鞘脂激活蛋白C促进前列腺癌细胞增殖、上调AR功能的分子机制研究
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摘要
鞘脂激活蛋白原(Prosaposin)是一种高度保守的热稳定糖蛋白(65-72kDa,527氨基酸残基),其基因位于10号染色体长臂(q21—22),全长17kb,有14个外显子。鞘脂激活蛋白原经水解后产生4种热稳定糖蛋白——鞘脂激活蛋白A、B、C、D(SaposinA、B、C、D),它们在溶酶体酶解鞘脂的过程中发挥重要作用。另外,作为一种分泌蛋白,鞘脂激活蛋白原还具有神经营养作用,能够促进神经胶质源性细胞突触增长、细胞增殖以及增加其抗凋亡的能力,其神经营养活性位于Saposin C的N端氨基酸序列(LIDNNRTEELLY)。Prosaposin、Saposin C以及包含这一功能区域的合成肽,如Prosaptide TX14A都可以与神经细胞膜上百日咳毒素敏感的细胞膜G蛋白偶联受体(GPCR)结合,刺激神经细胞中乙酰胆碱酯酶的活性,促进神经细胞的生长和分化。
鞘脂激活蛋白原分布于神经细胞和体液,除了其神经营养活性之外,在外周组织的广泛分布,提示可能还有其它生物活性。2004年Koochekpour S等报道鞘脂激活蛋白原在前列腺癌组织中高表达;并通过化学合成的Saposin C以及Prosaptide/X14A进行研究,发现其可以通过MAPK途径、P13K/Akt途径等信号通路促进前列腺癌细胞的生长、增殖、转移和侵袭,并且抑制癌细胞的凋亡。
前列腺癌是死亡率很高的恶性肿瘤,潜伏期长,易复发,且复发后往往转变为雄激素非依赖性前列腺癌(Androgen—independent prostate cancer,AIPC),临床上缺乏有效的治疗手段。目前发现有多种机制参与AIPC的演变过程,其中雄激素受体(androgen receptor,AR)相关途径的异常活化是AIPC发生发展的主要分子机制。AR作为核转录因子,通过介导雄激素的作用,结合于靶基因的特异元件AREs(androgen receptor elements)后,调节靶基因如前列腺特异抗原(Prostate-specific antigen,PSA)、人腺激肽释放酶2(human glandular kallikrein,hk2)等的表达,从而对前列腺的正常发育、调控其分泌功能发挥重要作用。而AR基因的扩增、突变,AR的辅助激活因子过表达,一些细胞因子、生长因子对AR的激素非依赖性激活,以及雄激素通过非基因组的(nongenomiC)信号途径对AR功能的调节等,使AR在激素非依赖性前列腺癌中持续活化而发挥功能。
由于Saposin C以及Prosaptide TX14A能够促进前列腺癌细胞增殖,而AR在前列腺癌细胞的增殖中起重要作用,本研究旨在探讨Saposin C及其功能结构域——鞘脂激活肽NP(Neurotrophic Peptide,NP)对AR表达和功能影响。我们首先构建含有Saposin C或NP与Tat-PTD蛋白转导结构域序列的融合蛋白的真核表达载体及其对照载体。Tat-PTD蛋白转导域具有不依赖ATP和膜通道、高效穿过生物膜的功能,它能将与其共价连接的多肽、蛋白质及DNA等分子跨膜运输出入几乎所有的组织和细胞。由于Saposin C为分泌蛋白,并通过膜受体介导其功能,我们在Saposin C/NP前面引入Tat-PTD,使Saposin C能通过Tat-PTD的蛋白转导功能实现跨膜转运并发挥作用。利用脂质体法转染雄激素依赖性前列腺癌细胞(LNCaP)和雄激素非依赖性前列腺癌细胞(PC3、DU145),通过MTT、RT-PCR、Western Blot、荧光素酶活性分析、免疫荧光技术及免疫共沉淀等方法分析Saposin C/NP对前列腺癌细胞生长的影响以及对AR基因表达和功能的影响。我们的研究结果如下:
(1)载体构建:以pcDNA3.1(+)为母载体,构建真核表达载体pcDNA-TAT-NP和pcDNA-NP:以带有myc和His标签的pcDNA3.1/myc-HisB(-)为母载体,插入SaposinC或NP序列分别得到pcDNA-TAT-Saposin C、pcDNA-Saposin C、pcDNA-NP/His的表达载体,测序分析序列正确。RT—PCR以及利用His标签进行Western Blot实验,分别在mRNA和蛋白水平检测所构建载体的表达,结果表明TAT-Saposin C、Saposin C、TAT—NP、NP均能够在前列腺癌细胞中表达;
(2)确定Saposin C和NP蛋白在细胞中的定位:免疫荧光技术检测结果表明,TAT-Saposin C定位于细胞内及细胞膜外,而Saposin C和NP只分布于细胞内;
(3)确定Saposin C/NP对细胞增殖的影响:流式细胞术结果表明,NP及TAT-NP能够促使细胞进入S和G_2/M期:MTT结果表明,TAT-Saposin C、Saposin C、TAT-NP和NP均能刺激激素依赖型及激素非依赖型前列腺癌细胞的增殖;
