Clarkson University
Examining the Effects of Human Papillomavirus on Cripto Expression
A Thesis Proposal by
Bethanny Smith-Packard
Department of Biology
March 2006
Signature
Advisor
Date
Goal
To determine the effect of the Human Papillomavirus on the expression of cripto.
Purpose
Cripto is a growth factor that is found only in vertebrates. It induces cell
proliferation, increases cell migration, and reduces apoptosis. Cripto has been found
expressed in many human cancers, including cervical cancer. Human Papillomavirus
(HPV) is the major risk factor for cervical cancer. The purpose of this research is to
investigate how the HPV genes effect cripto expression.
Background
Cripto and Cancer
The human cripto gene is a growth factor of the EGF-CFC family that is found
only in vertebrates. It is a small protein that is rich in cysteines. (1) It has an EGF-like
domain and a Cripto/Frl/Criptic (CFC) domain that have been found to be conserved
across species. It was the first member of the EGF-CFC family and was named for its
apparent lack of association to known proteins and pathways. Cripto is expressed in
early embryonic development in the myocardium of the developing heart. It is also
necessary for the development of the anterior-posterior axis in embryos. (2) This indicates
that cripto is necessary for movement that will orient the cell correctly. Embryos lacking
cripto will die in utero because of their inability to gastrulate. (3) Expression of cripto has
been found to be restricted after birth.
Cripto contributes to cancer progression and the deregulated growth of cancer
cells by its effect on cell shape, adhesivity, and motility. In addition, cervical cancer cells
over expressing cripto will become more invasive. (3) Cripto also induces cell
proliferation, increases cell migration, and reduces apoptosis (programmed cell death).
Cripto has been found over expressed in many human cancers, including breast, lung,
ovarian, colon, pancreatic, and cervical cancer (2), which is the second most common
cancer in women worldwide.
Human Papillomavirus
Human Papillomavirus (HPV) is the major risk factor for cervical cancer. It is
part of a family of viruses that normally cause benign warts on the hands and feet. Some
“low risk” HPV strains are sexually transmitted and cause genital warts. Only a few of
the many different strains of HPV are “high risk” for cervical carcinogenesis. Strain 16 is
most commonly found in cervical cancer.
HPV has a circular double-stranded genome that is about 8.0 kb in length. It has
two sections, the late region and the early region. (Figure 1) The genes in the late (L)
region code for the viral capsid proteins; regulation of these proteins is tightly controlled.
The genes in the early (E) region are involved in maintenance and replication of the HPV
genome. The E6 and E7 genes are responsible for producing proteins that lead to
carcinogenesis. (4) The LCR is the long control region, which is important in regulating
replication of the viral DNA and in transcription of the early genes. (5)
Figure 1: Diagram of the HPV-16 genome. The axis is labeled in the number of base pairs.
The HPV-16 E6 and E7 proteins are selectively retained and expressed in most cervical
cancers, and they contribute to carcinogenic progression. E6 degrades p53, which is a
tumor suppressor that is activated in response to DNA damage and prevents the cell cycle
from continuing until the damage is repaired. (6) E7 binds to the Rb, or retinoblastoma
protein; this protein controls movement into the synthesis phase of the cell cycle. (7)
Proposed Research
The hypothesis that this research project is focusing on is whether HPV genes
cause increased cripto activation. To examine this question, cervical epithelial cells are
isolated from human biopsies and grown in cell culture. Reporter gene assays will be
performed by transiently cotransfecting cervical cells with the HPV-16 E6 and E7 genes
or an HPV genome, and the reporter genes cripto-luciferase and renilla-luciferase. The
cripto reporter gene has the cripto promoter cloned in front of the firefly luciferase gene
and gives readings of cripto activation. The renilla reporter gene has a promoter for a
gene equally expressed in all cells cloned in front of the renilla gene (another luminescent
protein). This gives a reading that can be used to normalize the activity of cripto. These
activation readings will be measured with a luminometer.
Transient transfections are being performed using lipofection. In this process,
lipids surround the plasmid DNA to facilitate penetration through the cell membrane.
