STUDIES ON RIBONUCLEOTIDE REDUCTASE FROM NORMAL SENESCING HUMAN DIPLOID FIBROBLASTS

Human diploid fibroblasts grown in culture more closely resemble the in vivo state than cultures of permanent cell lines and therefore are an important model system in which to study complex regulatory processes. Ribonucleotide reductase is a key rate limiting enzyme in the regulation of DNA synthesis and is involved in regulating a variety of cellular functions. The experiments outlined in this thesis provide, for the first time, a general characterization of ribonucleotide reduction in normal human diploid fibroblasts. A novel permeabilized cell assay was developed and used to examine the regulation of ribonucleotide reductase in low and high passage cells. Several important changes in the regulation of reductase activity from high passage cells were observed. There was a marked decrease in the level of reductase activity in high passage cells. Furthermore, detailed kinetic studies indicated that the allosteric regulation of ribonucleotide reductase by dATP was altered with enzyme from high passage cells. In keeping with the age-related decline in reductase activity high passage cells were found to contain reduced deoxyribonucleotide pools. A variety of age-related alterations in the ribonucleotide di- and triphosphate pools were also observed. These observations suggested that changes in ribonucleotide reductase activity may alter the replicative lifespan of normal cells grown in culture. This idea was tested by developing a unique selection system for the isolation of hydroxyurea resistant human diploid fibroblasts. The lifespan of the variants which were isolated by this procedure was significantly reduced by 20 to 40%. It was found that the variant cells overproduced reductase activity and exhibited altered levels of deoxyribonucleotide pools. These studies provided evidence, for the first time, that there is a link between senescence, ribonucleotide reductase activity and altered deoxynucleotide pools. Somatic cell hybridization techniques were used to isolate hybrids between normal human fibroblasts and CHO cells. It was not possible to identify any intact human chromosomes in the hybrid cell lines. However, a variety of chromosomal alterations were observed in the hybrid lines; these alterations may have resulted in the translocation of a piece of human DNA, carrying the gene(s) for reductase onto the hamster chromosomes. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI