We describe a simple method for characterizing a frequent polymorphism (that substitutes an arginine for a proline) in the coding sequence of the Tp53 gene in patients with colonic cancer and in a control population.
The results of extensive analysis of p53 mutations in non-small cell lung cancers (NSCLCs) have revealed that p53 is mutated in 45% of NSCLC with base changes different from those of colon cancer.
Mutations changing the p53 coding sequence were found in 14 of 30 tumor samples (47%), while G:C to T:A transversions which are uncommon in other cancers such as colon cancer were the most frequently observed mutations, in agreement with an earlier report on non-small cell lung cancer in American patients.
To demonstrate the program, the mutational spectra of single base substitutions in the p53 gene are compared in (i) bladder cancers from smokers and non-smokers, (ii) small-cell lung cancers, non-small-cell lung cancers and colon cancers and (iii) hepatocellular carcinomas from high- and low-aflatoxin exposure groups. p53 mutations differ in several important aspects from a typical mutational spectra experiment, where a homogeneous population of cells is treated with a specific mutagen and mutations at a specific locus are recovered by phenotypic selection.
Human colon cancer development is associated with the accumulation of mutations and deletions in the suppressor genes DCC, APC and p53 and mutations in the dominant oncogene K-ras, with loss of wild type alleles.
In this study, we have examined p53 mutations in six cases of colon cancer cell lines, 22 cases of flat-type colorectal tumors, and 27 cases of polypoid-type colorectal tumors using the polymerase chain reaction (PCR) and temperature-gradient gel electrophoresis (TGGE); the latter has recently been developed as a screening method for gene mutations. p53 mutations were observed in four colon cancer cell lines, six flat-type colorectal tumors, and three polypoid-type colorectal tumors, all of which were analyzed by direct sequencing.
In comparison to the 41 RER-negative proximal colonic cancers, RER-positive cancers had more frequent exophytic growth (P = 0.04), large size (P = 0.03), poor differentiation (P = 0.0004), extracellular mucin production (P = 0.003) and Crohn's-like lymphoid reaction (P = 0.003), and a trend toward less frequent p53 gene product overexpression by immunohistochemistry (3/17, 18%, versus 18/41, 44%, P = 0.06).
For example, both the RB gene of retinoblastoma and the p53 gene, which is commonly mutated in breast and colon cancer among others, produce proteins involved in distinct steps of cell cycle control, while the nm23 product prevents metastasis.
Polymerase chain reaction-single-strand conformation polymorphism analysis of p53 exons 5-8 revealed 33 mutations in 20 of 182 colon tumors, the incidence being lower than that in human colon cancers.
These results demonstrate that in a human colon epithelial cell background, wild-type p53 is functionally dominant over this mutant p53 and thus provides a mechanism for the observed inactivation of both copies of the p53 gene in most colon cancers.
Differential GADD45, p21CIP1/WAF1, MCL-1 and topoisomerase II gene induction and secondary DNA fragmentation after camptothecin-induced DNA damage in two mutant p53 human colon cancer cell lines.
We also find that induction of wild-type p53 potentiates the cytotoxicity of topotecan, a member of the camptothecin family of drugs that also has clinical activity against colon cancer.
The high point mutation rate of replication error-prone (RER+) cells could theoretically lead to inactivation of the p53 gene by polyclonal mutations, which might explain the conflicting results that have been published on the p53 status of RER+ colon cancers.
In contrast, a mutant p53 bearing human colon cancer cell line, sw480, lacked spontaneous apoptosis, and upon irradiation neither induction of apoptosis nor increase expression of p53 and WAF-1 were seen.
The second form of genetic instability is characterized by the development of p53 gene abnormalities that result in gross aneuploidy and multiple structural chromosomal changes. p53/aneuploidy affects most colon cancers, breast cancers, and many other solid tumors.
Other studies have suggested an inverse relationship between p53 and bcl-2 protein expression in breast and colonic cancers and in a variety of subtypes of non-Hodgkin's lymphoma.