Characteristic synthesis and redistribution of 70 kd heat shock protein in thermotolerant Chinese hamster V79 cells

Upon exposure to heat shock the increased rate of hsp70 synthesis decreased more rapidly in thermotolerant V79 cells than in the non-thermotolerant cells. However, the levels of hsp70 in the thermotolerant cells at 12 h after a heat shock were almost the same as those in the non-thermotolerant cells. On the other hand, the migration of hsp70 from cytoplasm to nucleoli after a heat shock was very rapid in both thermotolerant and non-thermotolerant cells, but hsp70 in the nucleoli disappeared faster in the thermotolerant cells than in the non-thermotolerant cells, and this coincided with the faster decline of hsp70 synthesis in the thermotolerant cells. For the characteristic distribution of hsp70, protein synthesis was not required. Furthermore, the induction and expression of thermotolerance by the cells were little affected by the inhibition of protein synthesis. Thus, the synthesis of hsp70 itself seemed not to be essential for the induction and expression of thermotolerance of the cells, although hsp70Maybe essential for thermoresistance of cells. The rapid decrease of hsp70 synthesis and the rapid disappearance of hsp70 from the nucleoli after a heat shockMaybe essential for the expression of thermotolerance of the cells.     

Characteristic synthesis and redistribution of 70 kd heat shock protein in thermotolerant Chinese hamster V79 cells.

Upon exposure to heat shock the increased rate of hsp70 synthesis decreased more rapidly in thermotolerant V79 cells than in the non-thermotolerant cells. However, the levels of hsp70 in the thermotolerant cells at 12 h after a heat shock were almost the same as those in the non-thermotolerant cells. On the other hand, the migration of hsp70 from cytoplasm to nucleoli after a heat shock was very rapid in both thermotolerant and non-thermotolerant cells, but hsp70 in the nucleoli disappeared faster in the thermotolerant cells than in the non-thermotolerant cells, and this coincided with the faster decline of hsp70 synthesis in the thermotolerant cells. For the characteristic distribution of hsp70, protein synthesis was not required. Furthermore, the induction and expression of thermotolerance by the cells were little affected by the inhibition of protein synthesis. Thus, the synthesis of hsp70 itself seemed not to be essential for the induction and expression of thermotolerance of the cells, although hsp70 may be essential for thermoresistance of cells. The rapid decrease of hsp70 synthesis and the rapid disappearance of hsp70 from the nucleoli after a heat shock may be essential for the expression of thermotolerance of the cells.     

Mutagenicity of oxidative DNA damage in Chinese hamster V79 cells

We have investigated the mutagenicity of oxidative DNA damage induced in V79 Chinese hamster lung fibroblast, and measured 8- hydroxydeoxyguanosine (8OHdG) levels as an indicator of this damage. A hydroxyl radical generator, N,N'-bis(2-hydroxyperoxy-2-methoxyethyl)- 1,4,5,8-naphthalene-tetra -carboxylic-diimide (NP-III), induced 8OHdG in V79 upon irradiation with 366 nm ultraviolet light (UV) for 15 min. 8OHdG was determined by HPLC with electrochemical detection after anaerobic sample processing. The 8OHdG level in the cells treated without NP-III was 0.49 per 10(5) dG, whereas levels in the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 1.84, 4.06 or 6.95 per 10(5) dG, respectively. The 8OHdG induced by 20 microM NP-III with UV irradiation decreased rapidly, and the half-life of the induced 8OHdG was approximately 6 h. NP-III with UV irradiation also induced DNA strand breaks in all cells uniformly, as determined by single cell gel assay. Mutant frequencies at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in V79 were determined as the number of 6-thioguanine-resistant cells per 10(6) cells. Mutant frequency of the cells without NP-III was 8.0, and frequencies of the cells treated with 5, 10 or 20 microM NP-III and UV irradiation were 14.9, 20.6 or 24.7 respectively. Treatment with 20 microM NP-III and UV irradiation decreased the cell number, determined 3 days after the treatment, to 20.8%. These findings indicate that acutely induced oxidative DNA damage including mutagenic 8OHdG is only weakly mutagenic in V79.      

