Ernest H. Y. Chu

Effects of DNA damaging agents on cultured fibroblasts derived from patients with Cockayne syndrome

The cytotoxic action of physical and chemical agents on 10 skin fibroblast strains in culture derived from individuals with Cockaynes syndrome was measured in terms of colony-forming ability. As compared to fibroblasts from normal donors, all Cockayne cell strains tested exhibited a significantly increased sensitivity to UV light and a normal sensitivity to X-rays. Cells from two sets of parents of unrelated Cockayne children showed an intermediate level of UV sensitivity. There was no effect of 0.5 mM caffeine on UV survival in normal and two Cockayne strains tested, indicating that postreplicational repair in Cockayne cells as measured by caffeine sensitivity was probably normal. Sensitivity of normal and Cockayne cells to the chemical carcinogens and mutagens 4NQO, N-AcO-AAF, ICR-170 and EMS was also compared. An increased sensitivity of Cockayne cells to 4NQO or N-AcO-AAF, but not the ICR-170 or EMS, was observed. However, unlike the intermediate UV sensitivity, the cell strains from two parents of Cockayne patients showed the same sensitivity to N-AcO-AAF or 4NQO as fibroblasts from normal individuals. Quantiation of damage to the DNA after 20 J . m-2 UV irradiation indicates normal levels of [3H] thymidine incorporation in the Cockayne cells, in contrast to UV-irradiated xeroderma pigmentosum cells (XP 12BE) in which there was a very low level of repari synthesis. Moreover, we have shown previously that excision of UV-induced pyrimidine dimers in 2 of the 10 Cockayne cell strains was normal.

Inhibition of repair of UV-damaged DNA by caffeine and mutation induction in Chinese hamster cells

Abstract The effect of caffeine on UV-irradiated Chinese hamster cells in vitro was studied on the cellular and molecular levels. Caffeine (1 m M ) was shown to decrease the colony-forming ability and the frequencies of spontaneous and UV-induced mutations in Chinese hamster cells. The effect of caffeine in reducing the frequency of UV-induced mutations was demonstrated only if caffeine was present in the culture medium during the first post-irradiation cell division. Using alkaline sucrose gradient centrifugation, both parental and newly synthesized DNA in UV-irradiated and unirradiated cells were studied in the presence and absence of caffeine. Caffeine affected the sedimentation profile of DNA synthesized in UV-irradiated cells but not in unirradiated cells. Caffeine had no apparent effect on the incorporation of [ 3 H]-thymidine into DNA of control or UV-irradiated cells, nor on the small amount of excision of UV-induced pyrimidine dimers. These results may be interpreted by a hypothesis that caffeine inhibits a certain S-phase specific, post-replication, dark-repair mechanism. The hamster and perhaps other rodent cells exposed to low doses of UV are capable of DNA replication, by-passing the non-excised pyrimidine dimers. This postulated repair process probably involves de novo DNA synthesis to seal the gaps in the nascent strand. This repair may be also responsible for the enzymatic production of mutations.

The role of DNA repair and somatic mutation in carcinogenesis.

Publisher Summary This chapter discusses the role of DNA repair and somatic mutation in carcinogenesis. A highly speculative and heuristic scheme has been developed to account for a wide range of observations that make it appear that the carcinogenic process has several heritable components. Based on the assumptions (1) that germ-line mutations on several levels could influence carcinogenesis and (2) that DNA repair and somatic mutagenesis are related in mammalian systems in a manner similar to bacteriological systems, a model is presented in the chapter that links various genetic and environmental factors influencing DNA repair with observations on the initiation and promotion of tumors. Within this model, genes that influence (1) the level of genetic damage, (2) the level and efficiency of the repair of DNA damage, and (3) the proliferation of the altered cells, are those that are subjected to wide variation in expression owing to individual genetic or environmental predispositions. Currently, only circumstantial evidence links DNA repair to carcinogenesis in human beings. A critical component of the somatic mutation theory of cancer will be an elaboration of the role of the repair of carcinogen-induced DNA damage to mutagenesis in eukaryotic systems.

Staining Method for the Banding Patterns of Human Mitotic Chromosomes

SUMMARYA method is described which, through the combined use of warm phosphate saline and a Giemsa-trypsin mixture, consistently produces G-bands in metaphase chromosomes of human leukocytes.

Chromosomal Location of Human Genes Encoding Major Heat-Shock Protein HSP70

The HSP70 family of heat-shock proteins constitutes the major proteins synthesized in response to elevated temperatures and other forms of stress. In eukaryotes members of the HSP70 family also include a protein similar if not identical to bovine brain uncoating ATPase and glucose-regulated proteins. An intriguing relation has been established between expression of heat-shock proteins and transformation in mammalian cells. Elevated levels of HSP70 are found in some transformed cell lines, and viral and cellular gene products that are capable of transforming cells in vitro can also stimulate transcription of HSP70genes. To determine the organization of this complex multigene family in the human genome, we used complementary approaches: Southern analysis and protein gels of Chinese hamster-human somatic cell hybrids, and in situ hybridization to human chromosomes. We demonstrate that functional genes encoding HSP70 proteins map to human chromosomes 6, 14, 21, and at least one other chromosome.

