MOD # 136
Thursday, May 22, 2003, 3:00
Jennifer Elmore for Sue Fair
Molecular Basis of Cancer (part two)
Dr. Eisenberg Page 1 of 7
Friday (5/23) we will have class from 9:00 – 10:00. Picture is still at 10:00.
Quiz has been moved to Tuesday (5/27) at 11:00.
Oncogenes that we have been previously talking about work in a dominant fashion. Ex.
If have a mutation in maternal or paternal gene, that mutation works in a dominant fashion (mutations in ras or in any oncogene in Growth Factors or Growth factor receptors)
Cancer Suppressor Genes

While proto-oncogenes encode proteins that promote cell growth, the products of tumor-suppressor genes apply brakes to cell proliferation .
(prevent uncontrolled cell proliferation and it is through the loss of function of these genes that you see a transforming phenotype).
Cloned Tumor Suppressor Genes

Important ones are listed on chart in power points –

DCC – colon carcinoma

APC – familial adenomatous polyposis

P53 – Li-Fraumeni tumor (most commonly mutated oncogene and found in vast majority of cancers)

NF-1 – Neurofibromatosis type 1

NF-1 codes for Gap protein (GTPase activating protein) – binds to activated form of ras and cleaves phosphate off GTP (making
GDP) and phosphorylates ras (causing inactivation of ras).
Mutations in NF-1 result in constitutive expression of ras leading to neurofibromas.

NF-2 – Neurofibromatosis type 2

RB - Retinoblastoma

WT1 - Wilms’ tumor
Retinoblastoma – 2 types:
 Familial – 40 %
This appears as autosomal dominant , because a person inherits only one copy of the defective RB gene, but the second (normal) gene is inactivated by a somatic event (mutation). The frequency of a second event is very high (almost all develop tumors), resulting in the appearance as autosomal dominant. Actually this disorder is autosomal recessive because it requires both copies of gene to be inactivated.

Sporadic – (60%)
Inherit both normal functional genes, and two separate somatic events take place; converting genes to inactive form. The

MOD # 136
May 22, 2003 3:00 p.m.
Page 2 of 7 incidence for sporadic form is rare because need two events to take place. There is a single, usually late onset tumor.
Identification and Isolation of RB Gene

Retinoblastoma susceptibility gene = RB

A deletion in 13q14.1 (in tumor suppressor genes, common to see a deletion of region where suppressor genes found).

Cloned deleted region

Identify open reading frame deleted in RB tumors
Retinoblastoma Susceptibility Gene

Expression – o p105-RB present in normal retina absent in tumor o expressed in all normal adult and fetal tissues o other tumors associated with inactivated RB
 Function o regulates cell cycle (negatively) through interaction with transcription factors (required for DNA synthesis) o prevents movement from G1

S phase of cell cycle (normally it functions as a block = negatively = suppressor)
Loss of Heterozygosity (LOH)
When looking at these oncogenes, normally see in familial form: you inherit one gene that is functional and one that is nonfunctional and the functional gene is lost. First copy inactivated by somatic mutation and second copy is replaced by duplication. o occurs at high frequency o duplication is regional o if linked marker heterozygous = also reduced to homozygous
Pathogenesis of retinoblastoma (schematic)

Inherited form

Sporadic form – two separate mutations
The cell cycle

G1 into S phase (where dna synthesis occurs). What controls transition?
There are a number of proteins involved: cyclins and cyclin dependent kinases (phosphorylate and can also dephosphorylation).
Molecular Nature of Cell Cycle Regulation

Phosphorylation/dephosphorylation

Mitosis Promoting Factor (MPF)

Cyclically activated by protein kinases o Cdks (cyclin dependent kinases) o Cyclins
Role of RB gene, cyclins, and cyclin dependent kinases (fig. 8-26 p. 283)

Cdks are produced always, but need to bind to cyclin D and become phosphorylated to become activated (thus allowing the transition from G1 to S phase).

Cdk – catalytic subunit, always present (in inactive form)

MOD # 136
May 22, 2003 3:00 p.m.
Page 3 of 7

Cyclin – regulatory subunit, cyclic appearance (produced at different stages of cell cycle).
 MPF – mitosis promoting factor - cdk, mitotic cyclin
Regulation of Kinase Activity by Cyclin o accumulation of cyclin o activation of cyclin o degradation of cyclin
Growth factors and the release of cell cycle brakes

Rb protein may be normal brake to cell proliferation between G1 and
S phases. (Rb binds up transcription factors when it is dephosphorylated
– so transcription factors not able to stimulate DNA synthesis).
 Growth Factors:
 override brakes
 cell surface receptor
 intracellular signaling pathways
 phosphorylation of Rb
Molecules that regulate Nuclear Transcription and cell cycle (fig. 8-31 p. 289)

Phosphorylated form of Rb (inactive form) releases E2F and allows activation of transcription and G1 progresses into S phase.

