Expression and imprinting status of human PEG8/IGF2AS, a paternally expressed antisense transcript from the IGF2 locus, in Wilms' tumors.
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A large imprinted gene cluster in human chromosome 11p15.5 has been implicated in Beckwith-Wiedemann syndrome and Wilms' tumor. We have identified a paternally expressed imprinted gene, PEG8/IGF2AS, in this locus. It is transcribed in the opposite direction to the IGF2 transcripts and some genomic regions are shared with the IGF2 gene, as in the case of the mouse imprinted Igf2as gene reported previously by T. Moore et al. As to the relationship between these genomic regions, the human and mouse genes are very similar but there is no homology in their middle parts. Interestingly, PEG8/IGF2AS and IGF2 were found to be overexpressed in Wilms' tumor samples, at levels over ten and a hundred times higher than that in normal kidney tissues neighboring the tumors, respectively. These findings indicate that PEG8/IGF2AS is a good marker of Wilms' tumor and also suggest the possibility of PEG8/IGF2AS being one of the candidate Wilms' tumor genes. (+info)
Antisense suppression of proline-directed protein kinase FA enhances chemosensitivity in human prostate cancer cells.
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Initial clinic studies revealed that proline-directed protein kinase FA (PDPK FA) is overexpressed manyfold in various human cancerous tissues relative to the normal control. However, the role of overexpressed PDPK FA in cancers remains unknown and needs to be established. To determine whether PDPK FA is associated with drug sensitivity, we investigated the effects of partial inhibition of this kinase on the human prostate carcinoma cell line (PC-3). PDPK FA antisense expression vector and its specific antibody were successfully developed. Two stable transfected antisense clones (PA7 and PA3) of human prostate carcinoma cell were subcloned, and they expressed approximately 75% and approximately 35% of the total PDPK FA existing in the control-transfected clone as determined by both immunoprecipitate activity assay and immunoblot analysis. In sharp contrast, the PDPK FA antisense clones expressed no significant suppression of any other related proline-directed protein kinase member expression, demonstrating the specificity of these two antisense clones. When compared with parental or control-transfected cells, the low-PDPK FA-expressing antisense clones displayed an enhanced sensitivity to carboplatin, 5-fluorouracil, paclitaxel, and hydroxyurea. Estimation of the IC50 index further revealed that the antisense clones displayed up to > 100-fold drug sensitivity, and there was a correlation between suppressed levels of PDPK FA and drug sensitivity. Taken together, the results demonstrate that specific antisense suppression of overexpressed PDPK FA in human prostate cancer cells is sufficient to enhance various drug sensitivity, indicating that PDPK FA is an important regulator in controlling multiple drug resistance of human prostate cancer cells. (+info)
Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis.
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Plants are sessile organisms, and their ability to adapt to stress is crucial for survival in natural environments. Many observations suggest a relationship between stress tolerance and heat shock proteins (HSPs) in plants, but the roles of individual HSPs are poorly characterized. We report that transgenic Arabidopsis plants expressing less than usual amounts of HSP101, a result of either antisense inhibition or cosuppression, grew at normal rates but had a severely diminished capacity to acquire heat tolerance after mild conditioning pretreatments. The naturally high tolerance of germinating seeds, which express HSP101 as a result of developmental regulation, was also profoundly decreased. Conversely, plants constitutively expressing HSP101 tolerated sudden shifts to extreme temperatures better than did vector controls. We conclude that HSP101 plays a pivotal role in heat tolerance in Arabidopsis. Given the high evolutionary conservation of this protein and the fact that altering HSP101 expression had no detrimental effects on normal growth or development, one should be able to manipulate the stress tolerance of other plants by altering the expression of this protein. (+info)
Inhibition of PC cell-derived growth factor (PCDGF, epithelin/granulin precursor) expression by antisense PCDGF cDNA transfection inhibits tumorigenicity of the human breast carcinoma cell line MDA-MB-468.
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PC-cell derived growth factor (PCDGF) is an 88-kDa growth factor originally purified from the highly tumorigenic teratoma PC cell line and corresponds to the epithelin/granulin precursor. In teratoma cells, PCDGF expression was shown to be essential for tumorigenicity. We have reported that PCDGF was expressed in estrogen receptor-positive (ER(+)) human mammary epithelial cells in an estrogen-dependent fashion. In this study, we have investigated PCDGF expression in human mammary epithelial cell lines ranging from immortalized nontumorigenic cells to ER(+) and ER(-) breast carcinoma cells. Northern and Western blot analyses indicated that PCDGF mRNA and protein expression was low in nontumorigenic cells and increased in human breast carcinomas cell lines in a positive correlation with their tumorigenicity. Treatment of the ER(-) MDA-MB-468 cells with anti-PCDGF neutralizing antibody resulted in a dose-dependent inhibition of their proliferation, suggesting that secreted PCDGF acted as an autocrine growth factor for breast carcinoma cells. We then examined the in vitro and in vivo growth properties of MDA-MB-468 cells, where PCDGF expression had been inhibited by antisense PCDGF cDNA transfection. Inhibition of PCDGF expression resulted in a reduced proliferation rate in vitro and a 60-80% reduction in colony formation. Tumor formation in vivo was dramatically inhibited in antisense cells with a 90% inhibition of tumor incidence and tumor weight. These results demonstrate the importance of PCDGF overexpression for the proliferation and tumorigenicity of ER(-) breast carcinomas and suggest that PCDGF overexpression may play an important role in human breast cancer. (+info)
Epidermal growth factor (EGF) suppresses staurosporine-induced apoptosis by inducing mcl-1 via the mitogen-activated protein kinase pathway.
