Nkx3.1与p27KIP1在激素抵抗前列腺癌细胞中的结合效应研究
摘要
研究背景
前列腺癌可从局灶性病变，向局部进展期和进展期发展。前列腺癌的治疗方案取决于疾病的分期，对早期和局部进展期患者可通过根治性前列腺切除或放射治疗方法治愈。对进展期和发生转移的前列腺癌最初可用手术去势或药物去势等雄激素剥夺治疗，或在以上基础上加用雄激素受体(Androgen receptor；AR)阻止剂达到最大雄激素剥夺。大多数患者对雄激素剥夺治疗起初反应良好，18～24月后，几乎所有患者不可避免地出现循环血前列腺特异抗原(Prostate specific antigen；PSA)升高、肿瘤重新生长为特征的激素抵抗前列腺癌(Hormone refractory prostate cancer；HRPC)。HRPC治疗是世界性难题，目前尚无理想治疗方法。通过对HRPC细胞生物学行为进行修饰的策略可能对HRPC治疗有潜在的临床意义。
同源框基因是一类与发育相关的基因，该类基因都有一段共同的DNA序列，长约180bp，即同源框，该序列编码的蛋白质含60个氨基酸，这段氨基酸被称为同源域。具有同源域的蛋白通过对目标基因的表达调节来控制机体的发育程序，使机体在发育时按正确的时间、空间和组织类型发育。同时该类基因也能参与对细胞癌化过程的调节。但是该类基因并不足以直接造成癌化，而只是对癌化起促进或者抑制作用。由于同源框基因在发育和分化中具有的关键性调控作用以及在肿瘤时经常发生表达失常，目前认为，它们是研究肿瘤形成和胚胎发育间联系的理想靶点。
在前列腺癌，也有同源框基因的表达异常。Nkx3.1是受雄激素调节的前列腺特异表达的同源框基因。人类Nkx3.1定位于染色体8p21，约85％前列腺癌患者该区域缺损。通过对前列腺癌样本的基因分析发现Nkx3.1蛋白编码区无肿瘤特异性突变。然而，在前列腺癌中Nkx3.1失表达与前列腺癌进展和激素抵抗疾病强烈相关(p＜0.0001)。在激素依懒前列腺癌LNCaP细胞中Nkx3.1表达且雄激素刺激下其表达水平增加，而在激素抵抗前列腺癌的PC3细胞株中Nkx3.1失表达。最近在复合的突变鼠模型中研究发现Nkx3.1失表达与脂质磷酸酶PTEN失表达一起协同激活Akt信号途径促进前列腺癌进展。
p27主要与细胞周期素(cyclin)结合而发挥对cyclin-CDK的抑制作用。p27对周期素依赖激酶(Cyclin-dependent kinase；CDK)的抑制作用有两方面，一方面p27能抑制已结合到cyclin并被激活的CDK活性；另一方面p27也可以抑制CDK的激活过程，最终抑制细胞周期G1→S的转变，故目前认为其是一个肿瘤抑制因子。
与Nkx3.1相似，在前列腺癌中大部分p27的表达丢失，而且p27的表达降低和丢失与差的临床预后相关。研究发现，外源性恢复前列腺癌细胞株的p27可引起细胞周期阻滞在G0／G1期，并引起细胞凋亡增加。在大鼠中p27缺损也可引起前列腺上皮增生，并可与PTEN缺损一起协同引起前列腺肿瘤的发展。
尽管Nkx3.1与p27都与前列腺癌抑制有关，但是二者的肿瘤抑制均存在存在单体失能，亦即单个等位基因缺损不足以引起前列腺癌发生。Gary等在Nkx3.1与p27复合突变的等位基因大鼠模型中研究发现，Nkx3.1和p27协同抑制前列腺上皮细胞增殖和类似前列腺上皮内瘤的瘤前病变形成。这说明在前列腺癌的发动中，Nkx3.1与p27二者存在协同联系作用。
随着前列腺癌的进展，向激素抵抗状态转化时Nkx3.1与p27的表达渐渐降低直至消失，如恢复激素抵抗前列腺癌PC3细胞株的p27表达可一定程度抑制其生长。如同时恢复二者在PC3细胞株中的表达获得协同的抗肿瘤效应吗?这方面研究目前国内外尚无报道，我们拟对此问题作进一步探讨。
第一部分人类Nkx3.1和p27基因全长读码框(ORF)的克隆及真核表达载体的构建
【研究目的】
为了对Nkx3.1与p27的功能及其二者在激素抵抗前列腺细胞株中结合效应进行全面的研究，我们构建了Nkx3.1和p27真核表达载体。利用这几套载体我们可以恢复PC3细胞的Nkx3.1或p27的表达，或二者共同表达。
【材料和方法】
我们在人类正常前列腺组织中使用RT-PCR技术扩增了Nkx3.1与p27全长读码框(ORF)，并将二者克隆到T载体。并进一步以T载体为模板，把Nkx3.1克隆到真核细胞高效表达载体pcDNA3.1(+)(Invitrogen)，用同样的方法把p27克隆到真核细胞高效表达载体pcDNA3.1(-)(Invitrogen)。特别要说明的是在Nkx3.1 ORF和p27 ORF上分别加上HisTag和HAtag并构建在载体内，这样对表达产物可以用抗HisTag和抗HAtag单抗进行鉴定。
此外，为了评估质粒的转染效率和基因核定位，我们还构建了报告基因表达载体。分别把Nkx3.1 ORF和p27 ORF构建到pEGFP-C1和pEGFP-N1(Clontech)。所有构建质粒得到测序(上海博亚生物公司)证实。
【结果】
我们成功地构建了Nkx3.1和p27真核系列表达载体，这二套载体可在真核细胞内高效表达、His或HA标签纯化等。我们还成功构建Nkx3.1和p27的报告基因质粒系统，可以鉴定质粒的转染效率，基因定位等。通过以上二套质粒系统，我们可对Nkx3.1或／和p27的功能进行较为全面的评价。
【结论】
1．本部分成功构建了Nkx3.1和p27真核表达载体和报告载体系列。利用这二套载体我们可对Nkx3.1、p27的功能进行全面的评估。
2．本部分在PCR扩增技术上，发展了“富含GC-缓冲液”，能有效降低Tm值。与二甲基亚砜(DMSO)、甲酰胺等经典的PCR扩增添加试剂比较，该缓冲液具有更多的优势。
第二部分质粒表达分析、报告基因分析和基因表达定位分析
【研究目的】
为了评估构建质粒在真核细胞中转染效率、表达水平，从而为进一步研究Nkx3.1和p27生物学功能打好基础。
【材料和方法】
我们用Lipofectamine2000瞬时转染PC3细胞，转染后24、48小时时间点用western blot法检测Nkx3.1，p27蛋白表达水平。在报告基因分析中，我们用荧光显微镜观察GFP融合蛋白表达状况，并用流式细胞术检测质粒转染效率。同时我们在用4％多聚甲醛固定细胞后，用DAPI染色对Nkx3.1与p27基因表达进行定位。
【结果】
Western blot检查发现转染后Nkx3.1和p27蛋白在PC3细胞中高表达，并且表达水平与HisTag和HAtag表达相一致。在荧光显微镜下Nkx3.1-GFP和p27-GFG融合蛋白大量表达，用流式细胞检测转染效率为50％-55％(24小时)和46％-50％(48小时)。用DAPI染色基因表达定位发现Nkx3.1与p27均定位于细胞核。
【结论】
1．在真核细胞中pcDNA3.1-Nkx3.1-His和pcDNA3.1-P27-HA高表达；
2．Lipofectamine法转染效率在24h时间点约50％-55％，48h时间点约为46％-50％；
3．基因表达定位分析示Nkx3.1和p27均为核蛋白。
第三部分Nkx3.1、p27及二者联合对PC3细胞增殖影响的研究
【研究目的】
通过对不同转染组别细胞增殖指标的检测，分析Nkx3.1，p27抗增殖效应及联合应用Nkx3.1与p27是否存在抗PC3细胞增殖结合效应。
【材料和方法】
用空质粒、pcDNA3.1-Nkx3.1，pcDNA3.1-p27和Nkx3.1+p27分别转染PC3细胞，以空质粒组为对照组。分别在0h，24h，48h时间点用MTT方法检测细胞增殖。在相同时间点用台盼蓝染色行活细胞计数和死亡细胞计数。
【结果】
MTT分析发现，24h时间点Nkx3.1+p27组与对照组相比存在差异(p＜0.05)；在48h时间点p27组与对照组存在差异(p＜0.05)，而Nkx3.1+p27组与对照组存在显著差异(p＜0.01)。
台盼蓝排斥试验活细胞计数，24h时间点Nkx3.1+p27组与对照组相比存在差异(p＜0.05)，48h时间点Nkx3.1+p27组与对照组存在显著差异(p＜0.01)。
台盼蓝死亡细胞计数，在24h和48h时间点Nkx3.1+p27组与对照组相比存在差异(p＜0.05)。
【结论】
1．Nkx3.1对PC3细胞增殖无明显抑制作用；
2．p27对PC3细胞增殖存在抑制作用；
3．Nkx3.1与p27存在协同的抗PC3细胞增殖作用。
第四部分Nkx3.1、p27及二者联合对PC3细胞周期影响的研究
【研究目的】
通过对不同转染组细胞周期检测，分析Nkx3.1、p27及Nkx3.1+p27对PC3细胞周期影响。
【材料和方法】
用空质粒、pcDNA3.1-Nkx3.1，pcDNA3.1-p27和Nkx3.1+p27分别转染PC3细胞，在24h、48h时间点分别收集细胞，用流式细胞仪(PI染色法)检测各组细胞周期分布。
【结果】Nkx3.1，p27，Nkx3.1+p27转染组，G0／G1期细胞比例渐渐升高，但S期和G2／M期比例相对下降。在24h时间点p27和Nkx3.1+p27组与对照组相比，G0／G1期存在差异(p＜0.05)。在48h时间点，p27组与对照组相比G0／G1期存在差异(p＜0.05)，而Nkx3.1+p27组与对照组相比G0／G1期存在显著差异。
【结论】
1．Nkx3.1虽然可使PC3细胞的G0／G1期细胞比例增加，但与对照组比较无统计意义；
2．p27可使PC3细胞周期阻滞在G0／G1期；
3．Nkx3.1与p27使PC3细胞阻滞在G0／G1期具有协同性。
第五部分Nkx3.1、p27及二者联合对PC3细胞凋亡及其凋亡相关蛋白影响的研究
【研究目的】
通过对转染各组细胞的不同时间点细胞凋亡率及凋亡相关蛋白检测，分析Nkx3.1、p27及Nkx3.1+p27对PC3细胞凋亡影响及对凋亡机制作初步探讨。
【材料和方法】
用空质粒、pcDNA3.1-Nkx3.1，pcDNA3.1-p27和Nkx3.1+p27分别转染PC3细胞，在24h，48h时间点收集细胞。用AV+PI双染法流式细胞术检测细胞凋亡率。用western blot方法检测各组不同时间点的凋亡相关蛋白(bcl-2，bax，caspase-3，PARP)表达变化。
【结果】
与对照组相比，Nkx3.1与p27引起PC3细胞凋亡率均增加。当Nkx3.1与p27结合处理后，引起明显细胞凋亡协同效应。与我们的调亡率检测结果一致的是，在Nkx3.1+p27组与p27及Nkx3.1组相比，bcl-2表达明显增加，bax表达上升，caspase-3表达被激活，PARP劈裂片断表达增加。
【结论】
1．Nkx3.1与p27均可引起PC3细胞凋亡率增加；
2．Nkx3.1与p27引起PC3凋亡存在协同效应；
3．凋亡协同性产生主要是原因是凋亡拮抗物bcl-2表达下降、凋亡促进物bax表达增加，最终引起caspase-3激活。产生协同凋亡效应主要通过线粒体途径来实现。
Prostate cancer now represents a serious health problem worldwide. Despite intense investigations, it has remained a daunting task to elucidate the mechanisms underlying disease progression. Androgen ablation, however, remains the only effective therapy for patients with advanced disease. Approximately 80% of patients achieve symptomatic and /or objective response after androgen withdrawal, but progression to androgen independence ultimately occurs in almost all cases. Because androgen-independent prostate cancer cells eventually lead to death, successful strategies to modify the biological behavior of these cells may potentially have the most significant clinical impact.
Nkx3.1 is an androgen-regulated homeobox gene that is largely specific to prostate for expression[1,2]. The human Nkx3.1 has been mapped to chromosome 8p21, and has been located in a region of the chromosome deleted in approximately 85%of prostate cancer[3,4]. However, no tumor-specific mutations of the Nkx3.1 protein-coding region have been identified by genetic analysis of human prostate cancer samples[5]. Nevertheless loss of Nkx3.1 expression is strongly associated with hormone-refractory disease and advanced tumor stage in prostate cancer (p<0.0001) [6]. In the LNCaP androgen-dependent prostate cancer cell line, Nkx3.1 is expressed at a basal level that was increased upon androgen stimulation. In contrast, there was no Nkx3.1 expression in androgen-independent PC3 cells[7]. More recently it was shown that in compound mutant mice the loss of Nkx3.1 along with the loss of the lipid phosphatase Pten cooperates in prostate cancer progression through synergistic activation of the Akt pathway[8].
Similar to Nkx3.1, expression of the cyclin-dependent kinase inhibitor p27~(KIP1) is lost in a large fraction of human prostate cancer, and reduced or absent expression of p27~(KIP1) correlates with poor clinical outcome[9]. Exogenous p27~(KIP1) overexpression results in cell cycle regulation and an increase in cell apoptosis in the human prostate carcinoma cell lines[10]. Deletion of p27~(KIP1) in mice also results in prostatic epithelial hyperplasia and cooperates with loss of Pten to promote prostate tumor development[11]. Gary et al found that Nkx3.1 and p27~(KIP1) cooperate to suppress the proliferation of prostatic epithelial cells and the formation of preneoplastic lesions resembling prostatic intraepithelial neoplasia[12], whereas there was no literature report about combined effect of Nkx3.1 and p27~(KIP1) in androgen independent prostate cancer.
Consequently, we hypothesized that the anticancer effect by combination therapy of Nkx3.1 and p27~(KIP1) would be superior to single gene in PC3 prostate cancer cells. The objectives of this study were to test whether induction of apoptotic cell death by p27~(KIP1) or Nkx3.1 gene transfer is enhanced by Nkx3.1 or p27~(KIP1), and to determine whether combined use of Nkx3.1 and p27~(KIP1) exist synergistic antitumor effect in the androgen-independent human prostate PC3 cells.
We use two kinds of prostate cell line LNCaP and PC3 to demonstrate our hypothesis. LNCaP is an androgen-depedent prostate cacer cell line and expresses NKX3.1 and p27, and PC3 is an androgen-independent prostate cancer cell line without expressing NKX3.1 and p27. These cell were maintained at 37℃ in a humidified atmosphere of 5% CO_2/95% air and serially passaged in RPMI-1640 medium (Hyclone), supplemented with 10% fetal bovine serum.
RT-PCR method was used to obtain the amplified Nkx3.1-HisTag and p27-HAtag DNA, Nkx3.1-HisTag fragment was directionally cloned into the EcoR I and BamH I restriction sites of plasmid pcDNA3.1(+) (Invitrogen) and p27-HAtag was cloned into the same restriction sites of pcDNA3.1(-) (Invitrogen).In addition, Nkx3.1 was cloned into pEGFP-C1(Clontech) at Bgl II and EcoR I sites, p27 was cloned into pEGFP-N1 (Clontech) at BamH I and EcoR I sites. The successful cloning was confirmed by sequencing the plasmid.
Plasmid DNAs were delivered into cells with LipofectamineTM 2000 (Invitrogen) following the manufacturer's protocol. Reporter gene expression was monitored by fluorescence microscopy and quantification of GFP fusion protein by flow cytometry
Cell viability was evaluated by MTT assay. The viable cell number was monitored with absorbance at 570nm. At the same time, We used a trypan blue dye exclusion assay to determine
cell viability after the treatments. After the cells were collected by trypsinization, they were stained with trypan blue, and the total number cells and the number of viable cells in each well were counted.
We used flow cytometric to analysis the cell cycle and apoptosis. The cells transfected with various plasmid DNA for 24 h or 48 h were assayed for cell cycle progression by the propidium iodide (PI) staining method. However, Annexin V (Annexin V-FITC) and PI double staining was used to determine apoptosis.
