Using in vitro model systems, we have found that there is a striking difference between-normal, diploid cells and tumorigenic cells in their ability to amplify a given endogenous gene. Amplification frequency is high in tumorigenic cells (approximately 10(-3)), lower in nontumorigenic, preneoplastic cells (approximately 10(-5)) and undetectable in normal,- diploid cells (< 10(-9)) in both human and rodent species. To investigate the genetic control of gene amplification, amplification frequency was measured in hybrids formed between tumorigenic cells and normal, diploid cells. The ability to amplify an endogenous gene behaved as a recessive genetic trait. Amplification frequency in the hybrid cells was suppressed by over 5 orders of magnitude (10(-3) to < 10(-8)). In the previous funding period, we identified the p53 tumor suppressor gene product as one of the components of a pathway that regulates gene amplification. In this application, we wish to address three questions that are logical extensions of the previous specific aims. 1) What are the identities of other components of the pathways that control gene amplification? In Specific Aim 1, we wish to identify other members of the pathway(s) that modulate amplification ability in human cells. We possess cell populations that differ by one protein when compared to the parent population yet amplification ability is altered by 3 to 5 orders of magnitude. These model systems will allow the identification and analysis of cellular molecules that are altered during tumor progression and result in increased genomic instability. 2) Can genetic instability, as measured by changes in gene copy number, be detected in preneoplastic human tissue without culture in vitro? In Specific Aim 2, we wish to extend our previous studies to an in vivo analysis. The use of CGH will be used to analyze the control abnormalities that occur in the chromosomal instability diseases.
In Specific Aim 3, we wish to study amplification ability and altered cell cycle control in cells from patients with chromosomal instability syndromes. The chromosomal instability syndromes exhibit elevated numbers of chromosomal abnormalities, a marked predisposition to neoplasia, and defects in DNA metabolism. In our initial studies, we determined that cells from patients with the chromosomal instability syndrome, Ataxia Telangiectasia, exhibited an increased propensity to amplify DNA sequences. The third specific aim describes experiments to further test the idea that genomic instability, as monitored by gene amplification, may be a hallmark of tumorigenic potential. Based on these hypotheses it is predicted that cells from patients with Bloom's Syndrome, Fanconi's anemia, or Xeroderma pigmentosum will demonstrate an increased potential to amplify the CAD gene when compared with diploid wild-type cells.
