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Biomedical subjects

R S Gupta

Publications and source records attributed to R S Gupta.

At least 199 records · Page 11Linked to original sources
PubMed1984

Griseofulvin resistance mutation of Chinese hamster ovary cells that affects the apparent molecular weight of a congruent to 200,000-dalton protein.

A single-step griseofulvin-resistant mutant (GrsR-4) of CHO cells which exhibit very specific cross-resistance towards certain microtubule inhibitors showed the absence of a protein of molecular weight congruent to 200,000 (designated P5) and the concomitant presence of a new protein spot, M5, of lower molecular weight (Mr congruent to 180,000) which is not present in other cell lines. Peptide mapping studies showed that proteins P5 and M5 are related to each other and that M5 may be missing a peptide fragment present in P5. In GrsR-4 X GrsS cell hybrids, both P5 and M5 were present in equal amounts, which provided evidence against post-translation mechanisms in the origin of M5 and indicated that the GrsR-4 mutant most likely contains a nonsense mutation in the structural gene for protein P5, which causes its premature termination and leads to the formation of M5. Our studies also showed that in different Chinese hamster cell lines the two alleles of the protein P5 are nonidentical and make protein products which differ from each other in isoelectric points. It is suggested that protein P5 and its isoelectric variant P6 may constitute microtubule-associated proteins.

Animals↗
PubMed1984

4'-Demethylepipodophyllotoxin-beta-D-glucoside: a new podophyllotoxin derivative with VM-26-like and VP-16-213-like activities.

Mutants of Chinese hamster ovary (CHO) cells independently selected for resistance to the anticancer drug 4'-demethylepipodophyllotoxin thenylidene-beta-D-glucoside (VM-26) and the microtubule inhibitor podophyllotoxin exhibit mutually an exclusive cross-resistance pattern toward various podophyllotoxin derivatives that possess either VM-26-like or podophyllotoxin-like activities, respectively. The cross-resistance studies with these mutants have led to the identification of a new podophyllotoxin derivative, 4'-demethylepipodophyllotoxin-beta-D-glucoside (compound No. 13), which possesses VM-26-like and 4'-demethylepipodophyllotoxin ethylidene-beta-D-glucoside (VP-16-213)-like activity. This inference is strongly supported by our observation that, like VM-26 and VP-16-213, compound No. 13 showed no antimitotic activity (as seen by the effect on the mitotic index) but was highly active in inducing DNA strand breaks, sister chromatid exchanges, and mutations at the hypoxanthine-guanine phosphoribosyl transferase and adenosine kinase loci in CHO cells. On the basis of structure-activity data of various podophyllotoxin derivatives, three structural features have been identified that appear essential for VM-26-like activity. These are: 1) the presence of an hydroxyl group at the 4'-carbon, 2) an epi-configuration at the C-4 position, and 3) the presence of a glycoside moiety on the hydroxyl group at the C-4 position. In addition, the nature of the glycoside substituents at the C-4 position greatly affects the relative VM-26-like activity of various compounds.

Animals↗
PubMed1983

Comparison of the mutagenic responses of 12 anticancer drugs at the hypoxanthine-guanine phosphoribosyl transferase and adenosine kinase loci in Chinese hamster ovary cells.

5he mutagenic responses of 12 anticancer drugs--lomustine, dacarbazine, mitomycin C, chlorambucil, cis-diamminedichloroplatinum (II), busulfan, ellipticine, daunomycin, adriamycin, VM-26, VP16-213, and bleomycin--in Chinese hamster ovary (CHO) cells at two independent genetic loci which affect purine salvage pathway enzymes, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and adenosine kinase (AK) have been compared within the same experiments. The cellular mutants which lack either HGPRT (HGPRT- mutants) or AK (AK- mutants) activity can be directly selected in CHO cells using 6-thioguanine and 6-methyl mercaptopurineriboside (6-MeMPR) respectively, and optimal conditions for their selection have been described. Results of our studies show that all of the above drugs caused a concentration-dependent increase in the frequencies of both 6-thioguanine- and 6-MeMPR-resistant mutants. However, for a number of these drugs, namely adriamycin, daunomycin, bleomycin, and dacarbazine, which are only weakly mutagenic at the HGPRT locus, a relatively strong and clear mutagenic response was observed at the AK locus. In contrast, VM-26 showed a more pronounced effect at the HGPRT locus. For the remaining compounds, the mutagenic responses of the two loci (as indicated by the slopes of the dose-response curves) were comparable. The observed increase in the frequency of AK- mutants after treatment with various drugs, many of which predominantly induce frameshift and chromosome-breakage type of genetic lesions, suggests that like the HGPRT locus the AK locus is also capable of detecting all the different types of genetic lesions. The favorable response of the AK locus as compared to that of HGPRT locus in these studies indicates that the selection system of AK- mutants should provide a very useful genetic marker for mutagenesis studies in CHO cells.

