New Perspectives On Dna Mutations

Studies on hereditary nonpolyposis colorectal cancer (HNPCC) have shown that affected individuals may inherit an inactive copy of one of the DNA mismatch repair genes. The main function of these genes is to produce proteins whose primary function is to iden

TABLE 1.2. Tumor Suppressor Genes."

Genes for cytoplasmic proteins

APC (colon and stomach cancers)

DPC4 codes for relay molecule in cell division inhibitory pathway (pancreatic cancer)

NF1 codes for protein that inhibits a stimulatory (Ras) protein (brain, nerve, and leukemia) NF2 (brain and nerve cancers)

Genes for nuclear proteins

MTS1 codes for p16 protein, brake on cell cycle clock (many cancers)

RB codes for pRB protein, master brake on cell cycle (retinoblastoma, bone, bladder, lung, and breast cancer) p53 codes for p53 protein, halts cell cycle in G1, and induces cell suicide (many cancers)

p16 inhibits cyclin D-dependent kinase activity WT1 (Wilms tumor of the kidney)

BRCA1 functions in repair of damage to DNA (breast and ovarian cancers) BRCA2 functions in repair of damage to DNA (breast cancer)

Location not clear

VHL (kidney cancer)

These genes are associated with inhibition of cell division. Cancers require both alleles of the gene to be altered.

These genes are associated with inhibition of cell division. Cancers require both alleles of the gene to be altered.

tify and correct DNA replication errors. Mismatch repair prevents spontaneous mutation; therefore, cells defective in the DNA mismatch repair enzymes system may accumulate mutations at rates several hundredfold higher than normal.

Randomly dispersed throughout the human genome are tens of thousands of microsatellites, long stretches of reiterated mono- or dinucleotides: for example An (A = adenine; n = repeated n times) or (C-A)n (C-A = cytosine-adenine dinucleotide; e.g., CACACACAn]. The accurate replication of such repetitive DNA is usually compromised by the tendency of template and daughter DNA strands to misalign during DNA synthesis. In cells with defective DNA mismatch repair enzyme system, the precise control of microsatellite length is lost, and therefore, the cells contain many thousands of altered microsatellites. This accumulation of mutated repeats is commonly known as microsatellite instability (MSI), which is the defining characteristic of mismatch-repair-deficient tumors.

MSI is a continuing process and is a direct consequence of failure to rectify replication errors. In practice, MSI is defined by differences in the lengths of several microsatellites between DNA from tumor and normal tissue of the same individual.30 In principle, the two alleles of a given chromosome of an individual contains microsatellite DNA made of repeats (e.g., CA repeats) of unequal length. In "genetic" terms this means that individuals are most frequently heterozygous (they have two different copies of the same DNA portion in the two homologue copies of the same chromosome) for most of the known microsatellite sequences within their genome. This implies that microsatellites are genetic markers of great value in detecting the LOH process, which characterizes cancers that are due to the loss or inactivation of TSGs. In routine laboratory activity, microsatellite DNA can be easily amplified by using the PCR. The amplified DNA is analyzed by resolving it through a polyacryl-amide gel, where it can be evidenced as a band pattern that is characteristic of any single microsatellite in each individual.

While microsatellites have long been considered to be genetic markers useful in detecting LOH in the tumor genotype, as opposed to the somatic genotype of the affected individual, their widespread use in the characterization of different tumors has revealed that MSI is another relevant process in carcinogenesis. In simplified terms, the difference between LOH and MSI resides in the qualitative difference of the DNA migration patterns one can find when the constitutional (nucleated blood cells) and the tumor genotypes of an affected individual are compared. LOH is the loss of one or more bands in the tumors with respect to the related constitutional genotype. MSI is not a loss but a band shift, as shown in Figure 1.3, which presents

MSI in embryonal RMS of the orbit N T

Band shift

MSI in embryonal RMS of the orbit N T

Band shift

FIGURE 1.3. Three examples of MSI in embryonal rhabdomyosarcoma of the orbit. MSI, like LOH, is a modification, in comparison to the normal constitutional cells (blood) of the affected individual, of the DNA migration pattern of tumor cells as revealed on agarose or Polyacrylamide gel. Unlike LOH, which is evidenced as the loss of one or more bands in the tumor DNA, MSI is a qualitative difference that is evidenced as a "band shift."

three cases of MSI in embryonal rhabdomyosarcoma of the orbit.

Cells with a defective mismatch repair system are said to carry a mutator phenotype, a phenotype in which many genes carry mutations because of uncorrected errors in DNA replication. These cells are also said to carry genome instability, the process leading, through accelerated somatic evolution, to a genomically heterogeneous population of cells naturally selected for their ability to proliferate and invade, while simultaneously evading host defenses.31 This picture leads to at least two different considerations.

1. It is likely that several mutations in different mismatch repair genes, rather than a single mutation in one gene, represent the most relevant early event in carcinogenesis.

2. The genome instability theory brings us back to Boveri's observations, since an unstable genome is more likely to produce unbalanced distribution of chromosomes between the daughter cells during mitosis, as observed by Boveri in his experiments on double-fertilized sea urchin eggs.

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