Differential responsiveness of prostatic acid phosphatase and prostate-specific antigen mRNA to androgen in prostate cancer cells

Androgens regulate the expression of both human prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), two major prostate epithelium-specific differentiation antigens. Due to the important role of these two enzymes as prostate epithelium differentiation markers, we investigated their regulation of expression at the mRNA level in LNCaP human prostate carcinoma cells. Interestingly, phenol red, a pH indicator in the culture medium, promoted cell growth. To eliminate this non-specific effect, a phenol red-free, steroid-reduced medium was utilized. When high-density cells were grown in that medium, 5alpha-dihydrotestosterone (DHT) suppressed PAcP but stimulated PSA. However, tumor promoter phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) functioned as a potent inhibitor of both PAcP and PSA expression. Prolonged treatment with DHT as well as TPA resulted in a similar down-regulation of protein kinase C and cellular PAcP activities. Thus, the levels of PAcP and PSA mRNA are differentially regulated by androgens in LNCaP cells.     

Multifunctionality of prostatic acid phosphatase in prostate cancer pathogenesis

The role of human prostatic acid phosphatase (PAcP, {"type":"entrez-protein","attrs":{"text":"P15309","term_id":"130730"}}P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum (ER), magnifying normally difficult to detect subsets of the protein of interest. For PAcP, this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wildtype PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed ^PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate ^PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.     

Transactivation of fibronectin promoter by HTLV-I Tax through NF-kappaB pathway

The facts that fibronectin (FN) mRNA is elevated in cells expressing human T cell leukemia virus type I (HTLV-I) Tax protein and that Tax is known to transactivate the cellular cAMP-response element (CRE) prompted us to examine whether Tax activates the FN promoter of which CRE is thought to play an important role. We showed that Tax transactivated the FN promoter in Jurkat cells. Deletion analyses showed that the response-element resides within the promoter region of -69 bp and that an NF-kappaB-binding site at -41 bp is involved in the Tax-activation of the FN promoter. Gel-shift assays showed that DNA-protein complexes binding to the NF-kappaB site, composed of NF-kappaB p50/p65, were induced on the NF-kappaB motif at -41 bp by Tax. Overexpression of NF-kappaB enhanced the Tax-activation of the FN promoter. Our study shows that the FN promoter is transactivated by Tax through the NF-kappaB pathway.     

Characterization of a prostate-specific tyrosine phosphatase by mutagenesis and expression in human prostate cancer cells

The cellular form of human prostatic acid phosphatase (PAcP) is a neutral protein-tyrosine phosphatase (PTP) and may play a key role in regulating the growth and androgen responsiveness of prostate cancer cells. The functional role of the enzyme is at least due in part to its dephosphorylation of c-ErbB-2, an in vivo substrate of the enzyme. In this study, we investigated the molecular mechanism of phosphotyrosine dephosphorylation by cellular PAcP. We mutated several amino acid residues including one cysteine residue that was proposed to be involved in the PTP activity of the enzyme by serving as the phosphate acceptor. The cDNA constructs of mutant enzymes were transiently transfected into C-81 LNCaP and PC-3 human prostate cancer cells that lack the endogenous PAcP expression. The phosphotyrosine level of ErbB-2 in these transfected cells was subsequently analyzed. Our results demonstrated that the phosphotyrosine level of ErbB-2 in cells expressing H12A or D258A mutant PAcP is similar to that in control cells without PAcP expression, suggesting that these mutants are incapable of dephosphorylating ErbB-2. In contrast, cells expressing C183A, C281A, or wild-type PAcP had a decreased phosphotyrosine level of ErbB-2, compared with the control cells. Similar results were obtained from in vitro dephosphorylation of immunoprecipitated ErbB-2 by these mutant enzymes. Furthermore, transient expression of C183A, C281A, or the wild-type enzyme, but not H12A or D258A, decreased the growth rate of C-81 LNCaP cells. The data collectively indicate that His-12 and Asp-258, but not Cys-183 or Cys-281, are required for the PTP activity of PAcP.     

人类RELM-beta基因启动子的结构与功能分析

为克隆人类抵抗素样分子β（resistin-like molecule beta, RELMβ）基因的上游启动子序列,并观察其不同截短片段的启动子活性,以人类基因组DNA为模板,通过PCR扩增方法获得-871～+50 bp、-729～+50 bp、-471～+50 bp、-438～+50 bp、-371～+50 bp大小的RELMβ启动子片段,将其定向克隆入pGL3-Basic载体,构建荧光素酶报告基因载体,并制备转录因子CDX-2结合位点的突变或缺失体.在阳离子脂质体的介导下,报告基因载体分别瞬时转染人胚肾293细胞、结肠癌HCT116和SW480细胞、宫颈癌HeLa细胞.结果发现,各RELMβ启动子片段在293、HCT116、SW480细胞中均有活性,但在HeLa细胞中活性缺失;-471～-438 bp区存在RELMβ启动子的核心调控元件.针对该区域CDX-2转录因子结合位点进行突变,能导致RELMβ启动子活性显著降低;凝胶电泳迁移率实验表明,该区段能结合CDX-2.结果提示,成功克隆了具有活性的RELMβ启动子序列,CDX-2为其重要的转录因子,为研究RELMβ基因的转录调控机制奠定了实验基础.     

