The majority of human cancers (including solid tumors, leukemias, and lymphomas) contain chromosomal abnormalities, consisting of either numerical changes (aneuploidy) and/or structural aberrations (102,103). These two general types of chromosomal damage might reflect two distinct mechanisms of chromosomal instability (9,104): (1) chromosome number instability and (2) chromosome structure instability. In some forms of cancer, chromosomal instabilities predominate over nucleotide sequence instabilities, suggesting that these mechanisms of genetic instability might not significantly overlap. Recent evidence suggests a genetic basis for chromosomal instability in cancer, involving mutational inactivation of certain types of gene in aneuploid tumors (105).
Detailed karyotypic studies have been carried out on a large number of tumor types. Many of these studies have examined chromosomal alterations in leukemia and lymphoma (102), partially reflecting the relative ease with which chromosomes can be prepared from these cancer cells. Traditional cytoge-netic analyses of solid tumors are more difficult. Nonetheless, a substantial body of literature on the chromosomal aberrations of solid tumors has emerged (25). Additional methods have also been applied to examination of chromosomal abnormalities in solid tumors (103). A large number of studies have investigated allelic loss of heterozygosity in various human solid tumors using Southern analysis or polymerase chain reaction (PCR) (106-110). Although these methods do not provide the same information as karyotypic analysis, the indication of large-scale deletions can be inferred from the loss of multiple markers on a specific chromosomal arm (111). In addition, flow cytometry is now widely employed for determination of tumor ploidy (112), and fluorescence in situ hybridization (FISH) is used to examine specific chromosome numbers and alterations (113,114). Spectral karyotyping combines FISH with a karyotypic analysis of chromosomes from tumor cells
(115), eliminating the requirement for chromosome banding in karyotype analysis. Derivative methods have been developed that facilitate chromosome banding analysis through FISH
(116). These powerful new techniques for chromosomal analysis have generated a revival of genetic investigations of cancer at the level of the chromosome. A detailed review of chromosomal alterations in human cancer is beyond the scope of this chapter. Several excellent reviews are available (25,117,118).
9.1. INSTABILITY OF CHROMOSOME NUMBER Numerical alterations of chromosomes can involve both loss of entire chromosomes or allelic losses, which might be accompanied by duplication of the opposite allele. This phenomenon results in the generation of a tumor with normal karyotype but an abnormal allelotype (106). Several studies have produced evidence that suggest that tumors arising in various tissues share a common chromosome number instability and might lose a significant number (25-50%) of alleles during neoplastic transformation and tumorigenesis (106,108,119,120). These large-scale genomic changes could be the result of some form of progressive chromosomal instability (121,122). In support of this suggestion are studies showing that gains and losses of multiple chromosomes occur in aneuploid colorectal cancer cell lines 10-fold to 100-fold more frequently than in diploid cancer cell lines of the same histological subtype (66,123). In other studies, the rate of loss of heterozygosity (LOH) at marker loci proximal to a selectable gene (adenine phosphori-bosyl transferase) was increased 10-fold in colorectal cancer cell lines that exhibit proficiency of mismatch repair compared with cell lines that lack mismatch repair (124,125). In addition to these results, numerous studies have combined to show that aneuploid cancers exhibit highly variable karyotypes (102,126), suggesting that new chromosomal variations are produced in a progressive manner during tumor outgrowth and evolution.
The absence of chromosomal instability in diploid cancers and/or cancers that exhibit nucleotide sequence alterations argues against a nonspecific mechanism for chromosomal instability related to abnormal properties of neoplastic cells (9). Further, the high rates of numerical chromosomal alterations in aneuploid cells do not simply reflect the ability of these cells to survive changes in chromosome number (66). Likewise, tetraploid cells resulting from the fusion of diploid cancer cells retain a stable tetraploid chromosome number (66), suggesting that the presence of a nondiploid chromosome number does not in and of itself precipitate progressive chromosomal instability. Rather, the evidence from the literature supports the existence of a specific form of genetic instability in cancer cells that results from dysfunction of normal chromosomal homeostasis producing numerical chromosomal abnormalities. Several possibilities have been investigated, including the involvement of (1) mutant p53 protein, (2) abnormal centrosomes, (3) abnormal mitotic spindle checkpoint function, or (4) abnormal DNA-damage checkpoint function (9,122,127).
