CHEMORESISTANCE RELATED TO HYPOXIA ADAPTATION IN MESOTHELIOMA CELLS FROM TUMOR SPHEROIDS.

BACKGROUND: Hypoxia has been noted as a key factor for induction and maintenance of cancer stemness thereby leading to therapy resistance. Three-dimensional (3D) spheroid models demonstrate a heterogeneity of hypoxic regions replicating the in vivo situation within tumors. Utilizing an established 3D spheroid model, we investigated whether extrinsic hypoxia reinforced chemoresistance in malignant pleural mesothelioma (MPM) spheroids. MATERIALS AND METHODS: Tumor spheres were generated from Meso-1 (a typical human MPM cell line) cells having high spheroid-forming ability. To induce hypoxia condition, we utilized a hypoxia chamber with regulation of O2 and CO2 levels. Cell viability was estimated by a WST-8 assay. Real-time polymerase chain reaction and Western blot were performed to evaluate the expression at mRNA and protein levels. RESULTS: Compared with cells cultured in the two-dimensional monolayer model, tumor sphere cells showed elevated mRNA levels of cancer stemness markers (CD26, CD44 and ABCG2) and protein levels of the stemness and hypoxia adaptation markers (ABCG2, ALDH1A1 and HIFs). Correlating with this, 3D spheroid cells were more resistant to permetrexed and topotecan than the two-dimensional cells, indicative of their potential for hypoxic adaptation. Furthermore, significantly stronger resistance to both chemotherapeutic agents was observed in spheroid cells upon hypoxic challenge compared to spheroid cells under normoxia. CONCLUSION: From the present data, it is concluded that hypoxia adaptation of MPM cells from tumor spheres could enhance their chemoresistance.

Overexpression of interferon-activated gene 202 (Ifi202) correlates with the progression of autoimmune glomerulonephritis associated with the MRL chromosome 1.

B6.MRLc1(82-100) congenic mice carrying the telomeric region of lupus-prone MRL chromosome 1 develop autoimmune glomerulonephritis (GN). The GN susceptibility locus of B6.MRLc1(82-100) contains the interferon activated gene 200 (Ifi200) family, which consists of Ifi202, 203, 204, and 205. Recently, Ifi202 was suggested as a candidate gene for murine lupus. In this study, we assessed the association between Ifi200 family and GN in several disease models. We compared the expression of Ifi200 family members in 24 organs between the C57BL/6 and B6.MRLc1(82-100). The expressions of Ifi200 family members differed between strains, and the most dramatic differences appeared in Ifi202 expression. Briefly, in the blood, immune organs, lungs, and testes mRNA expression was higher in B6.MRLc1(82-100) mice. In the kidney and immune organs, only Ifi202 expression increased with the development of GN in B6.MRLc1(82-100), and significant differences from C57BL/6 were observed even before disease onset. Ifi202 expression in the kidneys of BXSB, NZB/WF1, and MRL/lpr was also significantly high in the early- and late-disease stages. Furthermore, laser microdissection-reverse-transcriptase-polymerase chain reaction analysis confirmed the high Ifi202 expression in all areas of B6.MRLc1(82-100) kidneys. In conclusion, in the Ifi200 family, Ifi202 expressions in the kidney and immune organs significantly increased with GN progression.

Nonsense mutation of feline beta-hexosaminidase beta-subunit (HEXB) gene causing Sandhoff disease in a family of Japanese domestic cats.

G(M2) gangliosidoses are inherited metabolic disorders and are caused by severely reduced enzymatic activity of lysosomal beta-hexosaminidase. In the present study, the open reading frame (ORF) of the HEXB gene in a family of Japanese domestic cats with G(M2) gangliosidosis variant 0 (Sandhoff disease) was determined. Two types of abnormal cDNA clones were obtained from the liver of an affected cat tissue. One showed a single nucleotide substitution from C to T at nucleotide position 667 of the HEXB ORF. In the deduced amino acid sequence, the codon of arginine was altered to a stop codon. The genotyping, using PCR-primer introduced restriction analysis confirmed that Sandhoff disease in this family is associated with this nonsense mutation. Discovery of the nonsense mutation will permit the confirmation of the clinical diagnosis of Sandhoff disease in conjugation with the already established enzyme-based test.

