A temperature sensitive mutant of the human p53, Val138, arrests rat cell growth without induced expression of cip1/waf1/sdi1 after temperature shift-down.
To investigate functions of wild type p53 in human cells, we introduced a (Ala-->Val) mutation at the 138th codon of the human p53 (Val138), which corresponds to the Val135 mutation of the temperature sensitive mouse p53. The human Val138 mutant showed temperature-sensitive transformation of rat embryo fibroblasts (REFs) in collaboration assay with activated ras, and arrested cell proliferation of transformed clones in G1 at 32.5 degrees C. Transient CAT assay for transcriptional activation in human Saos2 cells revealed activity equivalent to that of wild type at 32.5 degrees C but undetectable at 37.5 degrees C. These results suggest that the human Val138 mutant also exhibited the wild type phenotype at the permissive temperature as is for the mouse Val135 mutant, although we observed differences between the two mutants such as in transactivational activities in CV-1 and HeLa cells. Further, the role of cip1/waf1/sdi1 in the cell growth arrest of the Val138/ras-transformed REFs and Val138-introduced Saos2 cells was studied by northern hybridization analysis. Although rapid induction of cip1/waf1/sdi1 mRNA was observed in the Saos2 cells, no detectable induction of mRNAs for cip1/waf1/sdi1 and gadd45 was observed in the transformed REFs upon temperature shift-down, while mdm2 mRNA was enhanced, suggesting that the p53 gene could arrest cell growth by a mechanism other than that with induced expression of the gene for p21 cdk-cycline inhibitor.
Hirano
Y
Y
Department of Molecular Cellular Oncology and Microbiology, Faculty of Dentistry, Tokyo Medical and Dental University, Japan.
Yamato
K
K
Tsuchida
N
N
D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D016158:Genes, p53 / Q000235:genetics / Q000502:physiology*; D000882:Haplorhini; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics / Q000502:physiology*; D051381:Rats; D013045:Species Specificity; D013696:Temperature
D015500:Chloramphenicol O-Acetyltransferase