Kazuko Kita

Stimulation of ultraviolet-induced apoptosis of human fibroblast UVr-1 cells by tyrosine kinase inhibitors

Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray‐induced apoptosis in ultraviolet‐resistant human UVr‐1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet‐induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet‐induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal‐regulated kinases and stress‐activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal‐regulated kinases but stimulated phosphorylation of stress‐activated protein kinases. Consequently, the ultraviolet‐induced concurrent increase in both phosphorylated extracellular signal‐regulated kinases and stress‐activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet‐induced apoptosis.

Involvement of human heat shock protein 90α in nicotine-induced apoptosis

There have been conflicting reports of the apoptotic effects of nicotine on human cells and those studies reporting nicotine‐induced apoptosis have not unequivocally clarified the molecular mechanisms underlying the effect. However, we found here that human RSa cells, established from embryonic fibroblastic cells doubly infected with Rous sarcoma virus and Simian virus 40, underwent apoptosis when cultured with medium containing 0.06–0.6 μM nicotine. The apoptosis was assessed by cellular DNA fragmentation and caspase‐3 protease activation. Viability of RSa cells was reduced by nicotine treatment, as analyzed by MTT assay and the reduction was lessened by combination treatment with a caspase‐3 inhibitor, acetyl‐L‐aspartyl‐L‐glutamyl‐L‐valyl‐L‐aspart‐1‐al (Ac‐DEVD‐CHO). Levels of expression of heat shock protein 90α (Hsp90α) were found to be increased 20 min after the nicotine treatment, as analyzed by polymerase chain reaction‐based mRNA differential display after Northern blotting analysis of mRNA amounts. Cellular contents of Hsp90α were furthermore increased in the nicotine‐treated RSa cells, as quantitated by Western immunoblot analysis. By contrast, in RSa cells treated with nicotine in combination with geldanamycin (GA), an inhibitor of Hsp90α function, DNA fragmentation was not detected and caspase‐3 protease activity levels were the same as those of mock‐treated cells. Nicotine‐induced caspase‐3 activation and Hsp90α expression, as well as suppression of the induction by GA, were also observed in a xeroderma pigmentosum patient‐derived cell line, XP2OS cells. Thus, it was suggested that nicotine induces apoptosis, possibly via Hsp90α expression, in human cells tested.

Mammalian phosphoribosyl-pyrophosphate synthetase

PRPP synthetase from rat liver exists as large molecular weight aggregates composed of at least three different components. Cloning of cDNA for the catalytic subunit revealed the presence of two highly homologous isoforms of 34 kDa, designated as PRS I and PRS II. Northern blot analysis showed tissue-differential expression of the two isoform genes. cDNA was expressed in E. coli and studies on the recombinant isoforms showed differences in sensitivity to inhibition by ADP and GDP and to heat inactivation. The rat gene for PRS I has 22 kb and is split into 7 exons. cDNAs for human enzymes were also cloned. Human genes for PRS I and PRS II are localized at different regions on the X-chromosome and their promoter regions were examined. Another component, PRPP synthetase-associated protein of 39 kDa (PAP39), was cloned from cDNA library of the rat liver. The deduced amino acid sequence of PAP39 is remarkably similar to those of PRS I and PRS II. Evidence indicated molecular interaction between PAP39 and the catalytic subunits and an inhibitory effect of PAP39 on the catalytic activity. Expression of the PAP39 gene is tissue-differential like the PRS genes, indicating that the composition of PRPP synthetase may differ with the tissue, hence properties of the enzyme would differ. Further studies on these components and their interaction are expected to reveal various mechanisms governing mammalian PRPP synthetase.

Involvement of LEU13 in interferon-induced refractoriness of human RSa cells to cell killing by X rays.

Abstract Kita, K., Sugaya, S., Zhai, L., Wu, Y. P., Wano, C., Chigira, S., Nomura, J., Takahashi, S., Ichinose, M. and Suzuki, N. Involvement of LEU13 in Interferon-Induced Refractoriness of Human RSa Cells to Cell Killing by X Rays. Radiat. Res. 160, 302–308 (2003). Culture of human cells with human interferon α and β (IFNA and IFNB) results in increased resistance of the cells to cell killing by X rays. To identify candidate genes responsible for the IFN-induced X-ray resistance, we searched for genes whose expression levels are increased in human RSa cells treated with IFNA, using an mRNA differential display method and Northern blotting analysis. RSa cells, which showed increased survival (assayed by colony formation) after X irradiation when they were treated with IFNA prior to irradiation, showed increased expression levels of LEU13 (IFITM1) mRNA after IFNA treatment alone. In contrast, IFr and F-IFr cells, both of which are derived from RSa cells, showed increased X-ray resistance and high constitutive LEU13 mRNA expression levels compared to the parental RSa cells. Furthermore, the IFNA-induced resistance of RSa cells to killing by X rays was suppressed by antisense oligonucleotides for LEU13 mRNA. LEU13, a leukocyte surface protein, was previously reported to mediate the actions of IFN such as inhibition of cell proliferation. The present results suggest a novel role of LEU13 different from that in the inhibition of cell proliferation, involved in IFNA-induced refractoriness of RSa cells to X rays.

Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UV^r-1 cells derived from ultraviolet-sensitive human RSa cells

Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14–18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CΘ, Src, Bax and μ-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated μ-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, μ-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells. Cell Death and Differentiation (2000) 7, 531–537

Cyclical Cell Stretching of Skin-Derived Fibroblasts Downregulates Connective Tissue Growth Factor (CTGF) Production

Delayed healing of skin wounds can be caused by wound instability, whereas appropriate massage or exercise prevents sclerosis and scar contracture. However, the mechanism by which wound healing is related to mechanical stress has not been fully elucidated. The present study aimed to identify whether mechanical stretching of fibroblasts reduces their production of extracellular matrix. We transferred skin fibroblasts into collagen-coated elastic silicone chambers, cultured them on a stretching apparatus, and used RT-PCR to examine the effects of mechanical stretching on the expression levels of 17 genes related to extracellular matrix production and growth factor secretion. We found that connective tissue growth factor (CTGF) was downregulated after 24 hr of cell stretching. Specifically, the CTGF mRNA and protein levels were 50% and 48% of the control levels, respectively. These findings suggest that cyclic stretching of fibroblasts contributes to anti-fibrotic processes by reducing CTGF production.

Suppression of okadaic acid-induced apoptosis by overexpression of calpastatin in human UVr-1 cells

Proteolytic systems have various involvements in apoptotic pathways. To understand the role of calpain in apoptosis, calpastatin, a specific inhibitor of calpain, was overexpressed in human UVr‐1 fibroblasts by transfection of its cDNA. The elevated expression of calpastatin resulted in decreased survival in the presence of okadaic acid (OA) but in no apparent alteration in the sensitivity toward other drugs such as 5‐fluorouracil, mitomycin C and methotrexate. After treatment with OA, a typical apoptotic DNA ladder was observed in control vector‐transfected cells but not in calpastatin‐transfected cells. This indicates that OA‐induced apoptosis was suppressed by overexpression of calpastatin. Further immunoblot analysis showed that the OA‐induced hyperphosphorylation of c‐Jun was inhibited in calpastatin‐transfected cells. This might be involved in the resistance to OA‐induced cell death in calpastatin‐overproducing cells.

Protease activation following UV irradiation is linked to hypomutability in human cells selected for resistance to combination of UV and antipain

In order to examine the relationship between activation of an antipain-sensitive protease and suppression of mutability in UV (UVC)-irradiated human cells, a human cell variant with the high protease activity induced by UV was established and characterized for its susceptibility to UV-induced mutagenicity. Cells of a hypermutable cell strain, RSa, were mutagenized with ethyl methanesulfonate and irradiated with 10 J/m2 UV, followed by exposure to 20 mM antipain for 34 h. Whereas the combined treatment was totally lethal to RSa cells not treated with ethyl methanesulfonate, one surviving clone was isolated from the mutagenized cells and designated UVAP-1. When fibrinolytic protease activity was measured from extracts of the cell, it was found that the protease activity was elevated promptly after UV irradiation, reaching the maximum at 10 min post-irradiation. This protease activity was inhibited by antipain. After UV irradiation the phenotypic mutation frequencies of UVAP-1 cells were much lower than those of the parent RSa cells, as evaluated by the generation of clones resistant to ouabain-killing. Furthermore, mutation at the K-ras codon 12 in genomic DNA was detected in RSa cells but not in UVAP-1 cells. Thus, the protease activation was correlated with the decreased levels of UV-mutagenicity in UVAP-1 cells, supporting the possible involvement of the antipain-sensitive protease activity in the regulation of cellular mutability following UV irradiation.

Cloning and sequencing of rat cDNA for the 41-kDa phosphoribosylpyrophosphate synthetase-associated protein has a high homology to the catalytic subunits and the 39-kDa associated protein

Rat liver phosphoribosylpyrophosphate synthetase is a complex aggregate of 34-kDa catalytic subunits (PRS I and II) and 39- and 41-kDa associated proteins (PAP39 and 41). When the rat cDNA encoding PAP41 was isolated, the deduced protein sequence was seen to contain 369 amino acids with a calculated molecular mass of 41130. PAP41 has a 79 and 49% identity with PAP39 and PRSs, respectively. When conservative substitutions are included, PAP41 and the three other components have a 66% homology. PAP41 shares some common features with PAP39 and the two proteins form the PAP subfamily. The mRNA of PAP41 is present in all rat tissues we examined.

Increase in the levels of chaperone proteins by exposure to β-estradiol, bisphenol A and 4-methoxyphenol in human cells transfected with estrogen receptor α cDNA

We examined changes in the levels of chaperone proteins to evaluate the toxic effects of environmental chemicals in human cells in vitro. Some chaperones are up-regulated by estrogenic chemicals, but the effect is not necessarily dependent on the receptor. Thus we also investigated whether a chemical-induced change in chaperone protein expression is human estrogen receptor (hER)-dependent or not, using cultured human cell lines transfected with hERalpha cDNA or an empty vector. In the hERalpha-expressed cells, the protein levels of the heat shock protein 27 (HSP27), the glucose-regulated protein 78 (GRP78/BiP), and GRP94 increased after exposure to beta-estradiol (E(2)) (from 10(-9)M to 10(-6)M) and bisphenol A (BPA) (from 10(-6)M to 10(-5)M). On the other hand, the increase was not observed in the cells without hERalpha expression. These results suggest that the E(2)- and BPA-induced increase in the protein levels were hERalpha dependent. We next examined the effect of four phenolic chemicals similar in structure to BPA, and found that among them, 4-methoxyphenol (from 10(-6)M to 10(-5)M) increased the levels of the chaperone proteins with hERalpha dependency. Thus the human cultured cells would be suitable for evaluating whether an increase in chaperone proteins occurs upon exposure to environmental chemicals and whether the effect is ER-dependent.