Screening the p53 status of human cell lines using a yeast functional assay.
We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast. This assay enables us to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53-responsive GAL1 promotor in Saccharomyces cerevisiae. Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%). Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only. Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature-sensitive growth. p53 cDNA from eight cell lines showed p53-dependent temperature-sensitive growth, growing at 30 degrees C but not at 37 degrees C. Four temperature-sensitive p53 mutations were isolated: CAT-->CGT at codon 214 (H214R), TAC-->TGC at codon 234 (Y234C), GTG-->ATG at codon 272 (V272M), and GAG-->AAG (E285K). Functionally wild-type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels. These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization.
Jia
L Q
LQ
Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
Osada
M
M
Ishioka
C
C
Gamo
M
M
Ikawa
S
S
Suzuki
T
T
Shimodaira
H
H
Niitani
T
T
Kudo
T
T
Akiyama
M
M
Kimura
N
N
Matsuo
M
M
Mizusawa
H
H
Tanaka
N
N
Koyama
H
H
Namba
M
M
Kanamaru
R
R
Kuroki
T
T
D000483:Alleles; D016158:Genes, p53; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013696:Temperature; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis
D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53