Tumor Suppressor Genes 1

IRA & NF1

-IRA=yeast protein which is a Ras GAP protein -sequence similar to NF1

What is a newer way to find regions of frequent loss of heterozygosity?

-PCR -can test thousands of polymorphic sequences in normal & cancer cells and identify regions that frequently undergo loss of heterozygosity in tumors

Cancer alleles are what?

-Recessive -evidence for tumor suppressing genes

Futile cycle

-VHL provokes degradation of the HIF-1 alpha subunit of HIF-1. Consequently HIF-1alpha synthesized and then degraded quickly. -under normal conditions HIF-1 transcription factor accumulates to only a very low level and mostly inactive

"cafe au laid spots"

-areas of hyperpigmentation on the skin -patients with NF sometimes have these benign lesions on the iris

Adenomatous polyposis coli

-called familial adenomatous polyposis -associated with an inherited susceptibility to develop colon polyps -tumor suppressor -frequently associated with colon cancer

Chromosal Instability

-cells lacking APC have been shown to have an increase in chromosomal instability (CIN) -results from increases & decreases in chromosome number -can lead to accumulation of too many chromosomes

Oncogenes

-cellular genes are the source of most cancers when mutated -also known as tumor suppressor genes

B-catenin

-controls out migration of colonic epithelial cells -high in the stem cells in the crypt

Neurofibromatosis 1 (NF1)

-familial cancer associated with the development of benign tumors of the cell sheaths around nerves -can become malignant called neurofibrosarcomas

RFLP & SNP

-found within a restriction site -occur normally in normal tissue

Trichostatin A (TSA)

-histone deacetylase inhibitor -reverses deacetylation and reverts the chromatin to a state that favors transcription -treatment of breast cancer with TSA can restore sensitivity to RA

what happens to Schwann cells once they lose NF1?

-lose control of proliferation -induce co-proliferation of other cell types -thus multiple cell types can be affected

Mechanisms for inactivation of tumor suppressor genes?

-loss of heterozygosity -promoter methylation: methylation in the promoter can suppress transcription of the gene

2 different ways hypermethylation can occur

-loss of one allele by LOH and the second allele is inactivated by hypermethylation, followed by duplication of the methylated allele -both alleles could be inactivated independently by hypermethylation

Oncogene

-mutant (activated) or overexpressed version of the proto-oncogene -found in cancer -mutations that involve activation cause cancer

Colonic crypt contains replicating stem cells where?

-near the bottom -cells further up are more differentiatied and have specialized functions

Sporadic Retinoblastoma

-no family history -once tumor (usually only in 1 eye) is removed, no further risk of cancer

Histones

-nuclear proteins involved in packaging DNA -contain acetyl groups

Gene conversion

-occurs during DNA replication (S phase) -DNA polymerase begins replication on template strand, jumps to template strand of homologous chromosome, and then back to template strand of original chromosome

Oncogenes and tumor suppressor genes and alleles

-oncogenes: mutations on one allele sufficient to cause cancer -tumor suppressor genes: mutations must occur on both alleles

Methylation

-one factor that controls which genes are expressed in tumors -considered to be epigenetic: heritable trait but does not alter the nucleotide sequence of the DNA -inactivates some tumor suppressor genes -makes it hard to identify new tumor suppressor genes -only affects transcription when it is in the promoter

Tumor suppressor gene

-only causes cancer if it is inactivated or deleted -can be considered the opposite of an oncogene -mutations that cause inactivation cause cancer

Familial Retinoblastoma

-parent also had the disease -multiple tumors in both eyes -patient has increased risk for cancer after removal

Retinoic Acid

-potent inducer of cell cycle arrest -has been used to try and half proliferation of breast cancer cells -RARB2 encodes the RA receptor-often silenced due to proliferation in cancer patients, and thus unresponsive to RA

p16INK4A tumor suppressor gene

-promoter found to be methylated in 44% of smokers -indicates that methylation can be a very early event occurring long before any tumors form

How does loss of heterozygosity occur?

-random mutation -mitotic recombination -gene conversion -hemizygosity (an allele is missing)

Mitotic recombination

-recombination that occurs in mitosis rather than meiosis -occurs at low frequency

Arginine finger

-region that contacts Ras -fits into cleft and contributes to GTP hydrolysis

Ras oncogene

-single point mutation -one of the most well known oncogenes

Protective system of colon against cancer

-stem cells protected in the crypt area -some of the progeny is dispatched upward and out of the crypts to maintain a CONSTANT number of cells -these function briefly to form the lining of the gut, then diet by apoptosis and shed into colonic lumen -process of out migration to cell death takes 3-4 days

2 types of tumor suppressors

-suppress cell cycle progression (proliferation) or induce differentiation (ex. Rb) -inhibit proliferation in response to metabolic imbalances or genomic damage (ex. p53)

What happens in cell fusion experiments when cells growing adjacent to each other in culture are exposed to a fusogenic agent?