OH-induced free radicals in uridine studied by a method combining ESR, spin-trapping, and liquid chromatography.

Free radicals produced by the reactions of OH radicals with uridine were investigated by a method combining ESR, spin-trapping, and liquid chromatography. A N2O-saturated aqueous solution of uridine, containing 2-methyl-2-nitrosopropane as a spin-trap, was X-irradiated and the resulting spin-adducts were separated by gel permeation chromatography and reverse-phase HPLC. ESR and uv-absorbance spectra obtained from the separated spin-adducts show that 5-yl and 6-yl radicals are produced by OH addition to the 5,6 double bond of the base moiety. It is also shown that radicals due to H abstraction from the sugar moiety at the C-4' and C-5' positions are produced.

Hypertonic treatment inhibits radiation-induced nuclear translocation of the Ku proteins G22p1 (Ku70) and Xrcc5 (Ku80) in rat fibroblasts.

The effects of X irradiation and hypertonic treatment with 0.5 M NaCl on the subcellular localization of the Ku proteins G22p1 (also known as Ku70) and Xrcc5 (also known as Ku80) in rat fibroblasts with normal radiosensitivity were examined using confocal laser microscopy and immunoblotting. Although these proteins were observed mainly in the nuclei of human fibroblasts, approximately 80% of the intensities of immunofluorescence from both G22p1 and Xrcc5 was observed in the cytoplasm of rat fibroblasts. When the rat cells were X-irradiated with 4 Gy, the intensities of the fluorescence derived from G22p1 and Xrcc5 in the nuclei increased from 20% to 50% of the total cellular fluorescence intensity at 20 min postirradiation. No significant differences were observed between the total intensities of the cellular fluorescence from the proteins in unirradiated and irradiated rat fibroblasts. The results showed that the proteins were translocated from the cytoplasm to the nucleus in the rat cells after X irradiation. The nuclear translocation of the proteins from the cytoplasm was inhibited by hypertonic treatment of the cells with 0.5 M NaCl for 20 min, which inhibits the fast repair process of potentially lethal damage (PLD). When the rat cells were treated with 0.5 M NaCl immediately after X irradiation, the repair of DNA DSBs was inhibited. The surviving fraction was approximately 60% of that of irradiated cells that were not treated with 0.5 M NaCl. The surviving fraction increased with incubation time in the growth medium before treatment with NaCl. The proportions of the intensities of fluorescence from G22p1 in the nuclei of X-irradiated cells also increased from 20% to 50% with increasing interval between X irradiation and treatment with NaCl. These results suggest that nuclear translocation of G22p1 and Xrcc5 is important for the fast repair process of PLD in rat cells.

Expression of renin in coagulating glands.

The presence of an extrarenal or local reninangiotensin system has been noted in several tissues, although their functions have not yet been clarified. Renin from the coagulating gland (CG), is the most recently discovered local renin, and is a significant subject for investigation with histological and molecular biological techniques. Dot-like reactions for renin are detected immunohistochemically in the epithelial cells of CGs of the strains C57BL/6 mice. Excretory products of some terminal lumina are also found to be positive for renin. Colloidal gold particles, indicating the presence of renin, are detected in the lysosomal granules, in which they are especially located on the crystalline structure. They are also observed in the production of series of exocrine granules. At the apical region, both reninpositive exocrine and lysosomal granules are secreted by exocytosis. In the development, immunoreactivity for renin is first detected at 6 weeks after birth. After that time, the number of renin-containing cells gradually increase throughout the development. In adults, several patterns of renin immunoreactivity are demonstrated in almost all epithelial cells of CGs. At 4 weeks after castration, renin-containing cells in terminal ducts are decreased and remain at very low levels. After testosterone injection, the numerical value of renin-containing cells is high at 1 week. Finally, renin mRNA is detected in the CGs by Northern blot analysis and hybridohistochemistry. These findings suggest that renin is synthesized depending on testosterone, and released by exocrine secretion.

