzymes can compete with PSA for the molecular probe, thereby ensuring a specific reaction. Several substrate concentrations were tested against PSA to select an optimal concentration and also to compare substrate efficiency. In Figure 4-1, it is clear that the reaction velocity favors FT19 regardless of substrate concentration. The maximum velocity occurs at 0.5 mM and declines monotonically for more dilute samples. Also, it can be seen that FT15
-61-

performs better than FT14 with respect to the reaction velocity. This may indicate that the spacer residues do provide a unique advantage in FRET probe design. Figure 4-2 represents one measure of signal-to-background, which is a comparison of activated and inactivated forms of the same substrate. FT19 exhibits the greatest contrast over time, with the highest signal-toreference ratio. Although it has the highest reference fluorescence, FT19 is cleaved fast enough
by PSA that the reference signal does not hinder signal contrast. FT15 exhibits a strangely constant signal-to-background ratio in which the reference fluorescence does negatively impact the relative significance of the activated fluorescence. FT14 does not share this same trend; instead, it has an increasing signal-to-reference signal that is nearly parallel to that of FT19. If one examines Figure 4-3 in which the signal-to-background ratio is evaluated as the fluorescence ratio between FAM and TMR, the observations regarding substrate performance are more consistent with those seen in Figure 4-1 regarding reaction velocity. The FAM:TMR ratio is quite different between FT14, FT15, and FT19 and again, indicates that FT19 would provide the best image contrast for an optical detection scheme. Regardless of the choice of definition for signal-to-background ratio, FT19 emerges as the best FRET substrate based on the selected criteria. This is perhaps due to its intermediate sequence length that allows PSA sufficient access to the cleavage site without reducing the FRET efficiency between FAM and
TMR that make the substrate both specific and sensitive.
4.4 Conclusions
Three FRET substrates, FAM-QFYSSNK(TMR), FAM-6GQFYSSN6GK(TMR), and FAM-
SSIYSQTEEQK(TMR), referred to as FT14, FT15, and FT19, respectively, were evaluated against purified human PSA to determine the optimal peptide sequence and the most discerning metric for enzyme kinetics. This set of experiments examined initial reaction velocity, the ratio between FAM and reference fluorescence, and the ratio between FAM and TMR fluorescence as measures of the PSA enzyme reaction. FT19 emerged as the most specific substrate for PSA by exceeding the other two peptides in both the initial reaction velocity and signal-to-background ratios. As corroborating evidence, PSA was shown in the literature to have a higher kcat:Km
-62-

value for the base peptide sequence in FT19 (SSIYSQTEEQ) than for the base peptide sequence in FT14 and FT15 (QFYSSN). The addition of donor and acceptor fluorophores to the substrate structure did not appear to modify the relative reaction kinetics of the substrate with PSA. In the comparison between the two ratiometric parameters, only the FAM:TMR fluorescence ratio demonstrated the substrate specificity as FT19 > FT15 > FT14, which was the expected outcome as predicted by the initial reaction velocity. After the enzyme reaction had proceeded at room temperature for 24 hours, FAM fluorescence in the reaction with FT14 had increased more than
5-fold over the reference FAM fluorescence. Conclusions about substrate specificity derived from this metric indicated that FT14 experienced the most proteolysis by PSA. However, this result did not concur with the relative substrate specificity suggested by the initial reaction velocities, and thus, the FAM-to-reference fluorescence ratio is not a reliable measure of enzyme kinetics.
-63-

5
The next step after substrate evaluation with purified protein was to expose these same probes to human cell culture. Characterization of the specific reaction kinetics in this assay was of less importance than the ability of the substrates to distinguish between PSA-producing and non-
PSA-producing prostate cancer cell lines. Although specificity assays could have been performed with a variety of purified enzymes overexpressed by cancer cells, there is value in examining these substrates in a system that more closely resembles conditions in vivo. LNCaP and PC-3 were chosen as the experimental cell lines, primarily because they are human prostate cancer cells that are well-characterized in the literature. LNCaP cells are androgen-responsive and secrete PSA into the media. However, only 30% of the PSA secreted by LNCaP cells in vitro are considered active in forming complexes with ACT
98
. This population of PSA is presumably enzymatically active, enabling unaided binding to ACT. LNCaP tumor xenografts in vivo produce a small amount of enzymatically active PSA that accounts for only 18% of total secreted
PSA (Denmeade et al., 2001). On the other hand, PC-3 cells are androgen-insensitive and do not produce PSA. However, these cells can be transfected with human PSA as a model system.
Prior to enzyme assays, I performed Western blot analysis on the conditioned media from both cell lines to ascertain the presence (or absence, in the case of PC-3 cells) of PSA with the corresponding molecular weight. Since PSA can exist as a pro-form and also as complexes with several protease inhibitors, it was necessary to verify the main form of PSA in both cell lines.
-64-

