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.

DNA damage in peripheral blood mononuclear cells and neutrophils of dairy cows during the transition period.

This study was designed to investigate the apoptotic process in peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophil leukocytes (PMN) in dairy cattle during the transition period. Blood samples were collected from 4 dairy cattle at 3 weeks before the expected parturition (wk -3), parturition (wk 0) and 3 weeks after parturition (wk +3). The DNA damage of PBMC and PMN was evaluated based on the comet assay using visual scoring (arbitrary units). Undamaged DNA remained within the core (score 0) and the broken DNA migrated from the core towards the anode forming the tail of a comet (scores 1-4). Significantly higher scores in PBMC at wk 0 and wk +3 were observed compared with those in PMN although there were no significant changes of scores in either cell type during the experimental period. It is suggested that the apoptotic rate of PBMC is accelerated compared with that of PMC during the transition period.

Pathological correlations between podocyte injuries and renal functions in canine and feline chronic kidney diseases.

Podocytes cover the glomerulus and their adjacent foot processes form a principal barrier called the slit diaphragm. Podocyte dysfunctions, including podocyte loss and slit diaphragm disruptions, induce chronic kidney diseases (CKD). In this study, we analyzed the correlations between podocyte injuries and renal dysfunctions in domestic carnivores. Dogs and cats were divided into normal and CKD groups according to renal histopathology and plasma creatinine values. Immunostaining results showed that linear reactions of slit diaphragm molecules, e.g., nephrin, podocin, and ACTN4, were parallel to glomerular capillaries in all animals. However, in dogs, reactions of nephrin and ACTN4 were changed to a granular pattern in the CKD group, and their intensities significantly decreased with the number of podocytes in the glomerulus. Moreover, the expression of nephrin and ACTN4 negatively correlated with creatinine. Real-time PCR analysis showed that nephrin mRNA expression in the kidneys of CKD dogs was significantly lower than that in normal animals, and negatively correlated with creatinine. Although no significant correlation between renal dysfunction and podocyte injury was detected in cats, histoplanimetric scores of tubulointerstitial lesions in CKD cats were higher than those in both normal cats and diseased dogs. Furthermore, mRNAs of WT1 and SD molecules were detected in urine from CKD animals. In conclusion, podocyte injuries such as podocytopenia and decreased expression of nephrin and ACTN4 in the glomerulus were more strongly correlated with renal dysfunction in dogs than in cats. These findings suggest that the CKD pathogenesis, especially susceptibilities to podocyte injuries, differed between dogs and cats.

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.

Onset of autoimmune glomerulonephritis derived from the telomeric region of MRL-chromosome 1 is associated with the male sex hormone in mice.

Female B6.MRLc1(82-100) congenic mice develop more severe autoimmune glomerulonephritis (AGN) than males. We assessed the effects of gonadectomy on the pathogenesis of AGN in these mice. One-month-old male and female mice were divided into sham-operated group (SG) and gonadectomized group (GG), and the pathological changes were investigated at 8 months. SG females showed higher spleen and thymus weights, serum total IgG and autoantibody levels, glomerular damage scores and percent IgG- and CD3-positive glomeruli as compared with SG males. Gonadectomy showed more remarkable effects in males than in females. Spleen and thymus weights, urinary albumin excretion, glomerular damage scores, percent IgG- and CD3-positive glomeruli, and CD3-positive areas in the spleen were significantly higher in GG males than in SG males. CD3-positive cells were observed in both the thymic cortex and medulla in all animals except SG males. The expression ratio of active Fc gamma receptor (Fcgr) 3 to inhibitory Fcgr2b in the kidneys, which we have previously demonstrated to have a great impact on pathogenesis in B6.MRLc1(82-100), was significantly higher in GG males than in SG males. These results suggested that the differences in the pathogenesis of AGN are primarily because of the inhibitory roles of the male sex hormones.

Autoimmune glomerulonephritis induced in congenic mouse strain carrying telomeric region of chromosome 1 derived from MRL/MpJ.

