Masakatsu Horikawa

Analyses of differential sensitivities of synchronized hela S3 cells to radiations and chemical carcinogen during the cell cycle: (II) Ultraviolet light

Abstract UV-induction of thymine dimers in cellular DNA and their excision during different phases of the cell cycle of HeLa S3 cells were studied. Induction of thymine dimers was higher in the mitotic phase and the middle of the S phase than in the G 1 phase and from the late S phase to the early G 2 phase which are rather insensitive to UV. However, there is no significant difference in excision rate of UV-induced thymine dimers from the irradiated cells through the cell cycle. These findings indicate that the cyclic variation of UV-survivals during the cell cycle may be due to differences in the amount of thymine dimers in cellular DNA induced by UV-irradiation.

Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle. Part IV. X-rays.

Radiation-induction and rejoining of single-strand breaks (SSBs) in the DNA of synchronized HeLa S3 cells were investigated by alkaline sucrose density gradients. We could not find any significant differences in the extent of SSBs induced in cellular DNA and in the extent of their rejoining throughout the cell cycle, including mitosis. The cyclic variation curve of the content of non-protein sylfhydryls (NPSH) during the cell cycle is similar to that of X-ray survivals except in mitosis, although there was no close correlation between the content of apparent total sulfhydryls (APSH) and X-ray survivals. Radiation-induced mutants resistant to 8-azaguanine (8AG) occurred in higher frequency in the radio-sensitive G1-S boundary phase than in the radio-resistant G1, S and early G2 phases. Further, the pre-irradiation treatment with 50 mM cysteamine prevented reproductive death and induction of 8AG-resistant mutants by X-rays throughout the cell cycle. These findings seem to indicate that there is a close correlation between the extent of lethal radiation damage to the cells and their mutability, and that sulfhydryls may play an important role as a factor governing cellular radio-sensitivity.

Analyses of differential sensitivities of synhronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle III. 4-nitroquinoline I-oxide and its derivatives

Sensitivity to the chemical carcinogens 4-nitroquinoline 1-oxide (4-NQO) and 4-hydroxyaminoquinoline I-oxide (4-HAQO), during the cell cycle of synchronized HeLa S3 cells, decreases from the late S to the early G2 phases. Cells in other phases are relatively sensitive to both carcinogens. [3-H]4-NQO and [ 3-H]4-HAQO seem to be bound preferably more with cellular DNA of the mitotic phase to the middle of the S phase than with that of the late S phase in which the cells are rather insensitive to these carcinogens. However, we found no significant difference in the excision rates of these carcinogens from the DNA of HeLa S3 cells through the cell cycle. These findings indicate that the cyclic variation of 4-NQO and 4-HAQO cell survivals during the cell cycle may be due to the differences in the amounts of 4-NQO and 4-HAQO bound with cellular DNA.

Chapter 5: A Replica Plating Method of Cultured Mammalian Cells

Publisher Summary This chapter discusses a replica plating method of cultured mammalian cells. The chapter describes the technical procedure for replica plating and illustrates an example of the application of the technique to the characterization of mutants of cultured mammalian cells. The replica plating method described is useful for the replica plating of various cultured mammalian cell lines in vitro. Genetic and biochemical studies of single somatic mammalian cells in vitro were developed by Puck and his co-worker who introduced the colony-forming technique as used in the field of microbial genetics. The lack of a replica plating technique, as used in the field of microbial genetics, for cultured mammalian cells has delayed more detailed genetic analysis in somatic mammalian cells. This procedure isuseful for the isolation of useful mutants, for further purification of a mutant cell line, and for the investigation of mutagenesis at the somatic cell level similar to that at bacterial and fungal levels.

Repair of the sublethal and mutational damage induced by X-rays in Chinese hamster hai cells in vitro

The ability of Chinese hamster hai cells to repair damage related to cell death and mutational change induced by X-rays were examined by using a sensitive forward mutation system from prototrophic CH-hai Cl 23 cells to auxotrophs. The results obtained from the split dose experiments seem to suggest that Chinese hamster hai cells have the repair mechanisms for the sublethal and mutational damage induced by X-rays and that the repair mechanisms act in common for the repair of both cases of damage.

Chapter 6 Isolation of Metaphase Chromosomes from Synchronized Chinese Hamster Cells

Publisher Summary This chapter focuses on the isolation of metaphase chromosomes. Isolated metaphase chromosomes with excellent preservation of morphology and biological activities are very useful for analyzing the fine structure and physicochemical properties of chromosomes. The chapter describes a method for obtaining a large mitotic cell population from cultured Chinese hamster Don cells (CH-Don-13 clone) by two treatments with the combination of Colcemid and harvesting techniques, by which a relatively large number of metaphase chromosomes can be isolated. The mitotic cell population obtained by this method shows no significant biological or cytological damage, and the chromosomes isolated are morphologically intact. The chapter accounts for the optimum concentration of Colcemid and duration of treatment for accumulating mitotic cells. The collection of a large highly purified mitotic cell population by repeated treatments with a combination of Colcemid and harvesting techniques are also described. The chapter also discusses the biological and cytological effects of single and repeated treatments with Colcemid and of chilling on Chinese hamster cells.