Normal mammalian cells respond to DNA damage or arrest of DNA synthesis by activating pathways that protect themselves or the organism. These controls are lost in cancer cells, allowing them to tolerate a high rate of genetic variation. Understanding the pathways involved is an important part of understanding the mechanisms that underlie the generation and evolution of cancer cells. Dr. Stark will investigate pathways of response to DNA damage or arrest of DNA synthesis that lead to induction of the tumor suppressor p53 in a functional form. Mutant cell lines will be obtained in which genes encoding signaling components have been inactivated, and the genes will be cloned by functional complementation. Dr. Stark's lab has established a system for selecting mutant human HT1080 fibrosarcoma cell lines defective in induction of functional p53, based on p53-dependent transcription. Expression of a gene encoding the cell-surface protein cd2 is controlled by a p53-regulated element. There is strong induction of cd2 in response to arrest of DNA synthesis (using N-(phosphonacetyl)-L-aspartate (PALA) or aphidicolin) or DNA damage (using adriamycin, camptothecin or radiation). Two independent mutant cell lines have been obtained in which the induction is defective, using the fluorescence- activated cell sorter (FACS).
In Specific Aim 1, Dr. Stark proposes to obtain more mutant cell lines, to place the mutants into complementation groups, and to analyze the phenotypes of each complementation group. (For example, at what level is the formation of functional p53 inhibited? Are cells selected for defective p53 induction in response to PALA also defective in response to DNA damage?) The few specific candidate genes currently available will be investigated to determine if they are defective in the mutants. cDNA or genomic DNA libraries will be introduced to clone genes encoding unknown components by functional complementation. When specific genes are identified, they will be reintroduced into the null background of the mutant cell lines in order to define structure-function relationships in the signaling pathways.
In Specific Aim 2, mutant cell lines will be isolated that express cd2 constitutively (as an indication of constitutive p53 activity), without induction. Such cells may be defective in pathways that maintain the p53 protein at a low level in unstimulated cells or that maintain the endogenous low levels of p53 protein in an inactive state. Complementation of the defects with cDNA or genomic DNA libraries, as in Specific Aim 1, should again lead to identification of unknown components.
| Agarwal, M L; Ramana, C V; Hamilton, M et al. (2001) Regulation of p53 expression by the RAS-MAP kinase pathway. Oncogene 20:2527-36 |
| Stark, G R (1997) Genetic analysis of interferon and other mammalian signaling pathways. Harvey Lect 93:1-16 |
