High sensitivity of thymocytes of LEC strain rats to induction of apoptosis by X-irradiation

It is known that physical disruption of cell contacts induces apoptosis of thymocytes. When thymocytes from LEC and WKAH rats were incubated in vitro at 37 degrees C for 0-6 hr and then the proportion of apoptotic cells was determined using a flow cytometer, it was found that the percentages of apoptotic thymocytes from both LEC and WKAH rats increased with incubation time and that the proportion of apoptotic cells from LEC rats was significantly higher than that from WKAH rats at each incubation time. The fact that cycloheximide, an inhibitor of protein synthesis, did not show significant inhibitory effects on induction of apoptosis of thymocytes indicates that induction of apoptosis during in vitro cultivation did not require de novo protein synthesis. When thymocytes from LEC and WKAH rats were X-irradiated in vitro at 4 and 8 Gy, the percentages of radiation-induced apoptotic cells increased with post-incubation time after X-irradiation in both LEC and WKAH rat thymocytes and the proportions of apoptotic cells from LEC rats were significantly higher than those from WKAH rat cells at 2 and 4 hr post-incubation after X-irradiation. When thymocytes from LEC and WKAH rats were X-irradiated in the presence of cycloheximide, the induction of apoptosis was substantially inhibited, indicating that radiation-induced apoptosis of thymocytes from LEC and WKAH rats required de novo protein synthesis. The present results showed high sensitivities of thymocytes of LEC rats to induction of apoptosis during in vitro cultivation and by X-irradiation.     

Higher sensitivity in induction of apoptosis in fibroblast cell lines derived from LEC strain rats to ultraviolet B radiation

When lung fibroblast cell lines from LEC and WKAH rats were irradiated with ultraviolet B (UVB) and assayed for colony formation, LEC rat cells showed a higher sensitivity than did WKAH rat cells. The LEC rat cells were approximately 1.5-fold more sensitive to UVB radiation than were the WKAH rat cells in terms of D37 values, which are the doses of UVB required to reduce cell survival to 37%. When the rat cells were irradiated with UVB in the presence of 0.5 M dimethyl sulfoxide (DMSO), which efficiently scavenges free radicals such as hydroxyl radicals, no significant difference was observed between the survival curves of either LEC or WKAH rat cells irradiated with UVB in the presence of 0.5 M DMSO and those irradiated with UVB in the absence of DMSO. Therefore, formation of free radicals may not be involved in cell death induced by UVB radiation. Flow cytometry showed that the percentage of apoptotic cells in the LEC rat cell population increased with post-incubation time after UVB radiation. The proportion of apoptotic cells in the UVB-irradiated LEC rat cell population increased as the dose of UVB was increased. In contrast, no significant proportion of apoptotic cells was observed in the UVB-irradiated WKAH rat cell population. These results showed a higher sensitivity in induction of apoptosis by UVB radiation in LEC rat cells than in WKAH rat cells.     

High sensitivity of fibroblast cell lines derived from LEC rats to heat treatment

A fibroblast cell line derived from LEC rat was approximately twofold more sensitive to heat treatment at 45 degrees C than were that from WKAH rat in terms of heating time required to attain 50% loss of survival in a colony forming assay. The present study was carried out for understanding the mechanism underlying the higher sensitivity of LEC rat cells to heat treatment. Although apoptosis was not found in WKAH rat cells, the percentages of apoptotic cells in LEC rat cells significantly increased after heat treatment. LEC rat cells showed significantly lower sensitivity in induction of cell death and apoptosis to ceramide, a lipid signaling molecule that is associated with heat-induced apoptosis, than did WKAH rat cells. SP600125, an inhibitor of JNK suppressed the induction of cell death in both heated LEC and WKAH rat cells, but SB203580, an inhibitor of p38 mapk, did not. The relative surviving fractions of heated LEC and WKAH rat cells in the presence of both SB203580 and SP600125 were higher than those of cells in the presence of SP600125 alone. The amounts of hsp70 protein in WKAH rat cells increased from 4 to 12 hr after heat treatment, but did not in LEC rat cells. These results suggest that higher thermosensitivity in the fibroblast cell line from LEC rat is due to low inducibility of hsp70 protein after heat treatment.     

Abnormal accumulation of G2/M-phase cells from LEC strain rats after X-irradiation at S phase.

The effect of X-irradiation on the progression of the cell cycle in cell lines from LEC and WKAH rats was investigated by a flow cytometer. When the cells were exposed to 5 Gy of X-rays at S phase, the proportion of S-phase cells in both cell populations decreased with incubation time and that of G2/M-phase cells was approximately 80% at 6 hr post-irradiation. At 12 hr post-irradiation, approximately 45% of the WKAH rat cells appeared in the G1 phase. However, 80-90% of LEC rat cells remained in the G2/M phase and less than 5% in the G1 phase during 6-12 hr post-irradiation. Thus, the LEC rat cells irradiated at S phase remained in the G2/M phase for at least 6 hr longer than did the WKAH rat cells.     