In lupus erythematosus-prone mice, including the BXSB, NZW and NZB strains, telomeric regions of chromosome 1 (Chr.1) contain major glomerulonephritis susceptibility loci such as Bxs3, Sle1, and Nba2. To assess whether strain MRL, a model for lupus erythematosus, had glomerulonephritis susceptibility loci on Chr.1, we created B6.MRLc1(82-100) congenic mice carrying MRL/MpJ Chr.1 (82-100 cM) based on the C57BL/6 background and investigated renal pathology. From 6 months of age, B6.MRLc1 (82-100) showed the onset of diseases such as splenomegaly due to proliferation of CD3- or B220-positive cells, glomerular damage, and an increased serum anti-dsDNA antibody concentration, and these were earlier and severer in females. The score for glomerular damage was higher in B6.MRLc1(82-100) mice over 12 months old than in C57BL/6 or even in wild-type MRL/MpJ. Immune-complex depositions were demonstrated on glomerular basement membrane in B6.MRLc1(82-100) by immunohistochemistry and electron microscopy. For the percentage of IgG1-positive glomeruli, B6.MRLc1 (82-100) had significantly higher values than C57BL/6. In evaluations of clinical parameters, serum levels of blood urea nitrogen and the anti-dsDNA antibody in B6.MRLc1(82-100) were significantly higher than those in C57BL/6. In conclusion, B6.MRLc1(82-100) clearly developed autoimmune-mediated glomerulonephritis, and we demonstrated that MRL Chr.1 contained a novel glomerulonephritis susceptibility locus. We named this locus Mag (MRL autoimmune glomerulonephritis) and it provided new insights into the genetic basis and pathogenesis of lupus nephritis.

An improved procedure for rapid determination of viral RNA sequences of avian RNA viruses.

A system for rapid determination of viral RNA sequences, RDV, was improved for detection of avian RNA virus in allantoic fluids. We detected avian paramyxovirus nucleotide sequences using RDV method ver 2.0.

Ovarian cysts in MRL/MpJ mice originate from rete ovarii.

In MRL mice aged more than 1 year, but not in C57BL/6 mice, ovaries had grossly visible cysts presenting unilaterally or bilaterally. Postnatally, all MRL mice developed ovarian cysts by 8 months of age. Observations by light microscopy, including lectin histochemistry, indicated that the cysts sometimes included papillomatous tissues located at the hilar region and were similar to the rete ovarii system, but not to follicles. Two types of epithelial cells, ciliated and non-ciliated, were arranged on the cysts, in which both cell types had many microvilli projecting in various directions and random ramifications in the cystic lumen. These characteristics suggest that ovarian cysts developing in MRL mice originate mostly from the rete ovarii. Cysts derived from the rete ovarii at 8 months of age were histologically detected in all C3H mice as well as MRL mice, with variable incidence in ICR, AKR, CBA/N and ddY, and none in C57L/6, DBA/2, BALB and A/J mice. However, measurement of the maximum diameters of the ovarian cysts indicated that MRL mice regularly possessed the largest cysts visible to the naked eye. This is the first report of ovarian cysts in this inbred strain, suggesting that ovarian cysts in MRL mice appear with stable incidence and development.

Enhancement of cytotoxicity against Vero E6 cells persistently infected with SARS-CoV by Mycoplasma fermentans.

We previously reported that cells with persistent severe acute respiratory syndrome coronavirus (SARS-CoV) infection were established after apoptotic events. In the present study, we investigated the cytopathic effects of dual infection with SARS-CoV and Mycoplasma fermentans on Vero E6 cells. Dual infection completely killed cells and prevented the establishment of persistent SARS-CoV infection. M. fermentans induced inhibition of cell proliferation, but the cells remained alive. Apoptosis was induced easily in M. fermentans-infected cells, indicating that they were primed for apoptosis. These results indicated that M. fermentans enhances apoptosis in surviving cells that have escaped from SARS-CoV-induced apoptosis.

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.

Metabolic profiles and bile acid extraction rate in the liver of cows with fasting-induced hepatic lipidosis.

This study was designed to monitor lipid profile in the portal and hepatic blood of cows with fasting-induced hepatic lipidosis, and to compare the results with those in the jugular blood. The work was also carried out to investigate bile acid (BA) in these vessels, and further to investigate BA extraction rate in the liver. Five cows were equipped with catheters in the portal, hepatic and jugular veins (day 0), fasted for 4 days (day 1-day 4) and then refed (day 5-day 11). Before morning feeding, blood was sampled before, during and after fasting from the catheterized vessels. In the portal blood, the concentration of non-esterified fatty acids (NEFA) showed a progressive increase and at day 5 there was an approximate twofold rise. Increased NEFA concentrations were also found similarly in the other two veins. At day 5, beta-hydroxybutyrate (BHBA) in the portal, hepatic and jugular blood rose to 197, 190 and 186% of the pre-fasting value, respectively. However, the concentrations of NEFA and BHBA in the three veins gradually returned to pre-fasting concentration during the refeeding period. Compared with the pre-fasting value at day 0, the content of liver triglyceride (TG) increased significantly at day 5 (P < 0.01). In the liver, the hepatic extraction rate of BA dropped from 3.1 times pre-fasting to 2.2 times during fasting. There were no significant differences in the concentrations of glucose, TG, total cholesterol, cholesterol esters, free cholesterol and phospholipids. The results of the current study show that metabolic alterations occur in the portal, hepatic and jugular veins during induction of hepatic lipidosis in cows, and mostly metabolites, with exception of BA concentration, run parallel. The decreased BA extraction rate in the liver of fasted cows was considered to reflect hepatic cell impairment caused by TG accumulation. Hopefully, the findings, at least in part, contribute to the explanation of the pathophysiology of hepatic lipidosis in dairy cows.

