Once you have a tumor cells there is just a massive accumulation of more mutations. Tumors replicate more, and faster — each division is an opportunity for mutation. Sporadic cnacers are more common — you inherit susceptibility but most important mutations are somatic during your lifetime. There can be strong, almost mendellian predisposition. Mutations in tumor suppressor, tumor promoter (proto-oncogenes, which mutate to become oncogens) and DNA mismatch repair genes. Cancer is any tumor which can spread — a tumor develops in the following steps:

1. Initiating mutation.
2. Uncontrolled proliferation, dramatically increasing likelihood of additional mutations.
3. Reduced dependence on growth signals and even more mutations.
4. Anchorage, independence and metastasis.

Chromosome abnormalities (like translocations), loss of heterozygosity, positional cloning of familial cancer genes and functional cloning all tip off which cancer genes are involved in a cancer.

1. Tumor suppressor genes, which prevent inappropriate division and must be mutated in both alleles for a deleterious effect. The two-hit model is: in sporadic cancers, both tumor-suppressor alleles mutate somatically; in familial cancers, one defective tumor-suppressor allele is inherited and the other mutates somatically.
2. Tumor promoter genes (proto-oncogenes) which promote division during development but are normally silent in adults. They become oncogenes through mutation and force the cell into continuous division.
3. DNA repair genes, which protect against DNA mutation due to replication or the environment. Mutation in a DNA repair genes leads to increased mutation of tumor suppressor and proto-oncogenes.

Loss of heterozygosity is characterized by normal tissues showing two bands when probed — one for each allele — and tumor tissues only showing one band. This is an important mechanism of cancer, whereby a healthy cell with one faulty allele and one healthy allele suddenly has two faulty alleles. Loss of heterozygosity can occur via:

1. Local events, with one allele garnering point mutations or the like.
2. Somatic recombination, which is most likely.
3. Loss and duplication, where one allele is lost and the other is duplicated in its place.
4. Chromosome loss or partial deletion, leaving only the defective allele.

Recombination events are what usually lead to loss of heterozygosity. This happens via unequal crossing-over (improper recombiation) whereby part of one chromosome replaces the homologous region on the other chromosome, which leads to both chromosomes carrying the mutant region.

Unlike other tumor suppressor genes, just one defective p53 allele is detrimental because it is a tetramer. Just one mutant p53 allele means that only 1/16 of all p53 tetramers will be function. Most common detrimental mutations involve p53 core domain that interacts with DNA. Germ-line mutation of p53 (Li-Fraumeni) leads to oncogenesis amongst almost all relatives, and of different tissues (breast/epithelial/etc). p53 inducers and mtuant phenotypes are:

Normal cell require stimulatory signals to proliferate; tumor cells achieve autonomy where they proliferate without stimultion. Known proto-oncogenes include growth factors and their receptors (PDGF, FGF, EGF, CSF), signal transducers (ras, src, abl) and signal effectors (myc, fos, jun).

Oncogenes arise via:

1. Translocation, caused by breakpoints near proto-oncogene leading to inappropriate expression, or even fusion with another protein. Different fusions are associated with different cancers.
2. Structural mutation, caused by changes in coding regions of growth factor receptors and signal transducing proteins. A point mutation in ras leads to constitutive activation in the absence of growth factor stimulation.
3. Amplification or regulatoroy mutation, leading to structurally normal oncogenes that are just produced at high levels. For example, multiple copies of a single gene or insertion of a viral LTR before a gene.

Oncogenes are isolated via the DNA transformation assay:

1. Human tumor DNA transfected into mouse 3T3 cells, which are not immortal and are contact-inhibited (do not grow when crowded).
2. Oncogenically transformed cells form proliferating colonies. DNA from these colonies is purified and used to oncogenically transform another plate of 3T3 cells.
3. Step ‘b’ is repeated
4. DNA is isolated from proliferating colonies, and used to form a genomic library.
5. The genomic library is screened for human Alu sequences, which separates clones of murine DNA from clones of human DNA.
6. Remaining clones should contain the oncogene.

Protein coding changes alone are unlikely to determine human-chimp differences. Sequence differences in non-coding DNA that influences expression levels are likely to be critical.Which species comparison is most useful?