Analysis of the cell cycle of fibroblasts derived from the LEC rat after X-irradiation

The LEC rat is reported to exhibit hypersensitivity to X-irradiation, deficiency in DNA double-strand break repair, and radio-resistant DNA synthesis. This character of the LEC rat has been thought to be due to abnormal G1 arrest in cells after X-irradiation. In this report, we re-investigated the effect of X-irradiation on the cell cycle in primary-cultured fibroblasts. Primary-cultured fibroblasts derived from LEC and BN rats were exposed to 4 Gy of X-ray and their cell cycle analysis was performed with a flow cytometer. Fibroblasts derived from both rats showed normal response of the cell cycle, indicating the arrest at both G1--and G2/M-phase and no difference in the cell cycle population between fibroblasts derived from both rats. In contrast, when the same analysis was performed using the cell line, L7 and W8, which had been established from the lung fibroblasts of LEC and control WKAH rats, respectively, by immortalizing with SV40 T-antigen, L7 cells but not W8 cells showed impaired G1 arrest and abnormal cell cycle. These results suggest that fibroblasts derived from LEC rats possess the normal cell cycle response after X-irradiation, if they are kept naive as not immortalized with SV40 T-antigen.     

Wortmannin, a radiation sensitizer, enhances the radiosensitivity of WKAH rat cells but not that of LEC rat cells

No significant cytotoxic effect was observed in WKAH rat cells by the treatment of wortmannin, a radiation sensitizer, at concentrations lower than 30 microM for 24 hr. The relative surviving fractions of LEC rat cells were slightly, but significantly, lower than those of WKAH rat cells at each concentration of wortmannin. When the wortmannin-treated WKAH rat cells were X-irradiated, the relative surviving fractions decreased in a wortmannin concentration-dependent manner. On the contrary, no significant difference was observed between the survival curves of untreated and wortmannin-treated LEC rat cells after X-irradiation.     

Higher sensitivity of LEC strain rat in radiation-induced acute intestinal death.

LEC strain rats (LEC rats), which have been known to develop hereditarily spontaneous fulminant hepatitis 4-5 months after birth, were highly sensitive to whole-body X-irradiation as compared to WKAH strain rats (WKAH rats). Radiation-induced acute intestinal death occurred at doses higher than 6.5 Gy in LEC rats, and at doses higher than 12.8 Gy in WKAH rats, respectively. By the probit analysis of survival data, it was shown that the LD50/7 value of LEC rats was estimated to be 7.03 Gy which was significantly lower than that (12.99 Gy) of WKAH rats. Histopathological examinations of small intestines from LEC rats 2 days after irradiation at the dose of 8.5 Gy showed severe epithelial death together with edema, whereas little or no significant changes were noted in intestinal epithelium of 8.5 Gy-irradiated WKAH rats. These results suggest that the radiosensitivity of LEC rats to ionizing radiation appears to be higher than that of other strains of rats.     

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.

The amounts of DNA single strand breaks that are oxidative damage produced by copper were examined by comet assay in the liver cells of an inbred strain of Long-Evans Cinnamon (LEC) rats that spontaneously develops fulminant hepatitis. At 4 weeks of age, copper contents in the liver of LEC rats were approximately 30-fold higher than those of WKAH rats that are control rats used in the present study. Copper accumulated in the liver of LEC rats in an age-dependent manner and no significant differences were observed between copper contents in the livers of males and females at each week of age from 4 to 15 weeks. No significant amounts of DNA strand breaks were found in the liver cells of both male and female WKAH rats from 4 to 15 weeks of age. DNA strand breaks were produced in the substantial population of LEC rat liver cells at 10 weeks of age and induced in an age-dependent manner from 10 to 15 weeks of age. The amounts of DNA strand breaks produced by copper accumulation in the liver cells of female LEC rats are not more abundant than those in the cells of male rats, although it has been reported that hepatitis in female rats is more serious than that in male rats.     