(4)确定Saposin C/NP对AR表达和功能的影响:RT-PCR、Western Blot结果显示,在mRNA和蛋白水平,TAT-Saposin C、Saposin C、TAT-NP和NP均能增加AR基因的表达,并增强AR promoter的表达活性;通过检测PSApromoter、hk2-3ARE报告基因的表达活性,证实TAT-Saposin C、Saposin C、TAT-NP和NP均能够增强AR的转录激活活性,促进AR的靶基因PSA、hk2的表达;
(5)确定SaposinC/NP对AR核定位的影响:免疫荧光结果显示,TAT-SaposinC、Saposin C和NP在雄激素缺乏的情况下,能够使AR激活并定位于细胞核;
(6)确定SaposinC/NP激活AR可能的机制:AR蛋白的磷酸化是AR活化、定位于细胞核的重要方式,MAPK和P13K/Akt信号途径均是使AR磷酸化的重要途径。由于无TAT蛋白转导结构域的SaposinC和NP定位于细胞内,但仍然能够促进细胞增殖,促进AR的表达、功能以及促进AR的核定位。首先通过采用百日咳毒素封闭细胞膜上的G蛋白受体,发现Saposin C、NP仍能增强AR的转录激活功能,促进PSA promoter和hk2-3ARE的表达活性,说明膜受体可能不参与Saposin C、NP激活AR的过程;采用蛋白酶抑制剂封闭P13K/Akt途径后,并不影响胞内Saposin C对ERK的磷酸化,继而不影响AR的磷酸化活化;采用免疫荧光和免疫共沉淀技术进一步证实Saposin C、NP分别与Src蛋白产生蛋白一蛋白间相互作用,同时促进Src与AR蛋白—蛋白间的相互作用。上述结果提示Saposin C/NP活化AR的过程可能不依赖于细胞膜G蛋白受体,可能通过与Src蛋白相互作用,促进Src与AR的结合进而激活Src活化AR的信号途径,最终使AR磷酸化激活。
总之,真核表达载体Saposin C/NP在细胞内的表达能够促进前列腺癌细胞增殖,增强AR的表达和功能,促进AR核定位,其机制可能不依赖于细胞膜上的G蛋白受体途径,而是通过与非受体酪氨酸蛋白激酶Src产生蛋白—蛋白相互作用,活化Src激活AR的信号途径,使AR磷酸化激活,促进下游靶基因的表达,进而促进细胞增殖。另外,我们的实验结果也表明,Saposin C/NP上调AR的作用与雄激素具有协同效应,但雄激素活化AR的作用要强于Saposin C/NP。综上所述,神经营养因子Saposin C可能是在AIPC中促进AR活化的一个新的蛋白因子,其中Saposin C的功能结构域NP可能具有重要作用。
Prosaposin is a highly conserved glycoprotein(65-72kDa,527 amino acids),the gene was localized to the long arm of chromosome 10(q21-22)and encompasses 17 kb of genomic sequence with 14 exons.Prosaposin is the precursor of four small heat-stable sphingolipid activator proteins(saposins A,B,C and D,which are required for the enzymatic hydrolysis of sphingolipids in lysosomes).In addition to its intracellular presence and function,Prosaposin is also expressed as a secretory protein and also a well-known neurotrophic factor in vivo and in vitro.As a secretory protein, Prosaposin could promote survival,prevent apoptosis and stimulate synapse growth of neuroglial-derived cells.Prosaposin functional sequence is localized to a 12-amiono acid stretch at NH_2-terminal end of the saposin C domain (LIDNNRTEELLY).Several synthetic peptides(14-22 residues,e.g.TX14A)derived from this region are equally as bioactive as Prosaposin.Prosaposin,saposin C,and prosaptides exert their neurotrophic effects by binding to a putative high affinity G protein-coupled receptor(GPCR)which could be blocked by Pertussis Toxin.