Partial Trisomy of the Long Arm of Human Chromosome 1 as Demonstrated by in situ Hybridization with 5S Ribosomal RNA

SummaryIn a newborn boy with multiple malformations, a tandem duplication was detected at the distal end of the long arm of one human chromosome 1. The Giemsa bands, 1q31 to 1q43–44, were repeated serially. Since 5S rRNA genes are located at 1q42–43, in situ hybridization of 125I 5S rRNA with fixed chromosome preparations was used to confirm the chromosomal duplication. The infant exhibited numerous developmental and clinical abnormalities as might be expected with an abnormality of chromosome structure relating to a ribosome component.

Selective Systems in Somatic Cell Genetics

Experimental mutagenesis, cell hybridization, and intercellular gene transfer appear to be the three most significant technical developments in recent years that have stimulated rapid advances in somatic cell genetics. The ability to induce mutations in animal and plant somatic cells in culture enriches the genetic variability of the cell populations for further genetic and biochemical analysis. The process of mutagenesis and mutation rates in high eukaryotic cells can thus be studied and compared with those known in prokaryotes. The success of cell fusion between diverse parental genomes into multinucleate heterokaryons or synkaryonic hybrids has also led to a variety of investigative possibilities, among which are the studies of genomic interactions and gene expression, genetic complementation, recombination, segregation, and mapping of genes. However, parasexual transfer of genetic material from one cell to another is not limited to the process of fusion between intact cells. Successful attempts have been reported in which isolated DNA, chromosomes, or nuclear fragments have been introduced into living mammalian somatic cells. The expression of heterologous gene(s) in a recipient cell and the transmission of the “acquired” character (s) to cell progeny are problems of fundamental interest and significance.

Pleiotropic mutants of Chinese hamster cells with altered cytidine 5′-triphosphate synthetase

Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine, deoxycytidine, or deoxyuridine but not for cytidine or uridine. All were resistant to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside at concentrations that were toxic to wild-type V79 cells. The cytidine 5′-triphosphate (CTP) and deoxycytidine 5′-triphosphate (dCTP) pools in the mutants were expanded, but the uridine 5′-triphosphate (UTP) pool either decreased or remained unchanged relative to the wild-type level. Furthermore, since the parental cells appear to be deficient in dCMP deaminase activity and CTP (or one of its metabolites) has been shown to inhibit uridine 5′-diphosphate (UDP) reduction, an elevated CTP level should lead to the observed thymidine auxotrophy. It also explains the joint resistance of mutant clones to thymidine and cytosine arabinoside. The change in the ratio of intracellular dCTP to thymidine 5′-triphosphate (dTTP) may be responsible for the elevation in the rates of spontaneous mutations in these mutants.

Purification of cytidine-triphosphate synthetase from rat liver, and demonstration of monomer, dimer and tetramer

Cytidine-triphosphate synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2) has been purified over 31,000-fold to homogeneity with 17% recovery from rat liver cytosol, using high-performance liquid chromatography (HPLC) techniques. The presence of CTP synthetase monomer, dimer and tetramer has been demonstrated in the ammonium sulfate fraction of rat liver cytosol. By gel-permeation HPLC, the molecular weights of the three molecular forms of the enzyme have been estimated as 240,000 (tetramer), 120,000 (dimer) and 60,000 (monomer). By gel-permeation chromatography on Bio-Gel A-1.5m column, the molecular weights of dimer and monomer were estimated as 100,000 and 50,000, respectively. The molecular weight of the monomeric subunit is determined to be 66,000 by SDS-polyacrylamide gel electrophoresis. Monomers isolated fresh from 0-30 (NH4)2SO4 fraction of rat liver cytosol are enzymatically active. Purified rat liver CTP synthetase exhibited sigmoidal kinetic plots as a function of the substrate UTP in the presence of the end-product, CTP. Partially purified CTP synthetase usually forms an inactive coagulum on freezing and subsequent thawing. Incubation of CTP synthetase dimer at 25 degrees C for 1 h in the presence of UTP, ATP and Mg2+ resulted in optimum conversion to tetramer with least inactivation. The purified tetramer dissociates to dimers when UTP, ATP and Mg2+ are removed by dialysis.

Estimation of mutation rates in cultured mammalian cells

The factors that affect reliable estimations of mutation rates (mu) in cultured mammalian somatic cell populations by fluctuation analysis are studied experimentally and statistically. We analyze the differential effect of the final cell population size in each culture (Nt) and the number of parallel cultures (C) on the variation in the rate estimates (mu) inferred from the P0 method. The analysis can be made after the derivation of the variance of mu, which is a measure of variation of mu for a given combination of Nt and C in a number of repeat experiments. The variance of mu is inversely proportional to C and to the square of Nt . Nt determines the probability of occurrence of mutations in a cell culture. By influencing the size of P0, Nt also determines whether a rate estimate is obtainable from the experiment. Since Po is estimated from the fraction of cultures containing no mutation in a set of C cultures, C becomes a determining factor for the accuracy of mu. The rate estimated from P0 is biased, but the bias is in general 2 orders of magnitude smaller than mu. By the selection of an appropriate combination of Nt and C for the experiment, this bias can be reduced even further. Based on the notion of comparing two proportions, we propose a test statistic and have applied it to experimental results for a test of equality of mutation rates in different cell lines. This development places the comparison of mutation rates on a statistical basis.