A prostate-derived cDNA that is mapped to human chromosome 19 encodes a novel protein

The epithelial cells of prostate gland secrete various secretory products that play an important role in the growth and differentiation of prostate gland. These secretory products have also been implicated in neuroendocrine differentiation of benign prostatic hyperplasia and prostate malignancy. We have cloned a prostate-derived cDNA encoding a novel protein with a predicted molecular weight of 78 kDa (P(78)), and precisely mapped the cDNA sequence to chromosome 19. The P(78) gene has a complex genomic structure with 18 exons and 17 introns. The P(78) contains two conserved structural domains with limited similarity to domain D of synapsin I. The P(78) mRNA was expressed in various human cell lines. Western blot analysis using antibody specific for the P(78) revealed the presence of the P(78) protein in the prostate cancer cell lines with much lower level in metastatic prostate cancer cell lines compared to that in a primary prostate cancer cell line.     

Expression of prostate specific membrane antigen (PSMA) in prostatic adenocarcinoma and prostatic intraepithelial neoplasia

The prostatic membrane antigen (PSMA) is a protein that is expressed in the prostatic epithelium. We studied the expression of PSMA in a series of 55 patients with different stages of prostate cancer and we compared the PSMA staining in prostate cancer cells, in high-grade prostatic intraepithelial neoplasia (PIN) and in histologically benign prostatic epithelium for the same specimen. For this purpose archival paraffin-embedded specimens were studied by immunohistochemistry with a monoclonal antibody 7E11-C5.3 against PSMA using the streptavidin-biotin method. The mean percentage of PSMA immunoreactivity was 56.67% in prostate cancer (CaP) cells, and 48.6% in PIN cells, which was significantly higher than benign-appearing prostatic epithelium (5.72%) (for each pair, p<0.001). PSMA expression was greater in CaP with a higher Gleason score (p=0.01), but no relationship was found with serum PSA value. We conclude that PSMA overexpression is detected in high-grade PIN and is associated with a higher Gleason score of prostate cancer. It is a potential marker for studying carcinogenesis and progression of prostate cancer.     

Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression.

Ectopical expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the development and malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. We cloned and characterized the human MUC18 (huMUC18) cDNA gene from three human prostate cancer cell lines and three human melanoma cell lines. The cDNA sequences from the six human cancer cell lines were identical except differences in one to five nucleotides. The deduced amino acid sequences of the longest ORF were 646 amino acids that were identical in these cDNAs except for one to three amino acid residues. The amino acid sequences of all our huMUC18 cDNA genes are similar to that cloned by other group (GenBank access #M28882) except differences in the same seven amino acids. We conclude that huMUC18 cDNA gene reported here represents the gene product from a major allele. The MUC18 mRNA and protein was expressed in three metastatic prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one non-metastatic prostate cancer cell line (LNCaP.FGC). The expression of huMUC18 in these four cell lines is positively related to their extent of in vitro motility and invasiveness and in vivo metastasis in nude mice. HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from cultured primary normal prostatic epithelial cells and the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas, and in cells of a perineural invasion, a lymph node, and a lung metastases compared to that in normal or benign hyperplastic epithelium (BPH). We therefore conclude that MUC18 expression is increased during prostate cancer initiation (high grade PIN) and progression to carcinoma, and in metastatic cell lines and metastatic carcinoma. Increased expression of MUC18 is implicated to play an important role in developing and malignant progression of human prostate cancer. Furthermore, the lacking of predominant cytoplasmic membrane expression of MUC18 appeared to correlate with malignant progression of prostate cancer.     

Immunohistochemical localization of human protein 1 in the female prostate (Skene's gland) and the male prostate

Mouse monoclonal anti-urine protein 1 antibody and the biotin-streptavid in-peroxidase technique were used for the immunohistochemical demonstration of human protein 1 in prostatic tissue of both sexes. In the female prostate (Skene's gland), like the male prostate, high expression of human protein 1 was observed on the luminal surface and in the apical cytoplasm of secretory cells of prostatic glands, as well as on the luminal surface of the epithelium of the large ducts of the female prostate and urethra. Expression was also found in the membranes of secretory and basal cells of the glands, in membranes of the urethral uroepithelium and of the female prostate ducts, in the content of glands and ducts, as well as in vascular endothelium and smooth muscle. Human protein 1 (urine protein 1) expression in the secretory cells of the male and female prostate and its incorporation into the surface of cells lining the lumina of the female urethroprostatic complex is indicative not only of the secretory role of protein 1 but also of its potential protective properties operative in shielding the uroepithelium from the aggressive urinary environment. All genito-urinary tissue, and especially the female prostate, were found to be a potential source of urine protein 1 (human protein 1), refuting the notion held so far that it is exclusively the genito-urinary prostatic tissue of the male that participates in its production. The corresponding immunohistochemical distribution of human protein 1 in the same structures of the male and female prostate provides yet another analogous functional-morphological parameter of prostatic tissue in both sexes and further evidence supporting the non-vestigial concept of the prostate in the female. This revised version was published online in November 2006 with corrections to the Cover Date.     

