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.

Micronucleus test using cultured new born rat astrocytes.

Micronuclei is induced in cytoplasm as a consequence of the formation of chromosomal fragments or remaining chromosomes during cell division by the cause of clastogens or spindle poisons, and is used as an indicator of genotoxicity screening tests. There are few short-term genotoxicity screening tests using brain cells. We attempted to establish a new in vitro micronucleus test (MN test) system by use of central nervous system cells. Primary cultured astrocytes were prepared from newborn male Sprague-Dawley (SD) rats. In growth curve of astrocytes, doubling time was determined to be 31 h. In time study, the highest frequency of micronuclei was observed at 48 h, 72 h and 6 h-exposure-66 h-recovery by vincristine (VCR), mitomycin C (MMC) without metabolic activation system and cyclophosphamide (CPM) with metabolic activation system, respectively. Dose-response relationships between micronucleus frequency and concentrations of MMC, VCR and CPM were observed, respectively. It is suggested that the in vitro MN test using new born rat-astrocytes could be used as a screening test of environmental and occupational genotoxic chemicals in the central nervous system cells.

[Design and development of one-handed denture brush for bedridden people].

Oral care for elderly bedridden people is one of the most necessary forms of care, not only for prevention of oral infection or aspiration pneumonia, but also in order to savor the taste of food and to recover and maintain mental vitality through the improvement in oral function. We designed denture brush that can be handled with one-hand in order to support independence. People require cooperation between medical treatment, health, and welfare services. We introduced the newly designed denture brush as a means to support the improvement of QOL for elderly bedridden people, and we hope to see the spread and promotion of oral care.

Mutagenicity of p-aminophenol in E. coli WP2uvrA/pKM101 and its relevance to oxidative DNA damage.

It was recently reported that p-aminophenol (p-AP) induces DNA cleavage in mouse lymphoma cells, CHO cells and human lymphoblastoid cells. The mutagenicity of p-AP has not, however, been detected by reverse mutation assays. The purpose of this study was to assess the mutagenicity of p-AP by reverse mutation assay using Escherichia coli WP2uvrA/pKM101, which has a spectrum for detecting mutations different from those of other strains in the family with an AT base pair at the mutation site and has higher sensitivity to certain oxidative mutagens as compared to other strains. We found that p-AP was mutagenic to E. coli WP2uvrA/pKM101. The mutagenic activity of this compound was suppressed with the addition of dimethylsulfoxide or catalase, suggesting the involvement of active oxygen species in the mutagenic process induced by p-AP. To further elucidate the underlying mechanism, we used isolated DNA for the following experiments. It was revealed, by gel electrophoretic analysis, that p-AP induced DNA cleavage in the presence of Fe(III). However, p-AP alone did not induce this cleavage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by p-AP in calf thymus DNA was also detected in the presence of Fe(III) by HPLC with an electrochemical detector. ESR-spin trapping experiments using DMPO detected the production of hydroxyl radical (.OH) in the solution of p-AP with Fe(III). Both p-AP mediated DNA damages and .OH production by p-AP in the presence of Fe(III) were completely inhibited by .OH scavengers (ethanol, mannitol, sodium formate, dimethylsulfoxide) and catalase. These results suggest that .OH derived from the reaction between H2O2 and Fe(III) (Fenton reaction) participates in the oxidative DNA damage. Accordingly, the same mechanism might be working in E. coli WP2uvrA/pKM101 during induction of the mutation by p-AP.

Enhancement of lipid peroxidation in the liver of mice exposed to magnetic fields.

To elucidate the biological effect of static magnetic fields (SMF), we measured lipid peroxidation in the liver, kidneys, heart, lungs and brain of mice exposed to SMF and also evaluated the combined effect of SMF exposure on the hepatotoxicity induced by treatment with carbon tetrachloride (CCl4). Lipid peroxidation in the liver was significantly increased by exposure to 4.7 T of SMF for 3, 6, 24, or 48 h, whereas that in the kidneys, heart, lungs and brain was not changed compared to the control. The combination of CCl4 injection and SMF exposure caused an increase in lipid peroxidation in the liver exceeding that caused by either treatment alone. Furthermore, the increase in activities of both GOT and GPT caused by CCl4 administration were also enhanced by SMF exposure. These results indicate that the exposure to strong SMF induces lipid peroxidation in the liver of mice and enhances the hepatotoxicity caused by CCl4 administration.

[Follow-up study of vestibular neuronitis].

A follow-up study of 26 patients with vestibular neuronitis is reported. The disease is characterized by an acute attack of severe vertigo with complete loss of unilateral caloric response. The following results were obtained: 1) The average period of spontaneous nystagmus was 136 days and the standard error was 39 days. 2) No correlation could be found between age and the period of spontaneous nystagmus. 3) Six patients showed direction reversal in their spontaneous nystagmus (recovery nystagmus), and their outcome was good. 4) On the most recent caloric test, 42% of the patients had bilateral normal responses, 27% displayed partial improvement on the affected side, and no reaction was observed in 31% of patients. We suggest that three types of clinical courses may occur in vestibular neuronitis: i) complete recovery of the function of the affected vestibular nerve, ii) partial recovery of vestibular function, and iii) no recovery of the affected vestibular nerve, but central nervous system compensates for the vestibular imbalance.

Genetic variants of ribosomal DNA and mitochondrial DNA between swamp and river buffaloes.

To clarify the genetic relationship between Swamp and River buffaloes, the restriction fragment length polymorphisms (RFLPs) of nuclear genomic ribosomal DNA (rDNA) and cytoplasmic mitochondrial DNA (mtDNA) were analysed. Blood or liver samples from 73 Swamp and three River buffaloes were collected in East and South-east Asian countries. DNA samples from cattle, goats and sheep were used for comparisons. The analysis of rDNA allowed water buffaloes, cattle, goats and sheep to be characterized by four distinct repeat-types. However, swamp and river buffaloes showed the same repeat-type. Divergence of water buffalo and cattle is considered to have occurred approximately four to six million years ago. The RFLPs for mtDNA divided water buffaloes into three haplotypes, swamp-1, swamp-2 and river types. Swamp-1 accounted for 91% of all swamp buffaloes while swamp-2 was observed only in water buffaloes from Thailand (9%). All river buffaloes were of the same haplotype. No differences were observed between swamp and river buffaloes at the rDNA level. In contrast, a few distinct differences between them were found at the mtDNA level. Therefore, mtDNA polymorphisms provide an adequate means for classifying water buffaloes into either swamp or river buffaloes.

Pregnancy and delivery following cardiac operations.

For young female patients with cardiac diseases, to be married, pregnancy and delivery are serious problems. Ninety-two post-operative patients (138 deliveries) and 57 non-surgical patients (86 deliveries) were subjected to analysis of their cardiac and obstetric conditions. A stable course and successful delivery were observed in the patients treated whose cardiac diseases were treated with surgical therapy. Among the non-surgical patients, however, impairment of their clinical condition was experienced including 3 maternal deaths. Incidence of various complications occurring during the course of pregnancy and delivery was 12.3% in the surgical and 18.6% in the non-surgical groups. It was clinically demonstrated that women suffering from cardiac diseases might well tolerate their pregnancy and delivery if accorded proper surgical treatment.