To prodive further confirmation that the synergistic effect of combined use Nkx3.1 and p27 induces apoptosis in PC3 cells, we also analyzed apoptotic-related proteins using western blot method.
In the present study, significant anti-proliferative effect of p27~(KIP1) was shown on PC3 human prostate cancer cells in vitro. The combination of Nkx3.1 and p27~(KIP1) exhibited synergistic inhibitory effects on growth of PC3 cells: increase in cellular apoptosis and enhancement in G0/G1 phase arrest of cell cycle. These result suggest that the combining use of Nkx3.1 and p27~(KIP1) possesses novel anti-tumor action on PC3 human prostate cancer cells.
Proliferation status was assessed by MTT assay. Compared with control-plasmid group, cells in Nkx3.1plus p27 group experienced more greater growth inhibition than Nkx3.1 group and p27 group. Transfected PC3 cells fail to produce a measurable difference between control and Nkx3.1 group at any of the time point. However, transfection of PC3 cells with p27 did produce a significantly lower absorbance at the 48 h time point when compare to the control (p < 0.05). When Nkx3.1 and p27 were combined, the PC3 cell line exhibited a significant decrease in cell growth at 24 (p < 0.05) and 48 (p < 0.01) hours, respectively, compared with the control. These data were also supported by the results of the trypan blue assay for studying cell viability.
There were substantial increase in the fraction of cells in the G0/G1 phases and a corresponding decrease in the number of cells in S and G2/M phases after transfected with Nkx3.1 alone, p27 alone, or their combination. P27 group did produce a higher percentage of cells in G0/G1 at 24 (p < 0.05) and 48 (p < 0.05 ) hours when compare to control. We found that the combination of both Nkx3.1 and p27 synergistically induced increase in the G0/G1 phase at 48 h (p < 0.01) but not at 24 h time point (p < 0.05).
The results from an annexin-V FITC binding assay showed that the higher proportion of
annexinV-positive cells was observed in co-transfected group at the 24 and 48 hours time point, with values of 21.87% and 26.76%, respectively. Moreover, the combinatory effects of Nkx3.1 and p27 on cellular apoptosis correspond to the decrease in pro-caspase-3 protein expression (caspase-3 activation is presented by the loss of its pro-form), concurrent with decrease Bcl-2 protein and increased Bax protein expression in transfected PC3 cells. Consistent with the caspase-3 activation results.the combination of Nkx3.1 and P27 markedly increased the level of cleaved PARP.
In summary, this report describes a potentially useful approach, the combination of Nkx3.1 plus p27~(KIP1) exhibited significant anti-proliferative effects on PC3 cells. Especially, the combination of Nkx3.1 and p27~(KIP1) demonstrated synergistic multiple anticancer effects of activation of caspase-3 and PARP, down-regulating Bcl-2 protein and up-regulation Bax protein expression, triggering cellular apoptosis and inducing G0/G1 phase arrest. Our in vitro study suggests the potential combined use Nkx3.1 and p27~(KIP1) in gene therapy of androgen-independent prostate cancer.
References
1. Bieberich CJ, Fujita K, He WW, Jay G. Prostate-specific and androgen-dependent expression of a novel homeobox gene. J Biol Chem 1996; 271: 31779-31782
2. Prescott JL, Blok L, Tindall DJ. Isolation and androgen regulation of the human homeobox cDNA,Nkx3.1. Prostate 1998; 35: 71-80
3. Vocke CD, Pozzatti RO, Linehan WM, et al. Analysis of 99 microdissected prostate carcinoma reveals a high frequency of allelic loss on chromosome 8p21-22. Cancer res 1996; 56: 2411-2416
4. Swalwell JI, Vocke CD, Yang Y,et al. Determination of a minimal deletion interval on chromosome band 8p21 in sporatic prostate cancer. Genes Chromosomes Cancer 2002; 33: 201-205
5. Voeller HJ, Augustus M, Madlike V, et al. Coding region of Nkx3.1,prostate-specific homeo -box gene on 8P21,is not mutuated in human prostate cancers. Cancer Res 1997; 57: 4455-4459
6. Bowen C, Bubendorf L, Voeller HJ, et al. Loss of Nkx3.1 expression in human prostate cancers correlates with tumor progression. Cancer Res 2000; 60: 6111-6115
7. Korkmaz CG, Korkmaz KS, Manola J, et al. Analysis of androgen regulated homeobox gene Nkx3.1 during prostate carcinogenesis. J urol 2004; 172: 1134-1139
8. Kim MJ, Cardiff RD, Desai N, et al. Cooperativity of Nkx3.1 and Pten loss of function in a mouse modle of prostate carcinogenesis. Proc Natl Acad Sci USA 2002; 99:2884-2889
9. Eder IE, Bektic J, Haag P, Bartsch G, Klocker H. Genes differentially expressed in prostate cancer. BJU Int 2004;93:1151-1155
10. Katner AL, Hoang QB, Rayford W, et al. Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27KIP1. Prostate 2002; 53: 77-87
11. Di Cristofano A, De Acetis M, Koff A, et al. Pten and p27KIPl cooperate in prostate cancer tumor suppression in the mouse. Nat Genet 2001; 27: 222-224
12. Gary B, Azuero R, Mohanty GS, et al. Interaction of Nkx3.1 and p27KIP1 in prostate tumor initiation. Am J pathol 2004; 164: 1607-1614
前列腺癌可从局灶性病变，向局部进展期和进展期发展。前列腺癌的治疗方案取决于疾病的分期，对早期和局部进展期患者可通过根治性前列腺切除或放射治疗方法治愈。对进展期和发生转移的前列腺癌最初可用手术去势或药物去势等雄激素剥夺治疗，或在以上基础上加用雄激素受体(Androgen receptor；AR)阻止剂达到最大雄激素剥夺。大多数患者对雄激素剥夺治疗起初反应良好，18～24月后，几乎所有患者不可避免地出现循环血前列腺特异抗原(Prostate specific antigen；PSA)升高、肿瘤重新生长为特征的激素抵抗前列腺癌(Hormone refractory prostate cancer；HRPC)。HRPC治疗是世界性难题，目前尚无理想治疗方法。通过对HRPC细胞生物学行为进行修饰的策略可能对HRPC治疗有潜在的临床意义。
同源框基因是一类与发育相关的基因，该类基因都有一段共同的DNA序列，长约180bp，即同源框，该序列编码的蛋白质含60个氨基酸，这段氨基酸被称为同源域。具有同源域的蛋白通过对目标基因的表达调节来控制机体的发育程序，使机体在发育时按正确的时间、空间和组织类型发育。同时该类基因也能参与对细胞癌化过程的调节。但是该类基因并不足以直接造成癌化，而只是对癌化起促进或者抑制作用。由于同源框基因在发育和分化中具有的关键性调控作用以及在肿瘤时经常发生表达失常，目前认为，它们是研究肿瘤形成和胚胎发育间联系的理想靶点。
在前列腺癌，也有同源框基因的表达异常。Nkx3.1是受雄激素调节的前列腺特异表达的同源框基因。人类Nkx3.1定位于染色体8p21，约85％前列腺癌患者该区域缺损。通过对前列腺癌样本的基因分析发现Nkx3.1蛋白编码区无肿瘤特异性突变。然而，在前列腺癌中Nkx3.1失表达与前列腺癌进展和激素抵抗疾病强烈相关(p＜0.0001)。在激素依懒前列腺癌LNCaP细胞中Nkx3.1表达且雄激素刺激下其表达水平增加，而在激素抵抗前列腺癌的PC3细胞株中Nkx3.1失表达。最近在复合的突变鼠模型中研究发现Nkx3.1失表达与脂质磷酸酶PTEN失表达一起协同激活Akt信号途径促进前列腺癌进展。
p27主要与细胞周期素(cyclin)结合而发挥对cyclin-CDK的抑制作用。p27对周期素依赖激酶(Cyclin-dependent kinase；CDK)的抑制作用有两方面，一方面p27能抑制已结合到cyclin并被激活的CDK活性；另一方面p27也可以抑制CDK的激活过程，最终抑制细胞周期G1→S的转变，故目前认为其是一个肿瘤抑制因子。
与Nkx3.1相似，在前列腺癌中大部分p27的表达丢失，而且p27的表达降低和丢失与差的临床预后相关。研究发现，外源性恢复前列腺癌细胞株的p27可引起细胞周期阻滞在G0／G1期，并引起细胞凋亡增加。在大鼠中p27缺损也可引起前列腺上皮增生，并可与PTEN缺损一起协同引起前列腺肿瘤的发展。
尽管Nkx3.1与p27都与前列腺癌抑制有关，但是二者的肿瘤抑制均存在存在单体失能，亦即单个等位基因缺损不足以引起前列腺癌发生。Gary等在Nkx3.1与p27复合突变的等位基因大鼠模型中研究发现，Nkx3.1和p27协同抑制前列腺上皮细胞增殖和类似前列腺上皮内瘤的瘤前病变形成。这说明在前列腺癌的发动中，Nkx3.1与p27二者存在协同联系作用。
随着前列腺癌的进展，向激素抵抗状态转化时Nkx3.1与p27的表达渐渐降低直至消失，如恢复激素抵抗前列腺癌PC3细胞株的p27表达可一定程度抑制其生长。如同时恢复二者在PC3细胞株中的表达获得协同的抗肿瘤效应吗?这方面研究目前国内外尚无报道，我们拟对此问题作进一步探讨。
第一部分人类Nkx3.1和p27基因全长读码框(ORF)的克隆及真核表达载体的构建
【研究目的】
为了对Nkx3.1与p27的功能及其二者在激素抵抗前列腺细胞株中结合效应进行全面的研究，我们构建了Nkx3.1和p27真核表达载体。利用这几套载体我们可以恢复PC3细胞的Nkx3.1或p27的表达，或二者共同表达。
【材料和方法】
我们在人类正常前列腺组织中使用RT-PCR技术扩增了Nkx3.1与p27全长读码框(ORF)，并将二者克隆到T载体。并进一步以T载体为模板，把Nkx3.1克隆到真核细胞高效表达载体pcDNA3.1(+)(Invitrogen)，用同样的方法把p27克隆到真核细胞高效表达载体pcDNA3.1(-)(Invitrogen)。特别要说明的是在Nkx3.1 ORF和p27 ORF上分别加上HisTag和HAtag并构建在载体内，这样对表达产物可以用抗HisTag和抗HAtag单抗进行鉴定。
此外，为了评估质粒的转染效率和基因核定位，我们还构建了报告基因表达载体。分别把Nkx3.1 ORF和p27 ORF构建到pEGFP-C1和pEGFP-N1(Clontech)。所有构建质粒得到测序(上海博亚生物公司)证实。
【结果】
我们成功地构建了Nkx3.1和p27真核系列表达载体，这二套载体可在真核细胞内高效表达、His或HA标签纯化等。我们还成功构建Nkx3.1和p27的报告基因质粒系统，可以鉴定质粒的转染效率，基因定位等。通过以上二套质粒系统，我们可对Nkx3.1或／和p27的功能进行较为全面的评价。
【结论】
1．本部分成功构建了Nkx3.1和p27真核表达载体和报告载体系列。利用这二套载体我们可对Nkx3.1、p27的功能进行全面的评估。
2．本部分在PCR扩增技术上，发展了“富含GC-缓冲液”，能有效降低Tm值。与二甲基亚砜(DMSO)、甲酰胺等经典的PCR扩增添加试剂比较，该缓冲液具有更多的优势。
第二部分质粒表达分析、报告基因分析和基因表达定位分析
【研究目的】
为了评估构建质粒在真核细胞中转染效率、表达水平，从而为进一步研究Nkx3.1和p27生物学功能打好基础。
【材料和方法】
我们用Lipofectamine2000瞬时转染PC3细胞，转染后24、48小时时间点用western blot法检测Nkx3.1，p27蛋白表达水平。在报告基因分析中，我们用荧光显微镜观察GFP融合蛋白表达状况，并用流式细胞术检测质粒转染效率。同时我们在用4％多聚甲醛固定细胞后，用DAPI染色对Nkx3.1与p27基因表达进行定位。
【结果】
Western blot检查发现转染后Nkx3.1和p27蛋白在PC3细胞中高表达，并且表达水平与HisTag和HAtag表达相一致。在荧光显微镜下Nkx3.1-GFP和p27-GFG融合蛋白大量表达，用流式细胞检测转染效率为50％-55％(24小时)和46％-50％(48小时)。用DAPI染色基因表达定位发现Nkx3.1与p27均定位于细胞核。
【结论】
1．在真核细胞中pcDNA3.1-Nkx3.1-His和pcDNA3.1-P27-HA高表达；
2．Lipofectamine法转染效率在24h时间点约50％-55％，48h时间点约为46％-50％；
3．基因表达定位分析示Nkx3.1和p27均为核蛋白。
第三部分Nkx3.1、p27及二者联合对PC3细胞增殖影响的研究
【研究目的】
通过对不同转染组别细胞增殖指标的检测，分析Nkx3.1，p27抗增殖效应及联合应用Nkx3.1与p27是否存在抗PC3细胞增殖结合效应。
【材料和方法】
用空质粒、pcDNA3.1-Nkx3.1，pcDNA3.1-p27和Nkx3.1+p27分别转染PC3细胞，以空质粒组为对照组。分别在0h，24h，48h时间点用MTT方法检测细胞增殖。在相同时间点用台盼蓝染色行活细胞计数和死亡细胞计数。
【结果】
MTT分析发现，24h时间点Nkx3.1+p27组与对照组相比存在差异(p＜0.05)；在48h时间点p27组与对照组存在差异(p＜0.05)，而Nkx3.1+p27组与对照组存在显著差异(p＜0.01)。
台盼蓝排斥试验活细胞计数，24h时间点Nkx3.1+p27组与对照组相比存在差异(p＜0.05)，48h时间点Nkx3.1+p27组与对照组存在显著差异(p＜0.01)。
台盼蓝死亡细胞计数，在24h和48h时间点Nkx3.1+p27组与对照组相比存在差异(p＜0.05)。
【结论】
1．Nkx3.1对PC3细胞增殖无明显抑制作用；
2．p27对PC3细胞增殖存在抑制作用；
3．Nkx3.1与p27存在协同的抗PC3细胞增殖作用。
第四部分Nkx3.1、p27及二者联合对PC3细胞周期影响的研究
【研究目的】
通过对不同转染组细胞周期检测，分析Nkx3.1、p27及Nkx3.1+p27对PC3细胞周期影响。
【材料和方法】
用空质粒、pcDNA3.1-Nkx3.1，pcDNA3.1-p27和Nkx3.1+p27分别转染PC3细胞，在24h、48h时间点分别收集细胞，用流式细胞仪(PI染色法)检测各组细胞周期分布。
【结果】Nkx3.1，p27，Nkx3.1+p27转染组，G0／G1期细胞比例渐渐升高，但S期和G2／M期比例相对下降。在24h时间点p27和Nkx3.1+p27组与对照组相比，G0／G1期存在差异(p＜0.05)。在48h时间点，p27组与对照组相比G0／G1期存在差异(p＜0.05)，而Nkx3.1+p27组与对照组相比G0／G1期存在显著差异。
【结论】
1．Nkx3.1虽然可使PC3细胞的G0／G1期细胞比例增加，但与对照组比较无统计意义；
2．p27可使PC3细胞周期阻滞在G0／G1期；
3．Nkx3.1与p27使PC3细胞阻滞在G0／G1期具有协同性。
第五部分Nkx3.1、p27及二者联合对PC3细胞凋亡及其凋亡相关蛋白影响的研究
【研究目的】
通过对转染各组细胞的不同时间点细胞凋亡率及凋亡相关蛋白检测，分析Nkx3.1、p27及Nkx3.1+p27对PC3细胞凋亡影响及对凋亡机制作初步探讨。
【材料和方法】
用空质粒、pcDNA3.1-Nkx3.1，pcDNA3.1-p27和Nkx3.1+p27分别转染PC3细胞，在24h，48h时间点收集细胞。用AV+PI双染法流式细胞术检测细胞凋亡率。用western blot方法检测各组不同时间点的凋亡相关蛋白(bcl-2，bax，caspase-3，PARP)表达变化。
【结果】
与对照组相比，Nkx3.1与p27引起PC3细胞凋亡率均增加。当Nkx3.1与p27结合处理后，引起明显细胞凋亡协同效应。与我们的调亡率检测结果一致的是，在Nkx3.1+p27组与p27及Nkx3.1组相比，bcl-2表达明显增加，bax表达上升，caspase-3表达被激活，PARP劈裂片断表达增加。
【结论】
1．Nkx3.1与p27均可引起PC3细胞凋亡率增加；
2．Nkx3.1与p27引起PC3凋亡存在协同效应；
3．凋亡协同性产生主要是原因是凋亡拮抗物bcl-2表达下降、凋亡促进物bax表达增加，最终引起caspase-3激活。产生协同凋亡效应主要通过线粒体途径来实现。
Prostate cancer now represents a serious health problem worldwide. Despite intense investigations, it has remained a daunting task to elucidate the mechanisms underlying disease progression. Androgen ablation, however, remains the only effective therapy for patients with advanced disease. Approximately 80% of patients achieve symptomatic and /or objective response after androgen withdrawal, but progression to androgen independence ultimately occurs in almost all cases. Because androgen-independent prostate cancer cells eventually lead to death, successful strategies to modify the biological behavior of these cells may potentially have the most significant clinical impact.
