Genetic instability is a well known but poorly understood characteristic of both transformed cells and normal cells exposed to DNA-damaging agents. This property has been implicated in cell immortalization as well as in tumor formation and progression. An important contributor to such cytogenetic damage is thought to be the loss of normal regulation of mitosis.
The aim of this research project is to gain an understanding of the molecular events that control mitotic onset, to examine the effects of DNA damage and transformation on this control, and to determine whether aberrant regulation of mitosis promotes genetic instability and transformation. This goal will be pursued by 1) using microcell fusions to identify chromosomes that alter normal regulation of mitotic onset; 2) using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to identify specific proteins that are involved in preparing cells for mitosis; 3) cloning genes which undergo changes in expression during the interphase-to-mitosis transition; 4) determining whether the cell cycle regulation of these mitosis-related proteins and genes is altered in tumor cells or normal cells following DNA damage; and 5) determining whether chromosomal instability induced by chemical clastogens or ras oncogene proteins is caused, at least in part, by mitosis occurring before, or shortly after, the completion of DNA replication. The identification of chromosomes, genes and proteins involved in the control of mitosis will be aided greatly by recent findings in our laboratory that allow us to 1) examine the segregation of mitosis-promoting and mitosis-suppressing chromosomes in hamster-human hybrids; and 2) manipulate experimentally the onset of mitosis as well as the accumulation of as yet uncharacterized mitosis-related mRNAs and their protein products in synchronized mammalian cell populations. We have also refined a technique for isolating newly synthesized mRNA. This procedure will be used to enrich for mitosis-related transcripts that will be 1) translated in vitro and the protein products identified by 2-D PAGE; and 2) reverse transcribed, cloned into lambda gt10, and screened by differential hybridization for genes involved in mitotic onset. A monoclonal antibody to bromodeoxyuridine-substituted DNA will be used to visualize microscopically the replication state of DNA that surrounds chemically- and ras oncogene-induced chromosomal break sites. By manipulating the period of bromodeoxyuridine exposure, it will be possible to determine whether chromosomal breaks are located in regions where DNA has remained unreplicated or has replicated just prior to the onset of mitosis. These studies will improve our understanding of the normal control of mitosis, how this process is altered by exposure to carcinogens and oncogene products, and how aberrant regulation of mitosis contributes to genetic imbalances and neoplastic disease.