Some reports demonstrated that inactivation of prosaposin gene affected the development of the prostate gland.Koochekpour S et al affirmed that prosaposin gene amplified and overexpressed in prostate cancer cells,and also demonstrated that exogenous saposin C and Prosaptide TX14A stimulated growth,metastasis and invasion via activated MAPK,PI3K/Akt et al singling pathways in prostate cancer.
Prostate cancer is the highly strong lethal disease,once relapsing most of the patients will develop into hormone-resistant or hormone-independent prostrate cancer. At present,this is no effective treatment options for the patients.Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor(AR)has been shown to play a critical role because AR was the only overexpressed persistently molecular during the development and progression of the AIPC.The AR is a ligand-dependent transcription factor of the nuclear steroid hormone receptor superfamily.Binding androgen,AR can be activated by phosphorylation.The active AR enters into the nuclear and binds to the androgen-responsive element(ARE)of the target DNA in order to activate the target gene transcription and promote cells growth.During the development of AIPC the singling pathways AR mediating involved in AR gene mutation,AR gene amplification,the aberrant regulation of AR by growth factors and cytokines,and AR cofactors change.Evidences have been shown that ligand-independent activation of AR is concerned with the development of AIPC.
The present study is aimed to determine effect of Saposin C and its function domain neurotrophic peptide(NP)on androgen receptor(AR)expression and transcriptional activity and cells proliferation in prostate cancer.Then research deeply on molecular mechanism.We constructed DNA vectors that can express Saposin C/NP or a chimeric peptide of a viral TAT transduction domain and Saposin C/NP by gene cloning technology.The effect of ectopic expression of Saposin C/NP with or without the TAT transduction domain on cell growth was examined by MTT assay. Then reverse transcriptase-polymerase chain reaction(RT-PCR),Western blot analysis, transient transfection experiments and confocal laser microscopy were used to determine the effect of Saposin C/NP on AR expression and activation.To further approach the mechanism of Saposin C/NP on AR,we used pertussis toxin and inhibitor of MAPK or PI3K/Akt by Western Blot analysis,transient transfection experiments,confocal laser microscopy and co-immunoprecipitation.
Our research showed that the eukaryotic expression vectors pcDNA-NP/His, pcDNA-TAT-Saposin C/pcDNA-TAT-NP and pcDNA-Saposin C/pcDNA-NP were constructed and confirmed by sequencing,and they can normally express in prostate cancer cells.
Immunofluorescence staining demonstrated that TAT-Saposin C located on both cell membrane and interior of cell,Saposin C and NP located only in cell.
Flow cytometry analysis showed that expressions of TAT-NP and NP had a stimulating effect on prostate cancer cell proliferation,and promoted the cells to enter S and G_2/M phase.MTT analysis showed that expressions of TAT-Saposin C/TAT-NP and Saposin C/NP had a stimulating effect on prostate cancer cells proliferation.
The RT-PCR and Western blot analyses displayed that TAT-Saposin C/TAT-NP and Saposin C/NP induced AR gene expression.Dual-luciferace experiment results demonstrated that TAT-Saposin C/TAT-NP and Saposin C/NP activated transactivation of the AR.
Immunofluorescence staining showed that TAT-Saposin C,Saposin C and NP could activate AR and locate at cellular nucleus in the absence of androgens.
The effect of intracellular Saposin C/NP on the AR function and the proliferation of prostate cancer cells was not through the G-protein receptor pathway,but with Src and AR interaction,to form a complex,thereby activating non-receptor tyrosine protein kinase Src.The activated Src further activated MAPK and PI3K/Akt pathway. AR activation may also be directly activated Src.