Nkx3.1 is an androgen-regulated homeobox gene that is largely specific to prostate for expression[1,2]. The human Nkx3.1 has been mapped to chromosome 8p21, and has been located in a region of the chromosome deleted in approximately 85%of prostate cancer[3,4]. However, no tumor-specific mutations of the Nkx3.1 protein-coding region have been identified by genetic analysis of human prostate cancer samples[5]. Nevertheless loss of Nkx3.1 expression is strongly associated with hormone-refractory disease and advanced tumor stage in prostate cancer (p<0.0001) [6]. In the LNCaP androgen-dependent prostate cancer cell line, Nkx3.1 is expressed at a basal level that was increased upon androgen stimulation. In contrast, there was no Nkx3.1 expression in androgen-independent PC3 cells[7]. More recently it was shown that in compound mutant mice the loss of Nkx3.1 along with the loss of the lipid phosphatase Pten cooperates in prostate cancer progression through synergistic activation of the Akt pathway[8].
Similar to Nkx3.1, expression of the cyclin-dependent kinase inhibitor p27~(KIP1) is lost in a large fraction of human prostate cancer, and reduced or absent expression of p27~(KIP1) correlates with poor clinical outcome[9]. Exogenous p27~(KIP1) overexpression results in cell cycle regulation and an increase in cell apoptosis in the human prostate carcinoma cell lines[10]. Deletion of p27~(KIP1) in mice also results in prostatic epithelial hyperplasia and cooperates with loss of Pten to promote prostate tumor development[11]. Gary et al found that Nkx3.1 and p27~(KIP1) cooperate to suppress the proliferation of prostatic epithelial cells and the formation of preneoplastic lesions resembling prostatic intraepithelial neoplasia[12], whereas there was no literature report about combined effect of Nkx3.1 and p27~(KIP1) in androgen independent prostate cancer.
Consequently, we hypothesized that the anticancer effect by combination therapy of Nkx3.1 and p27~(KIP1) would be superior to single gene in PC3 prostate cancer cells. The objectives of this study were to test whether induction of apoptotic cell death by p27~(KIP1) or Nkx3.1 gene transfer is enhanced by Nkx3.1 or p27~(KIP1), and to determine whether combined use of Nkx3.1 and p27~(KIP1) exist synergistic antitumor effect in the androgen-independent human prostate PC3 cells.
We use two kinds of prostate cell line LNCaP and PC3 to demonstrate our hypothesis. LNCaP is an androgen-depedent prostate cacer cell line and expresses NKX3.1 and p27, and PC3 is an androgen-independent prostate cancer cell line without expressing NKX3.1 and p27. These cell were maintained at 37℃ in a humidified atmosphere of 5% CO_2/95% air and serially passaged in RPMI-1640 medium (Hyclone), supplemented with 10% fetal bovine serum.
RT-PCR method was used to obtain the amplified Nkx3.1-HisTag and p27-HAtag DNA, Nkx3.1-HisTag fragment was directionally cloned into the EcoR I and BamH I restriction sites of plasmid pcDNA3.1(+) (Invitrogen) and p27-HAtag was cloned into the same restriction sites of pcDNA3.1(-) (Invitrogen).In addition, Nkx3.1 was cloned into pEGFP-C1(Clontech) at Bgl II and EcoR I sites, p27 was cloned into pEGFP-N1 (Clontech) at BamH I and EcoR I sites. The successful cloning was confirmed by sequencing the plasmid.
Plasmid DNAs were delivered into cells with LipofectamineTM 2000 (Invitrogen) following the manufacturer's protocol. Reporter gene expression was monitored by fluorescence microscopy and quantification of GFP fusion protein by flow cytometry
Cell viability was evaluated by MTT assay. The viable cell number was monitored with absorbance at 570nm. At the same time, We used a trypan blue dye exclusion assay to determine
cell viability after the treatments. After the cells were collected by trypsinization, they were stained with trypan blue, and the total number cells and the number of viable cells in each well were counted.
We used flow cytometric to analysis the cell cycle and apoptosis. The cells transfected with various plasmid DNA for 24 h or 48 h were assayed for cell cycle progression by the propidium iodide (PI) staining method. However, Annexin V (Annexin V-FITC) and PI double staining was used to determine apoptosis.
To prodive further confirmation that the synergistic effect of combined use Nkx3.1 and p27 induces apoptosis in PC3 cells, we also analyzed apoptotic-related proteins using western blot method.
In the present study, significant anti-proliferative effect of p27~(KIP1) was shown on PC3 human prostate cancer cells in vitro. The combination of Nkx3.1 and p27~(KIP1) exhibited synergistic inhibitory effects on growth of PC3 cells: increase in cellular apoptosis and enhancement in G0/G1 phase arrest of cell cycle. These result suggest that the combining use of Nkx3.1 and p27~(KIP1) possesses novel anti-tumor action on PC3 human prostate cancer cells.
Proliferation status was assessed by MTT assay. Compared with control-plasmid group, cells in Nkx3.1plus p27 group experienced more greater growth inhibition than Nkx3.1 group and p27 group. Transfected PC3 cells fail to produce a measurable difference between control and Nkx3.1 group at any of the time point. However, transfection of PC3 cells with p27 did produce a significantly lower absorbance at the 48 h time point when compare to the control (p < 0.05). When Nkx3.1 and p27 were combined, the PC3 cell line exhibited a significant decrease in cell growth at 24 (p < 0.05) and 48 (p < 0.01) hours, respectively, compared with the control. These data were also supported by the results of the trypan blue assay for studying cell viability.
There were substantial increase in the fraction of cells in the G0/G1 phases and a corresponding decrease in the number of cells in S and G2/M phases after transfected with Nkx3.1 alone, p27 alone, or their combination. P27 group did produce a higher percentage of cells in G0/G1 at 24 (p < 0.05) and 48 (p < 0.05 ) hours when compare to control. We found that the combination of both Nkx3.1 and p27 synergistically induced increase in the G0/G1 phase at 48 h (p < 0.01) but not at 24 h time point (p < 0.05).
The results from an annexin-V FITC binding assay showed that the higher proportion of
annexinV-positive cells was observed in co-transfected group at the 24 and 48 hours time point, with values of 21.87% and 26.76%, respectively. Moreover, the combinatory effects of Nkx3.1 and p27 on cellular apoptosis correspond to the decrease in pro-caspase-3 protein expression (caspase-3 activation is presented by the loss of its pro-form), concurrent with decrease Bcl-2 protein and increased Bax protein expression in transfected PC3 cells. Consistent with the caspase-3 activation results.the combination of Nkx3.1 and P27 markedly increased the level of cleaved PARP.
In summary, this report describes a potentially useful approach, the combination of Nkx3.1 plus p27~(KIP1) exhibited significant anti-proliferative effects on PC3 cells. Especially, the combination of Nkx3.1 and p27~(KIP1) demonstrated synergistic multiple anticancer effects of activation of caspase-3 and PARP, down-regulating Bcl-2 protein and up-regulation Bax protein expression, triggering cellular apoptosis and inducing G0/G1 phase arrest. Our in vitro study suggests the potential combined use Nkx3.1 and p27~(KIP1) in gene therapy of androgen-independent prostate cancer.
References
1. Bieberich CJ, Fujita K, He WW, Jay G. Prostate-specific and androgen-dependent expression of a novel homeobox gene. J Biol Chem 1996; 271: 31779-31782
2. Prescott JL, Blok L, Tindall DJ. Isolation and androgen regulation of the human homeobox cDNA,Nkx3.1. Prostate 1998; 35: 71-80
3. Vocke CD, Pozzatti RO, Linehan WM, et al. Analysis of 99 microdissected prostate carcinoma reveals a high frequency of allelic loss on chromosome 8p21-22. Cancer res 1996; 56: 2411-2416
4. Swalwell JI, Vocke CD, Yang Y,et al. Determination of a minimal deletion interval on chromosome band 8p21 in sporatic prostate cancer. Genes Chromosomes Cancer 2002; 33: 201-205
5. Voeller HJ, Augustus M, Madlike V, et al. Coding region of Nkx3.1,prostate-specific homeo -box gene on 8P21,is not mutuated in human prostate cancers. Cancer Res 1997; 57: 4455-4459
6. Bowen C, Bubendorf L, Voeller HJ, et al. Loss of Nkx3.1 expression in human prostate cancers correlates with tumor progression. Cancer Res 2000; 60: 6111-6115
7. Korkmaz CG, Korkmaz KS, Manola J, et al. Analysis of androgen regulated homeobox gene Nkx3.1 during prostate carcinogenesis. J urol 2004; 172: 1134-1139
8. Kim MJ, Cardiff RD, Desai N, et al. Cooperativity of Nkx3.1 and Pten loss of function in a mouse modle of prostate carcinogenesis. Proc Natl Acad Sci USA 2002; 99:2884-2889
9. Eder IE, Bektic J, Haag P, Bartsch G, Klocker H. Genes differentially expressed in prostate cancer. BJU Int 2004;93:1151-1155
10. Katner AL, Hoang QB, Rayford W, et al. Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27KIP1. Prostate 2002; 53: 77-87
11. Di Cristofano A, De Acetis M, Koff A, et al. Pten and p27KIPl cooperate in prostate cancer tumor suppression in the mouse. Nat Genet 2001; 27: 222-224
12. Gary B, Azuero R, Mohanty GS, et al. Interaction of Nkx3.1 and p27KIP1 in prostate tumor initiation. Am J pathol 2004; 164: 1607-1614
引文
断表达增加。
【结论】
1．Nkx3.1与p27均可引起PC3细胞凋亡率增加；
2．Nkx3.1与p27引起PC3凋亡存在协同效应；
3．凋亡协同性产生主要是原因是凋亡拮抗物bcl-2表达下降、凋亡促进物bax表达增加，最终引起caspase-3激活。产生协同凋亡效应主要通过线粒体途径来实现。 mouse modle of prostate carcinogenesis. Proc Natl Acad Sci USA 2002; 99: 2884-2889
11. Eder IE, Bektic J, Haag P, Bartsch G, et al. Genes differentially expressed in prostate cancer. BJU Int 2004; 93: 1151-1155
12. Katner AL, Hoang QB, Gootam P, et al. Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27KIP1. The prostate 2002; 53: 77-87
13. Di Cristofano A, De Acetis M, Koff A, et al. Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse. Nat Genet 2001; 27: 222-224
14. Kim MJ, Bhatia-Gaur R, Banach-Petrosky WA, et al: Nkx3.1 mutant mice recapitulate early stages of prostate carcinogenesis. Cancer Res 2002; 62: 2999-3004
15. Abdulkadir SA, Magee JA, Peters TJ, et al: Conditional loss of nkx3.1 in adult mice induces prostatic intraepithelial neoplasia. Mol Cell Biol 2002; 22: 1495-1503
16. Di Cristofano A, De Acetis M, KoffA, et al: Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse. Nat Genet 2001; 27: 222-224
17. Fero ML, Randel E, Gurley KE, et al: The murine gene p27Kipl is haplo-insufficien for tumour suppression. Nature 1998; 396: 177-180
18. Gary B, Azuero R, Mohanty GS, et al. Interaction of Nkx3.1 and p27KIP1 in prostate tumor initiation. Am J pathol 2004; 164: 1607-1614
1. Feldman B J, Feldman D. The development of androgen-independent prostate cancer. Nat Rev Cancer 2001; 1: 34-5.
2. Grossman ME, Huang H, Tindall DJ. Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst 2001; 93: 1687-97.
3. Allay JA, Steiner MS, Zhang Y, et al. Adenovirus p16 gene therapy for prostate cancer. World J Urol 2000; 18: 111.
4. Bookstein R, Shew JY, Chen PL, et al. Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science 1990; 247: 712.
5. Eastham JA, Hall SJ, Sehgal I, et al. In vivo gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res 1995; 55: 5151.
6. Steiner MS, Anthony CT, Lu Y, et al. Antisense cmyc retroviral vector suppresses established human prostate cancer. Hum Gene Ther 1998; 9: 747.
7. Wei C, Willis R. A, Tilton BR, et al. Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy. Proc Natl Acad Sci USA 1997; 94: 6369.
8. Hay RT. The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo. EMBO J 1985; 4: 421.
9. Aberdam D, Negreanu V, Sachs L, et al, The oncogenic potential of an activated Hox2.4 homeobox gene in mouse fibroblast.Mol Cell Biol, 1991, 11: 554-557.
10. Cillo C, HOX gene expression in normal and neoplastic human kidney, Int J Cancer, 1992, 51: 892-897.
11. Abate-Shen C, deregulated homeobox gene expression in cancer: cause or consequence? Nat Rev Cancer. 2002, 2(10): 777-85.
12. Abate-Shen C, Shen MM. roles of the NKX3.1 homeobox gene in prostate organogeesis and carcinogenesis. Dev Dyn, 2003, 228: 767-778.
13. Bieberich CJ, Fujita K, He WW, et al, Prostate-specific and androgen-dependent expression of a novel homeobox gene. J Biol Chem. 1996, 271(50): 31779-82.
14. Bowen C, Bubendorf L, Voeller HJ, et al. Loss of NKX3.1 expression in human prostate cancers correlates with tumor progression. Cancer Res. 2000, 60(21): 6111-5.
15. Kuo MY, Hsu HY, Kok SH, et al. Prognostic role of p27(Kipl) expression in oral squamous cell carcinoma in Taiwan[J]. Oral Oncol, 2002, 38(2): 172-178
16. De Marzo AM, Meeker AK, Epstein JI, Coffey DS: Prostate stem cell com-partments: expression of the cell cycle inhibitor p27Kip1 in normal, hyperplastic, and neoplastic cells. Am J Pathol 1998; 153: 911-9
17. . Guo Y, Sklar GN, Borkowski A, Kyprianou N: Loss of the cyelin-dependent kinase inhibitor p27(Kip1) protein in human prostate cancer correlates with tumor grade. Clin Cancer Res1997; 3: 2269-2274
18. Yang RM, Naitoh J, Murphy M, Wang HJ, Phillipson J, deKemion JB, Loda M, Reiter RE: Low p27 expression predicts poor disease-free survival in patients with prostate cancer. J Urol 1998; 159: 941-5
19. Cote RI, Shi Y, Groshen S, Feng AC, Cordon-Cardo C, Skinner D, Lieskovosky G: Association of p27Kip1 levels with recurrence and survival in patients with stageC prostate carcinoma. J Natl Cancer Inst 1998; 90: 916-20
20. Cordon-Cardo C, KoffA, Drobnjak M, Capodieci P, Osmanl, Millard SS, Gaudin PB, Fazzari M, Zhang ZF, Massague J, Scher HI: Distinct altered patterns of p27KIP1 gene expression in benign prostatic hyperplasia and prostatic carcinoma. J Natl Cancer Inst 1998; 90: 1284-91
21. Tsihlias J, Kapusta LR, DeBoer G, Morava-Protzner I, Zbieranowski I, Bhattach-arya N, Catzavelos GC, Klotz LH, Slingerland JM: Loss of cyclin-dependent kinase inhibitorp27Kip1 is a novel prognostic factor in localized human prostate adenocarc-inoma. Cancer Res 1998; 58: 542-8
22. Katner AL, Hoang QB, Gootam P, et al. Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27KIP1.The prostate 2002; 53: 77-87
23. Kim, M. J., Cardiff, R. D., Desai, N., Banach-Petrosky, W. A., Parsons, R., Shen, M. M., and Abate-Shen, C. Cooperativity of Nkx3.1 and Pten loss of function in a mouse model of prostate carcinogenesis. Proc. Natl. Acad. Sci. USA, 2002; 99: 2884-2889
24. Di Cristofano A, De Acetis M, Koff A, ct al. Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse. Nat Genet 2001; 27: 222-224
25. Gary B, Azuero R, Mohanty GS, et al. Interaction of Nkx3.1 and p27KIP1 in prostate tumor initiation. Am J pathol 2004; 164: 1607-1614
1. Abdulkadir SA, Magee JA, Peters TJ, et al. Conditional loss of Nkx3.1 in adult mice induces prostatic intraepithelial neoplasia. Mol Cell Biol 2002;22:1495-1503.