Ataxia-telangiectasia-like Chinese hamster V79 cell mutants with radioresistant DNA synthesis, chromosomal instability, and normal DNA strand break repair

We have isolated three radiosensitive mutants (V-C4, V-E5, and V-G8) of the Chinese hamster V79 cell line which also show increased sensitivities to killing by bleomycin (approximately 2-5-fold) and ethyl methanesulfonate (approximately 2-fold). Genetic complementation analysis indicates that all three mutants belong to one complementation group. The mutants show a radioresistant DNA synthesis following X-ray irradiation when compared to wild-type V79 cells. Both the level and the rate of repair of DNA single- and double-strand breaks measured by DNA elution were similar to those observed in wild-type V79 cells. The level of spontaneously occurring chromosome aberrations in two of these mutants differs severalfold from the level observed in wild-type V-79 cells and in V-G8, to approximately 2- and 6-fold increase in V-E5 and V-C4, respectively. X-irradiation of the mutants resulted in consistently 3-4-fold higher levels of chromatid gaps, breaks, and exchanges than observed in wild-type V79 cells. In addition, G1 irradiation of the mutant cells yielded both chromosome and chromatid types of aberrations. The level and pattern of chromosomal aberrations induced by X-rays in V-C4, V-E5, and V-G8 are similar to those observed in ataxia-telangiectasia cells. These results indicate that our mutants represent the first rodent cell mutants which show phenotypic characteristics strongly resembling those in cells from ataxia-telangiectasia patients.     

Mechanism of epipodophyllotoxin-induced cell death in poly(adenosine diphosphate-ribose) synthesis-deficient V79 Chinese hamster cell lines

Mutant Chinese hamster V79 cells selected for alterations in poly(ADP-ribose) metabolism were shown to be resistant to epipodophyllotoxin (VP-16)-induced cytotoxicity. Cell lines ADPRT 54 and ADPRT 351 have reduced activity of poly(ADP-ribose) polymerase. N2, N3, and N4 cell lines grow in the absence of nicotinamide, with total NAD levels 1.5-3% of those found in parental V79 cells grown in complete medium. When grown in complete medium, the mutant cell lines are 2.3- to 9.6-fold resistant to VP-16-induced cytotoxicity. All of the cell lines respond to VP-16 treatment by formation of protein-cross-linked DNA strand breaks. Upon drug removal, all the cell lines reverse the DNA strand breaks at similar rates. Our studies show a clear dissociation between induction of DNA strand breaks and cytotoxicity. However, there is a good correlation between drug-induced sister chromatid exchanges and cytotoxicity. Thus, N3 cells, with low levels of VP-16-induced sister chromatid exchanges, show reduced levels of cytotoxicity relative to parental V79 cells, despite the fact that both cell lines show similar levels of VP-16-induced protein-cross-linked DNA strand breaks. Additional studies show that the time course of VP-16-induced cytotoxicity correlated better with the time course of sister chromatid exchange formation than with protein-cross-linked DNA strand break formation. These studies provide strong support for the proposal that VP-16-induced cytotoxicity involves the induction of sister chromatid exchanges. Thus, we suggest that drug-induced stabilization of topoisomerase II-DNA complexes stimulates induction of sister chromatid exchanges, which consequently lead to cell death.     

Ultrastructural changes induced by hyperthermia in Chinese hamster V79 fibroblasts

The effects of hyperthermic treatment on some intracellular components and on the general morphology of Chinese hamster V79 fibroblasts have been studied. After 1 h of heating at 42°C cells show small interruptions of the plasma membrane, dilation of the mitochondrial cristae and dissociation of the polyribosomes. These modifications become progressively more pronounced after 1 or 3 h treatment at 43°C. Severe alterations in the general morphology of the cells are evident after 1 h heating at 45°C.     