Collectively,our study showed that Saposin C,as a growth stimulating factor to PCa cells,exerted the activity by increasing the expression and activation of the AR in a ligand-independent manner.It is reasonably believed that Saposin C could stimulate proliferation of PCa by non-GPCR pathway,but interact with Src and AR form a complex.The Src and AR were activated in turn.It is certainly that the MAPK or PI3K/AKT pathway participate the course of AR activation.Furthermore,the synergistic effect of Saposin C and androgen on the expression and transactivation of AR was observed.
鞘脂激活蛋白原分布于神经细胞和体液,除了其神经营养活性之外,在外周组织的广泛分布,提示可能还有其它生物活性。2004年Koochekpour S等报道鞘脂激活蛋白原在前列腺癌组织中高表达;并通过化学合成的Saposin C以及Prosaptide/X14A进行研究,发现其可以通过MAPK途径、P13K/Akt途径等信号通路促进前列腺癌细胞的生长、增殖、转移和侵袭,并且抑制癌细胞的凋亡。
前列腺癌是死亡率很高的恶性肿瘤,潜伏期长,易复发,且复发后往往转变为雄激素非依赖性前列腺癌(Androgen—independent prostate cancer,AIPC),临床上缺乏有效的治疗手段。目前发现有多种机制参与AIPC的演变过程,其中雄激素受体(androgen receptor,AR)相关途径的异常活化是AIPC发生发展的主要分子机制。AR作为核转录因子,通过介导雄激素的作用,结合于靶基因的特异元件AREs(androgen receptor elements)后,调节靶基因如前列腺特异抗原(Prostate-specific antigen,PSA)、人腺激肽释放酶2(human glandular kallikrein,hk2)等的表达,从而对前列腺的正常发育、调控其分泌功能发挥重要作用。而AR基因的扩增、突变,AR的辅助激活因子过表达,一些细胞因子、生长因子对AR的激素非依赖性激活,以及雄激素通过非基因组的(nongenomiC)信号途径对AR功能的调节等,使AR在激素非依赖性前列腺癌中持续活化而发挥功能。
由于Saposin C以及Prosaptide TX14A能够促进前列腺癌细胞增殖,而AR在前列腺癌细胞的增殖中起重要作用,本研究旨在探讨Saposin C及其功能结构域——鞘脂激活肽NP(Neurotrophic Peptide,NP)对AR表达和功能影响。我们首先构建含有Saposin C或NP与Tat-PTD蛋白转导结构域序列的融合蛋白的真核表达载体及其对照载体。Tat-PTD蛋白转导域具有不依赖ATP和膜通道、高效穿过生物膜的功能,它能将与其共价连接的多肽、蛋白质及DNA等分子跨膜运输出入几乎所有的组织和细胞。由于Saposin C为分泌蛋白,并通过膜受体介导其功能,我们在Saposin C/NP前面引入Tat-PTD,使Saposin C能通过Tat-PTD的蛋白转导功能实现跨膜转运并发挥作用。利用脂质体法转染雄激素依赖性前列腺癌细胞(LNCaP)和雄激素非依赖性前列腺癌细胞(PC3、DU145),通过MTT、RT-PCR、Western Blot、荧光素酶活性分析、免疫荧光技术及免疫共沉淀等方法分析Saposin C/NP对前列腺癌细胞生长的影响以及对AR基因表达和功能的影响。我们的研究结果如下:
(1)载体构建:以pcDNA3.1(+)为母载体,构建真核表达载体pcDNA-TAT-NP和pcDNA-NP:以带有myc和His标签的pcDNA3.1/myc-HisB(-)为母载体,插入SaposinC或NP序列分别得到pcDNA-TAT-Saposin C、pcDNA-Saposin C、pcDNA-NP/His的表达载体,测序分析序列正确。RT—PCR以及利用His标签进行Western Blot实验,分别在mRNA和蛋白水平检测所构建载体的表达,结果表明TAT-Saposin C、Saposin C、TAT—NP、NP均能够在前列腺癌细胞中表达;
(2)确定Saposin C和NP蛋白在细胞中的定位:免疫荧光技术检测结果表明,TAT-Saposin C定位于细胞内及细胞膜外,而Saposin C和NP只分布于细胞内;
(3)确定Saposin C/NP对细胞增殖的影响:流式细胞术结果表明,NP及TAT-NP能够促使细胞进入S和G_2/M期:MTT结果表明,TAT-Saposin C、Saposin C、TAT-NP和NP均能刺激激素依赖型及激素非依赖型前列腺癌细胞的增殖;
(4)确定Saposin C/NP对AR表达和功能的影响:RT-PCR、Western Blot结果显示,在mRNA和蛋白水平,TAT-Saposin C、Saposin C、TAT-NP和NP均能增加AR基因的表达,并增强AR promoter的表达活性;通过检测PSApromoter、hk2-3ARE报告基因的表达活性,证实TAT-Saposin C、Saposin C、TAT-NP和NP均能够增强AR的转录激活活性,促进AR的靶基因PSA、hk2的表达;