2. Abate-Shen C, Banach-Petrosky WA, Sun XH,et al. NKX3.1 ;Pten mutant mice develop invasive prostate adenocarcinoma and lymph node metastases,Cance Res,2003,63:3886-3890.
3. Gary B, Azuero R, Mohanty GS, et al. Interaction of NKX3.1 and p27kip1 in prostate tumor initiation, Am J Patbol,2004,164:1607-1614.
4. Korkmaz KS, Korkmaz CG, Raghildstveit E, et al. Full-length cDNA sequence and genpmic organization of human NKX3A——alternative forms and regulation by both androgens and estrogens. Gene,2000,260:25-36.
5. Steadman D J, Giuffrida D, Gelmann EP. DNA-binding sequence of the human prostate-specific homeodomain protein NKX3.1, Nucl. Acids Res, 2000, 28: 2389-2395.
6. Katner AL, Gnarra JR, Rayford W. A recombinant adenovirus expressing p27(KIP) induces cell cycle arrest and apoptosis in human renal carcinoma cells. Jurol 2002; 168: 766-773
7. Katayose Y, Kim M, Rakkar AN, et al. Promoting apoptosis: a novel activity associated with cyclin dependent kinase inhibitor p27. Cancer Res 1997; 57: 5441-5445
8. Hiromura K, Pippin JW, Fero ML, et al. Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27Kip1. J Clin Invest 1999; 103:597-604
9. Wang X, Gorospe M, Huang Y, et al. p27Kip1 overexpression causes apoptotic death of mammalian cells. Oncogene 1997; 15: 2991-2997
10. Turturro F, Frist AY, Arnold MD, et al. Comparsion of the effects of recombinant adenovirus-mediated expression of wild-type P53 and p27Kip1 on cell cycle arrest and apoptosis in SUDHL-1 cells derived from anaplastic large cell lymphoma. Leukemia 2001; 15: 1225-1231
11. Schreiber M, Muller WJ, Singh G, et al. Comparison of the effectiveness of adenovirus vectors expressing cyclin kinase inhibitors p16INK4A, p18INK4C, p21WAFKIP1 and p27K1P1 in inducing cell cycle arrest, apoptosis and inhibition of tumorigenicity. Oncogene 1999; 18: 1663-1676
12. Naruse I, Hoshino H, Dobashi K, et al. Over-expression of p27Kip1 induces growth arrest and apoptosis mediated by changes of pRb expression in lung cancer cell lines. Int J Cancer 2000; 88: 377-383
13. Benita Y, Oosting RS, Lok MC, et al. Regionalized GC content of template DNA as a predictor of PCRsuccess. Nucleic Acids Research, 2003, 31(6): 99
14. Henke W, Herdel K, Jung K, et al. Betaine improves the PCR amplification of GC-rieh DNA sequences, 1997, 25: 3957-3958.
1. Breyer RM, Kennedy CR, Zhang Y, et al. Targeted gene disruption of the prostaglandin E2 EP2 receptor. Adv Exp Med Biol 2002; 507: 321-326.
2. Martinet W, Schrijvers DM, Kockx MM. Nucleofection as an efficient nonviral transfection method for human monocytic cells. Biotechnol Lett 2003; 25(13): 1025-1029.
3. Foley R, Lawler M, Hollywood D. Gene-based therapy in prostate cancer. Lancet Oncol 2004; 5(8): 469-479.
1. Abate-Shen C, Shen MM. roles of the NNKX3.1 homeobox gene in prostate organogeesis andcarcinogenesis. Dev Dyn, 2003, 228: 767-778.
2. Abdulkadir SA, Magee JA, Peters TJ, et al. Conditional loss of Nkx3.1 in adult mice induces prostatic intraepithelial neoplasia. Mol Cell Biol 2002; 22:1495-1503.
3. Abate-Shen C, Banach-Petrosky WA, Sun XH, et al. NKX3.1; Pten mutant mice develop invasive prostate adenocarcinoma and lymph node metastases, cance res, 2003, 63: 3886-3890.
4. Macleod K, Tumor suppressor genes, curr opin genet dev, 2000, 10: 81-93.
5. Payne SR, Kemp CJ. tumor suppressor genetics, carciogenesis, 2005, 12: 2031-2045.
6. Gary B, Azuero R, Mohanty GS, et al. interaction of NKX3.1 and p27kip1 in prostate tumor initiation. Am J Patho, 2004, 164: 1607-1614.
7. Kuo MY, Hsu HY, Kok SH, et al. Prognostic role of p27 (Kip1) expression in oral squamous cell carcinoma in Taiwan [J]. Oral Oncol, 2002, 38(2): 172-178
8. Yang RM, Naitoh J, Murphy M, Wang HJ, Phillipson J, deKemion JB, Loda M, Reiter RE: Low p27 expression predicts poor disease-free survival in patients with prostate cancer. J Urol 1998; 159: 941-5
9. Cote RI, Shi Y, Groshen S, Feng AC, Cordon-Cardo C, Skiuner D, Lieskovosky G: Association of p27Kip1 levels with recurrence and survival in patients with stageC prostate carcinoma. J Natl Cancer Inst 1998; 90: 916-20
10. Cordon-Cardo C, KoffA, Drobnjak M, Capodieci P, OsmanI, Millard SS,Gaudin PB, Fazzari M, Zhang ZF, Massague J, Scher HI: Distinct altered patterns of p27KIP1 gene expression in benign prostatic hyperplasia and prostatic carcinoma. J Natl Cancer Inst 1998; 90: 1284-91
11. Gary B, Azuero R, Mohanty GS, et al. Interaction of Nkx3.1 and p27~(KIPI) in prostate tumor initiation. Am J pathol 2004; 164: 1607-1614
1. Sherr CJ. Cancer cell cycles. Science, 1996, 274, 1672～1677
2. Bieberich CJ, Fujita K, He WW, et al, Prostate-specific and androgen-dependent expression of a novel homeobox gene. J Biol Chem. 1996, 271(50): 31779-82.
3. Macleod K, Tumor suppressor genes, curr opin genet dev, 2000, 10: 81-93.
4. Payne SR, Kemp CJ. tumor suppressor genetics, carciogenesis, 2005, 12: 2031-2045.
5. Katner AL, Gnarra JR, Rayford W. A recombinant adenovirus expressing p27(KIP) induces cell cycle arrest and apoptosis in human renal carcinoma cells. Jurol 2002; 168: 766-773
6. Katayose Y, Kim M, Rakkar AN, et al. Promoting apoptosis: a novel activity associated with cyclin dependent kinase inhibitor p27. Cancer Res 1997; 57: 5441-5445
7. Hiromura K, Pippin JW, Fero ML, et al. Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27Kip1. J Clin Invest 1999; 103: 597-604
8. Wang X, Gorospe M, Huang Y, et al. p27Kip1 overexpression causes apoptotic death of mammalian cells. Oncogene 1997; 15: 2991-2997
9. Turturro F, Frist AY, Arnold MD, et al. Comparsion of the effects of recombinant adenovirus-mediated expression of wild-type P53 and p27Kip1 on cell cycle arrest and apoptosis in SUDHL-1 cells derived from anaplastic large cell lymphoma. Leukemia 2001; 15: 1225-1231
10. Schreiber M, Muller WJ, Singh G, et al. Comparison of the effectiveness of adenovirus vectors expressing cyclin kinase inhibitors p16INK4A, p18INK4C, p21WAFKIP1 and p27KIP1 in inducing cell cycle arrest, apoptosis and inhibition of tumorigenicity. Oncogene 1999; 18: 1663-1676
11. Naruse I, Hoshino H, Dobashi K, et al. Over-expression of p27Kip1 induces growth arrest and apoptosis mediated by changes of pRb expression in lung cancer cell lines. Int J Cancer 2000; 88: 377-383
12. Katner AL, Hoang QB, Rayford W, et al. Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27KIP1. Prostate 2002; 53: 77-87
1. Thomberry NA and Lazebnik Y. Caspase: enemies within. Science 1998; 281: 1312-1316
2. Farrow SN and Brown R. New members of the Bcl-2 family and their protein partners. Curr Opin Genet Dev 1996; 6: 45-49
3. Raffo AJ, Perlman H, Chen MW, et al. Overexpression of Bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res 1995; 55: 4438-4445
4. Bauer JJ, Sesterhenn IA, Mostofi FK, et al. Elevated levels of apoptosis regulator proteins p53 and Bcl-2 arc independent prognostic biomarkers in surgically treated clinically localized prostate cancer. J Urol 1996; 156: 1511-1516
5. McDonnell TJ, Troncoso P, Brisbay SM, et al. Expression of the protooncogcnc bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res 1992; 52: 6940-6944
6. McConkey DJ, Greene G, Pettaway CA. Apoptosis resistance increases with metastatic potential in cells of the human LNCaP prostate carcinoma line. Cancer Res 1996; 56:5594-5599
7. Haldar S, Jena N, Croce CM. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci USA 1995; 92:4507-4511
8. Haldar S, Chintapalli J, Croce CM. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996; 56: 1253-1255
9. Yin XM, Oltvia ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994; 369: 321-323
10. Sato T, Hanada M, Bodrug S, et al. Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybird system. Proc Natl Acad Sci USA 1994; 91: 9238-9242
11. Wolf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 1999; 274: 20049-20052
1 Chen CD, Welsbie DS, Tran C, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med, 2004, 10: 33-39.
2 Feldman BJ, Feldman D. The development of androgen-independent prostate cancer. Nat Rev Cancer, 2001, 1: 34-35.
3 Grossman ME, Huang H, Tindall DJ. Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst, 2001, 93: 1687-1697.
4 Abrahamsson PA. Neuroendocrine differentiation in prostatic carcinoma. Prostate, 1999, 39: 135-48.
5 Berutti A, Dogliotti L, Mosca A, et al. Circulating neuroendocrine markers in patients with prostate carcinoma. Cancer, 2000, 88: 2590-2597
6 Adam RM, Kim J, Lin J, et al. Heparinbinding epidermal growth factor-like growth factor stimulates androgen-independent prostate tumor growth and antagonizes androgen receptor function. Endocrinology, 2002, 143: 4599-4608.
7 Jibom T, Bjartell A, Abrahamsson PA. Neuroendocrine differentiation in prostatic carcinoma during hormonal treatment. Urology, 1998, 51: 585-589.
8 Abrahamsson PA. Neurocndocrine cells in tumor growth of the prostate. Endocrine-related cancer, 1999, 6: 503-519.
9 Fixemer T, Remberger K, Bonkhoff H. Apoptosis resistance of neuroendocrine phenotypes in prostatic adenocarcinuma. Prostate, 2002, 53: 118-123.
10 Jongsma J, Oomen MH, Noordzij MA, et al. Androgen deprivation of the prohormone convertase-310 human prostate cancer model system induces neuroendocrine differentiation. Cancer Res, 2000, 60: 741-8.
11 Huss WJ, Gray DR, Werdin ES, et al. Evidence of pluripotent human prostate stem cells in a human prostate primary xenograft model. Prostate, 2004, 60: 77-90.
12 Chen X, Thakkar H, Tyan F, et al. Constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer. Oncogene, 2001, 20: 6073-6083.
13 Liao Y, Grobholz R, Abel U, et al. Increase of AKT/PKB expression correlates with Gleason pattern in human prostate cancer. Int J Cancer, 2003, 107: 676-680.
14 Ghosh PM, Malik SN, Bedolla R, et al. Akt in prostate cancer: Possible role in androgen-independence. Curr Drug Metab, 2003, 4: 487-496.
15 Graft JR, Konicck BW, McNulty AM, et al. Increased AKT activity contributes to prostate cancer progression by dramatically accelerating prostate tumor growth and diminishing p27Kip1 expression. J Biol Chem, 2000, 275: 24500-24505.
16 Lin HK, Hu YC, Yang L, et al. Suppression versus induction of androgen receptor functions by the phosphatidylinositol 3-kinase/Akt pathway in prostate cancer LNCaP cells with different passage numbers. J Biol Chem, 2003, 278: 50902-50907.
17 Murillo H, Huang H, Schmidt LJ, et al. Role of P13K signaling in survival and progression of LNCaP prostate cancer cells to the androgen refractory state. Endocrinology, 2001, 142: 4795-4805.
18 Wen Y, Hu MC, Makino K, et al. HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the AKT pathway Cancer Res, 2000, 60: 6841-6845.
19 Zhou BP, Hu MC, Miller SA, et al. HER-2/neu blocks tumor necrosis factorinduced apoptosis via the AKT/NF-κB pathway. J Biol Chem, 2000, 275: 8027-8031.
20 Campbell RA, Bhat-Nakshatri P, Patel NM, et al. Phosphatidylinositol 3-kinase/AKTmediated activation of estrogen receptor a: a new model for anti-estrogen resistance. J Biol Chem, 2001, 276: 9817-9824.
21 Krajewska M, Krajewski S, Epstein JI, et al. Immunohistochemical analysis of bel-2, bax, bcl-X, and mcl-1 expression in prostate cancers. Am J Pathol, 1996, 148: 1567-1576.
22 Furuya Y, Krajewski S, Epstein JI, et al. Expression of bcl-2 and the progression of human and rodent prostatic cancers. Clin Cancer Res, 1996, 2: 389-398.
23 Gleave M, Tolcher A, Miyake H, et al. Progression to androgen independence is delayed by adjuvant treatment with antisense Bcl-2 oligodeoxynucleotides after castration in the LNCaP prostate tumor model. Clin Cancer Res, 1999, 5: 2891-2898.
24 Catz SD, Johnson JL. Transcriptional regulation of bcl-2 by nuclear factor kappaB and its significance in prostate cancer. Oncogene, 2001, 20: 7342-7351.
25 Pugazhenthi S, Nesterova A, Sable C, et al. Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein. J Biol Chem, 2000, 275: 10761-10766.
26 Petrylak DP, Tangen CM, Hussain MH, et al. Docetaxel and Estramustine Compared with Mitoxantrone and Prednisone for Advanced Refractory Prostate Cancer. N Engl J Med, 2004, 351: 1513-1520.
27 Dizeyi N, Bjartel A, Nilsson E, et al. Expression of serotonin receptors in human prostate cancer tissue and cell lines. Prostate, 2004, 59: 328-336.
28 Abdul M, Anezinis PE, Logothetis CJ, et al. Growth inhibition of human prostatic carcinoma cell lines by serotonin antagonists. Anticancer Res, 1994, 14: 1215-1220.
29 Hansson J, Abrahamsson PA. Neuroendocrine differentiation in prostate carcinoma. Scand J Urol Nephrol, 2003, 37(Suppl 212): 28-36.
30 Levine L, Lucci Ⅲ JA, Pazdrak B, et al. Bombesin stimulates nuclear factor kB activation and expression of proangiogenic factors in prostate cancer cells. Cancer Res, 2003, 63: 3495-3502.
31 Xie S, Lin HK, Ni J, et al. Regulation of interleukin-6-mediated differentiation by androgen signaling in prostate cancer LNCaP cells. Prostate, 2004, 60: 61-67.
32 Wang Q, Horiatis D, Pinski J. Interleukin-6 inhibits the growth of prostate cancer xenografts in mice by the process of neuroendocrine differentiation. Int J Cancer, 2004, 111: 508-513.
