The interphase death of dividing cells. The kinetics of the death of cultured Chinese hamster fibroblasts after irradiation at various doses

In studying the kinetics of interphase death (ID) of cultured Chinese hamster cells after irradiation with doses of 100 to 800 Gy the authors showed an increase in the ID rate with increasing radiation dose; the presence of serum in the medium both during and after irradiation prevents the cell death.     

Hypoxanthine transport by cultured Chinese hamster lung fibroblasts.

The uptake of hypoxanthine by Chinese hamster lung fibroblasts grown in tissue culture was studied in wild type clones and 8-azaguanine-resistant mutant clones devoid of hypoxanthine-guanine phosphoribosyltransferase. Wild type fibroblasts rapidly accumulate [3H]hypoxanthine from the medium and over 80% of the intracellular radioactivity is found in acid-soluble nucleotides. The phosphoribosyltransferase-deficient clones accumulate much lower levels of hypoxanthine and over 85% of the intracellular 3H label is associated with chemically unaltered hypoxanthine. The internal level of hypoxanthine in the mutant clones rapidly approaches but does not exceed that present in the medium. Wild type and phosphoribosyltransferase-deficient cells take up hypoxanthine at almost identical initial rates at external hypoxanthine levels from 2 to 300 muM. Analysis of these data reveals two transport systems that obey the Michaelis-Menten relationship. These differ markedly in affinity, yielding average Km values of 20 and 600 muM for both cell types. Hypoxanthine transport by both low and high affinity transport systems is blocked by p-chloromercuriphenylsulfonate and N-ethylmaleimide. Counter-transport of hypoxanthine was demonstrated in phosphoribosyltransferase-deficient fibroblasts. It is concluded that hypoxanthine is transported into Chinese hamster cells by means of carrier-mediated processes (facilitated diffusion) that operate independently of phosphoribosylation.     

CFTR modulates programmed cell death by decreasing intracellular pH in Chinese hamster lung fibroblasts

To study the potentialinfluence of cystic fibrosis conductance regulator (CFTR) onintracellular pH regulation during apoptosis induction, we usedPS120 Chinese hamster lung fibroblasts devoid of theNa⁺/H⁺ exchanger (NHE1 isoform) transfectedwith constructs, allowing the expression of CFTR and/or NHE1. Kineticsof lovastatin-induced apoptosis were measured by orceinstaining, double staining with Hoechst-33258, propidium iodide, DNAfragmentation, and annexin V labeling. In PS120 control cells, thepercentage of apoptotic cells after 40 h of lovastatintreatment was 23 ± 3%, whereas in PS120 CFTR-transfected cells,this percentage was 40 ± 4%. In PS120 NHE1 cells, thetransfection with CFTR did not modify the percentage of apoptoticcells after 40 h (control: 19 ± 3%, n = 8;CFTR: 17 ± 1%, n = 8), indicating that blockingintracellular acidification by overexpressing theNa⁺/H⁺ exchanger inhibited the enhancement ofapoptosis induced by CFTR. In all cell lines, the initial pHvalues were identical (pH = 7.46 ± 0.04, n = 9), and treatment with lovastatin led to intracellular acidification.However, the pH value after 40 h was lower in PS120 CFTR-transfected cells (pH = 6.85 ± 0.02, n = 10) than in PS120 cells (pH = 7.15 ± 0.03, n = 10). To further investigate the origin of thisincreased intracellular acidification observed in CFTR-transfected cells, the activity of the DIDS-inhibitableCl/HCO exchanger was studied.8-Bromoadenosine 3',5'-cyclic monophosphate incubation resulted inCl/HCO exchanger activation in PS120 CFTR-transfected cells but had no effect on PS120 cells. Together, ourresults suggest that CFTR can enhance apoptosis in Chinese hamster lung fibroblasts, probably due to the modulation of the Cl/HCO exchanger, resulting in a more efficient intracellular acidification.

