Molecular Mechanisms of Mutagenesis in Human Cells as Related to Cancer

Many mutations are required to convert a normal cell to a cancer cell. Unfortunately, our understanding of how mutations occur in cancer cells is inadequate to explain the high incidence of human cancers. Moreover, a comprehensive understanding of this fundamental property of all cancer cells may enable us to develop new drugs aimed at controlling the cancer disease process. The long-term goal of our laboratory's research is to elucidate the molecular mechanisms by which mutations occur in normal human cells and to establish how these mechanisms are altered during cancer cell evolution. We use an in vitro/ex vivo mutagenesis system to study mutations induced at the herpes simplex virus type I thymidine kinase (HSV-tk) target gene. DNA damage may be introduced by chemical carcinogen treatment of purified DNA. We compare mutations arising during in vitro DNA synthesis by purified polymerases and during ex vivo DNA replication in cultured human cells.

Currently, the biochemical interactions between purified DNA polymerases and modified DNA are being explored using the in vitro assay. An early step in the mechanism of chemical carcinogenesis is the production of DNA lesions which result in mutations during DNA synthesis. We are interested in examining damage-induced mutagenesis from the perspective of the DNA polymerase as a variable. Human cells contain four known nuclear DNA polymerases that differ significantly in the mechanisms used to discriminate against errors. We are comparing how DNA lesions caused by known carcinogens are processed into mutations by human DNA polymerases a and b. Variant forms of DNA polymerase b which have a decreased accuracy for DNA synthesis are also being tested to determine the structural features of the polymerase that are critical for translesional DNA synthesis.

A second focus of our laboratory is to evaluate the significance of spontaneous and carcinogen-induced mutations at short tandem repeat (STR) DNA loci in human cells. Our working hypothesis is that mutations in repetitive DNA provide an important source of genotypic variation that drives neoplastic progression. Bimodal HSV-tk target sequences containing an STR motif and a unique sequence motif have been constructed, and are treated with various classes of chemical carcinogens. These targets are used as DNA templates during in vitro DNA synthesis catalyzed by human DNA polymerases and during DNA replication in human lymphoblastoid cells. This research will enhance our understanding of the mechanisms of human somatic cell mutagenesis by providing quantitation of mutation rates in repetitive DNA, and by establishing the degree to which repetitive DNA is destabilized by chemical carcinogens.