Dose and dose-splitting effects of X-rays on lung tumour induction in mice.

This paper reports lung-tumour induction 12 months after single or split doses of X-rays in C3H/He male mice. The early proliferative response of lung cells after doses which induced lung tumours was also examined after single X-irradiation. The lung-tumour incidence tended to increase with increasing dose after a single irradiation and peaked at 5 Gy. At more than 10 Gy it decreased sharply to the control level. The mean tumour diameters tended to increase with doses up to 7.5 Gy and then decreased beyond 10 Gy. These results suggested that suppression of tumour growth reduced the tumour incidence at doses of over 10 Gy. The lung-tumour incidence decreased with increasing intervals between two equal doses of 2.5 or 5 Gy. The decrease was thought to be caused by the repair of the tumorigenic injury. However, the tumour incidence after two 2.5 Gy irradiations at 1 day intervals or two 5 Gy irradiations at 6 h intervals was higher than that observed after a single dose. This phenomenon was regarded as a progression of the tumorigenic injury. The labelling indices of the lung cells, determined using tritiated thymidine after a single irradiation, were higher than those of non-irradiated control cells. This response was delayed as the dose increased. The responses versus days after irradiation could be classified into three patterns on the basis of their peaks. The possibility that the larger delay after higher doses had some relation to the reduction of target cells for tumour incidence is suggested.

Life span and tumours in the first-generation offspring of the gamma-irradiated male mouse.

C57BL/6 male mice were exposed to 3 Gy 60Co gamma-rays and mated with unirradiated females after 15 days to produce F1 progeny produced following irradiation of the spermatids. After weaning the offsprings were allowed to live their normal life span. The mean litter size of the irradiated group significantly decreased from 7.1 to 4.9 (p < 0.01), but the sex ratio was not altered by the irradiation. No significant differences in the survival curve and mean life-span between the irradiated and control groups were noted. The only radiation effect in tumour incidence was a decrease of histiocytic sarcoma in female offspring of irradiated males. Except for this, there were no significant differences between the irradiated group and the control group in the incidence or age distribution of tumours.

Deficiency in fast repair process of potentially lethal damage induced by X-irradiation in fibroblasts derived from LEC strain rats.

The time course for the repair of PLD in LEC and WKAH rat cells irradiated at 5 Gy was examined. In the case of WKAH rat cells, the surviving fraction increased with increasing incubation times after X-irradiation. When hypertonic treatment was performed at each incubation time with 0.5 M NaCl for 20 min, increase in the surviving fractions was not shown. In contrast, no significant recovery of the surviving fraction in LEC rat cells was observed after incubation of irradiated cells with or without 0.5 M NaCl for 20 min. On dose-survival curves, hypertonic treatment with 0.5 M NaCl enhanced radiosensitivity of WKAH rat cells, but not LEC rat cells. Although the surviving fraction of the cells from backcross mice with normal radiosensitivity reduced by treatment with 0.5 M NaCl, the survival fraction was not affected in the cells from backcross mice with higher radiosensitivity by treatment with 0.5 M NaCl. When the cells were X-irradiated and incubated with or without 0.225 M NaCl, the radiosensitivities of LEC and WKAH rat cells treated with 0.225 M NaCl for 4 h were approximately two-fold higher than those of untreated cells. Treatment with caffeine also reduced the surviving fractions of both X-irradiated LEC and WKAH rat cells, compared with those of untreated cells. These results indicated that the slow repair of PLD occurred in LEC rat cells but not the fast repair of PLD.

Radiation hypersensitivity of LEC strain rats controlled by a single autosomal recessive gene.