Adenosine Kinase↗
PubMed1983

Quantitative mutagenesis at the adenosine kinase locus in Chinese hamster ovary cells. Development and characteristics of the selection system.

Stable mutants exhibiting high degree of resistance (100-1000-fold) to various nucleoside analogs viz, toyocamycin, tubercidin, 6-methyl mercaptopurine riboside (6-MeMPR) and pyrazofurin, are obtained at similar frequency (congruent to 1 X 10(-4] in CHO cells. The mutants resistant to any of the above analogs exhibit similar degree of cross-resistance to the other three nucleoside analogs, and all of the mutants examined contained no measurable activity of the purine salvage pathway enzyme adenosine kinase (AK) which converts these analogs to their phosphorylated derivatives. These results indicate that very similar mutants are selected using any of these analogs. The recovery of AK- mutants in CHO cells is not affected by cell density (up to at least 5 X 10(5) cells per 100-mm diameter dish) and after treatment with mutagen(s) maximum mutagenic effect is observed after 7-8 days, which then remains unchanged for the next several days. Treatment of CHO cells with a number of mutagenic agents e.g. ethyl methanesulfonate, ICR170, ultraviolet light, and benzo[a]pyrene, led to a nearly linear concentration-dependent increase in the frequency of the AK- mutants in cultures. The mutagenic response of the AK locus to these agents compared favorably with that of the HGPRT locus (6-thioguanine resistance) within the same experiments. These results show that the selection system for AK- mutants provides an additional valuable genetic marker for quantitative mutagenesis studies in CHO cells.

Adenosine Kinase↗
PubMed1983

Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.

Adenosine↗
PubMed1983

Podophyllotoxin-resistant mutants of Chinese hamster ovary cells: cross-resistance studies with various microtubule inhibitors and podophyllotoxin analogues.

The cross-resistances of several mutants of Chinese hamster ovary cells which have been obtained after one and two selection steps in the presence of the microtubule inhibitor podophyllotoxin (PodRI and PodRII mutants, respectively) towards various other inhibitors of microtubule assembly (e.g., colchicine, Colcemid, vinblastine, griseofulvin, maytansine, steganacin, nocodazole, and taxol) have been examined. Based upon their specific patterns of cross-resistance/sensitivity to various microtubule inhibitors, both the PodRI and PodRII classes of mutants appear to be of more than one kind. Studies on the binding of [3H]podophyllotoxin to cytoplasmic extracts indicate that one of the PodRII mutants which has been shown previously to be affected in a Mr 66,000 to 68,000 microtubule-associated protein shows reduced binding of the drug in comparison to the parental PodS and PodRI cells. The different PodRI and PodRII mutants exhibited proportionally increased cross-resistances to various podophyllotoxin analogues (e.g., deoxypodophyllotoxin, epipodophyllotoxin, beta-peltatin, 4'-demethylpodophyllotoxin, alpha-peltatin, podophyllotoxin-beta-D-glucoside, beta-peltatin-beta-D-glucoside, picropodophyllotoxin, and podophyllic acid) which possess microtubule-inhibitory activity. However, with the exception of one PodRI class of mutant, none of the mutants exhibited any cross-resistance to 4'-demethylepipodophyllotoxin thenylidine-beta-D-glucoside and 4'-demethylepipodophyllotoxin ethylidine-beta-D-glucoside, the 2 podophyllotoxin analogues which lack microtubule-inhibitory activity. The cross-resistance studies with these mutants, which, based upon the biochemical studies and their highly specific patterns of cross-resistance, are presumably affected in microtubules, provide some very novel insights into the mechanisms of action of various microtubule inhibitors. The results presented in this paper also show that the cross-resistance studies with the set of podophyllotoxin-resistant mutants provide a sensitive and highly specific screening procedure for identifying compounds which possess podophyllotoxin-like activity and for investigating the structure-activity relationship among them. The results of structure-activity relationship studies for the various podophyllotoxin analogues examined are discussed.

