RB1CC1 protein suppresses type II collagen synthesis in chondrocytes and causes dwarfism.

RB1-inducible coiled-coil 1 (RB1CC1) functions in various processes, such as cell growth, differentiation, senescence, apoptosis, and autophagy. The conditional transgenic mice with cartilage-specific RB1CC1 excess that were used in the present study were made for the first time by the Cre-loxP system. Cartilage-specific RB1CC1 excess caused dwarfism in mice without causing obvious abnormalities in endochondral ossification and subsequent skeletal development from embryo to adult. In vitro and in vivo analysis revealed that the dwarf phenotype in cartilaginous RB1CC1 excess was induced by reductions in the total amount of cartilage and the number of cartilaginous cells, following suppressions of type II collagen synthesis and Erk1/2 signals. In addition, we have demonstrated that two kinds of SNPs (T-547C and C-468T) in the human RB1CC1 promoter have significant influence on the self-transcriptional level. Accordingly, human genotypic variants of RB1CC1 that either stimulate or inhibit RB1CC1 transcription in vivo may cause body size variations.

Therapeutic potential of SOX2 inhibition for embryonal carcinoma.

PURPOSE: Some nonseminomatous germ cell tumors are resistant to any type of chemotherapy. Control of embryonal carcinoma cells is crucial to manage nonseminomatous germ cell tumors. We established SOX2 targeting therapy in an embryonal carcinoma model. MATERIALS AND METHODS: SOX2 expression was evaluated in a series of testicular germ cell tumor tissue samples. The antitumor effect of SOX2 knockdown was analyzed in vitro and in vivo using an embryonal carcinoma model. RESULTS: In testicular germ cell tumor tissue SOX2 was expressed in the foci of embryonal carcinoma but negative in seminoma and yolk sac tumors. In an embryonal carcinoma model SOX2-siRNA induced apoptotic cell death in vitro and significant growth suppression in vivo. CONCLUSIONS: This study shows the therapeutic potential of SOX2 silencing for embryonal carcinoma. However, further improvements are needed in SOX2-siRNA delivery to the tumor.

Retinol dehydrogenase 10 contributes to cancer stemness and intracellular carbohydrate storage in ovarian clear cell carcinomas.

BACKGROUND: Ovarian clear cell carcinomas (OCCCs) have been recurrent and refractory among the present treatments, so novel therapeutics are urgently needed. OBJECTIVE: The present study accumulates the proof of concept to examine the feasibility of RDH10 as a therapeutic target for treating OCCCs. METHODS: Immunohistochemically, RDH10 expression was evaluated in 111 primary epithelial ovarian cancers, including 55 OCCCs, 31 ovarian endometrioid carcinomas and 25 ovarian serous carcinomas. The spherogenecity provoked by RDH10 was evaluated in OCCC cells. To analyze whether RDH10 promotes carbohydrate storage via the vitamin A-gluconeogenesis pathway, phosphoenolpyruvate carboxykinase 1 (PCK1) protein levels and intracellular carbohydrate content were measured in response to modified RDH10 expression. RESULTS: Abundant RDH10 was expressed specifically in OCCCs. RDH10 promoted spherogenecity and intracellular carbohydrate storage via modulation of PCK1 expression in OCCC cells. CONCLUSIONS: In the present study, abundant RDH10 contributed to cancer cell stemness and intracellular carbohydrate storage in OCCCs. RDH10 is a potentially, new therapeutic candidate for treating OCCC cases.

Effectiveness of COVID-19 vaccination in healthcare workers in Shiga Prefecture, Japan.

