The primary mechanisms of amplification involve recombination rather than over-replication. Unequal distribution of the recombined DNA into two daughter cells leads to an increase in gene copy number in one (amplification) and a decrease in the other (genetic loss). Thus, the same basic processes are likely to be responsible for the amplification of oncogenes and for the loss of tumor suppressor alleles in cancer. Region-specific recombinations between sister chromatids are important primary events in amplification. Both telomere-region and centromeric recombinations have been observed. The earliest amplified structures found will be studied to define the nature of the sequences involved. Cell lines can stimulated to increase rates of amplification, transiently by damaging DNA or arresting DNA synthesis, or stably in """"""""amplificator"""""""" mutant cells. An amplificator cDNA (clone 20) has been isolated and will be expressed in human and hamster cells to assess which of the several known amplification mechanisms are affected. Additional genes whose expression stimulates amplification will be cloned by a strategy related to the one used for clone 20. Normal cell strains do not give stable drug-resistant colonies containing amplified DNA, whereas most cell lines give such colonies readily. The tumor suppressor proteins, p53 and pRB, have already been shown to have a role in permissivity. These proteins will be conditionally regulated in order to address several questions. Are all of the several mechanisms of amplification that have been revealed so far regulated by the pathway involving p53 and pRB, which seems to require DNA damage? When nonpermissive human or rat cells are regulated in response to a gene amplification event, do they die by apoptosis or arrest in the cell cycle? lf the latter, is the arrest in Gl or G2? The experiments proposed should further define the several different primary events of amplification, reveal how each of these events is affected by external stimuli and through expression of amplificator genes, and reveal the nature of the damage-recognition pathways in normal, nonpermissive cells that deal with the different amplified structures produced by each mechanism.
