Our group has been using gene amplification, the localized increase in gene copy number, as a marker for genomic fluidity. We have found that there is a striking difference between normal, diploid cells and tumorigenic cell in their ability to amplify a given endogenous gene. Amplification frequency is high in tumorigenic cells (~10-3), lower in nontumorigenic, preneoplastic cells (~10-5) and undetectable in normal, diploid cells (<10-9). 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 hybrid cells was suppressed by over five orders of magnitude. In this application we wish to address two questions: 1) is there more than one genetic determinant that suppresses amplification potential in tumorigenic cells and 2) is the increased amplification potential indicative of an increase in other types of genomic changes which may occur in the genesis of neoplasia such as deletion formation, recombination, etc. We will approach these goals through the following specific aims.
In Specific Aim 1, we will fuse a panel of human tumorigenic cell lines in pairwise fashion, each of which has demonstrated a high rate of amplification (~10-4) and that have exhibited suppression of amplification potential when hybridized with normal fibroblast. We will analyze the hybrid progeny for their ability to amplify genomic sequences. The outcome of these experiments with five specifically chosen tumor x tumor hybrids will help to determine the nature of the genetic change and controls amplification. A high rate of gene amplification in the hybrid progeny would suggest that the lesion that controls amplification potential is common to both tumorigenic cells. A suppressed rate of gene amplification in the hybrid progeny would suggest that the lesion that controls amplification potential is different in the two parents. If the initial fusions don't indicate independent complementation groups, analysis of the remaining hybrids will allow us to uncouple the phenotype from tissue specificity, embryonic origin and complementation groups for immortality and tumor suppression. These studies will help define complementation groups for genes that control amplification potential in tumorigenic cells.
In Specific Aim 2, we will further characterize the """"""""fluidity"""""""" in three categories of cells: those that amplify at a high frequency (~10-3), those that amplify at a lower frequency (~10-4 to 10-5), and those cell lines where amplification is undetectable (<10-8). We will test the cell lines for manifestations of further fluidity in three different ways. (a) We will determine if the cells which spontaneously amplify the CAD locus at a high rate will also undergo spontaneous deletions, recombinations and other mutations at increased rates. (b) We will determine if the cells which spontaneously amplify the CAD locus at a high rate will also amplify other loci at a correspondingly high rate. (c) Finally, we will characterize deamplification in cell lines which amplify at a high and low rate.
| Hall, I J; Gioeli, D; Weissman, B E et al. (1997) Identification of additional complementation groups that regulate genomic instability. Genes Chromosomes Cancer 20:103-12 |
| Chen, C Y; Hall, I; Lansing, T J et al. (1996) Separate pathways for p53 induction by ionizing radiation and N-(phosphonoacetyl)-L-aspartate. Cancer Res 56:3659-62 |
