Stable expression of a cDNA encoding rat brain protein kinase C-beta I confers a multidrug-resistant phenotype on rat fibroblasts.
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Protein kinase C (PKC) is a Ca++- and phospholipid-dependent protein kinase that plays an important role in signal transduction pathways that regulate cell growth. Tumor cells selected for a multidrug resistant (MDR) phenotype often express elevated levels of PKC activity. To directly test whether PCK overexpression can produce an MDR phenotype, we studied rat embryo fibroblasts that were infected with the full-length cDNA clone RP58 encoding the beta I form of rat brain PKC. The PKC-beta I gene recipient R6-PKC3 cells are stable, overproduce PKC, and express an elevated level of PKC activity. R6-PKC3 cells exhibited significant resistance to adriamycin, actinomycin D, vinblastine, and vincristine but not to 5-fluorouracil. Intracellular accumulation of adriamycin, vinblastine, and vincristine was decreased in the R6-PKC3 cells, but this was not associated with an altered level of P-glycoprotein expression. Moreover, the reduction in drug accumulation appeared to be a consequence of a decreased rate of drug uptake. The data indicate that overexpression of PKC in rat fibroblasts produces an MDR phenotype without altering P-glycoprotein expression.Keywords:
P-glycoprotein
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Analysis of gene expression in human bullous keratopathy corneas containing limiting amounts of RNA.
To validate the use of polymerase chain reaction (PCR)-amplified full-length cDNA as a substitute for mRNA in nucleic acid array and gene expression analysis.Total RNA was isolated from age-matched normal autopsy corneas and pseudophakic bullous keratopathy (PBK) corneas. Full-length cDNA was generated and PCR amplified using the Smart cDNA synthesis technology. Southern blot analysis of this cDNA was compared with Northern blot analysis of the RNA. Amplified cDNA was used to probe a commercial gene array. By immunohistochemistry, the expression pattern of several adhesion molecules represented on the array was assessed.The cDNA produced by the Smart cDNA system gave results very similar to those of northern blot analysis when examined for beta2-microglobulin, Rab geranylgeranyl transferase, and tenascin-C. This cDNA obtained from normal or PBK corneas was labeled and used to probe a 588 gene array (Clontech). Among other differences, beta6 integrin was detected only with the PBK probe, beta-catenin was markedly elevated in PBK, and beta4 integrin appeared to be reduced in PBK. Immunohistochemical patterns of these proteins were consistent with the hybridization signals on the gene array.Smart cDNA synthesis and nucleic acid arrays were combined and validated for the first time to identify differential gene expression in normal and diseased corneas. These techniques require very little RNA such as that equivalent to a half of a single cornea, which is useful when the amount of tissue is limiting. Altered expression of adhesive proteins beta6 integrin and beta-catenin may be related to the formation of epithelial bullae and microcystic changes in PBK patients.
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Antiserum raised against purified protein kinase C (the Ca2+/phospholipid-dependent enzyme) (Ballester, R., and Rosen, 0
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AIM:To clone and identify the whole cDNA of MXR7 gene and to find out its expression in human HCC, and normal tissues.METHODS:The DNA primers were designed and synthesized according to the whole cDNA sequence of MXR7 gene. The cDNA of human HCC was taken as the template while the cDNA of MXR7 gene was synthesized by polymerase chain reaction (PCR). Recombinant DNA conforming to reading frame was constructed by connecting purified PCR product of the cDNA of MXR7 gene with expression vector pGEX-5X-1 of fusion protein. The plasmid MXR7/pGEX-5X-1 was identified by sequencing. Using (32)P labeled MXR7 cDNA as probe, MXR7 mRNA expression was detected by Northern blot analysis in 12 different human normal tissues, 7 preoperatively untreated non-liver tumor tissues, 30 preoperatively untreated HCC, the paracancerous liver tissues and 12 normal liver tissues samples.RESULTS:Restriction enzyme and sequence analysis confirmed that the insertion sequence in vector pGEX-5X-1 was the same as the cDNA sequence of MXR7 gene. Northern blot analysis showed no expression of MXR7 mRNA in 12 kinds of normal human tissues including liver, 7 tumor tissues in other sites and 12 normal liver tissues, the frequencies of MXR7 mRNA expression in HCC and paracancerous liver tissues were 76.6% and 13.3%, respectively. The frequency of MXR7 mRNA expression in HCC without elevation of serum AFP and in HCC <5cm was 90% (9/10) and 83.3% (5/6), respectively.CONCLUSION:MXR7 mRNA is highly expressed in human HCC, which is specific and occurs at an early stage of HCC, suggesting MXR7 mRNA can be a tumor biomarker for HCC. The detection of MXR7 mRNA expression in the biopsied liver tissue is helpful in discovering early subclinical liver cancer in those with negative serum AFP.