Unphosphorylated form (active form) binds E2F and blocks entrance to
S phase.
Rb and induction of cell proliferation (schematic) – showing phosphorylation inactivation of Rb protein allowing gene transcription to occur and therefore proliferation.
Role of Rb gene, cyclins, and cyclin-dependent kinases (schematic)

Synthesis of cyclin D protein

Cyclin-dependent kinases expressed throughout the cell cycle and must be activated by binding of cyclin D and complex is phosphorylated. This complex causes phosphorylation of RB gene and release of transcription factors allowing the progression to S phase. Loss of suppressor protein will ultimately turn on the cell cycle and lead to formation of tumors.
Degradation of cyclin

Most expression and activation of products and proteins are transient. So block must be put back into place after go through synthesis. This is done by degradation of cyclin D. Because if cyclin D is not available, complex deactivated and RB gene is not phosphorylated (converted back to its active form = dephosphorylated, where it sequesters transcription factors).
 Cdk activity is terminated by cyclin degradation once S phase is completed.

Same type of inactivation is true for other suppressor genes – loss of their activity will result in ability of tumors to form.
Molecules that regulate nuclear transcription of cell cycle p53 gene

MOD # 136
May 22, 2003 3:00 p.m.
Page 4 of 7
 p53 gene is located on chromosome 17p13.1 and it is the single most common target for genetic alteration in human tumors . A little over
50 % of human tumors contain mutations in this gene. Homozygous loss of the p53 gene is found in virtually every type of cancer, including carcinomas of the lung, colon, and breast (the three leading causes of cancer deaths).
 p53 mutations are common in a variety of human tumors suggests that the p53 protein serves as a critical gatekeeper against the formation of cancer. Indeed, it is evident that p53 acts as a “molecular policeman”
 p53 protein is localized to the nucleus

It functions primarily by controlling the transcription of several other genes.
Role of p53 (schematic) fig. 8-32 p. 291
P53 is gatekeeper – if cell becomes damaged, p53 will prevent DNA replication, essentially providing time for the cell to try and repair the DNA damage. It arrests DNA synthesis and then it upregulates proteins involved in DNA repair. If DNA repair fails, then it upregulates proteins involved in programmed cell death (apoptosis).
If DNA repair is successful, p53 ceases activity and allows normal replication.
Molecules that regulate nuclear transcription in cell cycle BRCA-1 and BRCA-2

BRCA-1 gene is located on chromosome 17q12-21 and BRCA-2 gene is located on 13q12-13.
 These genes are highly associated with the occurrence of breast and ovarian cancer.

Approximately 5-10% of breast cancers are familial. Mutations in
BRCA-1 and BRCA-2 are found in 80% of the familial cases .

In families with breast cancer, an individual that is diagnosed with disease, take DNA sample to see if other women in family have inherited mutations in BRCA-1 or BRCA-2. This test is useful in preventing women from undergoing unnecessary mastectomy procedures. Women who do not inherit mutations, still have same chance as any other woman of getting breast cancer, their risk is not increased.
Molecules that regulate signal transduction

Down regulation of growth-promoting signals is another potential area in which products of tumor-suppressor genes may be operative. The products of the NF-1 gene and the APC (adenomatous polyposis coli) gene fall into this category. Germ line mutation at the NF-1 (17q11.2) and the APC (5q21) loci are associated with benign tumors that are precursors of carcinomas that develop later.
 Picture of patient with neurofibromatosis Type 1
Cell Surface Receptors – said he would talk more about tomorrow

The binding of TGF-

to its receptor up regulates transcription of growth inhibitory genes.

MOD # 136
May 22, 2003 3:00 p.m.
Page 5 of 7

Cadherins are a family of glycoproteins that act as glue between epithelial cells. Loss of cadherins can favor the malignant phenotype by allowing easy disaggregation of cells, which can then invade locally or metastasize.

Deleted in colon carcinoma (DCC) is a gene located on chromosome
18q21. This chromosome is frequently deleted in human colon and rectum carcinomas. The DCC gene is considered a candidate tumor suppressor gene.
Other Tumor Suppressor Genes (here require the loss of both functional genes again in order to express transformed phenotype).