Western blot analysis was performed using goat polyclonal antibody to human PSA (Santa
Cruz Biotechnology, Santa Cruz, CA). LNCaP and PC-3 cells were grown in T75 flasks using
RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 1% glutaminepenicillin-streptomycin (GPS). Both cell lines were incubated at 37
0
C under 5% C02. Media was replaced with RPMI-1640 supplemented only with 1% GPS after 48 hours of incubation.
Conditioned media was collected 24 hours thereafter. Protein concentration was performed using Centricon YM-10 and Centricon YM-100 centrifuge filters (Millipore, Bedford, MA).
For the substrate assay, LNCaP and PC-3 cells were seeded in a CostarO 96-well ELISA plate (Corning Life Sciences, Corning, NY) at 10,000 cells per well in 90 pl, 95 pl, or 98 pl of serum-free RPMI-1640 media. These cells were incubated for 48 hours at 37*C under 5% C02.
Substrate in DMSO (from the 10 mM stock solution prepared for previous assays) was then added to each well based on the intended concentration (1 mM, 0.5 mM, or 0.2 mM). All assays were performed in triplicate for each cell line. Control samples were performed in duplicate.
FT14, FT15, and FT19 were tested in the same manner as described previously.
5.2 Results
LNCaP conditioned media (Figure 5-1) is shown to contain PSA as visualized by a goat polyclonal antibody to human PSA located along the gel at approximately 30 kDa. This molecular weight corresponds to the accepted range for PSA (30-35 kDa). PC-3 cells do not express PSA (Figure 5-2). However, PC-3 transfection with the PSA gene demonstrates PSA production as identified through Western blot. Although prior assays with purified human
PSA indicated that the FAM:TMR fluorescence ratio was more accurate in measuring enzyme kinetics, the data in these experiments were consistent between the two ratiometric parameters.
Consequently, normalized FAM fluorescence was used to determine the reaction rate of each substrate in LNCaP and PC-3 cell culture without sacrificing accuracy in data interpretation.
Moreover, future applications with optical sensors will require that the number of input signals
-65-

be minimal to facilitate ease of design and operation. A single input channel corresponding to the fluorescence parameters for one fluorophore (i.e., FAM) would be ideal. Normalized FAM fluorescence was derived by subtracting the control well fluorescence from the sample signal. It is not expected that this approach would greatly alter the reaction rates, since the control fluorescence is fairly steady. Figures 5-3 thru 5-5 show the time course of substrate cleavage for each of the three FRET probes.
LNCaP CM LNCaP Lysate
30 kDa
Figure 5-1 Western blot of LNCaP conditioned media (CM) and lysate
Transfected
CM
Control
CM
Transfected
Lysate
Control
Lysate e 2 W e bt o 35 kDa
Figure 5-2 Western blot of PC-3 conditioned media and lysate in wild-type and transfected cells
700
600 -
500 -
* LNCaP-LN3 o
PC-3
- Linear (LNCaP-LN3)
Linear (PC-3)
y = 4.1489x +55.684
R2= 0.9832
400
-
.00*
300 -
40i
..
200 z 100
-
0
T
17 -
0
......
20
L-9-1
L.-
40
I
60
Time (mi:
80 y = 0.0246x + 36.473
R =0.0418
100
Figure 5-3 FT14 in cell culture at 0.2 mM concentration
I
120 140
-
66-

7000
6000 a.' a.'
U, a.'
5000
4000
---
* LNCaP-LN3 o
PC-3
Linear (LNCaP-LN3)
Linear (PC-3)
y =42.03x +670.2
R2 = 0.9816
*
3000 a.'
N 2000 z 1000
0
-
20
,'
EM M 0
40 60 80
Time (min)
15 U y = 0.2573x + 322.05
R = 0.0608
0 E3
100 120
Figure 5-4 FT15 in cell culture at 0.2 mM concentration
14 0
3000
2500
2000 -
* LNCaP-LN3
O PC-3
- Linear (LNCaP-LN3)
Linear (PC-3)
Z 1500 y = 16.249x + 341.64
R= 0.9863
0 1000
y =-0.1929x + 423.74
R 2=0.0284
500
0
0
-i
20 40 60 80
Time (min)
100 120
Figure 5-5 FT19 in cell culture at 0.2 mM concentration
140
-67-

