49. Effect o f Caffeine on Chromosome Aberrations Induced by Mitomycin C in Human .T.cell Line (CCRFHSB2)
and Fanconi's Anemia Cells
By Yukimasa SHIRAISHI
Department of Anatomy, School of Medicine, Kanazawa University,
Kanazawa 920, Japan
(Communicated by Sajiro MAKINO, M. J. A., Nov. 12, 1977)
Mitomycin C (MMC) is well known as a powerful breaking agent, as a potential recombinogen, and as a dialkylating agent producing "cross-links" between the complementary DNA strands
(Cohen and Shaw 1964 ; Brogger and Johansen 1972 ; Bourgeois 1974) . Caffeine is noted to enhance the chromosome aberrations produced by UV irradiation (Trosko and Chu 1971) , mitomycin C
(Hartley-Asp and Kihlman 1971) , and thiotepa treatments (Sturelid 1976) in Chinese hamster cells. There is a possibility that caffeine can inhibit or reduce the repair of chromosome damages produced by mitomycin C. This leads to the view that a post-replication repair mechanism is involved here. According to Schuler et al. (1969) and Sasaki et al. (1973), Fanconi's anemia cells are excessively susceptible to MMC, suggesting a possibility for defective repair of DNA cross- links. Recent investigations indicated that a human leukemia T-cell line (CCRF-HSB2) was extremely radiosensitive with regard to both chromosome aberrations and cell survival (Shiraishi and Sand- berg 1976). A suggestion was made that there is a possible indica- tion of defective DNA repair in the CCRF-HSB2 cells (Shiraishi and Sandberg 1977). Here, investigations are needed to account for the above phenomenon with regard to the chromosome response of MMC alone, as well as MMC and caffeine in combined application. The present study was undertaken with the above view in mind.
Materials and methods. Heparinized blood samples from a normal male and a Fanconi's anemia patient were cultured for 72 hr in PHA-containing RPMI1640 medium supplemented with 15% heat- inactivated fetal calf serum at 37°C. CCRF-HSB2 cell line was also
grown in RPMI1640 medium supplemented with 15% heat-inacti- vated fetal calf serum at 37°C. The patient, 6 year-old-boy , has typical cytogenetical and clinical features as Fanconi's anemia show- ing a progressive hypoplastic pancytopenia associated with the short stature. CCRF-HSB2 cell line was previously described (Shiraishi et al. 1976). Various concentrations of MMC (5 x 10-8
, 1 x 10-7, 2 x
No. 6] Combination Effect of Caffeine and Mitomycin C 227 10-7, 5 x 10-7 g/ml) and 1 mM caffeine were applied on cell cultures as test chemical mutagens. The cultures were treated with MMC alone, or with MMC-caffeine in combination for the last 24 hrs before fixation. Colcemide (0.02 pg/ml) was added to the cultures at 90 min, prior to fixation. Chromosome preparations were processed according to the conventional air-drying method.
Findings and discussion. In Table I are presented the frequen- cies of metaphases showing chromosomal aberrations produced by the application of MMC alone, or by MMC-caffeine in combination.
In normal cells treated with MMC at levels of 1 x 10-7 and 5 x 10-7 g/ml, there was a significant increase in chromatid exchanges, in fair agreement with the results of previous studies (Cohen and Shaw 1964, Brogger and Johansen 1972). It was noted that the
Table I. Effects of mitomycin C (MMC) and caffeine on the frequency of chromosome aberrations in normal lymphocytes from human T-cell
line (CCRF-HSB2), and in Fanconi's anemia cells. The test chemicals were present for the last 24 hr in the culture
maj ority of exchanges occurred in the regions of secondary constric- tions of chromosomes Nos. 1, 9 and 16, or in the so-called C-band regions. However, when caffeine was added to MMC, the effect was much higher than that produced by MMC alone, or otherwise by caffeine alone. This picture was remarkable in the application of MMC at 1 x 10-7 and 5 x 10-7 g/ml level. The exchange became in- complete in the MMC plus 1 mM caffeine treatment, showing a significant reduction of the exchanges, while in contrast, there was a remarkable increase of breaks in frequency. The most startling effect is the appearance of so many cells with multiple aberrations. The cells with more than 50 aberrations were recorded as cells with multi- ple aberrations in the present study. The majority of them was chromatid breaks, a few being incomplete chromatid exchanges.
Probably those multiple aberrations make unable the cells for rejoin- ing which may be dealt with repairment.