Adriamycin uptake in V79 and adriamycin resistant Chinese hamster cells

Total intracellular and DNA-associated uptake of Adriamycin (ADR) was studied in V79 Chinese hamster cells and in a derived subline which is resistant to ADR killing. For both sublines, uptake of ADR was dependent on cell density with greater uptake seen with less dense cultures. Drug uptake was markedly less in the ADR-resistant cells compared to parent V79 cells. These results suggest that the resistance observed is in large part due to membrane changes that result in decreased drug uptake.     

Ultrastructural changes induced by hyperthermia in Chinese hamster V79 fibroblasts

The effects of hyperthermic treatment on some intracellular components and on the general morphology of Chinese hamster V79 fibroblasts have been studied. After 1 h of heating at 42 degrees C cells show small interruptions of the plasma membrane, dilation of the mitochondrial cristae and dissociation of the polyribosomes. These modifications become progressively more pronounced after 1 or 3 h treatment at 43 degrees C. Severe alterations in the general morphology of the cells are evident after 1 h heating at 45 degrees C.     

V79 Chinese hamster lung cells resistant to the bis-alkylator bizelesin are multidrug-resistant

Bizelesin (U-77779) is a highly potent bis-alkylating antitumor agent that is effective against several tumor systems in vitro and in vivo. V79 cells that were 125-to 250-fold resistant to bizelesin developed after constant exposure to gradually increasing concentrations of the drug. Resistant cells exhibited a multidrug-resistant phenotype and genotype as indicated by cross-resistance to several structurally and functionally unrelated drugs, e. g., colchicine, actinomycin D, and Adriamycin, and overexpression of mdr mRNA. Very low levels of cross-resistance to the alkylating, agents cisplatin and melphalan were seen. Multidrug-resistant mouse leukemia (P388/Adriamycin-resistant) and human (KB/vinblastine-resistant) cells were also resistant to bizelesin. Bizelesin resistance was unstable and decreased when cells were grown in the absence of the drug. Resistant and sensitive cell lines had similar levels of glutathione, and bizelesin cytotoxicity for resistant cells was not markedly affected by treatment with buthionine sulfoximine. Cross-resistance between bizelesin and several of its analogs is reported.     

Differential reduction by caffeine of adriamycin induced cell killing and cell cycle delays in Chinese hamster V79 cells

Exponentially growing Chinese-hamster V79-cells were treated with various doses of adriamycin (ADR) for 1 hr in the presence or absence of 2 mM caffeine and were subsequently incubated for 24 hr in fresh medium with or without caffeine (2 mM) before plating to assay for survival. The results indicated a reduction in killing when caffeine was present during treatment with ADR (e.g., reduction in killing from 0.03 to 0.3 after exposure to 0.5 microgram/ml ADR). This reduction in killing was even more pronounced after a 24 hr treatment with ADR in the presence of caffeine (e.g., reduction from 0.005 to 0.5 after exposure to 0.08 microgram/ml ADR). Incubation with caffeine after ADR treatment (1 hr) caused only a comparably small increase in cell survival. Presence of caffeine either simultaneously or after treatment with ADR caused a reduction of the inhibition of growth and mean-cell-volume increase, and a reduction of the accumulation of cells in G2-phase. Qualitatively similar results were also obtained after continuous treatment with ADR in the presence or absence of caffeine. Reduction in growth inhibition and accumulation of cells in G2-phase was observed under conditions only slightly affecting cell survival, thus suggesting that caffeine may affect these two phenomena by independent mechanisms. Flow cytometry measurement of the intracellular ADR content indicated a reduction in the presence of caffeine. Furthermore, post-treatment incubation with caffeine was found to increase the rate of decay of ADR-related fluorescence. Quantitative comparison between the effect of caffeine in the intracellular ADR accumulation and cell survival suggested that the observed reduction in killing could be attributed to a decrease in the intracellular drug levels. The reduction by caffeine of the ADR-induced cell cycle delays is attributed to either the decrease in the intracellular ADR dose in the presence of caffeine, or to an effect of caffeine similar to that exerted after exposure of cells to ionizing radiation. Trifluoperazine, which had only a small effect on cell survival of cells treated with ADR alone, potentiated killing when cells were treated with ADR in the presence of caffeine. This effect can be partly attributed to the observed modification in the intracellular ADR content under these conditions but, as a quantitative comparison suggests, other effects might also be involved.     