LEC strain rats (LEC rats), which are known to develop hereditarily spontaneous fulminant hepatitis 4-5 months after birth, were highly sensitive to whole-body X-irradiation when compared to WKAH strain rats. The radiosensitivity of F1 hybrids of LEC and WKAH rats was similar to that of WKAH rats and significantly lower than that of LEC rats. Segregation data of backcross hybrids (F1 x LEC and LEC x F1) suggested that the hypersensitivity of LEC rats to whole-body irradiation is controlled by a single autosomal recessive gene. The radiosensitivity of fibroblasts from LEC rats was higher than that of fibroblasts from WKAH rats. The repair process of DNA double-strand breaks in LEC cells was slower than that in WKAH cells. LEC rats could provide a useful animal model to assist in understanding the mechanism of radiation-induced DNA damage and repair.

A high frequency of induction of chromosome aberrations in the bone marrow cells of LEC strain rats by X-irradiation.

LEC strain rats, which have been known to develop hereditarily spontaneous fulminant hepatitis 4 to 5 months after birth, are highly sensitive to whole-body X-irradiation when compared to WKAH strain rats. The present results showed that the frequencies of all types of chromosome aberrations induced by X-irradiation in the bone marrow cells of LEC rats were approximately 2- to 3-fold higher than those of WKAH rats, though no significant difference was observed in the frequency of spontaneous chromosome aberrations between LEC and WKAH rats.

An analysis of experimental radiation carcinogenesis with model setting for competing risks.

The mean life span of animals killed by a specific type of tumor and the incidence of that particular tumor are influenced by competing risks. A parametric approach for censored samples was applied to correct for the competing risks. The life span distribution under each risk wherein the other risks were thought to be eliminated is assumed to be a Weibull distribution. Our data were from an experiment on tumor induction by X-rays in mice: whole body irradiation with 600 R; head with 800 R; trunk with 800 R; or lower body with 800 R. Analysis of the results shows that the mean appearance time of malignant lymphomas was accelerated by whole body irradiation. Acceleration of lung tumors was relatively large after trunk irradiation. Mammary tumors were accelerated by whole body, trunk or lower body irradiation.

Statistical test of the shortening of latent periods in experimental radiation carcinogenesis under competing risks.

The mean life span of animals killed by a specific type of tumor is influenced by competing risks. The parametric inference method of Noda et al. was applied to a set of mouse experimental data in order to correct for the appearance of observed life span distribution under competing risks and to test statistical significances of difference between corrected mean life spans. Data were from an experiment on tumors induced in mice by X-rays: whole body irradiated with 600 R; head with 800 R; trunk or lower body with 800 R. The statistically significant changes were found: Whole body irradiation shortened the mean latent period of malignant lymphomas whereas the head irradiation shortened the latent periods of none of tumors. Trunk irradiation shortened mean latent periods of lung tumors. Lower body irradiation showed a tendency to shorten the mean latent periods of mammary tumors. Results that differed from the above were obtained when there was no correction for competing risks.

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.

A high frequency of induction of chromosome aberrations in the fibroblasts of LEC strain rats by X-irradiation.

The LEC strain of rats (LEC rats), originally developed as a model for hereditary fulminant hepatitis, is highly sensitive to whole-body X-irradiation when compared to WKAH strain of rats (WKAH rats). The present results showed that frequencies of certain types of chromosome aberrations induced by in vitro X-irradiation in the fibroblasts of LEC rats were higher than those of WKAH rats. In particular, frequencies of chromatid gaps and chromosome exchanges in LEC cells were higher approximately 4- to 5-fold and 6- to 8-fold, respectively, than those of WKAH cells.

Cross-sensitivity of X-ray-hypersensitive cells derived from LEC strain rats to DNA-damaging agents.