33 Persad S, Attwell S, Gray V, et al. Inhibition of integrin-linked kinase(ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer. Proc Natl Acad Sci USA, 2000, 97: 3207-3212.
34 Neshat MS, Mellinghoff IK, Tran C, et al. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci USA, 2001, 98: 10314-10319.
35 Klasa RJ, Gillum AM, Klem RE, et al. Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment. Antisense Nucleic Acid Drug Develop, 2002, 12: 193-213.
36 Tortora G, Caputo R, Damiano V, et al. Combined blockade of protein kinase A and bcl-2 by antisanse strategy induces apoptosis and inhibits tumor growth and angiogenesis. Clin Cancer Res, 2001, 7: 2537-2544.
37 Miyake H, Tolcher A, Gleave ME. Antisense Bcl-2 oligodeoxynuclentides inhibit progression to androgen-independence after castration in the Shionogi tumor model. Cancer Res, 1999, 59: 4030-4034.
38 Tocher AW. Preliminary phase I results of G3139 (bcl-2 antisense oligonucleotide) therapy in combination with docetaxel in hormone-refractory prostate cancer. Semin Oncol, 2001, 28: 67-70.
39 Tocher AW, Kuhn J, Schwartz G, et al: A Phase I pharmacokinetic and biological correlative study of oblimersan sodium (genasense, g3139), an antisense oligonucleotide to the bcl-2 mRNA, and of docetaxel in patients with hormone-refractory prostate cancer. Clin Cancer Res, 2004, 10: 5048-5057.
1. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin 2002; 52(1): 23-47.
2. Lieberman R. Evidence-based medical perspectives: The evolving role of PSA for early detection, monitoring of treatment response, and as a surrogate end point of efficacy for interventions in men with different clinical risk states for the prevention and progression of prostate cancer. Am J Ther 2004; 11(6): 501-506.
3. Cataloua WJ, Smith DS. Cancer reurrence and survival rates after anatomic radical retropubic prostatectomy for prostate cancer: Intermediate-term results. J Urol 1998; 160(6 Pt 2): 2428-2434.
4. Roscigno M, Sangalli M, Mazzoccoli B, et al. Medical therapy of prostate cancer. Areview. Minerva Urol Nefrol 2005; 57(2): 71-84.
5. Breyer RM, Kennedy CR, Zhang Y, et al. Targeted gene disruption of the prostaglandin E2 EP2 receptor. Adv Exp Med Biol 2002; 507: 321-326.
6. Martinet W, Schrijvers DM, Kockx MM. Nucleofection as an efficient nonviral transfection method for human monocytic cells. Biotechnol Lett 2003; 25(13): 1025-1029.
7. Foley R, Lawler M, Hollywood D. Gene-based therapy in prostate cancer. Lancet Oncol 2004; 5(8): 469-479.
8. Greco O, Scott SD, Marples B, Dachs GU. Cancer gene therapy: 'Delivery, delivery, delivery'. Front Biosci 2002; 7: d1516-1524.
9. Rauen KA, SudilovskyD, Le JL, et al. Expression of the coxsackie adenovirus receptor in normal prostate and in primary and metastatic prostate carcinoma: Potential relevance to gene therapy. Cancer Res 2002; 62(13): 3812-3818.
10. Bischoff JR, Kim DH, Williams A, et al. An adenovirus mutant that replicates selectively in p53-deficient haman tumor cells. Science 1996; 274(5286): 373-376.
11. McCormick F. Interactions betwecn adenovirus proteins and the p53 pathway: The development of ONYX-015. Semin Cancer Biol 2000, 10(6): 453-459.
12. Rodriguez R, Schuur ER, Lim HY, et al. Prostate attenuated replication competent adenovirus (ARCA) CN706: A selective cytotoxic for prostatespecific antigen-positive prostate cancer cells. Cancer Res 1997; 57(13): 2559-2563.
13. DeWeese TL, van der Poel H, Li S, et al. Aphase I trial of CV706, a replication-competent, PSAselective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy. Cancer Res 2001; 61(20): 7464-7472.
14. Parks RJ, Chen L, Anton M, et al. A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Nail Acad Sci USA 1996; 93(24): 13565-13570.
15. Zhang WW. Development and application of adenoviral vectors for gene therapy of cancer. Cancer Gene Ther 1999; 6(2): 113-138.
16. Harvey BG, Hackett NR, El-Sawy T, et al. Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs. J Virol 1999, 73(8): 6729-6742.
17. Marshall E. Gene therapy death prompts review of adenovirus vector. Science 1999; 286(5448): 2244-2245.
18. Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol 1992; 158: 97-129.
19. Miller DG, Rutledge EA, Russell DW. Chromosomal effects of adeno-associated virus vector integration. Nat Genet 2002; 30(2): 147-148.
20. Kremer EJ, Perricaudet M. Adenovirus and adeno-associated virus mediated gene transfer. Br Med Bull 1995; 51(1): 31-44.
21. Verma IM, Somia N. Gene therapy—promises, problems and prospects. Nature 1997; 389(6648): 239-242.
22. Boris-Lawrie K, Temin HM. The retroviral vector. Replication cycle and safety considerations for retrovirusmediated gene therapy. Ann NY Acad Sci 1994; 716: 59-70; Discussion 71.
23. Hacein-Bey-Abina S, de Saint Basile G, Cavazzana-Calvo M. Gone therapy of X-linked severe combined immunodeficiency. Methods Mol Biol 2003; 215: 247-259.
24. Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003; 348(3): 255-256.
25. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302(5644): 415-419.
26. Frenkel N, Jacob RJ, Honess RW, et al. Anatomy of herpes simplex virus DNA Ⅲ. Characterization of defective DNA molecules and biological properties of virus populations containing them. J Virol 1975; 16(1): 153-167.
27. Huard J, Krisky D, Oligino T, et al. Gene transfer to muscle using herpes simplex virus-based vectors. Neuromuscul Disord 1997; 7(5): 299-313.
28. Harrow S, Papanastassiou V, Harland J, et al HSV1716 injection into the brain adjacent to tumour following surgical resection of high-grade glioma: Safety data and longterm survival. Gene Ther 2004; 11(22): 1648-1658.
29. Patel P, Ashduwn D, James N. Is gene therapy the answer for prostate cancer? Prostate Cancer Prostatic Dis 2004; 7(Suppl 1): S14-S19.
30. Hodge JW, Schlom J, Donohue SJ, et al. A recombinant vaccinia virus expressing human prostate-specific antigen (PSA): Safety and immunogenicity in a non-human primate, Int J Cancer 1995; 63(2): 231-237.
31. Somiari S, Glasspool-Malone J, Drabick JJ, et al. Theory and in vivo application of electroparative gene delivery. Mol Ther 2000; 2(3): 178-187.
32. Cemazar M, Sersa G, Wilson J, et al. Effective gene transfer to solid tumors using different nonviral gene delivery techniques: Electroporation, lipasomes, and integrin-targeted vector. Cancer Gene Ther 2002; 9(4): 399—406.
33. Yang NS, Burkholder J, Roberts B, et al. In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci USA 1990; 87(24): 9568-9572.
34. Rooney CM, Smith CA, Ng CY, et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998; 92(5): 1549-1555.
35. KastWM, Offringa R, Peters PJ, et al. Eradication of adenovirus El-induced tumors by EIA-specific cytotoxic T lymphocytes. Cell 1989; 59(4): 603-614.
36. Rodolfo M, Bassi C, Salvi C, Parmiani G. Therapeutic use of a long-term cytotoxic T cell line recognizing a common tumourassociated antigen: The pattern of in vitro reactivity predicts the in vivo effect on different tumours. Cancer Immunol Immunother 1991; 34(1): 53-62.
37. Riddell SR. Finding a place for tumor-specific T cells in targeted cancer therapy. J Exp Med 2004; 200(12): 1533-1537.
38. Knutson KL, Wagner W, Disis ML. Adoptive T cell therapy of solid cancers. Cancer Immunol Immunother 2005.
39. Yee C. Adoptive T cell therapy: Addressing challenges in cancer immunotherapy. J Transl Med 2005; 3(1): 17.
40. Clay TM, Custer MC, Sachs J, HwuP, Rosenberg SA, Nishimura MI. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes con fers anti-tumor reactivity. J Immunol 1999; 163(1): 507-513.
41. Clay TM, CusterMC, Spiess PJ, Nishimura Mi. Potential use ofT cell receptor genes to modify hematopoietic stem cells for the gene therapy of cancer. Pathol Oncol Res 1999; 5(1): 3-15.
42. Yotnda P, Savoldo B. Targeted delivery of adenoviral vectors by cytotoxic T cells. Blood 2004; 104(8): 2272-2280.
43. Leek RD, Harris AL. Tumor-associated macrophages in breast cancer. J Mammary Gland Biol Neoplasia 2002; 7(2): 177-189.
44. Bingle L, Brown NJ, Lewis CE. The role of tumour-associated macrophages in tumour progression: Implications for new anticancer therapies. J Patbol 2002; 196(3): 254-265.
45. Mantovani A, Bottazzi B. The origin and function of tumor-associated macrophages. Immunol Today 1992; 13(7): 265-270.
46. van Ravenswaay C, Kluin PM, Fleuren GJ. Tumour infiltratingn cells in human cancer. On the possible role of CD16t macrophages in antitumour cytotoxicity. Lab Invest 1992; 67(2): 166-174.
47. Satoh T, Saika T, Ebara S, Kusaka N, et al. Macrophages transduced with an adenoviral vector expressing interleukin 12 suppress tumor growth and metastasis in a preclinical metastatic prostate cancer model. Cancer Res 2003; 63(22): 7853-7860.
48. Lewis JS.. Macrophage responses to hypoxia: Relevance to disease mechanisms. J Leukoc Biol 1999; 66(6): 889-900.
49. Joseph IB, Isaacs JT. Macrophage role in the anti-prostate cancer response to one class of antiangiogenic agents. J Natl Cancer Inst 1998; 90(21): 1648-1653.
50. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991; 9: 271-296.
51. Wysocki PJ, Grabarczyk P, Mackiewicz-Wysocka M, et al. Genetically modified dendritic cells—a new, promising cancer treatment strategy? Expert Opin Biol Ther 2002; 2(8): 835-845.
52. Onaitis M, Kalady MF, Pruitt S, Tyler DS. Dendritic cell gene therapy. Surg Oncol Clin N Am 2002; 11(3): 645-660.
53. Collins MK, Cerundolo V. Gene therapy meets vaccine development. Trends Biotechnol 2004; 22(12): 623-626.
54. Dees EC, McKinnon KP, Kuhns JJ, et al. Dendritic cells can be rapidly expanded ex vivo and safely administered in patients with metastatic breast cancer. Cancer Immunol Immunother 2004; 53(9): 777-785.
55. Shalev M, Miles BJ, Thompson TC, et al. Suicide gene therapy for prostate cancer using a replication-deficient adenovirus containing the herpesvirus thymidine kinase gene. World J Urol 2000; 18(2): 125-129.
56. Ikegami S, Tadakuma T, Ono T, et al. Treatment efficiency of a suicide gene therapy using prostate-specific membrane antigen promoter/enhancer in a castrated mouse model of prostate cancer. Cancer Sci 2004; 95(4): 367-370.
57. Herman JR, Adler HL, Aguilar-Cordova E, et al. In situ gene therapy for adenocarcinoma of the prostate: A phase I clinical trial. Hum Gene Ther 1999; 10(7): 1239-1249.
58. Kubo H, Gardner TA, Wada Y, et al. Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer. Hum Gene Ther 2003; 14(3):227-241.
59. Yoshimura I, Ikegami S, Suzuki S, et al. Adenovirus mediated prostate specific enzyme prodrug gene therapy using prostate specific antigen promoter enhanced by the Cre-loxP system. J Urol 2002; 168(6): 2659-2664.
60. Freytag SO, Khil M, Stricker H, et al. Phase I study of replicationeompetent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer. Cancer Res 2002; 62(17): 4968-4976.
61. Freytag SO, Stricker H, Pegg J, Paielli D, et al. Phase I study of replication-competent adenovirus-mediated doublesuicide gene therapy in combination with conventional-dose three-dimensional conformal radiation therapy for the treatment of newly diagnosed, intermediate to high-risk prostate cancer. Cancer Res 2003; 63(21): 7497-7506.
62. Freytag SO, Paielli D, Wing M, Rogulski K, Brown S, Kolozsvary A, Seely J, Barton K, Dragovic A, Kim JH. Efficacy and toxicity of replication-competent adenovirus-modiatod double suicide gene therapy in combination with radiation therapy in an orthotopic mouse prostate cancer model, Int J Radiat Oncol Biol Phys 2002; 54(3): 873-885.
63. Miles BJ, Shalev M, Aguilar-Cordova E, et al. Prostate-specific antigen response and systemic T cell activation after in situ gene therapy in prostate cancer patients failing radiotherapy. Hum Gene Ther 2001; 12(16): 1955-1967.
64. Shalev M, Kadmon D, Teh BS, et al. Suicide gene therapy toxicity after multiple and repeat injections in patients with localized prostate cancer. J Urol 2000; 163(6): 1747-1750.
65. Satoh T, Teh BS, TimmeTL, et al. Enhanced systemic Tcell activation after in situ gene therapy with radiotherapy in prostate cancer patients, Int J Radiat Oncol Biol Phys 2004; 59(2): 562-571.
66. Sanda MG, Smith DC, Charles LG, et al. Recombinant vaccinia-PSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer. Urology 1999; 53(2): 260-266.
67. Eder JP, KantoffPW, Roper K, et al. A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in advanced prostate cancer. Clin Cancer Res 2000; 6(5): 1632-1638.
68. Vieweg J, Rosenthal FM, Bannerji R, et al. Immunotherapy of prostate cancer in the Dunning rat model: Use of cytokine gene modified tumor vaccines. Cancer Res 1994; 54(7): 1760-1765.
69. Griffith TS, Kawakita M, Tian J, et al. Inhibition of murine prostate tumor growth and activation of immunoregulatory cells with recombinant canarypox viruses. J Natl Cancer Inst 2001; 93(13): 998-1007.
70. NasuY, BangmaCH, HullGW, et al. Adenoviros-mediated interleukin-12 gene therapy for prostate cancer: Suppression of orthotopic tumor growth and pre-established lung metastases in an orthotopic model. Gene Ther 1999; 6(3): 338-349.
71. HullGW, McCurdyMA, Nasu Y, et al. Prostate cancer gene therapy: Comparison of adenovirus-mediated expression of interleukin 12 with interleukin 12 plus B4-1 for in situ gene therapy and gene-modified, cell-based vaccines. Clin Cancer Res 2000; 6(10): 4101-4109.
72. Nasu Y, Bangma CH, Hull GW, et al. Combination gene therapy with adenoviral vector-mediated HSV-tktGCV and IL-12 in an orthotopic mouse model for prostate cancer. Prostate Cancer Prostatic Dis 2001; 4(1): 44-55.
73. Nasu Y, Ebara S, Kumon H. Adenovirus-mediated interleukin-12 gene therapy for prostate cancer. Nippon Rinsho 2004; 62(6): 1181-1191.
74. Trodel S, Trachtenberg J, Toi A, et al. A phase I trial of adenovector-mediated delivery of interleukin-2 (AdIL-2) in high-risk localized prostate cancer. Cancer Gene Ther 2003; 10(10): 755-763.
75. KirschenbaumAIS, WangJP, et al. MUClexpression in prostate carcinoma: Correlation with grade and stage. Mol Urol 1999; 3: 163-168.
76. Oh YT, Chen DW, Dougherty G J, McBride WH. Adenoviral interleukin-3 gene-radiation therapy for prostate cancer in mouse model. Int J Radiat Oncol Biol Phys 2004; 59(2): 579-583.
77. Deliveliotis C, Skolarikos A, Karayannis A, et al. The prognostic value of p53 and DNA ploidy following radical prostatectomy. World J Urol 2003; 21(3): 171-176.
78. Ko SC, Gotoh A, Thalmann GN, et al. Molecular therapy with recombinant p53 adenovirus in an androgen-independent, metastatic human prostate cancer model. Hum Gene Ther 1996; 7(14): 1683-1691.
79. Cowen D, Salem N, Ashoori F, et al. Prostate cancer radiosensitization in vivo with adenovirus-mediated p53 gene therapy. Clin Cancer Res 2000; 6(11): 4402-4408.