Microsome-mediated mutagenesis in V79 Chinese hamster cells by various nitrosamines.

A microsome-mediated mutagenesis system has been established with the V79 Chinese hamster cell line. The cells, grown in monolayer, were treated with various nitrosamines in the presence of a postmitochondrial fraction ((S15) from rat liver and a reduced nicotinamide adenine dinucleotide-generating system for 1 hr, washed and incubated for 2 to 3 hr in fresh culture medium, and then plated for toxicity and mutagenicity assays. Mutation was determined by resistance to 20mug 8-azaguanine per ml. In this assay system, the S15 fraction and cofactors by themselves were not toxic to the cells; dose-related mutagenicity and cytotoxicity were induced by N-nitrosodimethylamine (DMN) only in the presence of the S15 fraction and cofactors. Pretreatment of rats with phenobarbitone led to an approximately 2-fold increase in the mutation rate over that with tissues from untreated rats with concentrations of DMN from 10 to 50 mM, while aminoacetonitrile pretreatment reduced the mutagenic effect. Methylcholanthrene pretreatment resulted in an increase in the mutation frequency with a higher concentration of DMN (50mM). Various other nitrosamines were also assayed in the presence or absence of the S15 fraction from phenobarbitone-pretreated rats and a reduced nicotinamide adenine dinucleotide phosphate-generating system. With the exception of N-nitrosomethylphenylamine, the carcinogenic nitrosamines (DMN, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-n-butylamine, N-nitrosodi-n-pentylamine, N-nitrosomethyl-n-propylamine, N-nitrosomorpholine, N-nitrosopyrrolidine, N-nitroso-N'-methylpiperazine, and N-nitrosomethylphenylamine) were mutagenic to the V79 Chinese hamster cells in the presence of the S15 fraction and cofactors. Neither N-nitrosodiphenylamine nor N-nitrosomethyl-tert-butylamine had a mutagenic effect. These findings show that chemical carcinogens can be tested for mutagenicity in cultured mammalian cells in the presence of a metabolic activation system. The results are discussed in relation to the carcinogenicity and mutagenicity of these compounds in other test systems.     

Further characterization of the in vitro assay for inhibitors of metabolic cooperation in the Chinese hamster V79 cell line

12-O-Tetradecanoylphorbol-13-acetate (TPA) has been previouslyshown to inhibit metabolic cooperation in Chinese hamster V79cells. An in vitro assay, based on this phenomenon, has beendeveloped to study tumor promoters. Several parameters concerningthe metabolic cooperation assay using V79 Chinese hamster cellswere further investigated in this report. Pretreatment of thecells with TPA in situ for different periods of time did notresult in any detectable change in the inhibition of metaboliccooperation. If cells were replated after TPA treatment, a differentresult was obtained. There was an apparent decrease in the abilityof TPA to inhibit metabolic cooperation when TPA was added backto the TPA-pretreated cultures. However, when TPA was omittedfrom the TPA pretreated cultures after replating, the inhibitionof metabolic cooperation remained high. It was also found thatpretreatment of the cells with another chemical, aldrin, exhibitedthe same pattern as the in situ TPA pretreatment effect on inhibitionof metabolic cooperation. In order to obtain a high level ofinhibition of metabolic cooperation when using aldrin in thisassay, it was determined that the chemical needed to be presentfor more than one day. Our studies also showed that a 24 h treatmentwith 6-thioguanine did not kill 6-thioguanine-sensitive cellsquickly, nor did it prevent them from performing metabolic cooperation.The relationship of cell density and TPA concentration was alsostudied. It was observed that a higher cell density requiredhigher TPA concentration to inhibit, maximally, metabolic cooperation.A ‘down regulation’ type effect was noted when culturewas challenged with different concentrations of TPA. These resultswere interpreted to be consistent with the hypothesis that inhibitedgap-junctional intercellular communication is one of the componentsof tumor promotion.     

