Introduction of wild-type p53 in a human ovarian cancer cell line not expressing endogenous p53.
Utilizing a temperature sensitive p53 mutant (pLTRp53cGval135) which expresses mutant p53 at 37 degrees C and a wild-type like p53 at 32 degrees C, we transfected a human ovarian cancer cell line (SKOV3) which does not express endogenous p53. Among the different clones obtained, we selected three clones. Two were obtained from simultaneous transfection of p53 and neomycin resistance expression plasmids (SK23a and SK9), the other was obtained from transfection experiments utilizing the neomycin resistance gene only (SKN). Introduction of mutant p53 did not alter the morphology or growth characteristics of this ovarian cancer cell line. Upon shifting to the permissive temperature, a dramatic change in morphology and growth rate was observed in SK23a and SK9 cells that is associated with the presence of a wild-type like p53. SKN and SKOV3 cells maintained at 32 degrees C did not change morphology and only slightly reduced proliferation. Both SK23a and SK9 cells did not show evidence of apoptosis when measured up to 72 hours of maintenance at 32 degrees C. In contrast to what observed in other cell lines, SK23a and SK9 cells maintained at 32 degrees C were not blocked in G1, but they were accumulated in G2-M. This accumulation was transient and could be due either to a blockade or to a delay in the G2 progression. No down-regulation of c-myc was observed in p53 expressing clones when shifted to the permissive temperature. In these conditions gadd45 mRNA expression was highly stimulated in SK9 and SK23a cells but not in SKN cells. In both clones Gas1 mRNA was not detected either at 37 degrees C or 32 degrees C. This system represents a new and useful model for studying the effect of the absence of p53 (SKOV3 or SKN), presence of mutated p53 (SK23a and SK9 kept at 37 degrees C) or wild type p53 (SK23a and SK9 kept at 32 degrees C) on the mechanism of response of cancer cells to DNA damaging agents.
Vikhanskaya
F
F
Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
Erba
E
E
D'Incalci
M
M
Broggini
M
M
D000818:Animals; D017209:Apoptosis; D002455:Cell Division; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D016158:Genes, p53; D006801:Humans; D051379:Mice; D009154:Mutation; D009355:Neomycin; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000473:pathology; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D013696:Temperature; D014162:Transfection; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D009355:Neomycin