80. Eastham JA, Chen SH, Sehgal I, et al. Prostate cancer gene therapy: Herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse and human prostate cancer models. Hum Gene Ther 1996; 7(4): 515-523.
81. Pisters LL, Pettaway CA, Troncoso P, et al. Evidence that transfer of functional p53 protein results in increased apoptosis in prostate cancer. Clin Cancer Res 2004; 10(8): 2587-2593.
82. Honda T, Kagawa S, Spurgers KB, et al. A recombinant adenovirus expressing wild-type Bax induces apoptosis in prostate cancer cells independently of their Bcl-2 status and androgen sensitivity. Cancer Biol Ther 2002; 1(2): 163-167.
83. Lowe SL, Rubinchik S, Honda T, McDonnell TJ, Dong JY, Norris JS. Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells. Gene Ther 2001; 8(18): 1363-1371.
84. Zhang Y, Yu J, Unni E, et al. Monogene and polygene therapy for the tratment of experimental prostate cancers by use of apoptotic genes bax and bad driven by the prostatespecific promoter ARR(2)PB. HumGene Ther 2002; 13(17): 2051-2064.
85. Krygier S, Djakiew D. Molecular characterization of the loss of p75(NTR) expression in human prostate tumor cells. Mol Carcinog 2001; 31(1): 46-55.
86. Fox ME, Lemmon MJ, Mauchline ML, et al. Anaerobic bacteria as a delivery system for cancer gene therapy: In vitro activation of 5-fluorocytosine by genetically engineered clostridia. Gene Ther 1996; 3(2): 173-178.
87. Yazawa K, Fujimori M, Amano J, et al. Bifidobaeterium longum as a delivery system for cancer gene therapy: Selective localization and growth in hypoxic tumors. Cancer Gene Ther 2000; 7(2): 269-274.
88. Fu GF, Li X, Hou YY, et al. Bifidobacterium longum as an oral delivery system of endostatin for gene therapy on solid liver cancer. Cancer Gene Ther 2005; 12(2): 133-140.
89. Yu YA, Shabahang S, Timiryasova TM, et al. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins. Nat Biotechnol 2004; 22(3): 313-320.
90. Zheng LM, Luo X, FengM, et al. Tumor amplified protein expression therapy: Salmonella as a tumor-selective protein delivery vector. Oncol Res 2000; 12(3): 127-135.
91. Luo X, Li Z, Lin S, et al. Antitumor effect of VNP20009, an attenuated Salmonella, in murine tumor models. Oncol Res 2000; 12(11-12): 501-508.
92. Rosenbarg SA, Spiess PJ, Kleiner DE. Antitumor effects in mice of the intravenous injection of attenuated Salmonella typhimurium. J Immunother 2002; 25(3): 218-225.
93. Lee KC, Zheng LM, Margitich D, Almassian B, King I. Evaluation of the acute and subehronic toxic effects in mice, rats, and monkeys of the genetically engineered and Escherichia coli cytosine deaminase gene-incorporated Salmonella strain, TAPET-CD, being developed as an antitumor agent, Int J Toxicol 2001; 20(4): 207-217.
94. Clairmont C, Lee KC, Pike J, et al. Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J Infect Dis 2000; 181(6): 1996-2002.
95. Lee CH, Wu CL, Shiau AL Endostatin gene therapy delivered by Salmonella choleraesuis in murine tumor models. J Gene Med 2004; 6(12): 1382-1393.
96. Zhao M, Yang M, Li XM, et al. Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci USA 2005; 102(3): 755-760.
97. Fong L, Brockstedt D, Benike C, et al. Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy. J Immunol 2001; 167(12): 7150-7156.
98. Small FJ, Fratesi P, Reese DM, Set al. Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells. J Clin Oncol 2000; 18(23): 3894-3903.
99. Burton J, Wells JM, Corke KP, et al. Macrophages accumulate in avascular, hypoxic areas of prostate tumours: Implications for the targeted therapeutic gene delivery to such sites. J Pathul 2000; 192(8A).
100. Vukanovic J, Isaacs JT. Linomide inhibits angiogenesis, growth, metastasis, and macrophage infiltration within rat prostatic cancers. Cancer Res 1995; 55(7): 1499-1504.
101. Franck-Lissbrant I, Haggstrom S, Damber JE, Bergh A. Testosterone stimulates angiogenesis and vascular regrowth in the ventral prostate in castrated adult rats. Endocrinology 1998; 139(2): 451-456.
102. Franck-Lissbrant 1. Tumour associated macrophages in human prostate cancer: Relation to clinicopathological variables and survival, Int J Oncul 2000; 17: 445-451.
103. Movsas B, Chapman JD, Horwitz EM, et al. Hypoxic regions exist in human prostate carcinoma. Urology 1999; 53: 11-18.
104. MacRae E, Brown NJ, Hamdy FC, Lewis CE. Use of macrophages to target gene therapy to hypoxic areas of prostate tumours. British Microcirculation Society Annual Meeting 2004 Abstract Booklet 2004.
105. Ma Q, Safar M, Holmes E, et al. Anti-prostate specific membrane antigen designer T cells for prostate cancer therapy. Prostate 2004; 61(1): 12-25.
【结论】
1．Nkx3.1与p27均可引起PC3细胞凋亡率增加；
2．Nkx3.1与p27引起PC3凋亡存在协同效应；
3．凋亡协同性产生主要是原因是凋亡拮抗物bcl-2表达下降、凋亡促进物bax表达增加，最终引起caspase-3激活。产生协同凋亡效应主要通过线粒体途径来实现。 mouse modle of prostate carcinogenesis. Proc Natl Acad Sci USA 2002; 99: 2884-2889
11. Eder IE, Bektic J, Haag P, Bartsch G, et al. Genes differentially expressed in prostate cancer. BJU Int 2004; 93: 1151-1155
12. Katner AL, Hoang QB, Gootam P, et al. Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27KIP1. The prostate 2002; 53: 77-87
13. Di Cristofano A, De Acetis M, Koff A, et al. Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse. Nat Genet 2001; 27: 222-224
14. Kim MJ, Bhatia-Gaur R, Banach-Petrosky WA, et al: Nkx3.1 mutant mice recapitulate early stages of prostate carcinogenesis. Cancer Res 2002; 62: 2999-3004
15. Abdulkadir SA, Magee JA, Peters TJ, et al: Conditional loss of nkx3.1 in adult mice induces prostatic intraepithelial neoplasia. Mol Cell Biol 2002; 22: 1495-1503
16. Di Cristofano A, De Acetis M, KoffA, et al: Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse. Nat Genet 2001; 27: 222-224
17. Fero ML, Randel E, Gurley KE, et al: The murine gene p27Kipl is haplo-insufficien for tumour suppression. Nature 1998; 396: 177-180
18. Gary B, Azuero R, Mohanty GS, et al. Interaction of Nkx3.1 and p27KIP1 in prostate tumor initiation. Am J pathol 2004; 164: 1607-1614
1. Feldman B J, Feldman D. The development of androgen-independent prostate cancer. Nat Rev Cancer 2001; 1: 34-5.
2. Grossman ME, Huang H, Tindall DJ. Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst 2001; 93: 1687-97.
3. Allay JA, Steiner MS, Zhang Y, et al. Adenovirus p16 gene therapy for prostate cancer. World J Urol 2000; 18: 111.
4. Bookstein R, Shew JY, Chen PL, et al. Suppression of tumorigenicity of human prostate carcinoma cells by replacing a mutated RB gene. Science 1990; 247: 712.
5. Eastham JA, Hall SJ, Sehgal I, et al. In vivo gene therapy with p53 or p21 adenovirus for prostate cancer. Cancer Res 1995; 55: 5151.
6. Steiner MS, Anthony CT, Lu Y, et al. Antisense cmyc retroviral vector suppresses established human prostate cancer. Hum Gene Ther 1998; 9: 747.
7. Wei C, Willis R. A, Tilton BR, et al. Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy. Proc Natl Acad Sci USA 1997; 94: 6369.
8. Hay RT. The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo. EMBO J 1985; 4: 421.
9. Aberdam D, Negreanu V, Sachs L, et al, The oncogenic potential of an activated Hox2.4 homeobox gene in mouse fibroblast.Mol Cell Biol, 1991, 11: 554-557.
10. Cillo C, HOX gene expression in normal and neoplastic human kidney, Int J Cancer, 1992, 51: 892-897.
11. Abate-Shen C, deregulated homeobox gene expression in cancer: cause or consequence? Nat Rev Cancer. 2002, 2(10): 777-85.
12. Abate-Shen C, Shen MM. roles of the NKX3.1 homeobox gene in prostate organogeesis and carcinogenesis. Dev Dyn, 2003, 228: 767-778.
13. Bieberich CJ, Fujita K, He WW, et al, Prostate-specific and androgen-dependent expression of a novel homeobox gene. J Biol Chem. 1996, 271(50): 31779-82.
14. Bowen C, Bubendorf L, Voeller HJ, et al. Loss of NKX3.1 expression in human prostate cancers correlates with tumor progression. Cancer Res. 2000, 60(21): 6111-5.
15. Kuo MY, Hsu HY, Kok SH, et al. Prognostic role of p27(Kipl) expression in oral squamous cell carcinoma in Taiwan[J]. Oral Oncol, 2002, 38(2): 172-178
16. De Marzo AM, Meeker AK, Epstein JI, Coffey DS: Prostate stem cell com-partments: expression of the cell cycle inhibitor p27Kip1 in normal, hyperplastic, and neoplastic cells. Am J Pathol 1998; 153: 911-9
17. . Guo Y, Sklar GN, Borkowski A, Kyprianou N: Loss of the cyelin-dependent kinase inhibitor p27(Kip1) protein in human prostate cancer correlates with tumor grade. Clin Cancer Res1997; 3: 2269-2274
18. Yang RM, Naitoh J, Murphy M, Wang HJ, Phillipson J, deKemion JB, Loda M, Reiter RE: Low p27 expression predicts poor disease-free survival in patients with prostate cancer. J Urol 1998; 159: 941-5
19. Cote RI, Shi Y, Groshen S, Feng AC, Cordon-Cardo C, Skinner D, Lieskovosky G: Association of p27Kip1 levels with recurrence and survival in patients with stageC prostate carcinoma. J Natl Cancer Inst 1998; 90: 916-20
20. Cordon-Cardo C, KoffA, Drobnjak M, Capodieci P, Osmanl, Millard SS, Gaudin PB, Fazzari M, Zhang ZF, Massague J, Scher HI: Distinct altered patterns of p27KIP1 gene expression in benign prostatic hyperplasia and prostatic carcinoma. J Natl Cancer Inst 1998; 90: 1284-91
21. Tsihlias J, Kapusta LR, DeBoer G, Morava-Protzner I, Zbieranowski I, Bhattach-arya N, Catzavelos GC, Klotz LH, Slingerland JM: Loss of cyclin-dependent kinase inhibitorp27Kip1 is a novel prognostic factor in localized human prostate adenocarc-inoma. Cancer Res 1998; 58: 542-8
22. Katner AL, Hoang QB, Gootam P, et al. Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27KIP1.The prostate 2002; 53: 77-87
23. Kim, M. J., Cardiff, R. D., Desai, N., Banach-Petrosky, W. A., Parsons, R., Shen, M. M., and Abate-Shen, C. Cooperativity of Nkx3.1 and Pten loss of function in a mouse model of prostate carcinogenesis. Proc. Natl. Acad. Sci. USA, 2002; 99: 2884-2889
24. Di Cristofano A, De Acetis M, Koff A, ct al. Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse. Nat Genet 2001; 27: 222-224
25. Gary B, Azuero R, Mohanty GS, et al. Interaction of Nkx3.1 and p27KIP1 in prostate tumor initiation. Am J pathol 2004; 164: 1607-1614
1. Abdulkadir SA, Magee JA, Peters TJ, et al. Conditional loss of Nkx3.1 in adult mice induces prostatic intraepithelial neoplasia. Mol Cell Biol 2002;22:1495-1503.
2. Abate-Shen C, Banach-Petrosky WA, Sun XH,et al. NKX3.1 ;Pten mutant mice develop invasive prostate adenocarcinoma and lymph node metastases,Cance Res,2003,63:3886-3890.
3. Gary B, Azuero R, Mohanty GS, et al. Interaction of NKX3.1 and p27kip1 in prostate tumor initiation, Am J Patbol,2004,164:1607-1614.
4. Korkmaz KS, Korkmaz CG, Raghildstveit E, et al. Full-length cDNA sequence and genpmic organization of human NKX3A——alternative forms and regulation by both androgens and estrogens. Gene,2000,260:25-36.
5. Steadman D J, Giuffrida D, Gelmann EP. DNA-binding sequence of the human prostate-specific homeodomain protein NKX3.1, Nucl. Acids Res, 2000, 28: 2389-2395.
6. Katner AL, Gnarra JR, Rayford W. A recombinant adenovirus expressing p27(KIP) induces cell cycle arrest and apoptosis in human renal carcinoma cells. Jurol 2002; 168: 766-773
7. Katayose Y, Kim M, Rakkar AN, et al. Promoting apoptosis: a novel activity associated with cyclin dependent kinase inhibitor p27. Cancer Res 1997; 57: 5441-5445
8. Hiromura K, Pippin JW, Fero ML, et al. Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27Kip1. J Clin Invest 1999; 103:597-604
9. Wang X, Gorospe M, Huang Y, et al. p27Kip1 overexpression causes apoptotic death of mammalian cells. Oncogene 1997; 15: 2991-2997
10. Turturro F, Frist AY, Arnold MD, et al. Comparsion of the effects of recombinant adenovirus-mediated expression of wild-type P53 and p27Kip1 on cell cycle arrest and apoptosis in SUDHL-1 cells derived from anaplastic large cell lymphoma. Leukemia 2001; 15: 1225-1231
11. Schreiber M, Muller WJ, Singh G, et al. Comparison of the effectiveness of adenovirus vectors expressing cyclin kinase inhibitors p16INK4A, p18INK4C, p21WAFKIP1 and p27K1P1 in inducing cell cycle arrest, apoptosis and inhibition of tumorigenicity. Oncogene 1999; 18: 1663-1676
12. Naruse I, Hoshino H, Dobashi K, et al. Over-expression of p27Kip1 induces growth arrest and apoptosis mediated by changes of pRb expression in lung cancer cell lines. Int J Cancer 2000; 88: 377-383
13. Benita Y, Oosting RS, Lok MC, et al. Regionalized GC content of template DNA as a predictor of PCRsuccess. Nucleic Acids Research, 2003, 31(6): 99
14. Henke W, Herdel K, Jung K, et al. Betaine improves the PCR amplification of GC-rieh DNA sequences, 1997, 25: 3957-3958.
1. Breyer RM, Kennedy CR, Zhang Y, et al. Targeted gene disruption of the prostaglandin E2 EP2 receptor. Adv Exp Med Biol 2002; 507: 321-326.
2. Martinet W, Schrijvers DM, Kockx MM. Nucleofection as an efficient nonviral transfection method for human monocytic cells. Biotechnol Lett 2003; 25(13): 1025-1029.
3. Foley R, Lawler M, Hollywood D. Gene-based therapy in prostate cancer. Lancet Oncol 2004; 5(8): 469-479.
1. Abate-Shen C, Shen MM. roles of the NNKX3.1 homeobox gene in prostate organogeesis andcarcinogenesis. Dev Dyn, 2003, 228: 767-778.
2. Abdulkadir SA, Magee JA, Peters TJ, et al. Conditional loss of Nkx3.1 in adult mice induces prostatic intraepithelial neoplasia. Mol Cell Biol 2002; 22:1495-1503.
3. Abate-Shen C, Banach-Petrosky WA, Sun XH, et al. NKX3.1; Pten mutant mice develop invasive prostate adenocarcinoma and lymph node metastases, cance res, 2003, 63: 3886-3890.
4. Macleod K, Tumor suppressor genes, curr opin genet dev, 2000, 10: 81-93.
5. Payne SR, Kemp CJ. tumor suppressor genetics, carciogenesis, 2005, 12: 2031-2045.
6. Gary B, Azuero R, Mohanty GS, et al. interaction of NKX3.1 and p27kip1 in prostate tumor initiation. Am J Patho, 2004, 164: 1607-1614.
