Leupeptin-sensitive proteases involved in cell survival after X-ray irradiation in human RSa cells

Proteases have received attention as important cellular components responsible for stress response in human cells. However, little is known about the role of proteases in the early steps of cell response after X-ray irradiation. In the present study, we first searched for proteases whose activity levels are changed soon after X-ray irradiation in human RSa cells with a high sensitivity to X-ray cell-killing. RSa cells showed an increased level of fibrinolytic protease activity within 10 min after irradiation with X-ray (up to 3 Gy). The induced protease activity was proved to be inhibited by leupeptin. We next examined whether this protease inducibility is related to the X-ray susceptibility of cells. Treatment of RSa cells with leupeptin prior to X-ray irradiation resulted in lowered colony survival and an increased ratio of G(2)/M-arrested cells and apoptotic cells. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human RSa cells to X-ray cell-killing.     

Enhanced gene expression of the murine ecotropic retroviral receptor and its human homolog in proliferating cells.

The receptor for gp70 envelope glycoprotein of murine ecotropic leukemia virus is essential for virus entry into the host cell and has been recently demonstrated to function as a cationic amino acid transporter. In the experiments reported herein, we compared the gene expression of the murine ecotropic retroviral receptor (ERR) and its human homolog (H13) in rapidly proliferating cells versus resting cells using four different systems. (i) The expression of ERR gene is enhanced during activation of T and B lymphocytes by concanavalin A and lipopolysaccharide, respectively. Similar enhancement is observed by adding phorbol 12-myristate 13-acetate (PMA) or calcium ionophore (A23187). These phenomena appear to involve protein kinase C; two PMA analogs, 4 alpha-phorbol and 4 alpha-PMA, lacking the ability to activate protein kinase C fail to induce elevated levels of gene expression, and the protein kinase C inhibitor, H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride[, inhibits the enhancement induced by PMA. (ii) Friend murine leukemia virus induces rapid splenomegaly, and acute erythroleukemia in sensitive mice. Concomitantly with splenomegaly, ERR gene expression in spleen cells increases dramatically. (iii) The level of expression of the ERR or H13 gene in a variety of tumor cells is highly elevated compared with the level in noncancerous cells. (iv) H13 gene expression decreases upon terminal differentiation of the human promyelocytic leukemia cell line HL-60 into granulocytes or macrophages by dimethyl sulfoxide or PMA, respectively. These results suggest that ERR and H13 genes play an important role in cellular proliferation.     

UVC mutagenicity is suppressed in Japanese miso-treated human RSa cells, possibly via GRP78 expression

Little is known about the ability of miso, to modulate mutability in human cells. We have observed increased levels of glucose-regulated protein 78 (GRP78) expression in association with suppression of mutation in human RSa cells irradiated with ultraviolet C (UVC). Here we examined to determine whether miso treatment results in increased GRP78 expression and suppression of UVC mutagenicity in RSa cells. Supernatants of water extracts of miso products and their components were tested. In the sample-treated cells, the amount of GRP78, as estimated by RT-PCR and immunoblotting analysis, increased, and the UVC-induced ouabain resistant mutation (Oua(R)) and the K-ras codon 12-base substitution mutation frequency decreased. This decrease was not observed in cells with downregulation of GRP78 by GRP78 siRNA transfection. The results suggest that miso suppresses UVC mutagenicity by increasing GRP78 expression in human cells.     

Changes in human pluripotent stem cell gene expression after genotoxic stress exposures