This study, which included serological and cellular immunity tests, evaluated whether coronavirus disease 2019 (COVID-19) vaccination adequately protected healthcare workers (HCWs) from COVID-19. Serological investigations were conducted among 1600 HCWs (mean ± standard deviation, 7.4 ± 1.4 months after the last COVID-19 vaccination). Anti-SARS-CoV-2 antibodies N-Ig, Spike-Ig (Roche), N-IgG, Spike-IgM, and -IgG (Abbott), were evaluated using a questionnaire of health condition. 161 HCWs were analyzed for cellular immunity using T-SPOT® SARS-CoV-2 kit before, and 52 HCWs were followed up until 138.3 ± 15.7 days after their third vaccination. Spike-IgG value was 954.4 ± 2282.6 AU/mL. Forty-nine of the 1600 HCWs (3.06%) had pre-existing SARS-CoV-2 infection. None of the infectious seropositive HCWs required hospitalization. T-SPOT value was 85.0 ± 84.2 SFU/106 cells before the third vaccination, which increased to 219.4 ± 230.4 SFU/106 cells immediately after, but attenuated later (to 111.1 ± 133.6 SFU/106 cells). Poor counts (< 40 SFU/106 cells) were present in 34.8% and 38.5% of HCWs before and after the third vaccination, respectively. Our findings provide insights into humoral and cellular immune responses to repeated COVID-19 vaccinations. COVID-19 vaccination was effective in protecting HCWs from serious illness during the original Wuhan-1, Alpha, Delta and also ongoing Omicron-predominance periods. However, repeated vaccinations using current vaccine versions may not induce sufficient cellular immunity in all HCWs.

Pro-GA, a Novel Inhibitor of γ-Glutamylcyclotransferase, Suppresses Human Bladder Cancer Cell Growth.

BACKGROUND/AIM: γ-Glutamylcyclotransferase (GGCT), a key enzyme involved in glutathione metabolism, catalyzes a specific reaction that generates 5-oxoproline and free amino acids from the γ-glutamyl peptide. Inhibition of GGCT is a promising therapeutic strategy for the treatment of various cancers. MATERIALS AND METHODS: Immuno-histochemistry was used to evaluate GGCT expression in bladder tumors. The growth inhibitory effect of pro-GA, a novel GGCT inhibitor, in the presence or absence of mitomycin C (MMC) was assessed in three distinct bladder cancer cell lines. RESULTS: Over half of the clinical bladder tumor samples overexpressed GGCT. Pro-GA reduced the growth of all bladder cancer cell lines in a dose-dependent manner, and increased the anti-tumor effect of MMC. CONCLUSION: Inhibition of GGCT using pro-GA provides a novel therapeutic strategy for the treatment of bladder cancers.

Prominent role of RAB39A-RXRB axis in cancer development and stemness.

In this study, we found that RAB39A, a member of the RAS oncogene family, was selectively expressed in cancer cells of different histotypes, by analyzing gene expression in human osteosarcoma cells and the cancer stem cells (CSCs) and by comparing them with normal cells through global transcriptomics and principal component analyses. We further validated RAB39A as a therapeutic target, by silencing its expression. The silencing impaired cancer stemness and spherogenic ability in vitro, as well as tumorigenesis in vivo. RNA-seq analyses in the silenced spheres suggested that RAB39A is associated downstream with RXRB and KLF4. Notably, RXRB expression was inhibited in RAB39A-silenced CSCs. Induced overexpression of RXRB in RAB39A-silenced cells restored spherogenic ability and tumorigenesis, confirming RXRB as a major effector of RAB39A. Quantitative RT-PCR analysis of ∼400 human cancer tissues showed that RAB39A was highly expressed in sarcomas and in malignancies of lymphoid, adrenal and testicular tissues. Our data provide the rationale for targeting of the RAB39A-RXRB axis as a therapy for aggressive cancers.

Dimethyl sulfoxide induces chemotherapeutic resistance in the treatment of testicular embryonal carcinomas.

