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272 Proc. Japan Acad., 53, Ser. B (1977) [Vol. 53(B),

58. Some Critical Exchange

Studies on in Human

the Sister Chromatid Chromosomes

By Yukimasa SHIRAISHI

Department of Anatomy, School of Medicine, Kanazawa University, Kanazawa 920, Japan

(Communicated by Sajiro MAKINO, M. J. A., Dec. 12, 1977)

The mechanism of the sister chromatid exchange (SCE) in mammalian chromosomes has not remained fully explored. There is evidence to show that the double-strand exchange appears to be more likely as the mechanism than the single-strand exchange. It has been postulated that the formation of SCE is associated with a process such as the post-replication repair that is possibly attributed to a single strand exchange (Kato 1973 ; Sobel 1972) . On the other hand, Kihlman (1975), in Vicia faba treated with thiotepa, failed to detect the single strand exchanges between the double DNA helices in the sister chromatids at chromosomal level. Thus, the possible relation- ship of SCE to post-replication repair of DNA damage has been a matter of controversy. Cells treated with mitomycin C ( MMC ) offer an opportunity for studying various facets of SCEs, since the high incidence of SCE is inducible in human cells. The present study was undertaken in order to inquire into the nature of SCE occurring

in non-treated and MMC-treated cells with a hope to contribute to the understanding of the mechanism of the sister chromatid ex-

change. The effect of caffeine, an effective inhibitor of post-replica- tion DNA repair was also examined on SCE frequency in the MMC-

treated cells.

Material and methods. Heparinized blood samples from normal subjects were cultured for 48 hr and 72 hr in PHA-containing RPMI1640 medium supplemented with 10% fetal calf serum. In PHA-stimulated human lymphocytes, the first and second mitoses are known to occur in 48 hr and 72 hr cultures, respectively. BrdU was used at a concentration of 5 x 10-~ g/ml in the culture media.

In order to induce SCE, the cultures were treated with 1 x 10-~ g/ml MMC alone, and also with MMC-1 mM caffeine in combination, in the presence of BrdU in total darkness. Chromosome preparations and differential sister chromatid staining were made following the method previously described (Wolff et al. 1974).

Results and discussion. Table I gives data on the effects of MMC and caffeine in the presence of BrdU on the differential stain-

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No. 71 Sister Chromatid exchanges in human Chromosomes

ing and SCE frequency. In the cells fixed at the first mitoses following exposure to BrdU, both sister-chromatids of metaphase chromosomes contained unifilarly substituted DNA, i.e., DNA of the TB constitu- tion (T: thymidine containing strand of the DNA complex, and B : bromouracil containing strand). No differential staining of the sister chromatids was detected in the non-treated and MMC-treated cells.

In the cells exposed to BrdU continuously for two replication cycles, bifilarly labeled (BB) and unifilarly labeled (TB) sister chromatids were demonstrated. Normal lymphocytes treated with 1 x 10-7 g/ml MMC for 72 hr were shown to have a multi-fold increase of SCE

Table I. Effect of mitomycin C (MMC) and caffeine on SCE frequency

Fig. 1. Metaphase chromosomes of MMC (1>< 10-7 g/ml) treated normal lymphocytes, stained first with 33258 Hoechst and then with Giemsa.

Fig. 2. Metaphase chromosomes of MMC (1 x 10-7 g/ml)-1mM caffeine treated normal lymphocytes. Note the increased SCEs.

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274 Y. SHIRAISHI {Vol. 53(B), with 57.2 per cell in an average (Fig. 1). Thus, the frequency was

more than 10-fold in treated cells when compared to that of untreated control cells showing 5.6 per cell (Table I). No gaps or unlabeled segments, as well as isolabeled segments were apparent along the chromatids (Fig. 1), though there was a multi-fold increase of SCE in the MMC-treated chromosomes. If the SCE were derived from the single-strand exchanges, gaps or unlabeled segments of chromo-

somes should appear along the chromatids in the second as well as in the first mitoses, as illustrated schematically in Fig. 3. Then the data here presented are sufficient to suggest that both strands of the DNA double helix are involved in the SCE at chromosomal level.

Since the exchanges connected with the post-replication repair of DNA damage involve single strands of DNA (Howard-Flanders 1973), the relationship of the SCE to the post-replication repair seems doubtful. Thus, the data obtained by me are consistent with those presented by Kihlman et al. (1975) in the V icia f aba experiment as well as with those of Wolff and Perry (1975) in Chinese hamster cells, without finding of heterolabeling in the first metaphase chromo- somes and isolabeling in the second ones. In addition, the effect of caffeine on the frequency of SCE in non-treated and MMC-treated

Fig. 3. Schematic illustrations of expected BrdU labeling patterns and Giemsa staining of sister chromatids and SCE of single-strand or

double-strand exchanges based on the semiconservative replication pattern of DNA. Unbroken lines represent original strands of DNA, and broken lines indicate BrdU labeling. Isolabeling of broken exhibits

staining gaps; the latter would be observed in the single-strand but not in the double-strand exchanges.

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No. 71 Sister Chromatid Exchanges in Human Chromosomes 275 cells was studied. Evidence was presented that, while caffeine alone exerted no effect on the SCE frequency in untreated cells, caffeine increased the frequency of SCE in MMC-treated chromo- somes in all specimens observed so far. As shown in Table I, the frequency of SCE in the MMC-caffeine combination experiment was significantly higher than that in the single MMC treatment. The results of the present study regarding SCE by caffeine are incon- sistent with the findings of Kato (1973, 1974) indicating that caffeine is an effective inhibition of post-replication repair, and that caffeine strongly reduces the frequency of SCE induced by the alkylating agent. The lack of the caffeine effect and the evidence for single strand exchanges in SCE provided by this study seem to allow the following statements that the mechanism of the sister chromatid exchange involves most likely exchanges of the double-strand of a DNA duplex present in each chromatid. Further investigations are now in progress and full data will be published elsewhere.

Acknowledgments. The author's sincere thanks are due to Emeritus Professor Dr. Saj iro Makino, M. J. A. for his kind advice and going over the manuscript. The work was supported in part by grant for Cancer Research from the Ministry of Education of Japan

(No. 201001).

References

Howard-Flanders, P. (1973) : Brit. med. bull., 29, 226.

Kato, H. (1973) : Exp. Cell Res., 82, 383.

(1974) : Ibid., 85, 239.

Kihlman, B. A. (1975) : Chromosoma (Berl.), 51, 11.

Sobel, H. M. (1972) : Proc. Natl. Acad. Sci. USA, 69, 2483.

Wolff, S., and Perry, P. (1974) : Chromosoma (Berl.), 48, 341.

-- (1975) : Exp. Cell Res., 93, 23.

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