Effects of ultraviolet radiation on intercellular communication in V79 Chinese hamster fibroblasts

The effects of ultraviolet (UV) radiation on gap junctionalintercellular communication (GJIC) in V79 Chinese hamster fibroblastswere studied by means of a dye transfer assay. Intercellularcommunication was shown to be altered by UVB (297/302 nm) andUVA (365 nm) radiation, the effect depending on the wavelengthof exposure and time between irradiation and microinjectionof the dye in the dye transfer assay. Exposure to 297/302 nmradiation induced a reduction in intercellular communication6 min after exposure. Incubation of the cells post-irradiationreversed the inhibition of GJIC. From 2 to 24 h after exposurean increase in GJIC over the control cells was seen, with amaximum at 8 h post-irradiation. UVA (365 nm) radiation, onthe other hand, induced an increase in the intercellular communication6 min after irradiation. Incubation of the cells post-irradiationled to a decrease in the number of communicating cells, witha minimum seen 4 h after exposure. The reduction in communicationobserved after exposure to UVB and UVA was not correlated withsimilar modifications in the gap junction protein connexin43as found when exposing the cells to the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate. For the higher fluences of UVA, a decrease in immunorecognizableconnexin43 was seen, concomitant with a markedly increased backgroundof higher mol. wt compounds. This may be due to UVA-inducedcrosslinking of connexin43. No correlation was found betweenchanges in communication induced by UV radiation and levelsof cyclic AMP.     

Bovine bladder urothelial cell activation of carcinogens to metabolites mutagenic to Chinese hamster V79 cells and Salmonella typhimurium

The ability of bovine bladder urothelial cells to activate genotoxic chemicals to mutagens was examined by cocultivating bladder cells with Chinese hamster V79 cells or Salmonella typhimurium as mutable targets. Activation of test chemicals to mutagenic intermediates by urothelial cells was detected by induction of 6-thioguanine resistance in V79 cells or by induction of histidine revertants in Salmonella. In the bladder cell-mediated V79 cell mutagenesis system, a significant increase in mutation frequency was induced by exposure to 7,12-dimethylbenz(a)anthracene and dimethylnitrosamine. The aromatic amines 2-aminofluorene, 2-acetylaminofluorene, and 4-aminobiphenyl were weakly mutagenic to V79 cells with bladder cell activation, while no mutagenic activity was detected with 1-naphthylamine, 2-naphthylamine, or benzidine. Because the mutagenic activity of the aromatic amines was low with V79 cells as the target, a bladder cell-mediated S. typhimurium system was developed for these chemicals. The aromatic amines 2-aminofluorene, 2-acetylaminofluorene, 4-aminobiphenyl and 2-naphthylamine were mutagenic to S. typhimurium TA98 and TA100 in the presence of bladder cells but not in their absence. Benzidine was mutagenic to TA98 but not to TA100. The putative noncarcinogen 1-naphthylamine was not mutagenic in the system. In contrast to the V79 data, 7,12-dimethylbenz(a)anthracene and dimethylnitrosamine were not mutagenic with either bacterial strain. Mutagenic responses were related to both the number of bladder cells used for activation and the concentration of test chemical in the Salmonella assay. The data demonstrate that bovine bladder urothelial cells can activate carcinogens from three chemical classes to mutagens and indicate the different sensitivities of V79 cells and S. typhimurium to genotoxic agents.     