(5)确定SaposinC/NP对AR核定位的影响:免疫荧光结果显示,TAT-SaposinC、Saposin C和NP在雄激素缺乏的情况下,能够使AR激活并定位于细胞核;
(6)确定SaposinC/NP激活AR可能的机制:AR蛋白的磷酸化是AR活化、定位于细胞核的重要方式,MAPK和P13K/Akt信号途径均是使AR磷酸化的重要途径。由于无TAT蛋白转导结构域的SaposinC和NP定位于细胞内,但仍然能够促进细胞增殖,促进AR的表达、功能以及促进AR的核定位。首先通过采用百日咳毒素封闭细胞膜上的G蛋白受体,发现Saposin C、NP仍能增强AR的转录激活功能,促进PSA promoter和hk2-3ARE的表达活性,说明膜受体可能不参与Saposin C、NP激活AR的过程;采用蛋白酶抑制剂封闭P13K/Akt途径后,并不影响胞内Saposin C对ERK的磷酸化,继而不影响AR的磷酸化活化;采用免疫荧光和免疫共沉淀技术进一步证实Saposin C、NP分别与Src蛋白产生蛋白一蛋白间相互作用,同时促进Src与AR蛋白—蛋白间的相互作用。上述结果提示Saposin C/NP活化AR的过程可能不依赖于细胞膜G蛋白受体,可能通过与Src蛋白相互作用,促进Src与AR的结合进而激活Src活化AR的信号途径,最终使AR磷酸化激活。
总之,真核表达载体Saposin C/NP在细胞内的表达能够促进前列腺癌细胞增殖,增强AR的表达和功能,促进AR核定位,其机制可能不依赖于细胞膜上的G蛋白受体途径,而是通过与非受体酪氨酸蛋白激酶Src产生蛋白—蛋白相互作用,活化Src激活AR的信号途径,使AR磷酸化激活,促进下游靶基因的表达,进而促进细胞增殖。另外,我们的实验结果也表明,Saposin C/NP上调AR的作用与雄激素具有协同效应,但雄激素活化AR的作用要强于Saposin C/NP。综上所述,神经营养因子Saposin C可能是在AIPC中促进AR活化的一个新的蛋白因子,其中Saposin C的功能结构域NP可能具有重要作用。
Prosaposin is a highly conserved glycoprotein(65-72kDa,527 amino acids),the gene was localized to the long arm of chromosome 10(q21-22)and encompasses 17 kb of genomic sequence with 14 exons.Prosaposin is the precursor of four small heat-stable sphingolipid activator proteins(saposins A,B,C and D,which are required for the enzymatic hydrolysis of sphingolipids in lysosomes).In addition to its intracellular presence and function,Prosaposin is also expressed as a secretory protein and also a well-known neurotrophic factor in vivo and in vitro.As a secretory protein, Prosaposin could promote survival,prevent apoptosis and stimulate synapse growth of neuroglial-derived cells.Prosaposin functional sequence is localized to a 12-amiono acid stretch at NH_2-terminal end of the saposin C domain (LIDNNRTEELLY).Several synthetic peptides(14-22 residues,e.g.TX14A)derived from this region are equally as bioactive as Prosaposin.Prosaposin,saposin C,and prosaptides exert their neurotrophic effects by binding to a putative high affinity G protein-coupled receptor(GPCR)which could be blocked by Pertussis Toxin.
Some reports demonstrated that inactivation of prosaposin gene affected the development of the prostate gland.Koochekpour S et al affirmed that prosaposin gene amplified and overexpressed in prostate cancer cells,and also demonstrated that exogenous saposin C and Prosaptide TX14A stimulated growth,metastasis and invasion via activated MAPK,PI3K/Akt et al singling pathways in prostate cancer.
