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