9.1.1. Inactivation of the p53 Tumor Suppressor Leads to Abnormalities of Chromosome Number The p53 tumor suppressor protein has long been suggested to play significant roles in cell cycle progression and cell cycle checkpoint function in response to DNA damage (82,128). The p53 gene is commonly mutated in human cancers (53), and these same cancers frequently exhibit abnormalities of chromosome number (117). Thus, numerous studies have been performed in order to define the role of p53 in the maintenance of chromosomal stability in normal cells and instability in neoplastic cells. In various forms of cancer, the presence of chromosomal abnormalities correlates with p53 mutation (129,130). Cells in culture often become aneuploid concurrent with mutation or inactivation of p53 (131,132). These observations suggests that loss of p53 function leads to abnormal regulation of mitosis and segregation of chromosomes (133). However, several other lines of evidence do not support a direct role for p53 mutation in the genesis of this form of chromosomal instability. For example, development of aneuploidy occurs very early in the process of neoplastic transformation and tumorigenesis (134), and p53 mutation typically occurs later in the process (135). In addition, some diploid tumor cell lines that exhibit a stable karyotype also contain mutant p53 (136). These observations combine to suggest that loss of normal p53 function could contribute significantly to chromosomal instability in certain forms of cancer but does not represent the primary cause of this form of genomic instability.
9.1.2. Abnormal Centrosome Function Leads to Chromosomal Abnormalities Aneuploid tumors demonstrate significant numbers of chromosomal imbalances, whereas such imbalances are rare in diploid or near-diploid tumors. The abnormalities of chromosome number observed in aneuploid tumors are consistent with a mechanism involving dysfunction of chromosome segregation during mitosis. Several lines of evidence support the idea that the integrity of the centrosome plays an integral role in the development of aneuploidy. Human tumors and tumor-derived cell lines have been characterized to contain abnormal numbers of centrosomes, abnormally sized and shaped centrosomes, and multipolar spindles in a number of human neoplasms, including tumors of the breast, lung, prostate, colon, pancreas, head and neck, bile duct, and brain (137,138). In addition, the numbers of centrosomes was elevated in six of the seven (85%) aneuploid colorectal carcinoma cell lines evaluated, compared to diploid tumor cell lines, which displayed normal centrosome numbers (123). Further, centrosome function was impaired in four of the five (80%) aneuploid colorectal cancer cell lines examined, whereas cen-trosome function was found to be intact in all diploid tumor cell lines (123). These observations suggest that abnormal cen-trosome number and/or function are common among neoplas-tic cells that display aneuploidy and might represent an essential component of chromosome number instability in human cancers.
The mechanism leading to the formation of increased numbers of centrosomes in cancer cells remains undefined. However, abnormal centrosome number and function has been linked to the STK15 kinase in some cancers (139,140) and to a related kinase (PLK1) in others (141). The STK15 gene was found to be amplified in approx 12% of primary breast cancers and in cell lines derived from neuroblastoma and tumors of the breast, ovary, colon, prostate, and cervix (140). Overexpression of STK15 (evidenced by immunostaining) was detected in 94% of invasive ductal carcinomas of the breast irrespective of histopathological subtype, suggesting that overexpression of this centrosome-associated kinase might be a common feature of breast cancers (142). In addition, overexpression of STK15 was found in cell lines that lacked evidence of gene amplification, and ectopic expression of STK15 in near-diploid human breast epithelial cells produced centrosome abnormality accompanied by induction of aneuploidy (140). An alternative mechanism suggests that mutational inactivation of p53 or functional inactivation of p53 through binding by mdm2 results in abnormal centrosome numbers and induction of chromosomal instability (143,144). Furthermore, there is evidence that loss of BRCA1 or BRCA2 can lead to centrosome amplification and chromosome segregation dysfunction (145,146). These studies combine to suggest that a number of different genes might contribute to centrosome function and homeostasis in normal cells and that inactivation or dysregulation of one or more of them can lead to abnormal centrosome number/function.