-they form a heterokaryon with multiple nuclei -when this cell passes through mitosis, the two sets ofo parental chromosomes are pooled in a single nucleus

Retinoblastoma

-tumor of the retina -one of the first tumor suppressor genes discovered -loss of heterozygosity causes cancer

when are tumor suppressor genes are involved in cancer

-when the genes are inactivated or lost -genes that constrain or suppress cell proliferation -antigrowth genes: tumor suppressors

The APC gene is frequently mutated in what?

FAP-familial adenomatous polyposis

What may be involved in DNA methylation inhibiting transcription?

Histone Deacetylase (HDACs)

Inactive vs. Active forms of the Ras oncogene

Inactive Form: Ras + GDP Active Form: Ras + GTP-initiates downstream signaling-can activate multiple pathways

RFLP

Restriction Fragment Length Polymorphism

T/F: Methylation specific PCR- Methylation specific primer will only bind to sequences that are methylated and will therefore only result in a PCR product in the sequence is methylated

True

T/F: Within a given tumor, often multiple genes are shut down by methylation

True

Wnt signaling and APC

Wnt signaling promotes proliferation

What is formed by fusion of a cancer cell with a normal cell?

a heterokaryon that i soften non-tumorigenic

loss of NF1 mimics what?

active Ras

Hemizygosity

an allele is missing

Loss of APC could lead to what?

aneuploidy (incorrect number of chromosomes) due to improper chromosome segregation

Phenochromocytomas

arising from the adrenal glands

In almost all patients who have pVHL

assocation with mutations in the VHL tumor suppressor gene

Additional APC function

can localize to components of the microtubule arrays that form the mitotic spindle which is responsible for segregating chromosomes during mitsos

Loss of APC causes what?

causes cells to stay crypt cells and to proliferate excessively -gives them chance to develop other mutations (ie. Ras mutations)

What experiments provided some of the first clues for the presence of tumor suppressor genes?

cell fusion experiments

Mutations in APC associated with what?

colon cancer often consist of premature termination of the Apc protein, thereby removing domains that are important for its ability to associate with B-catenin and the resulting degradation of B-catenin

what happens when histones get deacetylated?

converts the chromatin from a configuration favoring transcription to one blocking transcription

restriction sites

defined short sequences where restriction enzymes cut DNA

Patients with retinoblastoma frequently had what?

deletions in the Rb gene

What keeps B-catenin levels high?

enterocyte stem cells encounter Wnt factors released by stromal cells near the bottom of the crypt

RFLP analysis is used to identify what?

frequency of loss of heterozygosity in colorectal tumors

Von Hippel-Lindau Disease (pVHL)

hereditary predisposition to the development of a variety of tumors including clear-cell carcinomas of the kidney, pheochromocytomas (adrenal gland) and hemangioblastomas (blood vessel tumors) of the CNS and retina

BRCA1 gene

inactivation of this gene is associated with breast cancer risk

what happens when some of the progency migrate upward and no longer receive Wnt signal?

intracellular B-catenin goes down and transcription of stem cell genes stops

High levels of B-catenin does what in certain enterocytes (intestinal cells)?

leads to activation of genes that encourage the stem cell phenotype

Colon cancer

majority are sporadic but a small group arise due to inherited alleles

What are RFLPs used for?

mapping genes

what type of mutations lead to cancer in the colon?

mutations that block the out migration of colonic epithelial cells and the cell death that occurs after several days

Proto-oncogene

normal cellular gene that is associated with cancer only if it is improperly activated (usually by overexpression, mutation, or gene translocation)

Colon polyps

often nonmalignant but can grow into carcinomas

what occurs under normal oxygen?

proline hydroxoylase converts one or both prolines on HIF-1alpha to hydroxyproline residues

SNP

single nucleotide polymorphism

what happens when loss of heterozygosity occurs?

the entire chromosomal region is affected

Main function of VHL

to foster destruction of a subunit of a transccription factor called hypoxia-inducible factor-1 (HIF-1)

What happens in methylation specific PCR

treatment with sodium bisulfite under alkaline conditions converts nonmethylated cytosines (Cs) to uracil's (Us) while leaving methylated Cs untouched

What is APC critical for?

triggering the degradation of B-catenin

Glioblastomas

tumors of astroycte lineage in the brain