Animals↗
PubMed1983

Mutagenic responses of thirteen anticancer drugs on mutation induction at multiple genetic loci and on sister chromatid exchanges in Chinese hamster ovary cells.

The mutagenic responses of 13 antineoplastic drugs, namely, chlorambucil, busulfan, lomustine, dacarbazine, Adriamycin, daunomycin, bleomycin, VM-26, VP16-213, ellipticine, actinomycin D, mitomycin C, and cis-diamminedichloroplatinum(II) have been determined in two different assay systems in Chinese hamster ovary cells which measure mutation induction at multiple genetic loci and the frequencies of sister chromatid exchanges. The five genetic loci whose responses have been measured include those conferring resistance to 6-thioguanine (Thgr or TGr), ouabain, emetine, methylglyoxal bis(guanylhydrazone), and 5,6-dichlororibofuranosylbenzimidazole; of these, only the Thgr marker affects a function (hypoxanthine-guanine phosphoribosyltransferase, hgprt locus) which is not essential for cellular growth. All of these drugs showed a dose-dependent increase in mutation frequency at the hgprt locus, but their responses at other genetic loci differed greatly and showed marked specificity for different chemical classes of the drugs. The observed locus-specific differences in response to these drugs suggest that they may differ in terms of their accessibility or affinity to different chromosomal regions. All of these drugs also led to a significant increase in the frequency of sister chromatid exchanges, and a very good correlation was observed between the activity of these drugs in the sister chromatid exchange assay and the mutagenic response of the hgprt locus. Of the drugs which were examined, VM-26, VP16-213, chlorambucil, mitomycin C, and cis-diamminedichloroplatinum(II) showed a particularly strong response in both of these assay systems. In terms of the minimum concentration which gave a mutagenic response, the drugs differed from each other by a factor of about 100,000, with actinomycin D, VM-26, and daunomycin being mutagenic in the range of 3 x 10(-8) to 1 x 10(-7) M, whereas dacarbazine produced a weak mutagenic response only at about 2 x 10(-3) M.

Animals↗
PubMed1982

Mutagenic response to ultraviolet light and X-rays at five independent genetic loci in Chinese hamster ovary cells.

The mutagenic response to ultraviolet light and X-irradiation at a number of well characterized independent genetic loci, viz those conferring resistance to 6-thioguanine, ouabain, emetine, methylglyoxal (bis) guanylhydrazone (MGBG), and 5, 6-dichlororibofuranosyl benzimidazole (DRB), has been determined. Upon exposure of cells to UV light in the range of 70 to 180 ergs/mm2, the frequency of mutants at all of the above genetic loci increased in a linear dose-dependent manner. However, some genetic-locus-specific differences in response to UV light were observed in these studies. In contrast to UV light, exposure of cells to X-rays caused a linear dose-dependent increase in the frequency of mutants at only the thioguanine-resistant (Thgr) locus, which affects the purine salvage pathway enzyme hypoxanthine-guanine phosphoribosyltransferase, that is not essential for growth under these conditions. The other genetic loci that affect essential cellular functions (and hence should detect only specific base substitution types of mutations), however, showed no increase in the frequencies of mutants upon X-irradiation of cells. These results are in accordance with the nature of the genetic lesions which are caused by these agents and the properties of these genetic markers.

Animals↗
PubMed1982

Synergistic effect of purine derivatives on the toxicity of pyrazofurin and 6-azauridine towards cultured mammalian cells.

A novel synergistic effect of several purine derivatives such as adenine, adenosine, hypoxanthine, and guanine on the toxicity of nucleoside analogs pyrazofurin and 6-azauridine towards cultured Chinese hamster ovary (CHO) cells has been observed. The presence of the above purine derivatives enhanced the toxicity of pyrazofurin and 6-azauridine, in a dose dependent manner. The growth inhibitory effects of these nucleoside analogs either alone or in combination with the purine derivatives were reversed by uridine and cytidine, providing evidence that the synergistic effect of the purine derivatives was exerted at the level of pyrimidine nucleotide biosynthesis. Studies with mutant cells lacking various purine phosphorylating enzymes show that phosphorylation of purine derivatives through reactions utilizing phosphoribosylpyrophosate (PRPP) is essential for observing the synergistic response. It is suggested that the above purine derivatives (including adenosine, via conversion to hypoxanthine) exert their synergistic effects by depleting the cellular pool of PRPP by two separate mechanisms (direct utilization and feedback inhibition of its synthesis), which as a result becomes rate limiting in the synthesis of orotidine monophosphate (OMP). The reduced levels of OMP, which is a competing substrate with pyrazofurin- and 6-azauridine-5'-monophosphates for binding to the target enzyme OMP decarboxylase, could then account for the inhibition of the enzyme at lower concentrations of these analogs.