The cross-sensitivity of X-ray-hypersensitive lung fibroblasts from LEC strain (LEC) rats to other DNA-damaging agents was examined. The LEC cells were 2- to 3-fold more sensitive to bleomycin (BLM) that induces DNA double-strand breaks, and to a cross-linking agent, mitomycin C, than the cells from WKAH strain (WKAH) rats, while they were slightly sensitive to alkylating agents, ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, but not to UV-irradiation. Although no difference was observed in the initial yields of DNA double-strand breaks induced by BLM between LEC and WKAH cells, the repair process of DNA double-strand breaks was significantly slower in LEC cells than in WKAH cells.

Induction of lung tumors in C3H strain mice after single or fractionated irradiation with X-rays.

Murine model for lung tumor induction was studied in C3H/He male mice, a strain with low spontaneous incidence of lung tumors. Dose-response relationships in lung tumor induction were compared following irradiation with single doses and split doses of X-rays to the thorax either at night or in the daytime. The tumor incidence after a single 1.25 Gy dose at night during the period of nocturnal activity almost reached the maximum level after a 5 Gy dose in the daytime. Proliferative activity determined by observing the labeling index with tritiated thymidine in the normal lung was low as a whole, but tended to decrease in the daytime. When the proliferative response was induced by X-irradiation, significantly higher activity was observed at night. These circadian fluctuations were thought to affect radiosensitivity and lung tumor induction in mice. When split doses or fractionated doses of X-rays were applied to the thorax, lung tumor incidence definitely increased. The incidence after two 7.5 Gy doses with a 12 hr-interval was 41%, 3-fold higher than that after a single 15 Gy dose. Moreover, fractionated whole body irradiations (three times at 3 Gy with 3-month-interval) after a single 7.5 Gy thoracic irradiation was most effective in increasing not only the incidence (47%) but also the multiplicity of the lung tumor. More than 30% of tumor-bearing mice had two or more tumors following thoracic and whole body irradiations, while only 10% of tumor-bearers had multiple tumors after single or fractionated thoracic irradiation alone.

Higher sensitivity in LEC rat cells to a topoisomerase II inhibitor, ellipticine.

A concentration of ellipticine, an inhibitor of topoisomerase II, required to reduce cell survival to 37% (D37) is used as an index to compare the cellular sensitivity. D37 values of LEC and WKAH rat cells were 1.2 and 2.2 microM, respectively. Thus, LEC rat cells were approximately 1.8-fold more sensitive than WKAH rat cells to ellipticine. There was no significant difference between the topoisomerase II activities in nuclear extracts of LEC and WKAH rat cells. These results suggested that the high sensitivity of LEC rat cells to ellipticine is not associated with the level of topoisomerase II activity.

Effects of antioxidants on induction of apoptosis in bursal cells of Fabricius during in vitro cultivation.

After physically disrupting cell contacts, apoptosis of bursal cells of Fabricius was induced during in vitro cultivation. The percentage of apoptotic cells increased with incubation time and approximately 70% cells represented apoptosis after 6 hr of incubation. The induction of apoptosis was significantly inhibited by treatment of the cells with ascorbic acid (vitamin C), but not with trolox, a vitamin E analog. An intense DNA ladder pattern was shown at 6 hr post-isolation, which is a biochemical hallmark of apoptosis. Treatment of the cells with ascorbic acid inhibited the DNA fragmentation, but trolox did not. To monitor the intracellular production of reactive oxygen species (ROSs), the intensity of fluorescence emitted from DCFH-DA was measured. The intensity of fluorescence from cells incubated for 0.5-2 hr was approximately 2-fold higher than that from cells at 0 hr. The relative intensity of fluorescence decreased immediately after the addition of ascorbic acid to the cells. The intensity from the cells treated with ascorbic acid was 20-30% of that from the control cells at each incubation time. For trolox, the intensity was 50-70% of that from the control cells at each 1 to 2 hr incubation time. When ROSs-induced lipid peroxidation was assessed using cis-parinaric acid (PnA) as a monitor molecule, lipid peroxidation was found to occur in the control cells after isolation of the bursal cells. Treatment of the cells with trolox reduced lipid peroxidation, but treatment with ascorbic acid enhanced peroxidation.

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.