Human pluripotent stem cells (hPSCs) represent heterogeneous populations, including induced pluripotent stem cells (iPSCs), endogenous plastic somatic cells, and embryonic stem cells (ESCs). Human ESCs are derived from the inner cell mass of the blastocyst, and they are characterized by the abilities to self-renew indefinitely, and to give rise to all cell types of embryonic lineage (pluripotency) under the guidance of the appropriate chemical, mechanical and environmental cues. The combination of these critical features is unique to hESCs, and set them apart from other human cells. The expectations are high to utilize hESCs for treating injuries and degenerative diseases; for modeling of complex illnesses and development; for screening and testing of pharmacological products; and for examining toxicity, mutagenicity, teratogenicity, and potential carcinogenic effects of a variety of environmental factors, including ionizing radiation (IR). Exposures to genotoxic stresses, such as background IR, are unavoidable; moreover, IR is widely used in diagnostic and therapeutic procedures in medicine on a routine basis. One of the key outcomes of cell exposures to IR is the change in gene expression, which may underlie the ultimate hESCs fate after such a stress. However, gaps in our knowledge about basic biology of hESCs impose a serious limitation to fully realize the potential of hESCs in practice. The purpose of this review is to examine the available evidence of alterations in gene expression in human pluripotent stem cells after genotoxic stress, and to discuss strategies for future research in this important area.     

Identification of mono-ubiquitinated LDH-A in skeletal muscle cells exposed to oxidative stress

We previously reported that oxidative stress is associated with unloading-mediated ubiquitination of muscle proteins. To further elucidate the involvement of oxidative stress in ubiquitination, we examined the ubiquitination profile in rat myoblastic L6 cells after treatment with hydrogen peroxide. Hydrogen peroxide induced many ubiquitinated proteins with low molecular masses (less than 60 kDa) as well as high molecular masses (more than 160 kDa). Among them, a 42-kDa-ubiquitinated protein was abundantly accumulated and immediately disappeared after the treatment. Microsequencing revealed that the 42-kDa-protein was identical to the mono-ubiquitinated form of rat lactate dehydrogenase A (LDH-A), and we confirmed that hydrogen peroxide induced the mono-ubiquitination of LDH-A in COS7 cells overexpressing LDH-A and ubiquitin. Under unloading conditions, such as tail-suspension and spaceflight, mono-ubiquitinated LDH was accumulated in gastrocnemius muscle. Interestingly, E-64-d plus pepstatin, lysosomal protease inhibitors, further accumulated mono-ubiquitinated LDH-A in the cells after treatment with hydrogen peroxide, while they did not affect the amount of poly-ubiquitinated LDH. In contrast, epoxomicin, a potent proteasome inhibitor, did not change the amount of mono-ubiquitinated LDH-A in L6 cells treated with hydrogen peroxide, although it significantly increased the amount of poly-ubiquitinated LDH. Our results suggest that oxidative stress induces not only poly-ubiquitination but also mono-ubiquitination of LDH-A, which may be involved in its lysosomal degradation during unloading.     

Mutagenicity of bisphenol A and its suppression by interferon-alpha in human RSa cells

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1x10(-7) to 1x10(-5)M, base substitution mutations at K-ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1x10(-8)M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua(R)) phenotypic mutation was also found in cells treated with 1x10(-7) and 1x10(-5)M of bisphenol A. The induction of K-ras codon 12 mutations and Oua(R) mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-alpha prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1x10(-6)M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.     

Osmotic stress up-regulates aquaporin-3 gene expression in cultured human keratinocytes.

Of ten members of the aquaporin family (AQP), the mRNA expression and regulation of AQP1, AQP3, AQP4 and AQP9 in cultured human keratinocytes were examined by an RNase protection assay. AQP3 mRNA was expressed in growing and differentiating cells, while AQP9 mRNA was only detected in differentiating cells. The epidermis in skin-equivalent cultures expressed both AQP3 and AQP9 mRNA. However, neither AQP1 nor AQP4 mRNA was detectable in either monolayer or skin-equivalent cultures. Incubation of keratinocytes in sorbitol-added hypertonic medium increased AQP3 mRNA expression. This was confirmed using other solutes such as NaCl, mannitol, glucose and sucrose. The effect of sorbitol was reversible, dose-dependent and maximal at 24 h after addition. However, AQP1, AQP4 and AQP9 mRNA expression were unchanged under any of the hypertonic conditions examined. These findings indicated that osmotic stress up-regulates AQP3 gene expression in cultured keratinocytes.     