     

Expression of human and Chinese hamster hypoxanthine-guanine phosphoribosyltransferase cDNA recombinants in cultured Lesch-Nyhan and Chinese hamster fibroblasts

The results presented in this communication demonstrate that hypoxanthine-guanine phosphoribosyltransferase (HPRT) cDNA can be expressed in both Chinese hamster and human fibroblasts deficient in the endogenous gene product at levels permitting normal growth of the transformants. All the elements necessary for this expression are present in a pBR322-derived plasmid containing HPRT cDNA coding sequence and a retroviral long terminal repeat. These molecules function in both species investigated and, at least in the case of the Chinese hamster transformants, are efficient at the single copy level. Although the effects of the presence of intron sequences and a polyadenylation signal within the plasmids have yet to be evaluated, these studies demonstrate that neither is an absolute requirement for expression of HPRT cDNA sequences in cultured mammalian cells. We describe the construction of recombinant plasmids containing wild type human or Chinese hamster HPRT cDNA sequences in tandem with a retroviral LTR which confer the HPRT+ phenotype in HPRT-deficient V79 and Lesch-Nyhan fibroblasts. Both stable and unstable transformants, that expressed HPRT mRNA and protein, were isolated at high frequency.     

Independent fluctuations of cytidine and adenosine diphosphate reductase activities in cultured Chinese hamster fibroblasts.

The activities of CDP and ADP reductases were determined throughout the cell cycle of Chinese hamster fibroblasts synchronized by partial deprivation of isoleucine. Both enzyme activities were increased in S phase as compared to G1 phase. CDP reductase increased about 8-fold while ADP reductase increased about 2.5-fold. The ratio of CDP to ADP reductase was 0.26 at the G1 and early S phases of the cell cycle; the ratio was increased to 0.83 by late S phase. Addition of actinomycin D or cycloheximide to cell cultures in G1 phase prevented the increase of both CDP and ADP reductases activities in the latter part of the cell cycle, but the ratio of the two activities was not affected. The ratio of CDP to ADP reductase activities varied from 0.8 to 3 in different populations of exponentially growing DON cells. These results show that CDP and ADP reductase activities vary independently in growing cells. The independent variation with cell growth of CDP and ADP reductases suggests important individual functions of the deoxynucleotides during the cell cycle apart from their common role as precursors of DNA synthesis.     

The interphase death of dividing cells. The relation of interphase death to cell energy requirements

In experiments with irradiated cells of Chinese hamster and Ehrlich ascites tumor a study was made of the influence of energy provision on their interphase death rate. The presence of the uncoupler of respiration and oxidative phosphorylation--carbonyl cyanide-3-chlorophenylhydrazone--in a medium without glucose was shown to drastically increase the interphase death rate of cells of both types, whereas this effect was not observed in a medium with glucose.     

Effect of glutamine limitation on the death of attached Chinese hamster ovary cells.

The effect of glutamine depletion on the death of attached Chinese hamster ovary (CHO) cells was investigated. Experiments were performed using an anchorage dependent CHO cell line expressing gamma-IFN and a second cell line obtained by transfection of that cell line with the human bcl-2 (hbcl-2). Either cell line could grow in media devoid of glutamine with minimal cell death due to endogenous glutamine synthetase activity that allowed cells to synthesize glutamine from glutamic acid in the medium. However, compared to control cultures in glutamine-containing media, the cell growth rate in glutamine-free media was slower with an increased fraction of cells distributed in the G0/G1 phase. The slower rate of cell cycling apparently protected the cells from entering apoptosis when they were stimulated to proliferate in an environment devoid of other protective factors, such as serum or over-expressed hbcl-2. The depletion of both glutamine and glutamic acid did cause cell death, which could be mitigated by hbcl-2 over-expression.     

Factors affecting the photokilling of cultured Chinese hamster cells by phthalocyanines

Chloroaluminum phthalocyanine (CAPC) was recently shown to sensitize the inactivation of cultured Chinese hamster cells by visible light. Several factors affecting the photodynamic action of CAPC have been defined in the present study. Thus the photosensitized inactivation of Chinese hamster cells is not affected by superoxide dismutase, suggesting that O-2 radicals are not involved in the process. Postillumination treatments with D2O or heat (42 degrees C, 90 min) enhanced CAPC-induced photosensitivity, indicating the existence of a repair mechanism for photodamage. Preillumination treatments with sodium salicylate and 5-bromodeoxyuridine also enhanced photosensitivity. The later observation suggests that CAPC-induced DNA damage is potentially lethal. However, 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) synthesis which is involved in repair of DNA strand breakage, had no effect on the photosensitivity. Photosensitized inactivation by CAPC is dependent on the pH value of the medium during irradiation. Thus, in the range of pH values 6-8, the sensitivity was increased at the lower values.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.