© 1997 by Oxford University Press
Journal Of The National Cancer Institute, Vol 89, 66-71, Copyright © 1997 by Oxford University Press
PH Gumerlock, SG Chi, XB Shi, HJ Voeller, JW Jacobson, EP Gelmann and RW deVere White
BACKGROUND: The reported frequency of mutation of the p53 tumor suppressor
gene (also known as TP53) in human carcinomas of the prostate has varied
widely, ranging from 3% to 42%. This variability may be a consequence of
tumor heterogeneity and/or the use of different methods of analysis. Since
p53 mutation has been associated with clinical outcome for a number of
cancer types, determination of its true frequency in primary carcinomas of
the prostate is important. PURPOSE: The principal aims of this study were
as follows: 1) to validate the utility of detecting p53 gene mutations by
means of polymerase chain reaction-single-strand conformation polymorphism
(PCR- SSCP) analysis of complementary DNA (cDNA) (synthesized from prostate
tissue RNA and 2) to study the concordance of RNA- and DNA-based PCR- SSCP
assays in detecting p53 mutations in individual tumor fragments. METHODS:
RNA and genomic DNA were isolated by means of standard techniques from
specimens of 19 carcinomas of the prostate, selected on the basis of p53
data obtained in a previous analysis of cDNA (indicating that 14 were
mutant and five were wild-type). RNA was converted into cDNA by means of
reverse transcription (RT); the cDNA was then amplified by means of
nonisotopic (i.e., nonradioactive) PCR, and the PCR products were subjected
to SSCP analysis in polyacrylamide gels (RT-PCR-SSCP analysis). Genomic DNA
was examined by means of SSCP analysis of isotopically labeled (32PO4) PCR
products (DNA-PCR-SSCP analysis). In both approaches, the protein coding
region of the p53 gene was divided into multiple, smaller fragments for
study. PCR products exhibiting abnormal migration in SSCP gels were
subjected to direct nucleotide sequencing or to cloning and sequencing of
multiple clones. RESULTS: RT-PCR-SSCP and DNA-PCR-SSCP identified p53 gene
abnormalities in 15 of the 19 selected carcinomas, including one previously
reported to be wild-type for p53. Overall, PCR-SSCP analysis identified 18
p53 fragments with abnormalities; three carcinomas showed two abnormalities
each. Six (33%) of the 18 abnormalities were detected by both RT-PCR-SSCP
and DNA-PCR-SSCP, 10 (56%) were detected by RT-PCR- SSCP alone, and two
(11%) were detected by DNA-PCR-SSCP alone. The 18 abnormalities were caused
by 20 changes in the sequence of the p53 gene; in one carcinoma, double
mutations in two individual p53 exons were identified. CONCLUSIONS AND
IMPLICATIONS: PCR-SSCP analysis of both RNA and DNA allows the detection of
more mutations than the analysis of either alone. Some primary carcinomas
of the prostate contain more than one altered p53 gene, consistent with the
possibility of intratumoral heterogeneity of mutation of this gene. For
comprehensive analysis of p53 mutations in carcinomas of the prostate, and
perhaps in other tumor tissues, SSCP analysis of cDNA should be used in
combination with SSCP analysis of genomic DNA.
REVIEWS
p53 abnormalities in primary prostate cancer: single-strand conformation polymorphism analysis of complementary DNA in comparison with genomic DNA. The Cooperative Prostate Network
Department of Internal Medicine, University of California, Davis Cancer Center, Sacramento 95817, USA.
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