Dr Gihan E-H Gawish, MSc, PhD Molecular Biology and Clinical Biochemistry KSU Cytogenetics Understanding the Disease Progression Process, Classical and Molecular Cytogenetic Methods, Chromosome Segregation, the Abnormalities in Clinical Syndromes and Cancer 6 th Module Dr M.Alanazi1

Genomic instability=Accelerated mutations Does genomic instability explain the high rate of mutations in cancer? Is genomic instability the driving force for carcinogenesis? Is it a late or an early phenomenon in carcinogenesis?

Microsatellite instability -Caused by mutations in mismatch repair genes -increased rate of point mutations Microsatellite instability -Caused by mutations in mismatch repair genes -increased rate of point mutations Chromosomal instability -Increased rate of loss or gain of whole or fraction of chromosomes -cell to cell variability Chromosomal instability -Increased rate of loss or gain of whole or fraction of chromosomes -cell to cell variability

- Only limited number of repair mutations found in tumors. - Mismatch repair mutations in colon cancer (hMLH1 epigenetic silencing and mutations)does not generally occur before APC changes. - Tumor suppressive mutations are not really recessive in tumors. - Not all cancer have chromosomal instablity. - Chromosomal instability is not frequently found in adenomas.

Zygote to adulthood (15 yr) 5x10 13 cells ( 45 cell divisions) Mutation rate 5x10 9 /nucleotide/generation 5x10 9 bp/ genome = 25 mutations/generation = 1000 mutations in the stem cell in colonic crypt Mutations in cell of origin before tumorigenesis (using colonic crypt as an example) Between 15 and 65 yrs, colonic stem cells would have multiplied 5000 times= 125,000 mutations.

Chromosomal Instability in Cancer: Causes and Consequences Dr Gihan Gawish 6 Many human malignant tumours exhibit abnormal chromosomal segregation at cell division. these anomalies play a role in tumorigenesis by increasing the rate of chromosome mutations, including deletion and amplification of genes involved in cellular proliferation and/or survival. In vitro experiments have also shown that mitotic instability may be a mechanism for developing resistance to cytotoxic drugs. Abnormal mitotic mechanisms may result in numerical or structural aberrations in the daughter cells. Numerical aberrations can be caused either by the loss of chromosomes at metaphase/anaphase or by multipolar divisions associated with abnormal number or structure of centrosomes.

What is chromosomal instability? Dr Gihan Gawish 7  Chromosomal instability may be defined as a state of continuous formation of novel chromosome mutations, at a rate higher than in normal cells.  In practice, instability may be assessed by following the evolution of cytogenetic abnormalities in a tumour cell population over time and by comparing the rate of chromosome mutations with that in a normal cell population (Lengauer et al., 1997).  Chromosomal instability may be defined as a state of continuous formation of novel chromosome mutations, at a rate higher than in normal cells.  In practice, instability may be assessed by following the evolution of cytogenetic abnormalities in a tumour cell population over time and by comparing the rate of chromosome mutations with that in a normal cell population (Lengauer et al., 1997).

Dr Gihan Gawish 8 Mechanisms of chromosomal instability

2-Numerical instability Dr Gihan Gawish 9  Many epithelial tumours exhibit an asymmetrical segregation of chromosomes at the metaphase-anaphase transition, resulting in an aberrant distribution of the genetic material to the daughter cells (Steinbeck, 1998).  Also, abnormalities in the number and structure of centrosomes have been observed in malignancies with aneuploid chromosome numbers, including cancers of the breast (Lingle and Salisbury, 1999), colon (Ghadimi et al., 2000), and the head and neck region (Saunders et al., 2000).  Many epithelial tumours exhibit an asymmetrical segregation of chromosomes at the metaphase-anaphase transition, resulting in an aberrant distribution of the genetic material to the daughter cells (Steinbeck, 1998).  Also, abnormalities in the number and structure of centrosomes have been observed in malignancies with aneuploid chromosome numbers, including cancers of the breast (Lingle and Salisbury, 1999), colon (Ghadimi et al., 2000), and the head and neck region (Saunders et al., 2000).

Numerical instability Dr Gihan Gawish 10  changes in centrosomal configuration have been correlated with a number of molecular genetic abnormalities, including amplification of STK15 (Zhou et al., 1998), mutations in TP53 (Carroll et al., 1999), and inactivation of BRCA1 (Xu et al., 1999), BRCA2 (Tutt et al., 1999).  Abnormal centrosomal function may also be induced in vitro by expression of the papilloma virus genes E6 and E7, inhibiting normal TP53 and RB1 activity, respectively (Duensing et al., 2000).  It has also been suggested that inactivation of genes that control the timing of mitotic chromosome segregation may contribute to numerical instability. However, only rare examples of such aberrations have so far been identified (Cahill et al., 1998).  changes in centrosomal configuration have been correlated with a number of molecular genetic abnormalities, including amplification of STK15 (Zhou et al., 1998), mutations in TP53 (Carroll et al., 1999), and inactivation of BRCA1 (Xu et al., 1999), BRCA2 (Tutt et al., 1999).  Abnormal centrosomal function may also be induced in vitro by expression of the papilloma virus genes E6 and E7, inhibiting normal TP53 and RB1 activity, respectively (Duensing et al., 2000).  It has also been suggested that inactivation of genes that control the timing of mitotic chromosome segregation may contribute to numerical instability. However, only rare examples of such aberrations have so far been identified (Cahill et al., 1998).

Dr Gihan Gawish 11 A number of in vitro systems have shown that amplification of genes conferring resistance to cytotoxic drugs may occur through BFB events (Smith et al., 1992; Ma et al., 1993; Coquelle et al., 1997). This suggests that a state of chromosomal instability may not only be crucial for tumour development, but may also play a role in resistance to chemotherapy.

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Dr Gihan Gawish 20 Mitotic-spindle checkpoints: a barrier to CIN

oncogene Tumor suppressors oncogene Genomic instability: Colon Cancer

22 Genomic instability: Colon Cancer

Dr Gihan Gawish 23 Genomic instability: Colon Cancer

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Searching for the molecular mechanism of CIN Dr Gihan Gawish 25

Dr Gihan Gawish 26 Three degree of sister-chromatid separation

27 Three degree of sister-chromatid separation

Dr Gihan Gawish 28 The link between mitotic gene and CIN

Dr Gihan Gawish 29 The link between mitotic gene and CIN

Dr Gihan Gawish 30 Therapeutic strategies targeting CIN