• Human-mouse comparisons (80 Myr)
• Human-fish comparisons (400 Myr)
• Human-chicken comparisons (300 Myr)
• Human-primate comparisons
• Human-human comparisons

DNA methylation may mediate transcriptional repression by histone deacetylation and histone methylation. The methylation imprint established in early development is maintained by a “maintenance methylase” that recognizes hemimethylated DNA and methylates the CpG on the other strand. Methylation occurs most often in occurs most often in cytosines of CpG islands; these islands are relatively rich in CpG dinucleotides and are often associated with genes. Imprinted genes have CpG islands which are methylated differently on the maternal and paternal alleles.

During gametogenesis, the old imprint must be erased and a new sex-specific imprint must be established. Different genes can be active or silenced on the same parental homologue. Beckwith-Wiedeman syndrome arises when a patient has both Chromosome 11′s from the same parent — this leads to bilallelic expression of IGF2 (growth factor gene) and biallelic silencing of H19 (tumor suppressor gene).

Hypermethylation leads to chromatin condensation and gene silencing. Not all imprinted genes are hypermethylated at their promoters; some are hypermethylated at intergenic control regions called imprinting centers that control imprinted gene cluster (called enhancer blocking). Identical deletions of Chr 15 cause PWS when maternal UPD (silencing of matneral genes) inherited or AS when paternal UPD (paternal alleles silenced). So far all PWS cases are new due to sterility of patients; some AS cases are familial, with unaffected fathers passing a mutant gene to half their offspring of tiether sex. If a son passes the mutant on it will not show. If a daughter does then half her kids will have AS, leading to its appearance in multiple progeny over mutliple genrations.

Explains why monozygotic twins might have different phenotypes, with one child having an X-linked disorder and the other child not. In a normal female, only one of the two X chromosomes present is genetically active, the other being inactivated. X-inactivation occurs early in development. The inactive X can be either maternal or paternal in origin; the choice is random. X inactivation is irreversible in somatic cells, such that the inactive X in a particular cell remains inactive in all descendants of that cell. In most mammals, one X in females is inactivated. Alternatives are for one X in male hypertranscribes, two X’s in female hypotranscribe or the apternal X chromosome is inactive. In the early zygote in/activation, all descendant cells have inactivation or in alte blastocyst it is random and their is mosaicism.

Properties of the inactive X chromosome:

1. Most (but not all) genes are inactivated at the transcriptional level.
2. Heterochromatic
3. Late replicating
4. Highly methylated at CpG islands
5. Histone hypoacetylation
6. Enrichment for the variant histone macroH2A

Epigenetic regulation is stable transmission of gene expression to daughter cells in absence of change in DNA content or sequence. This is due to chromatin structure, DNA mod or both. Methylation imprint established in early dvelopment is maintined by maintenance methylase that recognizes hemi(one strand)methylated DNA and methylates CpG on other strand. CpG dinucleotides are targets for DNA methylation, and methlyated CpG are targets for specific binding by proteins like MeCP2. MeCP2 recruits histone deacetylases that remove acetyl groups from histone tail.

X inactivation involves a recognition step (referred to as counting counting) in which the number of X chr in a cells is counted relative to cell ploidy so that only a single X chr is functional per diploid adult cell.

1. Xist is initially expressed as an unstable transcript from both X chrs in undifferentiated ES cells and is perhaps prevented from coating the X chr by a blocking factor.
2. After counting and choice, Xist expression increases on the Xi and is repressed on the Xa. The coating of the Xi by Xist may involve long interspersed nuclear elements (L1 repeats).
3. Coating of the Xi establishes the inactive state. The coated Xi begins to replicate late in the S phase of the cell cycle (asynchronous replication), a hallmark of the Barr body (Xi) in adult cells.
4. MacroH2A recruitment and H3/H4 hypoacetylation stabilize the heterochromatic state. Widespread methylation of promoters further ensures faithful transmission of this state to all daughter cells. At this point Xist expression is not required although it is retained.

The choice of Xa and Xi always occurs in a mutually exclusive manner. What mechanism mediates the crosstalk?Hypothesis: Choice is mediated by a physical interaction of X-chromosomes (chromosome kissing).

Experiment: Measure distance between Xic regions during ES cell differentiation. During X^I establishment the X chromosomes become very near and then separate. This can be done by formaldehyde induced crosslinks of near DNAs, followed by digestion, ligation of the two DNA segments and then detect ligation product by quantitative PCR.