Dimethyl sulfoxide (DMSO) is an amphipathic molecule that is used as a solvent in biological studies and as a vehicle for drug therapy. The present study was designed to evaluate the potential effects of DMSO as a solvent in the treatment of testicular embryonal carcinomas (ECs). DMSO was applied to two human EC cell lines (NEC8 and NEC14), with the treated cells evaluated in relation to cisplatin (CDDP) resistance, differentiation (using Vimentin, Fibronectin, TRA-1-60, and SSEA-1 and -3 as markers) and stemness (denoted by expression of SOX2 and OCT3/4). Furthermore, DNA methyltransferase (DNMT-1, -3A and -3L) expression and methylation status were analyzed. DMSO induced resistance to CDDP, aberrant differentiation and reduction of stemness-related markers in each of the EC cell lines. The expression levels of DNMT-3L and -3A were reduced in response to DMSO, while this treatment also affected DNA methylation. The data demonstrated that DMSO perturbed differentiation, reduced stemness and induced resistance to CDDP in human EC cells. Therefore, DMSO could reduce drug efficacy against EC cells and its use should be carefully managed in the clinical application of chemotherapy.

Autonomic nervous system activity and the state and development of obesity in Japanese school children.

OBJECTIVE: The autonomic nervous system (ANS) plays an important role in regulating energy expenditure and body fat content; however, the extent to which the ANS contributes to pediatric obesity remains inconclusive. The aim of this study was to evaluate whether sympathetic and/or the parasympathetic nerve activities were altered in an obese pediatric population. We further examined a physiological association between the duration of obesity and the sympatho-vagal activities to scrutinize the nature of ANS alteration as a possible etiologic factor of childhood obesity. RESEARCH METHODS AND PROCEDURES: Forty-two obese and 42 non-obese healthy sedentary school children were carefully selected from 1080 participants initially recruited to this study. The two groups were matched in age, gender, and height. The clinical records of physical characteristics and development of the obese children were retrospectively reviewed to investigate the onset and progression of obesity. The ANS activities were assessed during a resting condition by means of heart rate variability power spectral analysis, which enables us to identify separate frequency components, i.e., total power (TP), low-frequency (LF) power, and high-frequency (HF) power. The spectral powers were then logarithmically transformed for statistical testing. RESULTS: The obese children demonstrated a significantly lower TP (6.77 +/- 0.12 vs. 7.11 +/- 0.04 ln ms(2), p < 0.05), LF power (6.16 +/- 0.12 vs. 6.42 +/- 0.05 ln ms(2), p < 0.05), and HF power (5.84 +/- 0.15 vs. 6.34 +/- 0.07 ln ms(2), p < 0.01) compared with the non-obese children. A partial correlation analysis revealed that the LF and HF powers among 42 obese children were negatively associated with the duration of obesity independent of age (LF: partial r = -0.55, p < 0.001; HF: partial r = -0.40, p < 0.01). The obese children were further subdivided into two groups based on the length of their obesity. All three spectral powers were significantly reduced in the obese group with obesity of >3 years (n = 18) compared to the group with obesity of <3 years. DISCUSSION: Our data indicate that obese children possess reduced sympathetic as well as parasympathetic nerve activities. Such autonomic depression, which is associated with the duration of obesity, could be a physiological factor promoting the state and development of obesity. These findings further imply that preventing and treating obesity beginning in the childhood years could be an urgent and crucial pediatric public health issue.

Raised intracellular glucose concentrations reduce aggregation and cell death caused by mutant huntingtin exon 1 by decreasing mTOR phosphorylation and inducing autophagy.