A novel mutation in the gene for canine acid beta-galactosidase that causes GM1-gangliosidosis in Shiba dogs.

A homozygous recessive mutation, causing GM1-gangliosidosis in Shiba dogs, was identified as a deletion of C nucleotide 1668 in the gene for canine acid beta-galactosidase, which was a novel mutation in canine GM1-gangliosidosis.

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.

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.

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.

Heat-shock resistance in experimental cryptorchid testis of mice.

Cryptorchidism is commonly used for research on spermatogenesis. However, there are few comparative investigations about the strain differences in mice, especially in long-term experiments. In the present study, the authors demonstrate its specific dynamics in the MRL/MpJ mouse strain, and discuss the cause of strain differences. In the mouse strains A/J BALB/c, C3H/He, and C57BL/6, after 2 weeks of experimental cryptorchidism, the ratios of the cryptorchid testis weight against the intact one were 0.38+/-0.05, 0.43+/-0.05, 0.38+/- 0.02, and 0.44+/-0.14, respectively. On the other hand, in the MRL/MpJ strain it was shifted to 0.69+/-0.08. The details of this strain difference were compared by calculation of germ cells with the Sertoli cell index at 2 weeks after operation. The indices of spermatogonia in all strains were not significantly different; however, in MRL/MpJ mice remarkable numbers of late spermatocytes and round spermatids were detected. The decrease of the testis weight ratio was similar until 10 days in the C57BL/6 and MRL/MpJ strains, but continued in C57BL/6 until 21 days, whereas in MRL/MpJ mice it plateaued after 10 days. Northern blot analysis for heat shock protein 70-2 using total RNA prepared from the cryptorchid and intact testes at 2 weeks after operation revealed that the expression was decreased in the cryptorchid testis of C57BL/6, but not MRL/MpJ mice. The results suggested that heat-resistant germ cells were present in MRL/MpJ, originating possibly from the genetic background.

Morphological study of metaphase-specific apoptosis in MRL mouse testis.

Apoptosis of male germ cells is a complex phenomenon in many animal species. Understanding its mechanisms could be useful in the diagnosis and therapy of male infertility. To examine the differences of distribution of apoptosis among mouse strains, the terminal transferase-mediated nick end labelling (TUNEL) method was employed. In the testes of MRL mice, many TUNEL-positive cells were identified at the metaphases of meiotic spermatocytes. Morphometrical analysis revealed that metaphase-specific apoptosis occurred at the region between secondary spermatocytes and step 1 spermatids in stage XII seminiferous tubules. In the investigation of the developing first-wave of seminiferous tubules, there were some metaphases showing apoptotic morphology prior to becoming secondary spermatocytes. Details of the apoptotic structure revealed by electron microscopy showed that cellular arrest occurred after the beginning of the M phase of the cell cycle. These results suggested that metaphase-specific apoptosis in the testis of the MRL mouse strain took place at least at the first meiotic division, perhaps showing the spindle assembly checkpoint of the cell cycle.

Application of representational difference analysis to genomic fragments of Marek's disease virus.

A rapid and simple method for isolation of DNA fragments of Marek's disease virus (MDV) based on representational difference analysis (RDA) was developed. Multiple viral DNA fragments, the sizes of which were restricted to 0.3 to 3.5 kbp, were simultaneously amplified after subtraction of chicken DNA from BamHI-, BglII-, EcoRI-, HindIII-, or XhoI-digested DNA fragments of MDV-infected cells. Nucleotide sequence of two RDA-derived fragments coincided with the sequence determined from direct sequencing of the MDV genome. We detected an interstrain difference in the size of restriction enzyme-digested fragments on agarose gel. This method was used on a single feather pulp to generate sufficient MDV DNA for cloning.

Hepatic copper accumulation induces DNA strand breaks in the liver cells of Long-Evans Cinnamon strain rats.

Effects of accumulation of copper and iron on the production of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) strain rats that spontaneously develop fulminant hepatitis. Copper and iron accumulated in the liver of LEC rats in an age-dependent manner from 4 to 15 weeks. Low-copper food prevented the accumulation of copper in the liver, but did not prevent accumulation of iron. When the amounts of DNA single strand breaks were estimated by comet assay, the number of DNA strand breaks in the liver cells of rats fed standard food increased with age from 4 to 15 weeks. The number of DNA strand breaks in the liver cells from rats fed low-copper food were the same as those of rats at 4 weeks of age. Thus, the copper accumulation in the liver of LEC rats induced DNA strand breaks, but accumulation of iron did not.