蛋白激酶C-α（PKC-α）在肾癌中与多药耐药相关性研究
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摘要
目的
蛋白激酶C(Protein KinaseC,PKC)是最早被确认的蛋白激酶之一,是结构相近、钙和磷脂依赖、甘油二酯(DAG)激活的丝氨酸/苏氨酸蛋白激酶家族,通过丝氨酸/苏氨酸磷酸化来调节各种细胞包括肿瘤细胞的不同功能,在介导细胞的生长、分化和死亡等信号传导中起重要作用。近年来发现PKC参与肿瘤的发生、发展,是肿瘤细胞粘附、运动、侵袭和转移的关键因素。
PKC同工酶的cDNA分析表明,该多肽链其氨基端一半序列为调节域(Regulatory Domain),其羧基端一半序列为催化域(Catalytic Domain),两者之间通过一个可被蛋白酶水解的铰链区(Hinge Domain)连接,该区在酶的激活过程中发生降解,从而使PKC活性部位暴露。在调节域还含有假底物位点,其与真底物位点的差别在于磷酸化位点的丙氨酸取代了丝氨酸/苏氨酸残基,因而不被磷酸化,从而封闭酶的活化中心。
由于假性底物的存在,静息状态下PKC均以无活性的形式存在于胞浆中,在外界刺激作用下,PKC移位(translocation)到胞膜而被激活。短暂的PKCs激活,在信号传导通路中所致的短期事件(如离子内流、分泌等)方面似乎起着重要作用;持续的PKC激活,可能在细胞增殖、分化及肿瘤发生等方面起着重要的作用。PKC是促癌剂PMA(phorbol,12-myristate-13-acetate,即佛波醇-12-豆蔻酰-13-乙酸)细胞内受体,PKC的过度表达导致细胞生长紊乱,并且触发肿瘤的形成。
Efferth等通过免疫细胞化学方法研究PKC在18种人肾细胞癌系中的表达。结果发现,PKC的高表达与癌细胞对阿霉素的耐药和P糖蛋白的高表达密切相关。从而证实PKC通过磷酸化和对P糖蛋白的调节参与肾细胞癌的内源性耐药。
ObjectiveProtein Kinase C is one of the earliest established Protein Kinase, made up of serine/threonine protien kinase family, whose construction close to each other , on which calcfium and phosphatide rely, activated by diacylglycerol (DG). PKC adjust different function of various kinds of cells, including tumor cell, though serine/threonine phosphorylation. PKC has important effect on cellular growth, differerntiate and such of signal conductions. For the past few years, it is known that participation of PKC in tumorous development and growth is key point factor of tumour cell adhesion, motion,invasion and transfusion.Study on cDNA of isozymes of PKC indicate that the polypeptide chain N -terminal sequence is regulatory domain, and the carboxyl terminal is catalytic domain is exposed. There is a hinge domain which can be hydrolyzed by protease between them. When enzyme were activated, the hinge domain is degradat-ed so thar PKC active site. The difference between pseudosubstrate site in regulatory domain and true substrate is that alanina in phosphorylation site displace serine/threonine residue, and close enzyme activation center instead of phosphorylation.PKC usually reside with inactivestyle in endochylema under quiescent condition. With surrounding stimulation, PKC is activated because of translation to cell membrane. Temporal PKC activation seems important effect in short - term events, ( such as ion inflow, secrete, and so on) , which induced in signal conduction access. Persistent PKC activation seems consequence effect in cell multiplication , differentiate, tumorigenesis and so on. PKC is tumor promoter phor-bol 12 - myristate - 13 - acetate intracellular receptor. Its over - expression in-
duces cell growth disorder and triggers tumorous form.Efferth et al. investigate PKC express in 18 kinds of human renal cell carcinoma system with immmuocytochemistry. There are intimate correlations between PKC high express and drug resistances of cancer cell for Adriamycin and P glucoprotein. PKC participate renal cell carcinoma endogenous drug resistances though PKC phosphorylation.PKC generally exist in animal, microbeorganism, and present in overall tissue and organ. There are four categories and thirteen subtypes in PKC based on different grades in isozyme construction, enzymic character, activating agent. Classical PKC (cPKC) : α、βⅠ、βⅡ、γ, which are activated by calcium , phosphatide, diacyglycerol (DAG) or phorbol ester. Novel PKC ( nPKC ) : (?) η、δ、ε、θ, which are activated by phosphatide, diacyglycerol( DAG) or phorbol ester instead of calcium. Atypical PKC (aPKC) : T/λ、ξ , which are only activated by phospholipin. Recently a kind of PKC is found , which is activated by phosphatidylinositol -4,5- diphosphate instead of calcium and DAG and is similar to the others in construction. So this kind of PKC is called the fourth kind of PKC. Various kinds of isozyme have different specificity of distribution and effects.We use research in vitro to discuss mechanism of MDR in renal cell carcinoma at the PKC subtype level, to provide the experiments about treating renal cell carcinoma.The experiment divides three parts:One; LR\BR recombination reactions were used to generate mammalian expression vectors of protein kinase C alpha cDNA with C - terminal fused GFP, and vectors were transfected into human RCC cell with Lipofectimine 2000. Western Blot and Inverted Fluorescent Microscope were used to determine the expression of PKC - a gene in RCC cells transferred by PKC - α cDNA.Two: LR\BR recombination reactions were used to generate mammalian expression vectors of protein kinase C alpha cDNA with C - terminal fused GFP, and vectors were transfected into human RCC cell with Iipofectimine 2000. Western Blot 、and Inverted Fluorescent Microscope were used to determine the expression of PKC - a in RCC cells transferred by PKC - α cDNA; RT - PCR
were used to determine the expression of MDR related gene in RCC cells transferred by PKC - αcDNA .Three: To investigated the expression of PKC - a in membrane and plasma of 30 cases renal cell carcinoma and normal adjacent tissue by Western blotting technology.MethodsCells culture786 - 0, was maintained in RPMI - 1640 medium, supplemented with 10% fetal bovine serum (FBS) , 2mM glutamine, 25mM HEPES at 37oC in a humidified, 5% CO_2incubator.Plasmid constructThe PKC - α cDNA was subcloned into pDONR~(TM)201 donor vector by PCR with Pyrobest DNA Polymerase using BP Clonase Reaction system. Entry clone was identified by DNA sequencing analysis. pcDNA - PKC - α - GFP recombi-nant plasmid expressing PKC - α with C - terminal fused Green Fluorescent Protein (GFP) was constructed with entry clone and GFP expression vector pcDNA - DEST47 using Gateway LR recombination reaction system. primers were designed as follows: 5 ' - ATG GCT GAC GTT TTC CCG GGC AA - 3 ' ;5 ' -CAT ACT GCA CTC TGT AAG ATG GGG -3'.Cell transfectionThe cells were transfected with pcDNA - PKC - α - GFP vector by Lipofec-timine2000 reagent. Positive clones were identified by GFP fluorescence microscopy.Selection of stable expressionThe stable expression clones (786 - 0/PKC - α - GFP) were established by the selection with G418 at a concentration of 400 μg/ml for four weeks. clone Isolated clones were analyzed for PKC - α - GFP expression by western blot analysis and fluorescence microscopy.
Multidrug resistance correlated genes assaysTo identify the alteration of Multidrug resistance correlated genes, MDR1, MRP1, LRP gene were assayed using RT - PCR , primers were designed as follows: MDR1:5' -ACACCCGACTTACAGATGATGTCTC-3' , 5' -CGAGAT-GGGTAACTGAAGTGAACAT - 3 ' , length of fragment:623bp; MRP1: 5 ' -AGTGACCTCTGGTCCTTAAACAAGG - 3 ' , 5 ' - GAGGTAGAGAGCAAG-GATGACTTGC - 3 ' , length of fragment:657bp; LRP: 5 ' - GAGCAGTTCA-CAGTGTTGTCC - 3 ' , 5 ' - GCGTGACGACAGAAACCGAAA - 3 ' ; length of fragment: 342bp.Expression and significance of PKC - a in renal cell carcinoma and normal adjacent tissueTo investigated the expression of PKC - a in membrane and plasma of 30 cases renal cell carcinoma and normal adjacent tissue by Western blotting technology.ResultsPKC - α cDNA was identified by DNA sequencing analysis.To investigate the effect