Hideko Suzuki

Effects of human interferon on cellular response to UV in UV-sensitive human cell strains.

Cells of a human RSa cell line, with high sensitivity to UV killing and low capacity for DNA repair, when pretreated with 1-100 units/ml of human interferon (HuIFN) preparations for more than 12 h before irradiation, acquired an enhancement of UV-induced DNA-repair replication synthesis in association with recovery from inhibition of total cellular DNA synthesis and UV survival. Prompt and transient induction of plasminogen activator activities was also found within 5 min after UV irradiation in the cells pretreated with HuIFN but not in the cells non-pretreated with HuIFN. The enhancement and induction effects of HuIFN were observed, irrespective of the kind of HuIFN preparation used (alpha, beta or gamma, and natural or recombinant) and in other UV-sensitive fibroblast cells which were derived from Cockayne syndrome and xeroderma pigmentosum fibroblasts (XP1KY). However, all of the enhancement of DNA-repair synthesis and the induction of plasminogen activator activities by HuIFN was suppressed by treatment with cycloheximide immediately after UV irradiation.

Orthophenylphenol mutagenicity in a human cell strain.

Orthophenylphenol (OPP), a widely used fungicide, induced ouabain-resistant (OuaR) mutants in a ultraviolet (UV)-sensitive human RSa cell strain and the frequencies increased in a dose-related fashion. OPP was a more potent mutagen than UV at doses related to equal survival. These results suggest that OPP has a mutagenic activity and that further experiments on this chemical are warranted.

Suppression of UV mutagenicity by human interferon.

Effects of human interferon (HuIFN)-alpha on UV mutagenicity were examined in a human cell strain, RSa, and xeroderma pigmentosum (XP)-derived fibroblasts (XP1KY). The frequency of ouabain-resistance mutation in UV-irradiated RSa cells was unusually high (Suzuki et al., 1985), but that in cells pretreated with HuIFN-alpha before irradiation was reduced. 6-Thioguanine-resistance mutation was also depressed in XP1KY cells treated with HuIFN-alpha before irradiation. However, the depression of UV mutagenicity by HuIFN-alpha was lessened by treatment with cycloheximide immediately after UV irradiation. The relationship between HuIFN-depressed UV mutagenicity and HuIFN-affected DNA-repair and repair-related functions is discussed.

Protease activation following UV irradiation is linked to hypomutability in human cells selected for resistance to combination of UV and antipain

In order to examine the relationship between activation of an antipain-sensitive protease and suppression of mutability in UV (UVC)-irradiated human cells, a human cell variant with the high protease activity induced by UV was established and characterized for its susceptibility to UV-induced mutagenicity. Cells of a hypermutable cell strain, RSa, were mutagenized with ethyl methanesulfonate and irradiated with 10 J/m2 UV, followed by exposure to 20 mM antipain for 34 h. Whereas the combined treatment was totally lethal to RSa cells not treated with ethyl methanesulfonate, one surviving clone was isolated from the mutagenized cells and designated UVAP-1. When fibrinolytic protease activity was measured from extracts of the cell, it was found that the protease activity was elevated promptly after UV irradiation, reaching the maximum at 10 min post-irradiation. This protease activity was inhibited by antipain. After UV irradiation the phenotypic mutation frequencies of UVAP-1 cells were much lower than those of the parent RSa cells, as evaluated by the generation of clones resistant to ouabain-killing. Furthermore, mutation at the K-ras codon 12 in genomic DNA was detected in RSa cells but not in UVAP-1 cells. Thus, the protease activation was correlated with the decreased levels of UV-mutagenicity in UVAP-1 cells, supporting the possible involvement of the antipain-sensitive protease activity in the regulation of cellular mutability following UV irradiation.

Piperonyl butoxide mutagenicity in human RSa cells

Piperonyl butoxide (PB) is used as a pesticide synergist and food additive. Its chemically induced mutagenicity was found in cultured human RSa cells by determination of ouabain-resistant (Ouar) phenotypic mutation, with the highest frequency at the concentration of 0.2 microgram/ml. Moreover, K-ras codon 12 mutations in genomic DNA, analyzed by polymerase chain reaction (PCR) and differential dot-blot hybridization using digoxigenin-labeled probes, were detected in RSa cells 6 days after exposure to PB (0.03-0.40 microgram/ml).

Mutagenicity of saccharin in a human cell strain

The mutagenicity of saccharin was investigated by the phenotypic changes of the sensitivity to ouabain lethality in human RSa cells with high susceptibility to UV mutagenicity. At concentrations ranging from 10.0 to 22.5 mg/ml, saccharin induced a dose-dependent increase in the number of mutations to ouabain resistance. This is the first report, to our knowledge, of saccharin having mutagenic activity in human cells.

UV-induced mutations affecting codon 12 of the K-ras gene are suppressed by interferon-α in human RSa cells

K-ras gene sequences mutant at codon 12 were recovered following differential dot-blot hybridization of genomic DNA from human RSa cells up to 12 days after the cells had been irradiated with far-UV (principally 254 nm). By contrast, no mutant codon 12 sequences were recovered from cells which had been treated with 50 IU/ml human interferon (HuIFN)-alpha for 24 h prior to their UV exposure. HuIFN-alpha treatment in combination with anti-HuIFN-alpha antibody did not lead to the loss of mutant sequences. However, culture of interferon-pretreated cells with medium containing the protease inhibitor antipain (0.01 mM) for 6 h immediately after UV irradiation led to the recovery of mutant codon 12 sequences. Thus, while treatment with HuIFN-alpha appeared to prevent any UV-induced mutations affecting codon 12 of the K-ras gene from being recovered, the putative antipain-sensitive protease responsible for this suppressive affect appeared to be significantly affected by the protease inhibitor antipain.