NF-2 gene mutation predispose to the development of neurofibromatosis type 2.
 WT-1 gene is located on 11p13 and is associated with the development of
Wilms tumor.
Inactivation of tumor suppressor proteins by oncogenic DNA viruses (schematic)

Another mechanism to cause transformation: protein produced by DNA virus bind up suppressor protein so it is not available to block cell cycle.

Viruses – for example retro-virus – contain within them an oncogene that has been transformed and if cell is infected by virus, the expression of oncogene under control of virus leads to transformation.
Levels of Control of Cell Growth (schematic)
 Starting with production of growth factor (overproduction or inappropriate expression) or expression of GF receptors – can be mutated to where they no longer require binding of growth factor.

Intracellular pathways can be turned on so they are constantly sending signals.

Signal sent can be affected and resulting in increased transcription

Also looked at several transcription factors required for control of cell division.
Checkpoint choices (schematic) when making decision whether or not to divide.

Everything is Okay – no damage detected, continuation through cell cycle occurs.

External signal (over expression of a growth factor)

Differentiate

Remain quiescent

Divide

Everything is not Okay
1.
Die – apoptosis (programmed cell death)
Apoptosis

Programmed cell death

Precisely regulated

Important functions

Distinct from necrosis (death due to other external factors)
Morphological changes seen in apoptosis

MOD # 136
May 22, 2003 3:00 p.m.
Page 6 of 7
Chromosome condensation (not available for cell division), cell body shrinkage, nucleus and cytoplasm fragments, apoptotic bodies, phagocytosis.
Regulation of apoptosis – under very tight control

Regulator: bcl 2

Adapter: Apaf 1

Effector: Caspase
Genes that regulate apoptosis – bcl-2

85% of B-cell lymphomas of the follicular type carry a characteristic t(14;18)(q32;q21) translocation. Many translocation that involve activation of oncogenes are amongst those genes that carry immunoglobulin genes. On chromosome 14 = Ig heavy chain gene. On chromosome 2-kappa light chain and on chromosome 22- lambda light chain. Predominantly when you see these translocations, they will involve the immunoglobulin heavy chain gene because in the normal progression of B cell, the heavy chain gene rearranges first. These rearrangements are normal cellular processes giving rise to antibodies.

Over expression of the bcl 2 protein protects lymphocytes from apoptosis and allows them to survive for extended periods; thus there is a steady accumulation of B lymphocytes (which can accumulate other mutations) and result in lymphadenopathy and marrow infiltration.
Bcl 2 as an oncogene

B-cell lymphoma
 myc – excess cells produced
 bcl 2 – excess cells do not die
Regulation of Apoptosis (schematic)
 bcl-2 prevents apoptosis when over expressed, prolonging their existence.
 bax – result ultimately in cell death
 caspases
Regulation of Cell death (schematic) fig. 8-33 p. 295
 teetering effect –excess bcl – allows cells to survive

If have over expression of bax – tilts cell towards apoptosis or cell death.

P53 detecting these mutations or DNA damage, can upregulate bax to stimulate apoptosis.
Regulation of Cell death (schematic)

P53 effects: cells with loss of p53 function = no G1 arrest and leads to
DNA damage and mutant or transformed cells.
 Cells with increased p53 there is G1 arrest leading to either DNA repair or apoptosis if a failure to repair occurs.
Sample Quiz Questions
1. A patient presents with lung cancer. Molecular analysis reveals a point mutation in codon 12 of the ras protein. This mutation is most likely to result in: inability of GAP to stimulate the intrinsic GTPase activity of ras.

MOD # 136
May 22, 2003 3:00 p.m.
Page 7 of 7
2. A patient presents with WBC count of 275,000 predominately immature and mature neutrophilic cells. Cytogenic analysis reveals a translocation involving c-abl protooncogene and the bcr region on chromosome 22. Which molecular event is most likely related to the translocation? Production of a 210 kd protein with an altered tyrosine kinase activity.
3. Activation of c-myc proto-oncogene in Burkitt’s lymphoma results from: translocation in the Ig heavy chain gene.
4. A patient has a needle biopsy that reveals t(14; 18)(q32; q21) translocation. The diagnosis is Follicular lymphoma. Which of the following mechanisms is most likely to produce neoplasia in this patient? Protection of B-lymphocytes from apoptosis.