In the FA cells treated with MMC at 5 x 10.8 and 1 x 10-7 g/ml levels, there was a striking increase of chromatid and chromosome breaks, in agreement with the previous findings (Sasaki et al. 1973).
The total frequency of aberrant cells was significantly higher than that of normal cells. The total aberration frequency at the level of 1 x 10 ' g/ml MMC was 100% in FA cells. Although the frequency of chromatid exchanges in MMC-treated FA cells was slightly elevated, it was extremely low compared to that of breaks (Table I). The most characteristic picture of MMC-treated FA cells is the appear- ance of cells with multiple aberrations ; the situation seems to well correspond to that of normal cells treated with both MMC plus 1 mM caffeine. FA cells treated with 5 x 10-7 g/ml MMC showed a mitotic reduction, together with an extreme reduction of viable cells. In contrast, caffeine exerted almost no effect on the chromosome aber- ration, even when added in the presence of MMC (Fig. 1). The aberration frequency in MMC-treated FA cells with and without caffeine showed no statistically significant difference (Table I).
In the CCRF-HSB2 cells treated with MMC at 5x 10-8 and 1 x 10-7 g/ml levels, the incidence of chromatid exchanges did not increase remarkably, whereas there was an increase in number of cells with the chromatid and chromosome breaks. Treatment with 5 x 10-7 g/ml MMC resulted in the extreme reduction of viable cells.
The situation was very similar to that obtained in MMC-treated FA cells at 5 x 10-7 g/ml. The total frequency of aberrant cells was significantly higher than that of normal cells following 1 x 10-7 and 5 x 10-7 g/ml MMC treatments. This indicates that CCRF-HSB2 cells are much more sensitive to MMC than normal cells. On the other hand, caffeine exerted no effect on the MMC-treated CCRF-HSB2
No. 61 Combination Effect of Caffeine and Mitomycin C 229 cells with regard to the appearance of chromosome aberrations
(Fig. 2). The picture is consistent with that observed in FA cells which were treated with 1 mM caffeine in the presence of MMC.
The present investigations resulted in that, while caffeine applied together with MMC produced a striking increase of chromosome breaks as well as of cells with multiple aberrations in normal cells, it exerted almost no influence on the MMC-induced aberration frequency in the FA and the CCRF-HSB2 cells. This finding is con- sistent with earlier reports that HeLa cells were deficient in the ability of caffeine to act synergistically in producing chromosome damage by UV-irradiation (Wilkinson et al. 1970), or to undergo post-replication repair (Roberts et at. 1973). The potentiating effect of caffeine might be the result of an inhibition by this agent of a post-replicative DNA repair process (Sturelid 1976). Then, it fol- lows that the inhibition by caffeine of the gap-filling process at the
molecular level is responsible for its potentiating effect on chromo- some damage and cell killing (Kihlman et at. 1974, Sturelid 1976).
Huttner et at. (1976) have suggested that cross-linking by MMC might involve a form of post-replication repair. The results of the present experiments with caffeine in combination with MMC are in- dicative of that the post-replication repair process in FA and CCRF-
Fig. 1. Fanconi's anemia cell treated with MMC(1 X 10-7 g/ml) plus 1 mM caffeine.
Fig. 2. CCRF-HSB2 cell treated with MMC(1x 10-7 g/ml) plus 1 mM caffeine. Breaks were mainly seen in the centromere regions of cromosomes No. 1, 9 and 16 (arrows).
HSB2 cells may be defective, although a correlation with defective cross-link repair was suggested in FA cells by Sasaki et al. (1973).
Further investigations have now been in progress along this line.
Acknowledgments. The author expresses his sincere thanks to Emeritus Professor Dr. Sajiro Makino, M. J. A., Hokkaido University, for his kind advice and going over the manuscript, and to Dr. Avery A. Sandberg, Buffalo, N.Y, for his keen interest in the problem. This work was supported in part by grant for Cancer Research from the Ministry of Education of Japan (No. 201001).
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Bourgeois, C. A. (1974) : ibid., 48, 203-211.
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Hartley-Asp, B., and Kihlman, B. A. (1971) : Hereditas, 69, 326-328.
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Shiraishi, Y., Minowada, J., and Sandberg, A. A. (1976) : In Vitro, 12, 495-509.
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Wilkinson, R., Kief er, J., and Nias, A. H. W. (1970) : Ibid., 10, 67-72.