of PKC - α on the drug resistance of renal cell carcinoma cells, we established stable cell clones expressing PKC - α - GFP using 786 -0 drug - sensitive RCC cell line. Three highest expression clones were i-dentified by western blot analysis. In order to further identify the character of expressed PKC - α - GFP, fluorescence microscopy was conducted to examine the distribution of PKC - a - GFP. As the result, these data demonstrated that stably expressed PKC - a - GFP has the same character as endogenous PKC -a.Effect of PKC - a on the drug resistance of 786 - 0MDR1,MRP1, LRP genes were detected in 786 - 0/PKC - α - GFP cells, and , for comparison, 786 - 0 cells. While 786 - 0 cells expressed MDR1, MRP1 and LRP were detected by RT - PCR , MDR1 expression in transfected 786 - 0 cells was significantly higher than that in homologous cells. Nevertheless for MRP1 and LRP, the diversification of expression were indistinguishable be-
蛋白激酶C(Protein KinaseC,PKC)是最早被确认的蛋白激酶之一,是结构相近、钙和磷脂依赖、甘油二酯(DAG)激活的丝氨酸/苏氨酸蛋白激酶家族,通过丝氨酸/苏氨酸磷酸化来调节各种细胞包括肿瘤细胞的不同功能,在介导细胞的生长、分化和死亡等信号传导中起重要作用。近年来发现PKC参与肿瘤的发生、发展,是肿瘤细胞粘附、运动、侵袭和转移的关键因素。
PKC同工酶的cDNA分析表明,该多肽链其氨基端一半序列为调节域(Regulatory Domain),其羧基端一半序列为催化域(Catalytic Domain),两者之间通过一个可被蛋白酶水解的铰链区(Hinge Domain)连接,该区在酶的激活过程中发生降解,从而使PKC活性部位暴露。在调节域还含有假底物位点,其与真底物位点的差别在于磷酸化位点的丙氨酸取代了丝氨酸/苏氨酸残基,因而不被磷酸化,从而封闭酶的活化中心。
由于假性底物的存在,静息状态下PKC均以无活性的形式存在于胞浆中,在外界刺激作用下,PKC移位(translocation)到胞膜而被激活。短暂的PKCs激活,在信号传导通路中所致的短期事件(如离子内流、分泌等)方面似乎起着重要作用;持续的PKC激活,可能在细胞增殖、分化及肿瘤发生等方面起着重要的作用。PKC是促癌剂PMA(phorbol,12-myristate-13-acetate,即佛波醇-12-豆蔻酰-13-乙酸)细胞内受体,PKC的过度表达导致细胞生长紊乱,并且触发肿瘤的形成。
Efferth等通过免疫细胞化学方法研究PKC在18种人肾细胞癌系中的表达。结果发现,PKC的高表达与癌细胞对阿霉素的耐药和P糖蛋白的高表达密切相关。从而证实PKC通过磷酸化和对P糖蛋白的调节参与肾细胞癌的内源性耐药。
ObjectiveProtein Kinase C is one of the earliest established Protein Kinase, made up of serine/threonine protien kinase family, whose construction close to each other , on which calcfium and phosphatide rely, activated by diacylglycerol (DG). PKC adjust different function of various kinds of cells, including tumor cell, though serine/threonine phosphorylation. PKC has important effect on cellular growth, differerntiate and such of signal conductions. For the past few years, it is known that participation of PKC in tumorous development and growth is key point factor of tumour cell adhesion, motion,invasion and transfusion.Study on cDNA of isozymes of PKC indicate that the polypeptide chain N -terminal sequence is regulatory domain, and the carboxyl terminal is catalytic domain is exposed. There is a hinge domain which can be hydrolyzed by protease between them. When enzyme were activated, the hinge domain is degradat-ed so thar PKC active site. The difference between pseudosubstrate site in regulatory domain and true substrate is that alanina in phosphorylation site displace serine/threonine residue, and close enzyme activation center instead of phosphorylation.PKC usually reside with inactivestyle in endochylema under quiescent condition. With surrounding stimulation, PKC is activated because of translation to cell membrane. Temporal PKC activation seems important effect in short - term events, ( such as ion inflow, secrete, and so on) , which induced in signal conduction access. Persistent PKC activation seems consequence effect in cell multiplication , differentiate, tumorigenesis and so on. PKC is tumor promoter phor-bol 12 - myristate - 13 - acetate intracellular receptor. Its over - expression in-