7. Kuo MY, Hsu HY, Kok SH, et al. Prognostic role of p27 (Kip1) expression in oral squamous cell carcinoma in Taiwan [J]. Oral Oncol, 2002, 38(2): 172-178
8. Yang RM, Naitoh J, Murphy M, Wang HJ, Phillipson J, deKemion JB, Loda M, Reiter RE: Low p27 expression predicts poor disease-free survival in patients with prostate cancer. J Urol 1998; 159: 941-5
9. Cote RI, Shi Y, Groshen S, Feng AC, Cordon-Cardo C, Skiuner D, Lieskovosky G: Association of p27Kip1 levels with recurrence and survival in patients with stageC prostate carcinoma. J Natl Cancer Inst 1998; 90: 916-20
10. Cordon-Cardo C, KoffA, Drobnjak M, Capodieci P, OsmanI, Millard SS,Gaudin PB, Fazzari M, Zhang ZF, Massague J, Scher HI: Distinct altered patterns of p27KIP1 gene expression in benign prostatic hyperplasia and prostatic carcinoma. J Natl Cancer Inst 1998; 90: 1284-91
11. Gary B, Azuero R, Mohanty GS, et al. Interaction of Nkx3.1 and p27~(KIPI) in prostate tumor initiation. Am J pathol 2004; 164: 1607-1614
1. Sherr CJ. Cancer cell cycles. Science, 1996, 274, 1672～1677
2. Bieberich CJ, Fujita K, He WW, et al, Prostate-specific and androgen-dependent expression of a novel homeobox gene. J Biol Chem. 1996, 271(50): 31779-82.
3. Macleod K, Tumor suppressor genes, curr opin genet dev, 2000, 10: 81-93.
4. Payne SR, Kemp CJ. tumor suppressor genetics, carciogenesis, 2005, 12: 2031-2045.
5. Katner AL, Gnarra JR, Rayford W. A recombinant adenovirus expressing p27(KIP) induces cell cycle arrest and apoptosis in human renal carcinoma cells. Jurol 2002; 168: 766-773
6. Katayose Y, Kim M, Rakkar AN, et al. Promoting apoptosis: a novel activity associated with cyclin dependent kinase inhibitor p27. Cancer Res 1997; 57: 5441-5445
7. Hiromura K, Pippin JW, Fero ML, et al. Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27Kip1. J Clin Invest 1999; 103: 597-604
8. Wang X, Gorospe M, Huang Y, et al. p27Kip1 overexpression causes apoptotic death of mammalian cells. Oncogene 1997; 15: 2991-2997
9. Turturro F, Frist AY, Arnold MD, et al. Comparsion of the effects of recombinant adenovirus-mediated expression of wild-type P53 and p27Kip1 on cell cycle arrest and apoptosis in SUDHL-1 cells derived from anaplastic large cell lymphoma. Leukemia 2001; 15: 1225-1231
10. Schreiber M, Muller WJ, Singh G, et al. Comparison of the effectiveness of adenovirus vectors expressing cyclin kinase inhibitors p16INK4A, p18INK4C, p21WAFKIP1 and p27KIP1 in inducing cell cycle arrest, apoptosis and inhibition of tumorigenicity. Oncogene 1999; 18: 1663-1676
11. Naruse I, Hoshino H, Dobashi K, et al. Over-expression of p27Kip1 induces growth arrest and apoptosis mediated by changes of pRb expression in lung cancer cell lines. Int J Cancer 2000; 88: 377-383
12. Katner AL, Hoang QB, Rayford W, et al. Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27KIP1. Prostate 2002; 53: 77-87
1. Thomberry NA and Lazebnik Y. Caspase: enemies within. Science 1998; 281: 1312-1316
2. Farrow SN and Brown R. New members of the Bcl-2 family and their protein partners. Curr Opin Genet Dev 1996; 6: 45-49
3. Raffo AJ, Perlman H, Chen MW, et al. Overexpression of Bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res 1995; 55: 4438-4445
4. Bauer JJ, Sesterhenn IA, Mostofi FK, et al. Elevated levels of apoptosis regulator proteins p53 and Bcl-2 arc independent prognostic biomarkers in surgically treated clinically localized prostate cancer. J Urol 1996; 156: 1511-1516
5. McDonnell TJ, Troncoso P, Brisbay SM, et al. Expression of the protooncogcnc bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer. Cancer Res 1992; 52: 6940-6944
6. McConkey DJ, Greene G, Pettaway CA. Apoptosis resistance increases with metastatic potential in cells of the human LNCaP prostate carcinoma line. Cancer Res 1996; 56:5594-5599
7. Haldar S, Jena N, Croce CM. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci USA 1995; 92:4507-4511
8. Haldar S, Chintapalli J, Croce CM. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996; 56: 1253-1255
9. Yin XM, Oltvia ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994; 369: 321-323
10. Sato T, Hanada M, Bodrug S, et al. Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybird system. Proc Natl Acad Sci USA 1994; 91: 9238-9242
11. Wolf BB, Green DR. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J Biol Chem 1999; 274: 20049-20052
1 Chen CD, Welsbie DS, Tran C, et al. Molecular determinants of resistance to antiandrogen therapy. Nat Med, 2004, 10: 33-39.
2 Feldman BJ, Feldman D. The development of androgen-independent prostate cancer. Nat Rev Cancer, 2001, 1: 34-35.
3 Grossman ME, Huang H, Tindall DJ. Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst, 2001, 93: 1687-1697.
4 Abrahamsson PA. Neuroendocrine differentiation in prostatic carcinoma. Prostate, 1999, 39: 135-48.
5 Berutti A, Dogliotti L, Mosca A, et al. Circulating neuroendocrine markers in patients with prostate carcinoma. Cancer, 2000, 88: 2590-2597
6 Adam RM, Kim J, Lin J, et al. Heparinbinding epidermal growth factor-like growth factor stimulates androgen-independent prostate tumor growth and antagonizes androgen receptor function. Endocrinology, 2002, 143: 4599-4608.
7 Jibom T, Bjartell A, Abrahamsson PA. Neuroendocrine differentiation in prostatic carcinoma during hormonal treatment. Urology, 1998, 51: 585-589.
8 Abrahamsson PA. Neurocndocrine cells in tumor growth of the prostate. Endocrine-related cancer, 1999, 6: 503-519.
9 Fixemer T, Remberger K, Bonkhoff H. Apoptosis resistance of neuroendocrine phenotypes in prostatic adenocarcinuma. Prostate, 2002, 53: 118-123.
10 Jongsma J, Oomen MH, Noordzij MA, et al. Androgen deprivation of the prohormone convertase-310 human prostate cancer model system induces neuroendocrine differentiation. Cancer Res, 2000, 60: 741-8.
11 Huss WJ, Gray DR, Werdin ES, et al. Evidence of pluripotent human prostate stem cells in a human prostate primary xenograft model. Prostate, 2004, 60: 77-90.
12 Chen X, Thakkar H, Tyan F, et al. Constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer. Oncogene, 2001, 20: 6073-6083.
13 Liao Y, Grobholz R, Abel U, et al. Increase of AKT/PKB expression correlates with Gleason pattern in human prostate cancer. Int J Cancer, 2003, 107: 676-680.
14 Ghosh PM, Malik SN, Bedolla R, et al. Akt in prostate cancer: Possible role in androgen-independence. Curr Drug Metab, 2003, 4: 487-496.
15 Graft JR, Konicck BW, McNulty AM, et al. Increased AKT activity contributes to prostate cancer progression by dramatically accelerating prostate tumor growth and diminishing p27Kip1 expression. J Biol Chem, 2000, 275: 24500-24505.
16 Lin HK, Hu YC, Yang L, et al. Suppression versus induction of androgen receptor functions by the phosphatidylinositol 3-kinase/Akt pathway in prostate cancer LNCaP cells with different passage numbers. J Biol Chem, 2003, 278: 50902-50907.
17 Murillo H, Huang H, Schmidt LJ, et al. Role of P13K signaling in survival and progression of LNCaP prostate cancer cells to the androgen refractory state. Endocrinology, 2001, 142: 4795-4805.
18 Wen Y, Hu MC, Makino K, et al. HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the AKT pathway Cancer Res, 2000, 60: 6841-6845.
19 Zhou BP, Hu MC, Miller SA, et al. HER-2/neu blocks tumor necrosis factorinduced apoptosis via the AKT/NF-κB pathway. J Biol Chem, 2000, 275: 8027-8031.
20 Campbell RA, Bhat-Nakshatri P, Patel NM, et al. Phosphatidylinositol 3-kinase/AKTmediated activation of estrogen receptor a: a new model for anti-estrogen resistance. J Biol Chem, 2001, 276: 9817-9824.
21 Krajewska M, Krajewski S, Epstein JI, et al. Immunohistochemical analysis of bel-2, bax, bcl-X, and mcl-1 expression in prostate cancers. Am J Pathol, 1996, 148: 1567-1576.
22 Furuya Y, Krajewski S, Epstein JI, et al. Expression of bcl-2 and the progression of human and rodent prostatic cancers. Clin Cancer Res, 1996, 2: 389-398.
23 Gleave M, Tolcher A, Miyake H, et al. Progression to androgen independence is delayed by adjuvant treatment with antisense Bcl-2 oligodeoxynucleotides after castration in the LNCaP prostate tumor model. Clin Cancer Res, 1999, 5: 2891-2898.
24 Catz SD, Johnson JL. Transcriptional regulation of bcl-2 by nuclear factor kappaB and its significance in prostate cancer. Oncogene, 2001, 20: 7342-7351.
25 Pugazhenthi S, Nesterova A, Sable C, et al. Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein. J Biol Chem, 2000, 275: 10761-10766.
26 Petrylak DP, Tangen CM, Hussain MH, et al. Docetaxel and Estramustine Compared with Mitoxantrone and Prednisone for Advanced Refractory Prostate Cancer. N Engl J Med, 2004, 351: 1513-1520.
27 Dizeyi N, Bjartel A, Nilsson E, et al. Expression of serotonin receptors in human prostate cancer tissue and cell lines. Prostate, 2004, 59: 328-336.
28 Abdul M, Anezinis PE, Logothetis CJ, et al. Growth inhibition of human prostatic carcinoma cell lines by serotonin antagonists. Anticancer Res, 1994, 14: 1215-1220.
29 Hansson J, Abrahamsson PA. Neuroendocrine differentiation in prostate carcinoma. Scand J Urol Nephrol, 2003, 37(Suppl 212): 28-36.
30 Levine L, Lucci Ⅲ JA, Pazdrak B, et al. Bombesin stimulates nuclear factor kB activation and expression of proangiogenic factors in prostate cancer cells. Cancer Res, 2003, 63: 3495-3502.
31 Xie S, Lin HK, Ni J, et al. Regulation of interleukin-6-mediated differentiation by androgen signaling in prostate cancer LNCaP cells. Prostate, 2004, 60: 61-67.
32 Wang Q, Horiatis D, Pinski J. Interleukin-6 inhibits the growth of prostate cancer xenografts in mice by the process of neuroendocrine differentiation. Int J Cancer, 2004, 111: 508-513.
33 Persad S, Attwell S, Gray V, et al. Inhibition of integrin-linked kinase(ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer. Proc Natl Acad Sci USA, 2000, 97: 3207-3212.
34 Neshat MS, Mellinghoff IK, Tran C, et al. Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci USA, 2001, 98: 10314-10319.
35 Klasa RJ, Gillum AM, Klem RE, et al. Oblimersen Bcl-2 antisense: facilitating apoptosis in anticancer treatment. Antisense Nucleic Acid Drug Develop, 2002, 12: 193-213.
36 Tortora G, Caputo R, Damiano V, et al. Combined blockade of protein kinase A and bcl-2 by antisanse strategy induces apoptosis and inhibits tumor growth and angiogenesis. Clin Cancer Res, 2001, 7: 2537-2544.
37 Miyake H, Tolcher A, Gleave ME. Antisense Bcl-2 oligodeoxynuclentides inhibit progression to androgen-independence after castration in the Shionogi tumor model. Cancer Res, 1999, 59: 4030-4034.
38 Tocher AW. Preliminary phase I results of G3139 (bcl-2 antisense oligonucleotide) therapy in combination with docetaxel in hormone-refractory prostate cancer. Semin Oncol, 2001, 28: 67-70.
39 Tocher AW, Kuhn J, Schwartz G, et al: A Phase I pharmacokinetic and biological correlative study of oblimersan sodium (genasense, g3139), an antisense oligonucleotide to the bcl-2 mRNA, and of docetaxel in patients with hormone-refractory prostate cancer. Clin Cancer Res, 2004, 10: 5048-5057.
1. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin 2002; 52(1): 23-47.
2. Lieberman R. Evidence-based medical perspectives: The evolving role of PSA for early detection, monitoring of treatment response, and as a surrogate end point of efficacy for interventions in men with different clinical risk states for the prevention and progression of prostate cancer. Am J Ther 2004; 11(6): 501-506.
3. Cataloua WJ, Smith DS. Cancer reurrence and survival rates after anatomic radical retropubic prostatectomy for prostate cancer: Intermediate-term results. J Urol 1998; 160(6 Pt 2): 2428-2434.
4. Roscigno M, Sangalli M, Mazzoccoli B, et al. Medical therapy of prostate cancer. Areview. Minerva Urol Nefrol 2005; 57(2): 71-84.
5. Breyer RM, Kennedy CR, Zhang Y, et al. Targeted gene disruption of the prostaglandin E2 EP2 receptor. Adv Exp Med Biol 2002; 507: 321-326.
6. Martinet W, Schrijvers DM, Kockx MM. Nucleofection as an efficient nonviral transfection method for human monocytic cells. Biotechnol Lett 2003; 25(13): 1025-1029.
7. Foley R, Lawler M, Hollywood D. Gene-based therapy in prostate cancer. Lancet Oncol 2004; 5(8): 469-479.
8. Greco O, Scott SD, Marples B, Dachs GU. Cancer gene therapy: 'Delivery, delivery, delivery'. Front Biosci 2002; 7: d1516-1524.
9. Rauen KA, SudilovskyD, Le JL, et al. Expression of the coxsackie adenovirus receptor in normal prostate and in primary and metastatic prostate carcinoma: Potential relevance to gene therapy. Cancer Res 2002; 62(13): 3812-3818.
10. Bischoff JR, Kim DH, Williams A, et al. An adenovirus mutant that replicates selectively in p53-deficient haman tumor cells. Science 1996; 274(5286): 373-376.
11. McCormick F. Interactions betwecn adenovirus proteins and the p53 pathway: The development of ONYX-015. Semin Cancer Biol 2000, 10(6): 453-459.
12. Rodriguez R, Schuur ER, Lim HY, et al. Prostate attenuated replication competent adenovirus (ARCA) CN706: A selective cytotoxic for prostatespecific antigen-positive prostate cancer cells. Cancer Res 1997; 57(13): 2559-2563.
13. DeWeese TL, van der Poel H, Li S, et al. Aphase I trial of CV706, a replication-competent, PSAselective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy. Cancer Res 2001; 61(20): 7464-7472.
14. Parks RJ, Chen L, Anton M, et al. A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Nail Acad Sci USA 1996; 93(24): 13565-13570.
15. Zhang WW. Development and application of adenoviral vectors for gene therapy of cancer. Cancer Gene Ther 1999; 6(2): 113-138.
16. Harvey BG, Hackett NR, El-Sawy T, et al. Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs. J Virol 1999, 73(8): 6729-6742.
17. Marshall E. Gene therapy death prompts review of adenovirus vector. Science 1999; 286(5448): 2244-2245.
18. Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol 1992; 158: 97-129.
19. Miller DG, Rutledge EA, Russell DW. Chromosomal effects of adeno-associated virus vector integration. Nat Genet 2002; 30(2): 147-148.
20. Kremer EJ, Perricaudet M. Adenovirus and adeno-associated virus mediated gene transfer. Br Med Bull 1995; 51(1): 31-44.
21. Verma IM, Somia N. Gene therapy—promises, problems and prospects. Nature 1997; 389(6648): 239-242.
22. Boris-Lawrie K, Temin HM. The retroviral vector. Replication cycle and safety considerations for retrovirusmediated gene therapy. Ann NY Acad Sci 1994; 716: 59-70; Discussion 71.