Radiofrequency radiation at 40 kHz induces hepatic injury in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease

In the present study, we examined effects of radiofrequency (RF) radiation at 40 kHz on hepatic injury in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease, which is a heritable disease of copper metabolism in the liver. The activities of ALT and AST in serum of LEC rats exposed to RF radiation for 2 weeks were approximately 3.8-fold and 2-fold higher than those in serum of sham-exposed rats, respectively. Although there were no significant differences in hepatic copper contents between LEC rats exposed to RF radiation for 2 weeks and sham-exposed rats, copper contents in the kidney and serum of exposed LEC rats were approximately 4.2-fold and 12.9-fold higher than those in sham-exposed rats, respectively. Relative O??-scavenging activities in the S-100 fraction of the liver of LEC rats exposed to RF radiation for 2 weeks were 1.6-fold higher than those in sham-exposed rats. No significant differences were observed in activities of AST and ALT in serum and relative O??-scavenging activity in the S-100 fraction of the liver of normal control WKAH rats that were sham-exposed and exposed to RF radiation. No significant differences were observed in copper contents in the liver, kidney and serum of WKAH rats that were sham-exposed and exposed to RF radiation for 2 weeks. The results show that RF radiation at 40 kHz induced hepatic injury in LEC rats.     

Inactivation of p53 and retinoblastoma family pathways in canine osteosarcoma cell lines

Canine osteosarcoma (OS) has been used as a model system for the study of cancer biology and treatment despite the lack of information regarding its pathogenesis. Expression of tumor suppressor genes known to participate in malignant transformation were studied in five different OS cell lines. Each of the cell lines exhibited properties of transformed cells, and those that were tested grew in soft agarose and formed osteoid-containing tumors when injected subcutaneously into nude mice. p53 function was determined to be defective in each cell line as indicated by the lack of induction of p53-responsive genes, p21 and mdm2, following treatment with 5-fluorouracil. p53 mRNA and protein levels were elevated in three cell lines and were extremely low in two cell lines. p53 protein overexpression correlated with the presence of mutations within the DNA binding domain. Four cell lines appeared to contain normal retinoblastoma (Rb) mRNA and Rb protein and no detectable p16 mRNA or protein. In contrast, the remaining cell line contained high levels of p16 mRNA and protein and significantly reduced levels of Rb, p107, and p130 proteins. These results underscore the importance of inactivating p53 and Rb family pathways in canine OS and suggest that unlike human OS, cells derived from canine OS contain mutations that simultaneously inactivate all three Rb family members.     

Restriction fragment length polymorphism for the Yc subunit gene of rat liver glutathione S-transferase

An altered expression of the Yc subunit gene of rat glutathione S-transferase (GST) in the liver of the LEC rat, which is a mutant strain with spontaneous hereditary hepatitis associated with severe jaundice, has been reported. To provide further information concerning the structure of the Yc subunit gene, we carried out the Southern blot hybridization analysis of DNA samples from rats of eight different inbred strains including LEC with cDNA complementary to mRNA specific for the Yc subunit of rat liver GST as a probe. The hybridization patterns of the DNA samples from rats belonging to the different inbred strains showed interstrain variation in the length of restriction fragments with four restriction endonucleases. Since the DNA samples prepared from several rats of one inbred strain gave an identical hybridization pattern, the restriction fragment patterns for the Yc gene could be used as markers for genetic monitoring of inbred rat strains. Although the altered expression of Yc-Yc activity of GST has been observed in the liver of the LEC rat, the characteristic changes in the gene structure of the Yc subunit of LEC rat were not detected in the present hybridization analysis.     

Identification of a locus for susceptibility to renal cell carcinoma in the Long-Evans Cinnamon rat

The Long-Evans Cinnamon (LEC) mutant rat shows higher incidence of renal cell carcinomas induced by a treatment with the chemical carcinogen N-diethylnitrosamine, as compared to the normal control rat. We performed the first genome-wide scan for genes responsible for susceptibility to chemically induced renal cell carcinoma in an F2 intercross obtained by mating the LEC and Fischer-344 (F344) rats. The genotype of 71 (F344 x LEC) F2 progenies was determined with the use of 338 simple sequence length polymorphisms (SSLPs) spread over the genome. The F2 rats which carried renal cell carcinoma were shown to possess the incidence of homozygosity of the LEC allele which is higher than that of the other genotypes at SSLP markers on chromosome 5 (chi2 = 17.5 for D5Rat21). Our linkage analysis has led to the revelation of a novel gene that influences susceptibility to renal cell carcinoma on rat chromosome 5.     