DNA single-strand breaks by nitropyrenes and related compounds in Chinese hamster V79 cells

The DNA single-strand breaks (SSB) induced by nitropyrenes and related compounds were studied in Chinese hamster V79 cells. 1-Nitropyrene (1-NP), 1,6-dinitropyrene (1,6-DNP), 1,8-dinitropyrene (1,8-DNP), 1-nitrosopyrene (1-NOP) and 1-aminopyrene (1-AP) caused DNA-SSB in the cells. 1-NP induced SSB most effectively among the compounds tested, though the mutagenic activity of 1-NP towards mammalian cells has been reported to be negative or marginal. 1-NOP induced SSB less effectively than 1-NP, though 1-NOP is assumed to be the intermediate to the ultimate metabolite. From in vitro experiments, this result could be explained, at least partly, by the detoxicating function of reduced glutathione (GSH) or other sulfhydryl materials.     

Studies on adaptation of V79 Chinese hamster cells to low doses of methylating agents

Pretreatment of V79 cells with N-methyl-N'-nitro-N-nitroso-guanidine(MNNG) or N-methyl-N-nitrosourea (MNU) significantly reducedthe frequency of mutations (6-thio-guanine resistance) inducedby a challenge dose of these agents. Mutagenic adaptation canbe observed after exposure to pretreatment doses which haveno measurable toxic or cytogenetic effects, and which do notchange DNA synthesis as measured at the moment of challenge.The influence of pretreatment on challenge dose-induced cellkilling, chromosomal aberrations, and sister chromatid exchangeshas also been studied. The effect of the challenge with respectto these end points was not in the same way dependent on thepretreatment dose as the challenge dose-induced mutation frequency.Therefore it is concluded that mutagenic adaptation and adaptationwith respect to the other end points studied have no identicalunderlying causes.     

Mutagenicity of nitrofluoranthenes, 3-aminofluoranthene and 1-nitropyrene in Chinese hamster V79 cells

1-Nitropyrene (1-NO₂Py), 3-nitrofluoranthene (3-NO₂Ft), 3-aminofluoranthene(3-NH₂Ft) and 8-nitrofluoranthene (8-NO₂Ft) were tested formutagenicity in cultured Chinese hamster V79 cells. Mutationsat the hypoxanthine-guanine phosphoribosyl transferase (HGPRT)gene locus were quantified. 1-NO₂Py had marginal direct-actingmutagenicity which was enhanced by a mixture of Aroclor 1254-and 1242-induced liver homogenates (S9) in the treatment medium.1-NO₂Py was more mutagenic with S9 activation than 3-NO₂Ft,3-NH₂Ft or 8-NO₂Ft. However, 8-NO₂Ft, 3-NO₂Ft and 3-NH₂Ft weremore mutagenic than 1-NO₂Py when a post-microsomal liver supernatant(S100) was used for metabolic activation. The results of theseinvestigations strongly support activation of some nitratedpolycyclic aromatic hydrocarbons to proximate mutagens by asequence of reductions and possible formation of polycyclicaromatic hydroxylamines.     

Mechanisms of hypoxic and aerobic cytotoxicity of mitomycin C in Chinese hamster V79 cells.