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Objective To explore the effects of protein kinase C alpha(PKCα) cDNA on the expression of genes of multidrug resistance (MDR) in renal cell carcinoma(RCC) 786-0 cell line.Methods LR/BR recombination reactions were used to generate mammalian expression vectors of PKCα cDNA with C-terminal fused green fluorescent protein(GFP),and vectors were transfected into human RCC cell with Lipofectimine 2000.Western blot method and inverted fluorescent microscope were used to determine the expression of PKCα in RCC cells transfected by PKCα cDNA.RT-PCR was used to determine the expression of MDR-related genes MDR1,MRP1 and LRP in RCC cells transfected by PKCα cDNA.Results After transfection of 786-0 cells with pcDNA-DEST47-PKCα-GFP vectors,the results of Western blot showed that PKCα was highly expressed in human RCC 786-0 cells;and inverted fluorescent microscopy showed that GFP was highly expressed in RCC 786-0 cells.The results of semi-quantitative RT-PCR analysis showed that the expression level of MDR1 was higher in RCC cells transfected by PKCα cDNA than in RCC cells.Conclusions The expression of MDR1 mRNA in 786-0 cell line can be up-regulated by PKCα cDNA,which suggests PKCα cDNA can induce the increase of MDR in renal cell carcinoma.
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Objective To observe the altered gene expression of ECV304 cell after treated with endostatin (ES) using cDNA microarray, and to investigate its molecular mechanism. Methods BiostarH40S gene expression DNA microarrays were used to profile changes in gene expression of ECV304 cells after treated with ES for 36 hours. To prepare the probes, mRNA from both control and treated cells were isolated and purified, and then reversely transcribed to cDNA with the incorporation of fluorescent-labeled dUTP: Cy3 and Cy5 respectively. The probes were hybridized with expressed cDNA microarray, the fluorescent signals of Cy3 and Cy5 were scanned and analyzed by a computer system. Results ES inhibited the proliferation of ECV304 cells. The result of electrophoresis indicated that the extracted total RNA had high quality. Approximately 5. 96% of all human genes examined on 4096 gene microarrays showed altered expression, with 225 down-regulated genes and 19 up-regulated genes. Conclusion The expression of some genes of ECV304 cells decrease after treated with ES, which may be related to the mechanism of inhibition of neovascularization.
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Gene expression profiling/utilization; Microchip analytical procedures/method; Endostatins/pharmacology; Gene expression
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Staurosporine
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ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMouse protein kinase C-.delta., the major isoform expressed in mouse hemopoietic cells: sequence of the cDNA, expression patterns, and characterization of the proteinHarald Mischak, Angelika Bodenteich, Walter Kolch, JoAnne Goodnight, Franz Hofer, and J. Frederic MushinskiCite this: Biochemistry 1991, 30, 32, 7925–7931Publication Date (Print):August 13, 1991Publication History Published online1 May 2002Published inissue 13 August 1991https://pubs.acs.org/doi/10.1021/bi00246a008https://doi.org/10.1021/bi00246a008research-articleACS PublicationsRequest reuse permissionsArticle Views97Altmetric-Citations69LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
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To study the gene expression during the chemical inducing erythroid differentiation of murine erythroleukemia cells (MEL).Differential display assay was used to analyze the gene expression before and after the induction differentiation of MEL cells by DMSO and hemin; and then, the flanking sequences of one of the cDNA fragment was amplified by modified rapid amplification of cDNA end (RACE) method, Northern blot analysis was adopted to characterize the expression of this gene.The expression of a new transcript similar to VL-30 retrotransposon family increased significantly during the chemical inducing erythroid differentiation in MEL cells. The cloned cDNA is 1883bp, and it is highly homologous to the internal region of murine BVL-1 (1,955-4,000 nt). At least three transcripts in MEL cells were detected by Northern hybridization and all of them increased after the induction.A new VL-30-like gene is identified for the first time in MEL cells during chemical inducing erythroid differentiation.
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Cross-resistance to anticancer drugs, termed multidrug resistance (mdr), has been functionally associated with the expression of a plasma membrane energy-dependent efflux pump, termed P-glycoprotein, the product of the mdr1 gene. When MCF-7 breast carcinoma cells were transfected with the human mdr1 gene (BC-19 cells), they expressed levels of P-glycoprotein equivalent to those of cells selected for resistance to doxorubicin (MCF-7/ADR) but exhibited 10- to 50-fold less resistance to doxorubicin and vinblastine. We have now demonstrated that when BC-19 cells were stably transfected with protein kinase C alpha (PKC alpha), resistance to doxorubicin and vinblastine was increased; wild-type MCF-7 cells transfected with PKC alpha did not exhibit any change in drug resistance. Increased resistance in PKC alpha-transfected BC-19 cells was associated with enhanced PKC activity and phosphorylation of P-glycoprotein and decreased drug accumulation. The PKC activator, phorbol dibutyrate, further increased resistance to doxorubicin and stimulated P-glycoprotein phosphorylation. These results demonstrate that transfection of P-glycoprotein-expressing cells with PKC resulted in increased mdr and that PKC may have served as an important modulator of this process.
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