The epidermal growth factor receptor from prostate cells is dephosphorylated by a prostate-specific phosphotyrosyl phosphatase.

Human prostatic acid phosphatase (PAcP) has been found to have phosphotyrosyl-protein phosphatase activity (H. C. Li, J. Chernoff, L. B. Chen, and A. Kirschonbaun, Eur. J. Biochem. 138:45-51, 1984; M.-F. Lin and G. M. Clinton, Biochem. J. 235:351-357, 1986) and has been suggested to negatively regulate phosphotyrosine levels, at least in part, by inhibition of tyrosine protein kinase activity (M.-F. Lin and G. M. Clinton, Adv. Protein Phosphatases 4:199-228, 1987; M.-F. Lin, C. L. Lee, and G. M. Clinton, Mol. Cell. Biol. 6:4753-4757, 1986). We investigated the molecular interaction of PAcP with a specific tyrosine kinase, the epidermal growth factor (EGF) receptor, from prostate carcinoma cells. Of several proteins phosphorylated in membrane vesicles from prostate carcinoma cells, PAcP selectively dephosphorylated the EGF receptor. The prostate EGF receptor was more efficiently dephosphorylated by PAcP than by another phosphotyrosyl phosphatase, potato acid phosphatase. Further characterization of the interaction of PAcP with the EGF receptor revealed that the optimal rate of dephosphorylation occurred at neutral rather than at acid pH. Thus, the enzyme that we formerly referred to as PAcP we now call prostatic phosphotyrosyl-protein phosphatase. Hydrolysis of phosphate from tyrosine residues in the immunoprecipitated EGF receptor catalyzed by purified prostatic phosphotyrosyl-protein phosphatase caused a 40 to 50% decrease in the receptor tyrosine kinase activity with angiotensin as the substrate. In contrast, autophosphorylation of the receptor was associated with an increase in tyrosine kinase activity.     

Protein kinase C pathway is involved in regulating the secretion of prostatic acid phosphatase in human prostate cancer cells

The stimulated secretion of prostatic acid phosphatase (PAcP) has been known to be a hallmark of androgen action on human prostate epithelial cells for the last five decades. The molecular mechanism of androgen action on PAcP secretion, however, has remained mostly unknown. We investigated the molecular mechanism that promotes PAcP secretion in LNCaP human prostate carcinoma cells which express PAcP and are androgen-responsive. Treatment with 12-o-tetradecanoyl phorbol-13-acetate (TPA), a protein kinase C (PKC) activator, resulted in an increased secretion of PAcP in a dose- and time-dependent fashion. 4Alpha-phorbol, a biologically inactive isomer of TPA, had no effect. This TPA stimulation of PAcP secretion was observed 2 h after exposure, while TPA did not have a significant effect on the mRNA level even with a 6 h treatment. A23187 calcium ionophore, known to mobilize cellular calcium which is a co-factor of PKC, also activated PAcP secretion. This TPA stimulation of PAcP secretion was more potent than the conventional stimulating agent 5alpha-dihydrotestosterone (DHT) at the same concentration of 50 nM. Furthermore, the action of TPA and DHT on PAcP secretion was blocked by five different PKC inhibitors. Results also showed that DHT, as well as TPA, could rapidly modulate PKC activity. Therefore, PKC can regulate PAcP secretion, and may also be involved in DHT action on PAcP secretion.     

Tyrosyl kinase activity is inversely related to prostatic acid phosphatase activity in two human prostate carcinoma cell lines.

Alterations in prostatic acid phosphatase (PAcP), a phosphotyrosyl phosphatase, corresponded to changes in overall tyrosyl kinase activity. PAcP added to extracts of prostate carcinoma cells with a low endogenous level of PAcP activity and elevated tyrosyl kinase activity decreased the tyrosyl kinase activity. On the other hand, when PAcP activity was decreased by the addition of androgens to cells, there was a corresponding increase in tyrosyl kinase activity.     

Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

The human β-like globin genes are arranged as a clusterof five genes (ε, Gγ, Aγ, δ and β) in the order of theirtemporal expression. The human embryonic ε-globin geneis expressed in the blood island of the embryonic yolk sacand is silenced completel