Adenine↗
PubMed1982

Species specific differences in the toxicity of mithramycin, chromomycin A3, and olivomycin towards cultured mammalian cells.

Three structurally related anticancer drugs, mithramycin, chromomycin A3, and olivomycin, showed large unexpected differences (up to more than 1000 fold) in their toxicity towards cultured cells from various species (human, Chinese hamster, Syrian hamster, and mouse). Among the cell types examined, human cells (both a diploid fibroblast cell strain and HeLa cells) were maximally sensitive to all these drugs, followed by the Syrian hamster kidney cells (BHK 21). The mouse (LMTK- cells) and Chinese hamster (CHO) cells, which were more resistant, showed interesting differences in their sensitivity towards these drugs. For example, whereas the mouse cells were more resistant to mithramycin than CHO cells, the sensitivity pattern was reversed for both chromomycin A3 and olivomycin. In cell extracts derived from human, mouse, and Chinese hamster cells RNA synthesis, which is the cellular target of these drugs, showed identical sensitivity to both mithramycin and chromomycin A3, indicating that the species specific differences in the toxicity to these drugs are at the level of cellular entry of these compounds. Based on the structures of these glycosidic antibiotics and their patterns of toxicity, it is suggested that the intracellular transport of these drugs involves specific interactions between the sugar residues on these compounds and some type of cell surface receptor(s), which differ among different cell types. Some implications of these results for toxicity studies are discussed.

Animals↗
PubMed1982

Mutagenic responses of five independent genetic loci in CHO cells to a variety of mutagens. Development and characteristics of a mutagen screening system based on selection for multiple drug-resistant markers.

With the aim of developing a sensitive mutagen screening system, the responses of 15 different chemical mutagens at 5 independent genetic loci in Chinese hamster ovary (CHO) cells have been determined. The genetic markers which have been employed include resistance to thioguanine (Thgr), ouabain (OuaR), the protein synthesis inhibitor emetine (Emtr), the polyamine synthesis inhibitor methylglyoxal bisguanylhydrazone (Mbgr) and the nucleoside analog 5,6-dichlororibofuranosyl benzimidazole (DrbR). The optimal selection conditions for all of these genetic markers in CHO cells have been described. The chemicals whose response was investigated in these studies include direct-acting alkylating agents (ethyl methane-sulfonate, methyl methanesulfonate, beta-propiolactone, ethyleneimine, N-nitrosomethylurea and 4-nitroquinoline-N-oxide), DNA intercalating and cross-linking agents (ICR-170, acridine orange, ethidium bromide, mitomycin C and actinomycin D), polycyclic hydrocarbons (benzo[a]pyrene (B(a)P) and 7,12-dimethylbenz[a]anthracene (DMBA)) and aromatic amines (benzidine and beta-naphthylamine). Simultaneous examination of the response of the set of genetic markers to these chemicals revealed that although all of these chemicals caused a dose-dependent increase in the frequency of mutations at many of the above genetic loci, the magnitude of the mutagenic response at different genetic loci varied greatly depending upon the chemical. Of the genetic loci examined, no one single locus showed higher response to all of the above chemicals, instead, depending upon the chemical, specific loci were found to be more responsive than others. The polycyclic hydrocarbons and aromatic amines were weakly mutagenic in this system at several genetic loci even without any exogenous microsomal activation, although in the presence of a rat liver S9 fraction similar toxic and mutagenic effects of B(a)P and DMBA were observed at 5-20-fold lower concentrations. These results indicate that CHO cells may possess significant capacity for the metabolic activation of many procarcinogens, and also underscore the merits of measuring the mutagenic response at multiple genetic loci in mutagen screening studies.

Animals↗