Establishment of ku70-deficient lung epithelial cell lines and their hypersensitivity to low-dose x-irradiation

In clinical situations, cellular resistance to chemotherapy and radiotherapy is a significant component of tumor treatment failure. The DNA repair protein Ku70 is a key contributor to chemoresistance to anticancer agents, e.g., etoposide and bleomycin, or radioresistance. Ku70 plays a key role as a sensor of DNA double-strand breaks (DSBs) induced following exposure to ionizing radiation as well as treatment with some chemotherapeutic drugs. The responses of different organs to radiation vary widely and likely depend on the cell population in the organs. However, it is not clear whether Ku70 plays a role in the low-dose radioresistance of lung epithelial cells. In this study, we established Ku70-deficient epithelial cell lines from murine lungs lacking Ku70. Ku70-/- lung epithelial cells exhibited reduced Ku80 expression. Moreover, Ku70-/- lung epithelial cells were more sensitive than controls (Ku70+/- lung epithelial cells) to low-dose X-irradiation (< 0.5 Gy). We also found that consistent with the Ku70 function as a sensor of DSBs, Ku70 mainly localized in the nuclei of murine lung epithelial cells. These findings clearly indicate that Ku70 plays a key role in regulation of the Ku80 expression level in and the radioresistance of lung epithelial cells. Our data also suggest that these cell lines might be useful not only for study of Ku70 functions and the DSB repair pathway, but also for study of the molecular mechanism underlying the sensitivity to chemotherapeutic drugs and radiation in lung epithelial cells.     

Analysis of radiosensitivity of cancer stem‐like cells derived from canine cancer cell lines

Cancer stem‐like cells (CSCs) are self‐renewing cells comprising a small subpopulation in tumours, and generate differentiated progeny through asymmetric division. It has been shown that CSCs are resistant to ionizing radiation, and this feature could be one of the mechanisms of tumour recurrence after radiation therapy. Much attention has been focused on to target CSCs; however, difficult of isolating CSCs and lack of knowledge on their radiosensitivity have limited this kind of research in veterinary medicine. In the present study, sphere‐forming cells (SC), cultured using sphere formation method, were isolated from four type of canine tumour cell lines and evaluated if they have CSCs‐like properties by expression of CSCs markers (real‐time polymerase chain reaction) and capacity of tumorigenesis (xenograft transplantation in nude mice), and were assessed radiosensitivity (clonogenic survival assay) and DNA repair kinetics (immunofluorescence staining for p53‐binding protein 1) after X‐ray irradiation in comparison with the corresponding normal adherent culture cells (AC). All SCs were isolated using sphere formation and showed high gene expression of CD133 and tumorigenic ability as compared with AC. All SCs were significantly resistant against X‐ray irradiation as compared with AC. In addition, the amount of DNA double‐strand breaks after X‐ray irradiation were significantly lower in SC compared with the corresponding AC. These results indicate that SC isolated through sphere formation possess CSCs‐like characteristics and CSCs are important factor that affect radiosensitivity in canine tumours. In addition, radioresistance of CSCs may depend on reaction of DNA double‐strand break after X‐ray exposure.     

Flow cytometric analysis of thymocyte subpopulations in mice after whole-body X-irradiation.

To determined the cellular kinetics of thymocyte subpopulations in DBA1 mice after whole-body 6.8 Gy X-irradiation, they were analyzed for the expression of several cell surface antigens using flow cytometry. The results show that i) The majority of thymocytes rapidly depleted by irradiation was CD4+8+ cells. ii) radioresistant CD4+8- and CD4-8+ survived 18-48 hr after X-irradiation were considered to be relatively mature type, since they expressed high levels of CD3 and LECAM-1. iii) CD3-positive cells were detected in CD4-8- cells at 72 hr after irradiation.     

N -ethyl- N -nitrosourea induces retinal photoreceptor damage in adult rats

Seven-week-old male Lewis rats received a single intraperitoneal injection of N-ethyl-N-nitrosourea (ENU) (100, 200, 400 or 600 mg/kg), and retinal damage was evaluated 7 days after the treatment. Sequential morphological features of the retina and retinal DNA damage, as determined by a TUNEL assay and phospho-histone H2A.X (γ-H2AX), were analyzed 3, 6, 12, 24 and 72 hr, 7 days, and/or 30 days after 400 mg/kg ENU treatment. Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) was analyzed immunohistochemically by poly (ADP-ribose) (PAR) expression in response to DNA damage of the retina. All rats that received ≥ 400 mg/kg of ENU developed retinal degeneration characterized by the loss of photoreceptor cells in both the central and peripheral retina within 7 days. In the 400 mg/kg ENU-treated rats, TUNEL-positive signals were only located in the photoreceptor cells and peaked 24 hr after ENU treatment. The γ-H2AX signals in inner retinal cells appeared at 24 hr and peaked at 72 hr after ENU treatment, and the PAR signals selectively located in the photoreceptor cell nuclei appeared at 12 hr and peaked at 24 hr after ENU treatment. However, degeneration was restricted to photoreceptor cells, and no degenerative changes in inner retinal cells were seen at any time points. Retinal thickness and the photoreceptor cell ratio in the central and peripheral retina were significantly decreased, and the retinal damage ratio was significantly increased 7 days after ENU treatment. In conclusion, ENU induced retinal degeneration in adult rats that was characterized by photoreceptor cell apoptosis through PARP activity.