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Biomedical subjects

J E Dick

Publications and source records attributed to J E Dick.

77 records · Page 5Linked to original sources
PubMed1984

Human diploid fibroblasts with alterations in ribonucleotide reductase activity, deoxyribonucleotide pools and in vitro lifespan.

Three drug resistant human diploid fibroblast clones were isolated which contained elevated levels of ribonucleotide reductase activity when compared to wild type fibroblasts. The drug resistant cells do not appear to possess an enzyme with altered affinity for hydroxyurea. The increase in enzyme activity can entirely account for cellular drug resistance. In keeping with the observed changes in reductase activity in drug resistant fibroblasts, deoxyribonucleotide pools were also found to be altered. Most significantly, there was a 1.8-fold expansion of the dCTP pool size, suggesting that elevation in intracellular dCTP concentrations plays an important role in cellular resistance. Furthermore, the drug resistant fibroblasts exhibited substantial reductions in their replicative abilities, suggesting that the regulation of ribonucleotide reductase and the accompanying deoxyribonucleotide pools in human diploid cells is involved in aspects of cellular senescence.

Cell Survival↗
PubMed1982

Involvement of ribonucleotide reductase activity in the senescence of normal human diploid fibroblasts.

The levels of intracellular ribonucleotide reductase activity, a highly regulated rate-limiting step in DNA synthesis, were investigated during serial subculture of normal human diploid fibroblasts in vitro. This key enzyme activity was found to decline significantly during cellular senescence. This observation along with previous findings of a mutator gene associated with mammalian ribonucleotide reductase suggests a possible mutation mechanism for aging which involves changes in reductase activity during cellular senescence. Furthermore, in keeping with the decrease in enzyme activity, we show that cell resistance to the antitumor agent hydroxyurea, whose site of action is ribonucleotide reductase, decreases progressively with increasing passage numbers. This indicates that an important factor to be considered in drug therapy aimed at the reductase is the increased sensitivity of normal cells to drug with cell age, due to a decline in enzyme activity. Much remains to be determined about age-dependent factors involved in drug therapy; cultured normal human diploid fibroblasts provide a useful system in which to investigate these important parameters.

Cell Survival↗
PubMed1980

Ribonucleotide reduction in intact human diploid fibroblasts.

There have been very few studies on ribonucleotide reductase activity in human tissue. In this report we describe a rapid and convenient procedure for determining purine and pyrimidine ribonucleotide reduction in normal human diploid fibroblasts and use the method to examine some general properties of the activity in these cells. ADP and CDP reductase was characterized for its response to the positive effectors, ATP and dGTP, the negative effector dATP, and the reducing agent dithiothreitol. Apparent Km values for ADP and CDP were determined to be 0.1 mM and 0.04 mM respectively. THe antitumor agent hydroxyurea inhibited both purine and pyrimidine reductase in a noncompetitive fashion, giving Ki value of 0.40 mM and 0.41 mM for ADP and CDP respectively. These Ki estimates are about four to five times higher than those reported for some permanent cell lines. An examination of the cytotoxic effects of hydroxyurea indicated a close correlation between the concentration of drug which inhibited enzyme activity and decreased colony-forming ability. Clearly the ability to investigate ribonucleotide reduction in low numbers of normal human diploid cells will be useful for genetic and biochemical studies.

Adenosine Diphosphate↗
PubMedDate not supplied

Expression of human adenosine deaminase in murine haematopoietic progenitor cells following retroviral transfer.

Adenosine deaminase (ADA) deficiency, an autosomal recessive inborn error of metabolism, leads to severe combined immune deficiency in man. This enzyme, although constitutively expressed in most tissues, is expressed at high level in immature T cells, and study of the pathophysiology of the disorder indicates that increased deoxyadenosine or altered methylation capacity have toxic effects on T-cell maturation. Although bone marrow transplantation can correct the immune deficiency, this therapy is associated with graft-versus-host disease and incomplete immune restoration, and so our laboratory and others have sought to develop a method of gene replacement as a possible treatment for the disease. Moreover, characterization of the complementary DNA of the human ADA gene and some of its mutants makes it possible to design gene transfer strategies. We have now subcloned a human adenosine deaminase cDNA into the retrovirus shuttle vector pZIP-SV(B), and in this way have isolated a cell line, 4.2T, which produces high titres of replication-defective retrovirus which have been used to transfer the gene for human ADA to mouse bone marrow cells. Transfer and expression of the neomycin-resistance gene (neo) and the ADA gene in murine bone marrow colony-forming units (CFU) was demonstrated by in vitro colony formation in the presence of the antibiotic G418 or 9-xylofuranosyladenine plus deoxycoformycin, respectively. Isoenzyme analysis also showed human ADA expression in the cultured mouse bone marrow.

Adenosine Deaminase↗