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Overexpression of epidermal growth factor receptor (EGFR) and establishment of transforming growth factor alpha (TGF alpha)/EGF autocrine system are frequently detected in tumor cells. In addition to mitogenic ability, we demonstrate in this report that EGF protects a human esophageal carcinoma (CE) cell line, CE81T/VGH, from staurosporine-induced apoptosis. The anti-apoptotic signal of EGF is alleviated by a MEK inhibitor PD98059 or an ERK2 dominant negative mutant but not by a phosphatidylinositol-3'-kinase (PI-3K) inhibitor wortmannin. Furthermore, v-raf blocks apoptosis induced by staurosporine. This evidence implies that the survival signal of EGF is mediated via the Raf-MEK-ERK pathway but not the PI3-K pathway. The survival effect of EGF is coincident with the induction of mcl-1, an antiapoptotic gene in the bcl-2 family. PD98059 also suppresses the induction of Mcl-1 by EGF, implying that EGF may up-regulate Mcl-1 via the MAP kinase pathway. Overexpression of mcl-1 is sufficient to protect against apoptosis, while transfection of a mcl-1 antisense plasmid causes cell death. The expression of mcl-1 antisense plasmid also suppresses the anti-apoptotic effect of EGF. Taken together, these results indicate that EGF may up-regulate Mcl-1 through the MAP kinase pathway to suppress apoptosis. (+info)
Enhancement of immunogenicity of tumor cells by cotransfection with genes encoding antisense insulin-like growth factor-1 and B7.1 molecules.
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Insulin-like growth factor-1 (IGF-1) is expressed in many tumor cell lines and has a role in both normal cell proliferation and in the growth of cancers. Tumor cells transfected with a vector encoding an IGF-1 antisense cDNA transcriptional cassette driven by the mouse metallothionein-1 promoter become immunogenic and lose their tumorigenicity in syngeneic animals. The enhanced immunogenicity is associated with an up-regulation in the expression of major histocompatibility complex class I molecule on cell surfaces. Blockade of the expression of IGF-1 in tumor cells by the IGF-1 antisense RNA approach is not uniformly effective in the induction of antitumoral protective immunity in low and nonimmunogenic tumor model systems. Here, we report that the immunogenicity of hepa 1-6 hepatoma and SMCC-1 colon carcinoma cells, which are poorly immunogenic and unresponsive to antisense IGF-1 gene transfer, can be induced by cotransfection with genes encoding antisense IGF-1 and mouse B7.1 molecules. The tumor cells modified in this manner become strongly immunogenic and can be used as a cellular vaccine to induce a protective immune response in vivo. Immunization with the transfected tumor cells also results in regression of the established hepa 1-6 hepatoma and SMCC-1 colon cancer. The immunity is tumor-specific and is mediated by CD3+ CD8+ T cells. Cytotoxic T lymphocytes generated in vitro by priming naive spleen cells and in vivo by immunizing mice with the double-transfected tumor cells specifically lysed autologous tumors cells and were effective in adoptive immunotherapy. The data suggest that modification of tumor cells in vitro by cotransfection with genes encoding antisense IGF-1 and B7.1 molecules may open a new avenue for cancer immunogene therapy. (+info)
Depending on the duration and severity, psychological tension and physical stress can enhance or suppress the immune system in both humans and animals. Although it is well established that stress alters the release of various hormones and neurotransmitters, the mechanisms by which stress affects immune responses remain elusive. We report here that mice subjected to chronic 12-hour daily physical restraint for two days exhibited a significant reduction in splenocytes, a process likely mediated by apoptosis as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. CD95 (Fas/APO-1) expression in splenic lymphocytes of stressed mice was substantially increased. Interestingly, Fas-immunoglobulin fusion protein and blocking antibodies against CD95 ligand inhibit stress-induced reduction in lymphocytes. The stress-induced changes in CD95 expression and lymphocyte number could be blocked by naltrexone or naloxone, specific opioid receptor antagonists, indicating a pivotal role of endogenous opioids in this process. In addition, the reduction of splenocytes in this model system seems to be independent of the hypothalamo-pituitary-adrenal axis, as both adrenalectomized and sham-operated mice exhibited similar responses to chronic stress. Moreover, chronic physical restraint failed to induce a decrease in lymphocyte numbers in CD95-deficient (Fas(lpr/lpr)) mice. Therefore, stress modulates the immune system through CD95-mediated apoptosis dependent on endogenous opioids. (+info)
The interferon-beta and tamoxifen combination induces apoptosis using thioredoxin reductase.
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Interferons (IFNs) suppress cell growth by inducing cellular genes. The anti-estrogen tamoxifen (Tam), binds to estrogen receptor and inhibits transcription of estrogen stimulated genes. In cells resistant to IFN-induced growth suppression, IFN/Tam combination causes cell death. We previously reported that the combination of IFN-beta and Tam was a more potent growth suppressor of human tumor xenografts than either agent alone. The IFN/Tam combination acts in a manner similar to the IFN/retinoic acid combination. Using a genetic technique, we have recently identified several genes associated with retinoid-IFN-induced mortality (GRIM). One such gene, GRIM-12, was identical to human thioredoxin reductase (TR). In the present study we have examined whether the IFN/Tam combination also requires GRIM-12 for inducing cell death. We report here that GRIM-12 is necessary for mediating the cell death effects of IFN/Tam, and its expression is induced by IFN/Tam at a post-transcriptional stage. Repression of GRIM-12 levels either by antisense expression or by dominant negative inhibitors caused resistance to IFN/Tam induced death and promoted cell growth. Overexpression of GRIM-12 increased IFN/Tam induced apoptosis. Thus, these studies have identified a critical role for GRIM-12 (TR) in apoptosis. (+info)