duces cell growth disorder and triggers tumorous form.Efferth et al. investigate PKC express in 18 kinds of human renal cell carcinoma system with immmuocytochemistry. There are intimate correlations between PKC high express and drug resistances of cancer cell for Adriamycin and P glucoprotein. PKC participate renal cell carcinoma endogenous drug resistances though PKC phosphorylation.PKC generally exist in animal, microbeorganism, and present in overall tissue and organ. There are four categories and thirteen subtypes in PKC based on different grades in isozyme construction, enzymic character, activating agent. Classical PKC (cPKC) : α、βⅠ、βⅡ、γ, which are activated by calcium , phosphatide, diacyglycerol (DAG) or phorbol ester. Novel PKC ( nPKC ) : (?) η、δ、ε、θ, which are activated by phosphatide, diacyglycerol( DAG) or phorbol ester instead of calcium. Atypical PKC (aPKC) : T/λ、ξ , which are only activated by phospholipin. Recently a kind of PKC is found , which is activated by phosphatidylinositol -4,5- diphosphate instead of calcium and DAG and is similar to the others in construction. So this kind of PKC is called the fourth kind of PKC. Various kinds of isozyme have different specificity of distribution and effects.We use research in vitro to discuss mechanism of MDR in renal cell carcinoma at the PKC subtype level, to provide the experiments about treating renal cell carcinoma.The experiment divides three parts:One; LR\BR recombination reactions were used to generate mammalian expression vectors of protein kinase C alpha cDNA with C - terminal fused GFP, and vectors were transfected into human RCC cell with Lipofectimine 2000. Western Blot and Inverted Fluorescent Microscope were used to determine the expression of PKC - a gene in RCC cells transferred by PKC - α cDNA.Two: LR\BR recombination reactions were used to generate mammalian expression vectors of protein kinase C alpha cDNA with C - terminal fused GFP, and vectors were transfected into human RCC cell with Iipofectimine 2000. Western Blot 、and Inverted Fluorescent Microscope were used to determine the expression of PKC - a in RCC cells transferred by PKC - α cDNA; RT - PCR
were used to determine the expression of MDR related gene in RCC cells transferred by PKC - αcDNA .Three: To investigated the expression of PKC - a in membrane and plasma of 30 cases renal cell carcinoma and normal adjacent tissue by Western blotting technology.MethodsCells culture786 - 0, was maintained in RPMI - 1640 medium, supplemented with 10% fetal bovine serum (FBS) , 2mM glutamine, 25mM HEPES at 37oC in a humidified, 5% CO_2incubator.Plasmid constructThe PKC - α cDNA was subcloned into pDONR~(TM)201 donor vector by PCR with Pyrobest DNA Polymerase using BP Clonase Reaction system. Entry clone was identified by DNA sequencing analysis. pcDNA - PKC - α - GFP recombi-nant plasmid expressing PKC - α with C - terminal fused Green Fluorescent Protein (GFP) was constructed with entry clone and GFP expression vector pcDNA - DEST47 using Gateway LR recombination reaction system. primers were designed as follows: 5 ' - ATG GCT GAC GTT TTC CCG GGC AA - 3 ' ;5 ' -CAT ACT GCA CTC TGT AAG ATG GGG -3'.Cell transfectionThe cells were transfected with pcDNA - PKC - α - GFP vector by Lipofec-timine2000 reagent. Positive clones were identified by GFP fluorescence microscopy.Selection of stable expressionThe stable expression clones (786 - 0/PKC - α - GFP) were established by the selection with G418 at a concentration of 400 μg/ml for four weeks. clone Isolated clones were analyzed for PKC - α - GFP expression by western blot analysis and fluorescence microscopy.