Ceruloplasmin gene expression in human cancer cells

The copper transport protein, ceruloplasmin, is suggested to have a role in cancer since it is involved in angiogenesis and neovascularization. In order to understand the role of ceruloplasmin in malignant cells, we have recently isolated and sequenced a human ceruloplasmin cDNA clone. In the present study, we have investigated the ceruloplasmin gene expression in human colon and breast cancer cell lines. The poly (A) RNA from human colon (WiDr) and human breast (MCF-7) cancer cell lines was analyzed for the presence of ceruloplasmin mRNA. The Northern blot analysis revealed the presence of a 3.7 kb band of ceruloplasmin mRNA in these cell lines. Dot blot analysis revealed that ceruloplasmin mRNA is at least three fold more abundant in tumor cells as compared to normal rat liver.     

Enhanced gene amplification in human cells knocked down for DNA-PKcs

Gene amplification, a key mechanism for oncogene activation and drug resistance in tumour cells, involves the generation and joining of DNA double-strand breaks. Amplified DNA can be carried either on intra-chromosomal arrays or on extra-chromosomal elements (double minutes). We previously showed that, in rodent cells deficient in DNA-PKcs, intra-chromosomal amplification is significantly enhanced. In the present work, we studied gene amplification in human HeLa cell lines in which the expression of the DNA-PKcs gene was constitutively inhibited by shRNAs. These cell lines showed an increased sensitivity to ionizing radiations, an enhanced frequency of chromosomal aberrations and an increased rate of occurrence of methotrexate resistant colonies compared to the control cell lines (6-18 times). The main mechanism of resistance to methotrexate was extra-chromosomal amplification of the dihydrofolate reductase gene. These results indicate that, in human cells, inhibition of DNA-PKcs gene expression favours gene amplification occurring via the production of double minutes. In addition, they show that cell lines constitutively expressing shRNAs are good model systems to study the role of specific functions in gene amplification.     

Biotin-dependent regulation of gene expression in human cells

The role of biotin as cofactor of carboxylases and its importance in metabolic homeostasis are well known. In recent years, different researchers have suggested the participation of biotin as a regulator molecule in the control of gene expression. Biotin-dependent gene expression requires of the transformation of biotin into biotinyl-5'-AMP by holocarboxylase synthetase and the activation of soluble guanylate cyclase and a cGMP-dependent protein kinase. The regulatory role of biotin is responsible for the correct expression of enzymes involved in biotin utilization in human cells. We propose that this mechanism protects the brain from biotin deficiency.     

Exercise affects the gene expression profiles of human white blood cells.

White blood cells (WBCs) express tens of thousands of genes, whose expression levels are modified by genetic and external factors. The purpose of the present study was to investigate the effects of acute exercise on gene expression profiles (GEPs) of WBCs and to identify suitable genes that may serve as surrogate markers for monitoring exercise and training load. Five male participants performed an exhaustive treadmill test (ET) at 80% of their maximal O(2) uptake (Vo(2 max)) and a moderate treadmill test (MT) at 60% Vo(2 max) for exactly the same time approximately 2 wk later. WBCs were isolated by the erythrocyte lysis method. GEPs were measured using the Affymetrix GeneChip technology. After scaling, normalization, and filtering, groupwise comparisons of gene expression intensities were performed, and several measurements were validated by real-time PCR. We found 450 genes upregulated and 150 downregulated (>1.5-fold change; ANOVA with Benjamini-Hochberg correction, P < 0.05) after ET that were closely associated with the gene ontology lists "response to stress" and "inflammatory response". Analysis of mean expression levels after MT showed that the extent of up- and downregulation was workload dependent. The genes for the stress (heat shock) proteins HSPA1A and HSPH1 and for the matrix metalloproteinase MMP-9 showed the most prominent increases, whereas the YES1 oncogene (YES1) and CD160 (BY55) were most strongly reduced. Despite different methodological approaches used, the consistency of our results with the expression data of another study (Connolly PH, Caiozzo VJ, Zaldivar F, Nemet D, Larson J, Hung SP, Heck JD, Hatfield GW, Cooper DM. J Appl Physiol 97: 1461-1469, 2004) suggests that expression fingerprints are useful tools for monitoring exercise and training loads and thereby help to avoid training-associated health risks.