23. Hacein-Bey-Abina S, de Saint Basile G, Cavazzana-Calvo M. Gone therapy of X-linked severe combined immunodeficiency. Methods Mol Biol 2003; 215: 247-259.
24. Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003; 348(3): 255-256.
25. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003; 302(5644): 415-419.
26. Frenkel N, Jacob RJ, Honess RW, et al. Anatomy of herpes simplex virus DNA Ⅲ. Characterization of defective DNA molecules and biological properties of virus populations containing them. J Virol 1975; 16(1): 153-167.
27. Huard J, Krisky D, Oligino T, et al. Gene transfer to muscle using herpes simplex virus-based vectors. Neuromuscul Disord 1997; 7(5): 299-313.
28. Harrow S, Papanastassiou V, Harland J, et al HSV1716 injection into the brain adjacent to tumour following surgical resection of high-grade glioma: Safety data and longterm survival. Gene Ther 2004; 11(22): 1648-1658.
29. Patel P, Ashduwn D, James N. Is gene therapy the answer for prostate cancer? Prostate Cancer Prostatic Dis 2004; 7(Suppl 1): S14-S19.
30. Hodge JW, Schlom J, Donohue SJ, et al. A recombinant vaccinia virus expressing human prostate-specific antigen (PSA): Safety and immunogenicity in a non-human primate, Int J Cancer 1995; 63(2): 231-237.
31. Somiari S, Glasspool-Malone J, Drabick JJ, et al. Theory and in vivo application of electroparative gene delivery. Mol Ther 2000; 2(3): 178-187.
32. Cemazar M, Sersa G, Wilson J, et al. Effective gene transfer to solid tumors using different nonviral gene delivery techniques: Electroporation, lipasomes, and integrin-targeted vector. Cancer Gene Ther 2002; 9(4): 399—406.
33. Yang NS, Burkholder J, Roberts B, et al. In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci USA 1990; 87(24): 9568-9572.
34. Rooney CM, Smith CA, Ng CY, et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998; 92(5): 1549-1555.
35. KastWM, Offringa R, Peters PJ, et al. Eradication of adenovirus El-induced tumors by EIA-specific cytotoxic T lymphocytes. Cell 1989; 59(4): 603-614.
36. Rodolfo M, Bassi C, Salvi C, Parmiani G. Therapeutic use of a long-term cytotoxic T cell line recognizing a common tumourassociated antigen: The pattern of in vitro reactivity predicts the in vivo effect on different tumours. Cancer Immunol Immunother 1991; 34(1): 53-62.
37. Riddell SR. Finding a place for tumor-specific T cells in targeted cancer therapy. J Exp Med 2004; 200(12): 1533-1537.
38. Knutson KL, Wagner W, Disis ML. Adoptive T cell therapy of solid cancers. Cancer Immunol Immunother 2005.
39. Yee C. Adoptive T cell therapy: Addressing challenges in cancer immunotherapy. J Transl Med 2005; 3(1): 17.
40. Clay TM, Custer MC, Sachs J, HwuP, Rosenberg SA, Nishimura MI. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes con fers anti-tumor reactivity. J Immunol 1999; 163(1): 507-513.
41. Clay TM, CusterMC, Spiess PJ, Nishimura Mi. Potential use ofT cell receptor genes to modify hematopoietic stem cells for the gene therapy of cancer. Pathol Oncol Res 1999; 5(1): 3-15.
42. Yotnda P, Savoldo B. Targeted delivery of adenoviral vectors by cytotoxic T cells. Blood 2004; 104(8): 2272-2280.
43. Leek RD, Harris AL. Tumor-associated macrophages in breast cancer. J Mammary Gland Biol Neoplasia 2002; 7(2): 177-189.
44. Bingle L, Brown NJ, Lewis CE. The role of tumour-associated macrophages in tumour progression: Implications for new anticancer therapies. J Patbol 2002; 196(3): 254-265.
45. Mantovani A, Bottazzi B. The origin and function of tumor-associated macrophages. Immunol Today 1992; 13(7): 265-270.
46. van Ravenswaay C, Kluin PM, Fleuren GJ. Tumour infiltratingn cells in human cancer. On the possible role of CD16t macrophages in antitumour cytotoxicity. Lab Invest 1992; 67(2): 166-174.
47. Satoh T, Saika T, Ebara S, Kusaka N, et al. Macrophages transduced with an adenoviral vector expressing interleukin 12 suppress tumor growth and metastasis in a preclinical metastatic prostate cancer model. Cancer Res 2003; 63(22): 7853-7860.
48. Lewis JS.. Macrophage responses to hypoxia: Relevance to disease mechanisms. J Leukoc Biol 1999; 66(6): 889-900.
49. Joseph IB, Isaacs JT. Macrophage role in the anti-prostate cancer response to one class of antiangiogenic agents. J Natl Cancer Inst 1998; 90(21): 1648-1653.
50. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991; 9: 271-296.
51. Wysocki PJ, Grabarczyk P, Mackiewicz-Wysocka M, et al. Genetically modified dendritic cells—a new, promising cancer treatment strategy? Expert Opin Biol Ther 2002; 2(8): 835-845.
52. Onaitis M, Kalady MF, Pruitt S, Tyler DS. Dendritic cell gene therapy. Surg Oncol Clin N Am 2002; 11(3): 645-660.
53. Collins MK, Cerundolo V. Gene therapy meets vaccine development. Trends Biotechnol 2004; 22(12): 623-626.
54. Dees EC, McKinnon KP, Kuhns JJ, et al. Dendritic cells can be rapidly expanded ex vivo and safely administered in patients with metastatic breast cancer. Cancer Immunol Immunother 2004; 53(9): 777-785.
55. Shalev M, Miles BJ, Thompson TC, et al. Suicide gene therapy for prostate cancer using a replication-deficient adenovirus containing the herpesvirus thymidine kinase gene. World J Urol 2000; 18(2): 125-129.
56. Ikegami S, Tadakuma T, Ono T, et al. Treatment efficiency of a suicide gene therapy using prostate-specific membrane antigen promoter/enhancer in a castrated mouse model of prostate cancer. Cancer Sci 2004; 95(4): 367-370.
57. Herman JR, Adler HL, Aguilar-Cordova E, et al. In situ gene therapy for adenocarcinoma of the prostate: A phase I clinical trial. Hum Gene Ther 1999; 10(7): 1239-1249.
58. Kubo H, Gardner TA, Wada Y, et al. Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer. Hum Gene Ther 2003; 14(3):227-241.
59. Yoshimura I, Ikegami S, Suzuki S, et al. Adenovirus mediated prostate specific enzyme prodrug gene therapy using prostate specific antigen promoter enhanced by the Cre-loxP system. J Urol 2002; 168(6): 2659-2664.
60. Freytag SO, Khil M, Stricker H, et al. Phase I study of replicationeompetent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer. Cancer Res 2002; 62(17): 4968-4976.
61. Freytag SO, Stricker H, Pegg J, Paielli D, et al. Phase I study of replication-competent adenovirus-mediated doublesuicide gene therapy in combination with conventional-dose three-dimensional conformal radiation therapy for the treatment of newly diagnosed, intermediate to high-risk prostate cancer. Cancer Res 2003; 63(21): 7497-7506.
62. Freytag SO, Paielli D, Wing M, Rogulski K, Brown S, Kolozsvary A, Seely J, Barton K, Dragovic A, Kim JH. Efficacy and toxicity of replication-competent adenovirus-modiatod double suicide gene therapy in combination with radiation therapy in an orthotopic mouse prostate cancer model, Int J Radiat Oncol Biol Phys 2002; 54(3): 873-885.
63. Miles BJ, Shalev M, Aguilar-Cordova E, et al. Prostate-specific antigen response and systemic T cell activation after in situ gene therapy in prostate cancer patients failing radiotherapy. Hum Gene Ther 2001; 12(16): 1955-1967.
64. Shalev M, Kadmon D, Teh BS, et al. Suicide gene therapy toxicity after multiple and repeat injections in patients with localized prostate cancer. J Urol 2000; 163(6): 1747-1750.
65. Satoh T, Teh BS, TimmeTL, et al. Enhanced systemic Tcell activation after in situ gene therapy with radiotherapy in prostate cancer patients, Int J Radiat Oncol Biol Phys 2004; 59(2): 562-571.
66. Sanda MG, Smith DC, Charles LG, et al. Recombinant vaccinia-PSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer. Urology 1999; 53(2): 260-266.
67. Eder JP, KantoffPW, Roper K, et al. A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in advanced prostate cancer. Clin Cancer Res 2000; 6(5): 1632-1638.
68. Vieweg J, Rosenthal FM, Bannerji R, et al. Immunotherapy of prostate cancer in the Dunning rat model: Use of cytokine gene modified tumor vaccines. Cancer Res 1994; 54(7): 1760-1765.
69. Griffith TS, Kawakita M, Tian J, et al. Inhibition of murine prostate tumor growth and activation of immunoregulatory cells with recombinant canarypox viruses. J Natl Cancer Inst 2001; 93(13): 998-1007.
70. NasuY, BangmaCH, HullGW, et al. Adenoviros-mediated interleukin-12 gene therapy for prostate cancer: Suppression of orthotopic tumor growth and pre-established lung metastases in an orthotopic model. Gene Ther 1999; 6(3): 338-349.
71. HullGW, McCurdyMA, Nasu Y, et al. Prostate cancer gene therapy: Comparison of adenovirus-mediated expression of interleukin 12 with interleukin 12 plus B4-1 for in situ gene therapy and gene-modified, cell-based vaccines. Clin Cancer Res 2000; 6(10): 4101-4109.
72. Nasu Y, Bangma CH, Hull GW, et al. Combination gene therapy with adenoviral vector-mediated HSV-tktGCV and IL-12 in an orthotopic mouse model for prostate cancer. Prostate Cancer Prostatic Dis 2001; 4(1): 44-55.
73. Nasu Y, Ebara S, Kumon H. Adenovirus-mediated interleukin-12 gene therapy for prostate cancer. Nippon Rinsho 2004; 62(6): 1181-1191.
74. Trodel S, Trachtenberg J, Toi A, et al. A phase I trial of adenovector-mediated delivery of interleukin-2 (AdIL-2) in high-risk localized prostate cancer. Cancer Gene Ther 2003; 10(10): 755-763.
75. KirschenbaumAIS, WangJP, et al. MUClexpression in prostate carcinoma: Correlation with grade and stage. Mol Urol 1999; 3: 163-168.
76. Oh YT, Chen DW, Dougherty G J, McBride WH. Adenoviral interleukin-3 gene-radiation therapy for prostate cancer in mouse model. Int J Radiat Oncol Biol Phys 2004; 59(2): 579-583.
77. Deliveliotis C, Skolarikos A, Karayannis A, et al. The prognostic value of p53 and DNA ploidy following radical prostatectomy. World J Urol 2003; 21(3): 171-176.
78. Ko SC, Gotoh A, Thalmann GN, et al. Molecular therapy with recombinant p53 adenovirus in an androgen-independent, metastatic human prostate cancer model. Hum Gene Ther 1996; 7(14): 1683-1691.
79. Cowen D, Salem N, Ashoori F, et al. Prostate cancer radiosensitization in vivo with adenovirus-mediated p53 gene therapy. Clin Cancer Res 2000; 6(11): 4402-4408.
80. Eastham JA, Chen SH, Sehgal I, et al. Prostate cancer gene therapy: Herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse and human prostate cancer models. Hum Gene Ther 1996; 7(4): 515-523.
81. Pisters LL, Pettaway CA, Troncoso P, et al. Evidence that transfer of functional p53 protein results in increased apoptosis in prostate cancer. Clin Cancer Res 2004; 10(8): 2587-2593.
82. Honda T, Kagawa S, Spurgers KB, et al. A recombinant adenovirus expressing wild-type Bax induces apoptosis in prostate cancer cells independently of their Bcl-2 status and androgen sensitivity. Cancer Biol Ther 2002; 1(2): 163-167.
83. Lowe SL, Rubinchik S, Honda T, McDonnell TJ, Dong JY, Norris JS. Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells. Gene Ther 2001; 8(18): 1363-1371.
84. Zhang Y, Yu J, Unni E, et al. Monogene and polygene therapy for the tratment of experimental prostate cancers by use of apoptotic genes bax and bad driven by the prostatespecific promoter ARR(2)PB. HumGene Ther 2002; 13(17): 2051-2064.
85. Krygier S, Djakiew D. Molecular characterization of the loss of p75(NTR) expression in human prostate tumor cells. Mol Carcinog 2001; 31(1): 46-55.
86. Fox ME, Lemmon MJ, Mauchline ML, et al. Anaerobic bacteria as a delivery system for cancer gene therapy: In vitro activation of 5-fluorocytosine by genetically engineered clostridia. Gene Ther 1996; 3(2): 173-178.
87. Yazawa K, Fujimori M, Amano J, et al. Bifidobaeterium longum as a delivery system for cancer gene therapy: Selective localization and growth in hypoxic tumors. Cancer Gene Ther 2000; 7(2): 269-274.
88. Fu GF, Li X, Hou YY, et al. Bifidobacterium longum as an oral delivery system of endostatin for gene therapy on solid liver cancer. Cancer Gene Ther 2005; 12(2): 133-140.
89. Yu YA, Shabahang S, Timiryasova TM, et al. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins. Nat Biotechnol 2004; 22(3): 313-320.
90. Zheng LM, Luo X, FengM, et al. Tumor amplified protein expression therapy: Salmonella as a tumor-selective protein delivery vector. Oncol Res 2000; 12(3): 127-135.
91. Luo X, Li Z, Lin S, et al. Antitumor effect of VNP20009, an attenuated Salmonella, in murine tumor models. Oncol Res 2000; 12(11-12): 501-508.
92. Rosenbarg SA, Spiess PJ, Kleiner DE. Antitumor effects in mice of the intravenous injection of attenuated Salmonella typhimurium. J Immunother 2002; 25(3): 218-225.
93. Lee KC, Zheng LM, Margitich D, Almassian B, King I. Evaluation of the acute and subehronic toxic effects in mice, rats, and monkeys of the genetically engineered and Escherichia coli cytosine deaminase gene-incorporated Salmonella strain, TAPET-CD, being developed as an antitumor agent, Int J Toxicol 2001; 20(4): 207-217.
94. Clairmont C, Lee KC, Pike J, et al. Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J Infect Dis 2000; 181(6): 1996-2002.
95. Lee CH, Wu CL, Shiau AL Endostatin gene therapy delivered by Salmonella choleraesuis in murine tumor models. J Gene Med 2004; 6(12): 1382-1393.
96. Zhao M, Yang M, Li XM, et al. Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci USA 2005; 102(3): 755-760.
97. Fong L, Brockstedt D, Benike C, et al. Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy. J Immunol 2001; 167(12): 7150-7156.
98. Small FJ, Fratesi P, Reese DM, Set al. Immunotherapy of hormone-refractory prostate cancer with antigen-loaded dendritic cells. J Clin Oncol 2000; 18(23): 3894-3903.
99. Burton J, Wells JM, Corke KP, et al. Macrophages accumulate in avascular, hypoxic areas of prostate tumours: Implications for the targeted therapeutic gene delivery to such sites. J Pathul 2000; 192(8A).
100. Vukanovic J, Isaacs JT. Linomide inhibits angiogenesis, growth, metastasis, and macrophage infiltration within rat prostatic cancers. Cancer Res 1995; 55(7): 1499-1504.
101. Franck-Lissbrant I, Haggstrom S, Damber JE, Bergh A. Testosterone stimulates angiogenesis and vascular regrowth in the ventral prostate in castrated adult rats. Endocrinology 1998; 139(2): 451-456.
102. Franck-Lissbrant 1. Tumour associated macrophages in human prostate cancer: Relation to clinicopathological variables and survival, Int J Oncul 2000; 17: 445-451.
103. Movsas B, Chapman JD, Horwitz EM, et al. Hypoxic regions exist in human prostate carcinoma. Urology 1999; 53: 11-18.
104. MacRae E, Brown NJ, Hamdy FC, Lewis CE. Use of macrophages to target gene therapy to hypoxic areas of prostate tumours. British Microcirculation Society Annual Meeting 2004 Abstract Booklet 2004.
105. Ma Q, Safar M, Holmes E, et al. Anti-prostate specific membrane antigen designer T cells for prostate cancer therapy. Prostate 2004; 61(1): 12-25.