Abnormal G1 Arrest in the Cell Lines from LEC Strain Rats after X-Irradiation.

Abstract

ABSTRACT. The effect of X-irradiation of cell lines from LEC and WKAH strain rats on a progression of cell cycle was investigated. When WKAH rat cells were exposed to 5 Gy of X-rays and their cell cycle distribution was determined by a flow cytometer, the proportion of Sphase cells decreased and that of G2/M-phase cells increased at 8 hr post-irradiation. At 18 and 24 hr post-irradiation, approximately 80% of the cells appeared in the G1 phase. On the contrary, the proportion of S-phase cells increased and that of G1-phase cells decreased in LEC rats during 8-24 hr post-irradiation, compared with that at 0 hr post-irradiation. Thus, radiation-induced delay in the progression from the G1 phase to S phase (G1 arrest) was observed in WKAH rat cells but not in LEC rat cells. In the case of WKAH rat cells, the intensities of the bands of p53 protein increased at 1 and 2 hr after X-irradiation at 5 Gy, compared with those of unirradiated cells and at 0 hr post-irradiation. In contrast, the intensities of the bands were faint and did not significantly increase in LEC rat cells during 0-6 hr incubation after X-irradiation. Present results suggested that the radioresistant DNA synthesis in LEC rat cells is thought to be due to the abnormal G1 arrest following X-irradiation. -KEY WORDS: cell cycle, G1 arrest, LEC strain rat, p53 protein, X-irradiation.J. Vet. Med. Sci. 59(9): 769-773, 1997 to S phase in the cell lines derived from lung fibroblasts of LEC rats and showed an abnormal G1 arrest following Xirradiation. MATERIALS AND METHODS Culture of the cells and X-irradiation:The rat fibroblast cell lines were established from lungs of LEC and WKAH rats by SV 40 immortalization as described previously [7]. Cells were grown in monolayer culture in Eagle's minimum essential medium (MEM) containing 10% fetal calf serum (FCS). Cell cultures were kept at ambient humidity and 37°C in an atmosphere containing 5% CO 2 .X-irradiation was carried out utilizing a Hitachi MBR-1520R X-ray generator operated at 150 kV and 15 mA with a 0.5 mm Cu + 1.0 mm Al filter at a dose rate of 0.95 Gy/ min.Flow cytometry: The cell cycle phase distribution of a cell population was determined by measuring the DNA content of individual cells by flow cytometry. In preparation for flow cytometry, cells (1 × 10 6 to 5 × 10 6 ) were exposed to 5 Gy of X-rays. After X-irradiation, the cells were incubated at 37°C for 0-24 hr, harvested by trypsinization, washed with PBS and then pelleted by centrifugation at 500 × g. The cells were fixed in 5 ml of cold 70% ethanol for 30 min at room temperature and then stored at 20°C. Just prior to flow cytometric analysis, individual samples were treated with RNase (1 mg/ml) for 30 min at room temperature and then stained with propidium iodide according to the manufacturer's instructions for the cellular DNA flow cytometric analysis reagent set (Boeringer Mannheim). Fluorescence was measured with a Coulter EPICS Elite flow sorter using a 610-nm filter. Cell cycle distribution was determined using multicycle software. ...

High Sensitivity of Thymocytes of LEC Strain Rats to Induction of Apoptosis by X-Irradiation.

“…A linkage study of deficiency in repair of DSBs to the high sensitivity of radiation-induced apoptosis in LEC rat thymocytes is now in progress. We have also reported that LEC rat cells show an abnormality of transient cell cycle arrests that occur in normal rat cells following ionizing radiation [7,8,10]. Since it is well known that a regulatory pathway of cell cycle progression is associated with induction of apoptosis [12,23,32], abnormality of transient cell cycle arrests may be associated with the high sensitivity of radiation-induced apoptosis in LEC rat cells.…”

“…In our previous studies using colony-forming assay to determine cell survival, we found that fibroblasts from LEC rats were more sensitive to X-irradiation than were cells from WKAH rats [9,17]. On the other hand, no significant difference was found in induction of apoptosis of LEC and WKAH rat fibroblasts by X-irradiation [10]. The present results showed that LEC rat thymocytes were more sensitive to induction of apoptosis by Xirradiation than were WKAH rat cells and that apoptosis was induced at an earlier incubation time after X-irradiation in LEC rat thymocytes than in WKAH rat thymocytes.…”

“…Other characteristics of LEC rats are a high incidence of spontaneous liver cancer in long-surviving individuals [33] and an increased sensitivity in vivo and in vitro to ionizing radiation [9]. In our previous studies using colonyforming assay for determination of cell survival, we found that primary fibroblasts and fibroblast cell lines from LEC rats were more sensitive to X-irradiation, but not to ultraviolet radiation in the short wavelength (ultraviolet C, UVC), than were cells from a control strain of WKAH rats [9][10][11]17]. On the other hand, LEC rat cells showed a higher sensitivity to induction of apoptosis by UVC radiation, but not X-irradiation [9][10][11].…”

“…In our previous studies using colonyforming assay for determination of cell survival, we found that primary fibroblasts and fibroblast cell lines from LEC rats were more sensitive to X-irradiation, but not to ultraviolet radiation in the short wavelength (ultraviolet C, UVC), than were cells from a control strain of WKAH rats [9][10][11]17]. On the other hand, LEC rat cells showed a higher sensitivity to induction of apoptosis by UVC radiation, but not X-irradiation [9][10][11]. Therefore, the effects of X-irradiation on induction of apoptosis in LEC rat cells are still not clear.…”

No Significant Differences Were Observed in the Amounts of DNA Strand Breaks Produced by Copper between Male and Female Liver Cells of Long-Evans Cinnamon (LEC) Strain Rats.

“…Furthermore, LEC rat cells show abnormalities of transient cell cycle arrests from the G1 to S phase and at the G2/M phase after treatment of the cells with DNA-damaging agents [7,8]. Many studies suggest that abnormalities of cell-cycle checkpoint can contribute to the process of neoplastic transformation of the cells.…”