9.1.3. Aberrant Mitotic Spindle Checkpoint Function Leads to Aneuploidy The mitotic spindle checkpoint governs proper chromosome segregation by ensuring that chro-matid separation does not occur prior to completion of alignment of all chromosomes along the mitotic spindle (147). It follows that if the mitotic spindle checkpoint is defective, chromosome segregation during mitosis will occur asynchronously, potentially producing an unequal distribution of chro-matids between the daughter cells (147). Evidence supporting a role for aberrant mitotic spindle checkpoint function in the development of aneuploidy includes the observation that aneu-ploid cells respond inappropriately to agents that disrupt the spindle apparatus, such as colcemid. Normal cells respond to colcemid treatment by arresting in metaphase, whereas cells that display instability of chromosome numbers prematurely exit mitosis and initiate another round of DNA synthesis (67). The hallmark of mitotic spindle checkpoint defect is the inability to inhibit entry into the S-phase when mitosis cannot be completed because of damage to the mitotic spindle (148). Mutation or aberrant expression of genes that encode proteins involved in mitotic spindle checkpoint function can eliminate proper checkpoint function in tumor cells, contributing to development of aneuploidy. A number of these genes have now been identified (149). Alterations in mitotic spindle checkpoint genes have been documented in several human cancers, including decreased expression of hMAD2 in breast cancers (150), and mutations in the hBUB1 gene in colorectal cancers (67,151). However, these mitotic spindle checkpoint genes are not implicated in all aneuploid cancers. Some aneuploid breast cancers lack mutations in hBUB1 and exhibit normal mRNA expression levels (152). Likewise, cancers of the respiratory tract, including head and neck cancers, small-cell lung carcinoma, and non-small-cell lung carcinoma, have not been shown to have significant numbers of mutations in hBUB1 (151,153,154), and sporadic tumors of the digestive tract rarely contain mutations of hBUB1 or hsMAD2 (155). The absence of mutations or significant alterations in expression of mitotic spindle checkpoint genes in aneuploid cells suggests that additional genes and/or mechanisms of checkpoint inactivation are operational in the majority of cancers that demonstrate chromosomal instability. Certain p53 mutations have been described that are associated with gain-of-function and relaxed spindle checkpoint function in response to mitotic inhibitors, suggesting that both mutational inactivation of p53 and dominant gain-of-function mutations in p53 can contribute to genomic instability and aberrant chromosome segregation (156). In addition, defective checkpoint function has been demonstrated in patients with ataxia telangiectasia who carry mutations of the ATM gene (157). These studies combine to suggest that a variety of genes might function in normal control of the mitotic spindle checkpoint, and when mutated or aberrantly expressed, they could contribute to chromosomal instability through inac-tivation of the mitotic spindle checkpoint.
9.1.4. Abnormal DNA Damage Checkpoint Function Leads to Aneuploidy The DNA damage checkpoint represents the major cellular mechanism that guards against the replication of damaged DNA or entry of cells with DNA damage into mitosis. The types of DNA damage that elicit checkpoint activation include polymerase errors remaining after DNA replication and other forms of incompletely repaired DNA, damage resulting from exposure to exogenous genotox-ins (ionizing radiation, chemical mutagens, and others), and damage related to endogenous genotoxic insult (such as reactive oxygen species). A number of genes have been implicated in the control of this checkpoint, including p53 (128), ATM (158), BRCA1 and BRCA2 (159), and some others (9). Functional inactivation of one or more of these genes through genetic or epigenetic mechanisms could result in a genomic instability related to the loss of the DNA-damage checkpoint. Loss of this checkpoint might then lead to development of ane-uploidy, directly resulting from abnormal segregation of damaged chromosomes (9).
9.2. INSTABILITY OF CHROMOSOME STRUCTURE The majority of human cancers exhibit chromosomal abnormalities, including marker chromosomes with altered structure. It is generally accepted that many (if not the majority) of the alterations of chromosome structure occurring in cancer cells confer some selective advantage to the evolving tumor. Thus, accumulation of a critical number of chromosomal aberrations or development of specific chromosomal abnormalities might represent essential steps in the process of neoplastic transformation. Three general forms of chromosomal alteration are observed in cancer cells: (1) gene amplifications, (2) rearrangements and translocations, and (3) large-scale deletions.