Mitomycin C (MMC) induced aerobic and hypoxic cytotoxicity in Chinese hamster V79 cells was studied to evaluate the role of the 1-electron versus 2-electron reductive bioactivation. Superoxide dismutase, catalase, and desferal had no protective effects on the aerobic or hypoxic cytotoxicity of MMC, whereas Tempol and Tempol-H, which are known to interrupt and terminate radical reactions, provided partial protection under aerobic conditions. However, under hypoxic conditions, Tempol provided complete protection whereas Tempol-H was ineffective. Electron paramagnetic resonance and spin-trapping investigations, designed to study the mechanisms of such protective effects, confirmed that MMC is activated by the human NADPH:cytochrome P-450 oxidoreductase to its semiquinone radical and that, under aerobic conditions, the semiquinone radical reduces molecular oxygen. Under hypoxic conditions, the semiquinone of MMC reduces H2O2 to produce OH radicals as detected by electron paramagnetic resonance-spin trapping with 5,5-dimethyl-1-pyrroline N-oxide. The 1-electron reduced product of MMC was also found to reduce Tempol to the hydroxylamine, Tempol-H, whereas oxidation of Tempol-H by MMC-. was negligible. Cell survival studies and electron paramagnetic resonance observations indicate that the hypoxic cytotoxicity of MMC is mediated by 1-electron activation to its semiquinone intermediate. Under aerobic conditions, the steady state concentration of this intermediate is low due to the facile autooxidation of the semiquinone producing O2-. and H2O2 which are capable of causing oxidative cytotoxicity. Tempol, which can accept an electron from reducing radical species, completely inhibited the hypoxic cytotoxicity of MMC indicating MMC-., the semiquinone of MMC as the species responsible for DNA alkylation and selective hypoxic cytotoxicity of MMC. Our results also indicate that the aerobic cytotoxicity is mediated by other processes in addition to the 1-electron mediated activation.     

Colostrinin decreases spontaneous and induced mutation frequencies at the hprt locus in Chinese hamster V79 cells

Colostrinin (CLN), a uniform mixture of low-molecular weight, proline-rich polypeptides, induces neurite outgrowth of pheochromocytoma cells, extends the lifespan of diploid fibroblast cells, inhibits beta amyloid-induced apoptosis and resulted in improved cognitive function when administered to Alzheimer's patients. Here we investigated CLN's antimutagenic activity in cells stressed oxidatively or exposed to chemical or physical agents. Our data show that CLN did not alter cell cycle kinetics and cloning efficiency, while it inhibited the development of spontaneous mutations at the coding region of the hypoxanthine phosphoribosyl-transferase (hprt) gene in Chinese hamster V79 cells. In a dose-dependent manner, CLN lowered reactive oxygen species (ROS)-induced frequency of cells resistant to 6-thioguanine (6-TG) to nearly background level. Likewise, CLN decreased the frequency of methyl methanesulfonate- or mitomycin C-induced mutations in V79 cells. Notably, CLN (at 100, 250, and 500 ng per ml concentrations) decreased UVA-induced mutation frequency, while only the highest dose of CLN also decreased significantly the number of UVB-induced 6-TG-resistant mutant cells. Similar results were obtained using cell cultures of human origin. Overall, our data show that CLN significantly lowers the mutation frequency that develops spontaneously or is induced by ROS, chemical and physical agents. CLN itself has no mutagenic activity. Therefore, CLN may be used in human therapies systemically and/or locally for the prevention of diseases associated with sequence alterations in genomic and mitochondrial DNA.     

Characterization of a low-molecular-weight growth inhibitor formed by density-inhibited, tumorigenic V79 Chinese hamster cells

A novel type of low-molecular-weight growth-inhibitory factor responsible for the density inhibition of tumorigenic V79 Chinese hamster cells has been purified, if not homogenously, by a series of reverse-phase and gel filtration high-performance liquid chromatography. The factor is an acid-stable, heat-labile substance distinct from antiproliferative nucleoside analogues or polyamines and has a molecular weight of approximately 2000. The biological activity of this inhibitor was enhanced nearly 5-fold by trypsin treatment, thereby suggesting that the inhibitor may be a precursor peptide which becomes an oligopeptide with intense biological activity by proteolysis, or that trypsin treatment allows resultant small molecules to efficiently transfer across the cytoplasmic membrane. This inhibitor reversibly inhibits the growth of a broad spectrum of cell types from neoplastic and nonneoplastic cells from various species. These data suggest that this inhibitor is primarily a growth-regulatory molecule common to mammalian cells and may play an important role in regulating growth of both normal and neoplastic cells.