Huntington's disease is caused by a CAG trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract. This gain-of-function mutation is associated with huntingtin aggregation and cell death. Autophagy is an important clearance route for mutant huntingtin exon 1. While mammalian target of rapamycin (mTOR) is a key regulator of autophagy, the upstream modifiers of this process are poorly understood. Our previous expression profiling studies in HD cell models observed changes in four genes associated with glucose metabolism, including the GLUT1 glucose transporter. A role for intracellular glucose as a modulator for polyglutamine toxicity was suggested as cell death was reduced by GLUT1 overexpression. Here we show that the protective effect of GLUT1 is associated with decreased huntingtin exon 1 aggregation in cell models. Consistent with this result, we also observed reduced aggregation and enhanced clearance of mutant huntingtin when cells were cultured in raised glucose concentrations (8 g/l). These effects were mimicked by 8 g/l 2-deoxyglucose (2DOG) (transported, phosphorylated but not metabolized further), but not with 8 g/l 3-O-methyl glucose (transported but not metabolized further). Thus, this phenomenon is probably mediated by glucose-6-phosphate. Increased clearance of mutant huntingtin by raised glucose (8 g/l) and 2DOG correlated with increased autophagy and reduced phosphorylation of mTOR, S6K1 and Akt. Thus, raised intracellular glucose/glucose 6-phosphate levels reduce mutant huntingtin toxicity by increasing autophagy via mTOR and possibly Akt. As mTOR and Akt regulate a diversity of crucial cellular processes, our data also suggest a major new set of targets for intracellular glucose signalling.

Modulation of polyglutamine-induced cell death by genes identified by expression profiling.

The majority of triplet-repeat diseases are caused by mutated genes with an extended polyglutamine tract, exemplified by Huntington's disease (HD). In order to model HD pathogenesis in a controlled system, we developed stable PC12 cell lines that express exon 1 fragments of the huntingtin gene with 23 or 74 polyglutamines driven by an inducible doxycycline (dox)-sensitive promoter (HD-23Q or HD-74Q). We aimed to identify early perturbations induced by the mutation by studying expression levels of 1824 genes at 0, 5, 10 and 18 hours after induction, using adaptor-tagged competitive PCR (ATAC-PCR). At these time points, the cells show no appreciable death or mitochondrial impairment and very low inclusion levels. A total of 126 genes, including 69 known genes, exhibited statistically significant alterations in the HD-74Q cell lines but no changes in the HD-23Q lines. We tested 11 of these genes for their abilities to modulate polyglutamine-induced cell death in transiently transfected cell models. Five genes [glucose transporter 1 (Glut1), phosphofructokinase muscle isozyme (Pfkm), prostate glutathione-S -transferase 2 (Gstm2), RNA-binding motif protein 3 (Rbm3) and KRAB-A interacting protein 1 (Krip-1)] significantly suppressed cell death in both neuronal precursor and non-neuronal cell lines, suggesting that these transcriptional changes were relevant to polyglutamine pathology. The efficient recovery of functionally relevant genes supports the utility of gene expression profiling for discovering pathways related to pathogenesis, and the importance of analyzing molecular events in the early stages of disease.

Molecular features of non-B, non-C hepatocellular carcinoma: a PCR-array gene expression profiling study.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) usually develops following chronic liver inflammation caused by hepatitis C or B virus. Through expression profiling in a rare type of HCC, for which the causes are unknown, we sought to find key genes responsible for each step of hepatocarcinogenesis in the absence of viral influence. METHODS: We used 68 non-B, non-C liver tissues (20 HCC, 17 non-tumor, 31 normal liver) for expression profiling with PCR-array carrying 3072 genes known to be expressed in liver tissues. To select the differentially expressed genes, we performed random permutation testing. A weighted voting classification algorithm was used to confirm the reliability of gene selection. We then compared these genes with the results of previous expression profiling studies. RESULTS: A total of 220 differentially expressed genes were selected by random permutation tests. The classification accuracies using these genes were 91.8, 92.0 and 100.0% by a leave-one-out cross-validation, an additional PCR-array dataset and a Stanford DNA microarray dataset, respectively. By comparing our results with previous reports on virus-infected HCC, four genes (ALB, A2M, ECHS1 and IGFBP3) were commonly selected in some studies. CONCLUSIONS: The 220 differentially expressed genes selected by PCR-array are potentially responsible for hepatocarcinogenesis in the absence of viral influence.