9.2.1. Gene Amplification The amplification of specific chromosomal segments or genes have been documented in some cancers and in many cancer cell lines (64,160), some of which involve cellular proto-oncogenes resulting in abnormal expression levels of the proto-oncogene products (161). In general, gene amplification occurs late in tumorigenesis and is associated with tumor progression. It is the recognized mechanism through which many tumors acquire resistance to chemothera-peutic agents. Thus, gene amplifications can profoundly affect tumor behavior and can have prognostic significance for some cancers. However, gene amplifications probably are not involved with early genetic alterations in preneoplastic lesions leading to neoplastic transformation. The mechanisms governing gene amplification have not been determined with any certainty. However, several studies suggest that gene amplification occurs at much higher rates in neoplastic cells than in normal cells (64). A role for the p53 tumor suppressor in gene amplification has been suggested by some investigators. Evidence supporting this suggestion includes the observation that gene amplification occurs more readily in cells following inactivation of p53 function (162,163). However, gene amplification can also occur in cells with normal p53 (162). One possibility for the role of p53 in this process is that amplification of a chromosomal segment in a normal cell might trigger apoptosis in response to perceived DNA damage (164), whereas in the absence of normal p53 function cells would not undergo apoptosis, but would continue to accumulate amplicons in subsequent rounds of replication (9). Thus, this form of chromosomal instability might involve a mechanism (or a mechanistic component) that increases the ability of an affected cell to survive the genetic alteration.
9.2.2. Chromosomal Rearrangements and Translocations Chromosomal rearrangements can take on several different forms, the most common of which are chromosomal translocations. Patterns of chromosomal translocation in human cancer can be classified as complex or simple (9). In some human cancers, no consistent pattern of chromosomal abnormality can be discerned (complex translocations). These tumors exhibit complex type translocations, which might appear to be random. Among individual tumors of one type, or individual cells of a single tumor, different chromosomal aberrations might be found. Very often, these rearrangements are accompanied by large-scale loss of chromosomal segments. Although it is possible that some of these chromosomal alterations are not essential to tumorigenesis, it is unlikely that any chromosomal alteration that does not confer a proliferative or adaptive advantage would be preserved in an evolving tumor. In some human cancers, specific chromosomal anomalies are consistently found in a high percentage of tumors (simple translocations). These recurrent chromosomal abnormalities might reflect molecular alterations that are essential and necessary to the molecular pathogenesis of the specific tumor type. The discovery of the Philadelphia chromosome [trans(9;22)(q34;q11)] in the cancer cells of patients with chronic myelogenous leukemia was the first report suggesting the involvement of nonrandom chromosomal changes in the molecular pathogenesis of the disease (23). Subsequent studies suggest that the neoplastic cells of 80-90% of leukemia and lymphoma patients contain some sort of demonstrable karyotypic abnormality, and many of these are uniquely associated with morphologically or clinically defined subsets of these cancers (102,126). Similar relationships between chromosomal alterations (and other genetic changes) and definable stages of tumor development and progression have been established for some human solid tumors, including colorectal carcinoma (5,14), and proposed for others, including ovarian carcinoma (165) and pancreatic carcinoma (166). The role of chromosomal translocation in cancer pathogenesis has been suggested to involve activation of proto-oncogenes by repositioning of the gene adjacent to a heterologous genetic control element. Evidence for this type of proto-oncogene activation includes studies of chromosome translocations in Burkitt's lymphoma (167). In this cancer, the c-myc proto-oncogene is translocated from chromosome 8 to chromosome 14, proximal to the immunoglobulin enhancer sequences, resulting in abnormal constitutive expression of c-myc (71).
9.2.3. Large-Scale Chromosomal Deletions Large-scale deletions of whole chromosomes or chromosomal arms have been documented in many cancers. These deletions can contribute to the abnormal allelotype of tumors and might accompany chromosomal rearrangements and/or translocations. In most cases, such deletions are thought to be related to the presence of a tumor suppressor locus on the affected chromosomal arm. Large-scale deletions affecting several chromosomes have been documented in sporadic colorectal carcinoma, including deletions of 5q, 17p, and 18q (14). Each of these chromosomal arms contains a known tumor suppressor locus; the adenomatous polyposis coli (APC) gene at 5q (168-171), the p53 gene at 17p (69,135), and the DCC (for "deleted in colorectal cancer") gene at 18q (172).
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