Figures 5-3 thru 5-5 demonstrate that all three FRET substrates can distinguish LNCaP cells from PC-3 cells. Although it is unclear whether these substrates are responding solely to PSA proteolysis or other enzymes expressed by the LNCaP cells, the data do indicate a significant increase in signal-to-background for all substrates. It is curious that FT15 has the highest reaction rate out of all the substrates as it did not perform well in the purified PSA assays.
However, the reaction rate for FT15 in PC-3 cell culture indicates that there may be non-specific proteolysis occurring to generate such a high velocity (0.26 RFU/min) as compared to the other
FRET probes. FT15 is the longest peptide sequence among the three sequences tested in vitro.
The substrate sequence is buffered on both termini with six glycine residues. Consequently, it is
highly possible that the addition of glycine spacers leads to a peptide conformation with greater enzyme accessibility to the cleavage site. Enzymes without sufficient specificity to access the
FT14 and FT19 cleavage sites may be hydrolyzing FT15. Previous studies have demonstrated low levels of enzymatically active PSA in the LNCaP cell line. The LNCaP-LN3 cell line was derived from three rounds of selection for a lymph node metastasis of an orthotopicallyimplanted LNCaP tumor in nude mice. It is not known whether LNCaP-LN3 cells produce a higher level of enzymatically-active PSA than the parental cell line, but there are indications that LNCaP-LN3 does produce a significantly greater amount of total PSA than LNCaP
(personal communication, J. Banyard).
Substrate assays with two human prostate cancer cell lines demonstrated that all three FRET substrates are preferentially activated in the conditioned media of a PSA-producing cell line.
FT15 experienced a nearly 15-fold increase in FAM fluorescence during the 2-hour reaction, making it the most labile substrate in this experiment. The conditioned media of the non-PSAproducing cell line did not activate any of the FRET substrates appreciably.
-
68 -

This thesis outlines the characterization of candidate fluorophores for use in a FRET-based scheme, the development of several FRET substrates for PSA based on peptide sequences from the literature, and substrate evaluation with purified PSA as well as human prostate cancer cell lines. Fluorescein and tetramethylrhodamine have been used historically in FRET studies of intermolecular interaction. Both fluorophores are available commercially in large quantities and at high purity. They exhibit high quantum yield and a large spectral overlap that enables
FRET application. Evaluation of near-infrared fluorophores suggested that AF680 has a higher quantum yield than Cy5.5, making it the prime candidate for in vivo imaging applications. A number of PSA substrates were culled from the literature, including HSSKLQ QFYSSN, and
SSIYSQTEEQ in order of lowest to highest kcat:Km value. There was concern regarding the presence of an internal lysine in the substrate reported by Denmeade et al., as it posed a challenge to synthetic modification with fluorophores. Consequently, QFYSSN and
SSIYSQTEEQ were chosen for this study. Prior experiments had suggested that peptide sequence length, and thus, the distance between the FRET pair, impacted the ability of PSA to access the cleavage site. The addition of glycine residues to both termini of QFYSSN was used to test the distance-dependent effect of the FRET pair on enzyme kinetics. As measured by initial reaction velocity, PSA kinetics for the lengthened version of QFYSSN was significantly higher than for the original sequence. FT19 demonstrated superior performance, as evinced by the initial reaction velocity and overall fluorescence increase, in assays with purified human
PSA. The modification of SSIYSQTEEQ with fluorophores necessarily changes the dynamics of
69 -

proteolysis, but the FRET substrate based on this sequence performed as expected. In experiments with human prostate cancer cell lines, LNCaP-LN3 and PC-3, all three FRET substrates were able to distinguish between the PSA-producing cell line (LNCaP-LN3) from the non-PSA-producing one (PC-3). However, FT15 outperformed FT19 in its reaction velocity, indicating possible non-specific proteolysis that is promoted by its elongation as compared to the FT19 sequence. These results point to several design considerations that must be addressed prior to in vivo experiments. Solubility continues to be a major issue, as many of the PSA substrates contain residues with hydrophobic side groups. In addition, this study has highlighted the importance of sequence length in the balance between non-specific proteolysis and increased PSA enzyme kinetics.
-70
-

7
This thesis highlights only one part of a novel optical imaging system that seeks to bring molecular imaging to in vivo applications. Several modifications to the FRET probes described here need to be made to yield substrates that can be useful in vivo. The next generation of molecular probes will feature near-infrared dyes paired with dark quenchers such as BHQ2.
Both Cy5.5 and AF680 were characterized to determine the better fluorophore for use in a FRET pair. The next generation of PSA FRET substrates will be designed to have increased solubility and increased reaction kinetics with PSA. In vitro studies will focus on characterizing PSA production by the two human prostate cancer cell lines that will be used in vivo. Results from this study indicated non-specific proteolysis of the FRET substrates, so future studies will aim to clarify this point with enzyme inhibitor experiments. In addition, to bring these contrast agents into in vivo applications, one must assess the animal models to be used. Overexpression of active PSA in a cell line such as PC-3 may be required to provide a robust experimental system with which to compare imaging data. In vivo studies also pose obstacles in terms of systemic reactions to contrast administration. The FRET substrates will need to be modified to be biocompatible with increased circulation times. The current investigation represents the beginning of an iterative process in substrate design that hopes to yield a specific and sensitive indicator for PSA enzyme activity in vivo.
-
71
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