(Figure 2) This technique allows the plasmid DNA to be taken up and expressed by the
cells.
Figure 2: Diagram showing the transfection technique. The DNA, shown in the bottom left hand tube, is
mixed with the lipofectAMINE, shown in the top left. The positively charged lipofectAMINE coats the
negatively charged DNA, forming a lipid complex. This complex is taken up by the cells.
Reporter gene assays will allow us to look at how cripto activation is changed
with the addition of the HPV genes and genome. We will also be able to look at how
cripto activation changes throughout carcinogenic progression.
Preliminary Results
Previously optimized protocols were followed for the reporter gene assays.
Preliminary results seem to show down-regulation of cripto by the HPV genes (Figure 3),
although very high renilla readings make it difficult to determine.
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Cripto Transfection-HCX28 Endo
Ratio of cripto to renilla
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0.8
Ratio of Activation
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pLXSN
E6
E7
E6/E7
HPV16
HPV18
HPV11
Figure 3. Activation of cripto with HPV genes and genomes, pLXSN is a control
Since we were obtaining low cripto readings and these results were not what we
had been expecting, we decided to look at different constructs of the cripto promoter
(Figure 4) to see if there is a specific section that significantly activates cripto expression.
1. -2479
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-2022
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-8 2.5kb construct (full length)
Nhe I construct
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Stu I construct
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Xba I construct
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Pvu II construct
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Nhe I/Eco RI construct
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Hind III construct
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Eco RI/Hind III construct
Figure 4. Deletion constructs of the cripto promoter
Deletion constructs of the cripto promoter were obtained from the NIH. These
were prepared and characterized. Reporter gene assays performed using the deletion
constructs showed that the whole promoter is necessary to active cripto. It also seems to
show that cripto expression is being down regulated by HPV16. (Figure 5)
Cripto Constructs (HCX72-TZ)
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Constructs with HPV16
Constructs with pLNSX
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Ratio of Activation
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Figure 5. Activation of cripto constructs with HPV16 (blue) or pLNSX (red)
These experiments need to be replicated to assure their validity. We want to
perform reporter gene assays with cells at different stages leading up to cancer. We will
also be extracting RNA and performing reverse transcription-polymerase chain reaction
(RT-PCR) in order to look at how cripto expression changes during carcinogenic
progression. We also want to look at the protein expression of cripto by performing
western blots.
Timeline
For the future I hope to repeat some of my past experiments and also begin
looking at cripto expression at different stages of cancer progression. I have already
extracted RNA and performed reverse transcription, but still need to do PCR. The rate at
which I am able to perform some of the reporter gene assays depends on when the lab
receives cervical samples.
References
1. Shen, M. M. Decrypting the role of Cripto in tumorigenesis. The Journal of Clinical
Investigation. 2003, 112: 500-502.
2. Adamson, E. D., Minchiotti, G., and Salomon, D. S. Cripto: A Tumor Growth Factor
and More. Journal of Cellular Physiology. 2002, 190: 267-278.
3. Bianco, C., Normanno, N., Salomon, D. S., and Ciardiello, F. Role of the Cripto (EGFCFC) Family in Embryogenesis and Caner. Growth Factors. 2004, 22: 133-139.
4. Stoler, M. H. Human Papillomaviruses and Cervical Neoplasia: A Model for
Carcinogenesis. International Journal of Gynecological Pathology. 2000, 19: 1628.
5. Sichero, L., Franco, E. L., and Villa, L. L. Different P105 Promoter Activities among
Natural Variants of Human Papillomavirus Type 18. The Journal of Infectious
Diseases. 2005, 191: 739-742.
6. Mantovani, F. and Banks, L. The Human Papillomavirus E6 protein and its
contribution to malignant progression. Oncogene. 2001, 20: 7874-7887.
7. Munger, K., J. Basile, S. Duensing, A. Eichten, S. Gonzalez, M. Grace, and V. Zacny.
Biological activities and molecular targets of the human papillomavirus E7
oncoprotein. Oncogene. 2001, 20: 7888-7898.