Mutant p53 protein as a biomarker of chemical carcinogenesis in humans.

Abstract

To the Editor: The production of specific point mutations in the p53 tumor suppressor gene, as can be induced by certain chemical carcinogens, is believed to contribute to the development of some occupational cancers. A model system for studying this process in vivo in humans is provided by workers who have been occupationally exposed to high levels of vinyl chloride (VC) and are at risk for the development of the sentinel neoplasm, angiosarcoma of the liver (ASL). This is because of the fact that the active metabolites of VC are known to form stable etheno-adenosine adducts in DNA that could result in A → T transversions. Such A → T transversions have been found in the DNA from two of four ASLs in VC-exposed workers studied to date (at the first base of codons 249 and 255),1 and similar mutations have not been identified in non-VC-induced ASLs.2 These mutations would result in amino acid substitutions in the encoded p53 proteins (Arg → Trp at 249 and Ile → Phe at 255). These amino acid substitutions occur in a highly conserved region of p53 that is believed to be critical to its tumor-suppressor function. Similar amino acid substitutions in this region of p53 occur frequently in human cancers and are thought to contribute to the development of the cancers. In certain cases, such amino acid substitutions produce a conformational change in the p53 protein that stabilizes the mutant protein from intracellular degradation (which is rapid for the wild-type protein, which has a short half-life), extending the half-life and resulting in intranuclear accumulations of the mutant protein. Such mutant p53 can be demonstrated by the use of a specific mouse monoclonal antibody (PAb240), which identifies an epitope between amino acids 212 and 217 that is normally concealed in the wild-type p53 but which is revealed in many mutant p53 proteins because of the aforementioned conformational changes produced in the mutants. We have been able to use a previously described enzyme-linked immunosorbent assay (ELISA) based on PAb240 3 to detect increased amounts of the mutant p53 protein (up to 0.84 ng/mL) in the extracellular supernatant of cells in culture (SW480) known to contain increased amounts of the mutant p53. This suggests that in a similar situation in vivo, in which individuals have tumors that contain p53 mutations, increased amounts of mutant p53 protein may be detectable in easily accessible extracellular biological fluids such as serum. Therefore, we have examined the expression of mutant p53 protein in 29 individuals from a previously described cohort of VC-exposed workers and unexposed control subjects.4 This group included: four VC-exposed workers with ASLs (two with p53 mutations and two without p53 mutations), one VC-exposed worker with hepatocellular carcinoma (HCC) without a p53 mutation, 18 VC exposed workers without ASLs, and five unexposed control subjects group-matched for age, sex, and race. In these individuals, tumor-tissue was analyzed by immunohistochemistry by using a modification of the technique of Cattoretti et al5 with mouse monoclonal antibody DO-1, and serum was analyzed by ELISA based on PAb240 as described previously.3 The two of four cases of ASL (50%) that were known to contain p53 mutations in their tumor DNA were found to have increased amounts of mutant p53 protein in their tumor tissue and serum, whereas the two cases of ASL and one case of HCC that were known not to contain p53 mutations in their tumor DNA were found not to have increased amounts of mutant p53 protein in their tumor tissue or serum. Because these results suggested that serum mutant p53 could be a valid surrogate for mutant p53 gene expression in tissue, we examined the serum mutant p53 in the 18 VC-exposed workers without ASLs and the five control subjects. Three of 18 exposed workers (16%) had elevated serum mutant p53, compared with none of the control subjects. All of the five p53 serum-positive individuals in this group had histories of potential high exposure to VC (greater than 10 years of work in an exposed job category and an estimated total dose greater than 1500 ppm-year), whereas the VC-exposed workers with lesser exposure were all serum-negative. These results suggest that the detection of serum mutant p53 protein may be a useful biomarker for the study of VC-induced carcinogenesis in exposed human populations. Steven J. Smith, MPH; Jiin-Chyuan Luo, MD, DrPH; Paul Brandt-Rauf, ScD, MD, DrPH Division of Environmental Health Sciences; Columbia University School of Public Health; New York, NY Marie-Jeanne Marion, PhD INSERM; Lyon, France