Multidrug resistance correlated genes assaysTo identify the alteration of Multidrug resistance correlated genes, MDR1, MRP1, LRP gene were assayed using RT - PCR , primers were designed as follows: MDR1:5' -ACACCCGACTTACAGATGATGTCTC-3' , 5' -CGAGAT-GGGTAACTGAAGTGAACAT - 3 ' , length of fragment:623bp; MRP1: 5 ' -AGTGACCTCTGGTCCTTAAACAAGG - 3 ' , 5 ' - GAGGTAGAGAGCAAG-GATGACTTGC - 3 ' , length of fragment:657bp; LRP: 5 ' - GAGCAGTTCA-CAGTGTTGTCC - 3 ' , 5 ' - GCGTGACGACAGAAACCGAAA - 3 ' ; length of fragment: 342bp.Expression and significance of PKC - a in renal cell carcinoma and normal adjacent tissueTo investigated the expression of PKC - a in membrane and plasma of 30 cases renal cell carcinoma and normal adjacent tissue by Western blotting technology.ResultsPKC - α cDNA was identified by DNA sequencing analysis.To investigate the effect of PKC - α on the drug resistance of renal cell carcinoma cells, we established stable cell clones expressing PKC - α - GFP using 786 -0 drug - sensitive RCC cell line. Three highest expression clones were i-dentified by western blot analysis. In order to further identify the character of expressed PKC - α - GFP, fluorescence microscopy was conducted to examine the distribution of PKC - a - GFP. As the result, these data demonstrated that stably expressed PKC - a - GFP has the same character as endogenous PKC -a.Effect of PKC - a on the drug resistance of 786 - 0MDR1,MRP1, LRP genes were detected in 786 - 0/PKC - α - GFP cells, and , for comparison, 786 - 0 cells. While 786 - 0 cells expressed MDR1, MRP1 and LRP were detected by RT - PCR , MDR1 expression in transfected 786 - 0 cells was significantly higher than that in homologous cells. Nevertheless for MRP1 and LRP, the diversification of expression were indistinguishable be-
引文
1 Takahashi M, Mukai H, Oishi K, et al. Association of immature hypophosphorylated protein kinase cepsilon with an anchoring protein CG - NAP. J. Biol.Chem.2000; 275(44) :34592 - 34596
2 Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature, 1984; 308(19) : 693 -670
3 Wu TT, Tsai JH, Tsai JH, et al. Augmented release of matrix metalloproteinase - 9 by PKC activation in organotypic cultures of human breast cancer and adjacent normal breast tissue and fibroadenoma. Chin J Physiol. 2004 Jun30; 47(2) :73 -78
4 Wang X, Wang Q, Hu W, et al. Regulation of phorbol ester - mediated TRAF1 induction in human colon cancer cells through a PKC/RAF/ERK/NF -kappaB -dependent pathway. Oncogene. 2004 Mar 11; 23(10) : 1885 - 1895
5 Sagawa N, Fujita H, Banno Y, et al. Gelsolin suppresses tumorigenicity through inhibiting PKC activation in a human lung cancer cell line, PC10. Br J Cancer. 2003 Feb 24; 88(4) :606 -612
6 Dempsey EC, Newton AC, Mochly - Rosen D, et al. Protein kinase C isozymes and the regulation of diverse cell responses. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000; 279(3) : L429 L438
7 Nakashima S. Protein kinase C alpha (PKC alpha) : regulation and biological function. J Biochem (Tokyo). 2002 Nov;132(5):669 -75
8 Engers R, Mrzyk S. Protein kinase C in human renal cell carcinomas: role in invasion and differential isoenzyme expression. Br J Cancer 2000 Mar;82 (5): 1063-1069
9 Lahn M, Sundell K, Moore S. Targeting protein kinase C - alpha (PKC - alpha) in cancer with the phosphorothioate antisense oligonucleotide aprinocarsen. Ann N Y Acad Sci. 2003 Dec; 1002:263 -270
2 Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature, 1984; 308(19) : 693 -670
3 Wu TT, Tsai JH, Tsai JH, et al. Augmented release of matrix metalloproteinase - 9 by PKC activation in organotypic cultures of human breast cancer and adjacent normal breast tissue and fibroadenoma. Chin J Physiol. 2004 Jun30; 47(2) :73 -78
4 Wang X, Wang Q, Hu W, et al. Regulation of phorbol ester - mediated TRAF1 induction in human colon cancer cells through a PKC/RAF/ERK/NF -kappaB -dependent pathway. Oncogene. 2004 Mar 11; 23(10) : 1885 - 1895
5 Sagawa N, Fujita H, Banno Y, et al. Gelsolin suppresses tumorigenicity through inhibiting PKC activation in a human lung cancer cell line, PC10. Br J Cancer. 2003 Feb 24; 88(4) :606 -612
6 Dempsey EC, Newton AC, Mochly - Rosen D, et al. Protein kinase C isozymes and the regulation of diverse cell responses. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000; 279(3) : L429 L438
7 Nakashima S. Protein kinase C alpha (PKC alpha) : regulation and biological function. J Biochem (Tokyo). 2002 Nov;132(5):669 -75
8 Engers R, Mrzyk S. Protein kinase C in human renal cell carcinomas: role in invasion and differential isoenzyme expression. Br J Cancer 2000 Mar;82 (5): 1063-1069
9 Lahn M, Sundell K, Moore S. Targeting protein kinase C - alpha (PKC - alpha) in cancer with the phosphorothioate antisense oligonucleotide aprinocarsen. Ann N Y Acad Sci. 2003 Dec; 1002:263 -270