Prostate cancer is the highly strong lethal disease,once relapsing most of the patients will develop into hormone-resistant or hormone-independent prostrate cancer. At present,this is no effective treatment options for the patients.Although the mechanisms involved in the progression of prostate cancer are not entirely understood, androgen receptor(AR)has been shown to play a critical role because AR was the only overexpressed persistently molecular during the development and progression of the AIPC.The AR is a ligand-dependent transcription factor of the nuclear steroid hormone receptor superfamily.Binding androgen,AR can be activated by phosphorylation.The active AR enters into the nuclear and binds to the androgen-responsive element(ARE)of the target DNA in order to activate the target gene transcription and promote cells growth.During the development of AIPC the singling pathways AR mediating involved in AR gene mutation,AR gene amplification,the aberrant regulation of AR by growth factors and cytokines,and AR cofactors change.Evidences have been shown that ligand-independent activation of AR is concerned with the development of AIPC.
The present study is aimed to determine effect of Saposin C and its function domain neurotrophic peptide(NP)on androgen receptor(AR)expression and transcriptional activity and cells proliferation in prostate cancer.Then research deeply on molecular mechanism.We constructed DNA vectors that can express Saposin C/NP or a chimeric peptide of a viral TAT transduction domain and Saposin C/NP by gene cloning technology.The effect of ectopic expression of Saposin C/NP with or without the TAT transduction domain on cell growth was examined by MTT assay. Then reverse transcriptase-polymerase chain reaction(RT-PCR),Western blot analysis, transient transfection experiments and confocal laser microscopy were used to determine the effect of Saposin C/NP on AR expression and activation.To further approach the mechanism of Saposin C/NP on AR,we used pertussis toxin and inhibitor of MAPK or PI3K/Akt by Western Blot analysis,transient transfection experiments,confocal laser microscopy and co-immunoprecipitation.
Our research showed that the eukaryotic expression vectors pcDNA-NP/His, pcDNA-TAT-Saposin C/pcDNA-TAT-NP and pcDNA-Saposin C/pcDNA-NP were constructed and confirmed by sequencing,and they can normally express in prostate cancer cells.
Immunofluorescence staining demonstrated that TAT-Saposin C located on both cell membrane and interior of cell,Saposin C and NP located only in cell.
Flow cytometry analysis showed that expressions of TAT-NP and NP had a stimulating effect on prostate cancer cell proliferation,and promoted the cells to enter S and G_2/M phase.MTT analysis showed that expressions of TAT-Saposin C/TAT-NP and Saposin C/NP had a stimulating effect on prostate cancer cells proliferation.
The RT-PCR and Western blot analyses displayed that TAT-Saposin C/TAT-NP and Saposin C/NP induced AR gene expression.Dual-luciferace experiment results demonstrated that TAT-Saposin C/TAT-NP and Saposin C/NP activated transactivation of the AR.
Immunofluorescence staining showed that TAT-Saposin C,Saposin C and NP could activate AR and locate at cellular nucleus in the absence of androgens.
The effect of intracellular Saposin C/NP on the AR function and the proliferation of prostate cancer cells was not through the G-protein receptor pathway,but with Src and AR interaction,to form a complex,thereby activating non-receptor tyrosine protein kinase Src.The activated Src further activated MAPK and PI3K/Akt pathway. AR activation may also be directly activated Src.
Collectively,our study showed that Saposin C,as a growth stimulating factor to PCa cells,exerted the activity by increasing the expression and activation of the AR in a ligand-independent manner.It is reasonably believed that Saposin C could stimulate proliferation of PCa by non-GPCR pathway,but interact with Src and AR form a complex.The Src and AR were activated in turn.It is certainly that the MAPK or PI3K/AKT pathway participate the course of AR activation.Furthermore,the synergistic effect of Saposin C and androgen on the expression and transactivation of AR was observed.
引文
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5. Hu X, Lazar MA. Transcriptional repression by nuclear hormone receptors. Trends in Endocrinology and metabolism, 2000; 11:6-10
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8. Snock R, Bruchovsky N, Kasper S, Matusik RJ, Gleave M, Sato N, Mawji NR, Rennie PS. Differential transactivation by the androgen receptor in prostate cancer cells. Prostate, 1998; 36: 256-63
9. Heinlein CA, Chang C. Androgen receptor coregulators: an overview.Endocrine Rev.2002;23: 175-200
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