Immunohistochemical localization of human protein 1 in the female prostate (Skene's gland) and the male prostate

Mouse monoclonal anti-urine protein 1 antibody and the biotin-streptavid in-peroxidase technique were used for the immunohistochemical demonstration of human protein 1 in prostatic tissue of both sexes. In the female prostate (Skene's gland), like the male prostate, high expression of human protein 1 was observed on the luminal surface and in the apical cytoplasm of secretory cells of prostatic glands, as well as on the luminal surface of the epithelium of the large ducts of the female prostate and urethra. Expression was also found in the membranes of secretory and basal cells of the glands, in membranes of the urethral uroepithelium and of the female prostate ducts, in the content of glands and ducts, as well as in vascular endothelium and smooth muscle. Human protein 1 (urine protein 1) expression in the secretory cells of the male and female prostate and its incorporation into the surface of cells lining the lumina of the female urethroprostatic complex is indicative not only of the secretory role of protein 1 but also of its potential protective properties operative in shielding the uroepithelium from the aggressive urinary environment. All genito-urinary tissue, and especially the female prostate, were found to be a potential source of urine protein 1 (human protein 1), refuting the notion held so far that it is exclusively the genito-urinary prostatic tissue of the male that participates in its production. The corresponding immunohistochemical distribution of human protein 1 in the same structures of the male and female prostate provides yet another analogous functional-morphological parameter of prostatic tissue in both sexes and further evidence supporting the non-vestigial concept of the prostate in the female. This revised version was published online in November 2006 with corrections to the Cover Date.     

Application of alpha-methylacyl coenzyme A racemase immunohistochemistry in the diagnosis of prostate cancer: a review

Alpha-methylacyl coenzyme A racemase (AMACR) is a recently discovered enzyme protein that has been shown to be increased at both the mRNA and protein levels in prostatic adenocarcinoma as compared with normal prostatic tissues. Since its discovery, AMACR has gained wide acceptance for use in the diagnosis of prostatic adenocarcinoma in conjunction with morphology and immunohistochemical staining for basal cell markers. Numerous studies have consistently shown high sensitivity and specificity of AMACR for prostate cancer. This review focuses on AMACR expression in prostate cancer and its morphologic variants, high grade prostatic intraepithelial neoplasia, adenosis and benign conditions of the prostate. In addition, we discuss AMACR expression in other tumors. We also focus on the utility and technical aspects of the now-popular "triple stain" immunohistochemical antibody cocktail, consisting of antibodies to high-molecular-weight keratin, p63 and AMACR. Finally, we emphasize diagnostic pitfalls in the application of AMACR to small, atypical foci of glands seen on prostate needle core biopsy and project future diagnostic as well as clinical applications for the protein.     

Zinc as an anti-tumor agent in prostate cancer and in other cancers

Human prostate glandular epithelial cells have the unique capability of accumulating high levels of zinc. This is essential to inhibit m-aconitase activity so that citrate can accumulate for secretion into prostatic fluid, which is a major function of the prostate gland. As a result, the Krebs cycle is truncated with the consequence of the lost ATP production that would result from citrate oxidation. The cellular accumulation of zinc also inhibits mitochondrial terminal oxidation and respiration. In addition to these metabolic effects, zinc accumulation exhibits anti-proliferative effects via its induction of mitochondrial apoptogenesis. Zinc accumulation also inhibits the invasive/migration activities in malignant prostate cells. The anti-proliferative effects and the effects on invasion and migration occur through zinc activation of specific intracellular signaling pathways. Consequently, these effects impose anti-tumor actions by zinc. The ability of prostate cells to accumulate zinc is due to the expression and activity of the zinc uptake transporter, ZIP1. To avoid the anti-tumor effects of zinc, in prostate cancer the malignant prostate cells exhibit a silencing of ZIP1 gene expression accompanied by a depletion of cellular zinc. Therefore we regard ZIP1 as a tumor suppressor gene in prostate cancer. In addition to prostate cells, similar tumor suppressor effects of zinc have been identified in several other types of tumors.     

Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer

The regulation of intracellular Ca(2+) plays a key role in the development and growth of cells. Here we report the cloning and functional expression of a highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene product of the CaT1 protein cloned from rat duodenum and is therefore called CaT-like (CaT-L). CaT-L is expressed in locally advanced prostate cancer, metastatic and androgen-insensitive prostatic lesions but is undetectable in healthy prostate tissue and benign prostatic hyperplasia. Additionally, CaT-L is expressed in normal placenta, exocrine pancreas, and salivary glands. New markers with well defined biological function that correlate with aberrant cell growth are needed for the molecular staging of cancer and to predict the clinical outcome. The human CaT-L channel represents a marker for prostate cancer progression and may serve as a target for therapeutic strategies.     

Phosphorylation of the PKG substrate, vasodilator-stimulated phosphoprotein (VASP), in human cultured prostatic stromal cells.

Nitric oxide (NO) is known to regulate contractility and proliferation of cells within the prostate, however, the mechanism by which this occurs is unknown. The cGMP-dependent protein kinase (PKG) signalling pathway may be involved, and recent work has shown that activation of this pathway can be assessed by analysis of phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The aim of the current study is to characterise the expression of VASP in the human prostate and human cultured prostatic stromal cells (HCPSCs), and to investigate whether NO activates PKG in these cells. Our studies revealed that VASP is expressed, and that incubation of HCPSCs with PKG-activating cGMP-analogues or the NO-donor, SNP, caused a significant PKG-dependent increase in VASP serine-239 phosphorylation. In addition, SNP elicited a reduction in intracellular K(+) in a time frame consistent with the phosphorylation of VASP and activation of PKG. These data demonstrate that VASP can be used to assess the NO/cGMP/PKG signalling pathway in HCPSCs. In addition, we demonstrate for the first time that SNP, probably via NO release, leads to phosphorylation of VASP in a manner consistent with PKG activation.     

Heat shock protein 70 is secreted from tumor cells by a nonclassical pathway involving lysosomal endosomes

Heat shock protein (HSP)70 can be released from tumor cells and stimulate a potent antitumor immune response. However, HSP70 does not contain a consensus secretory signal and thus cannot traverse the plasma membrane by conventional mechanisms. We have observed HSP70 release from intact human prostate carcinoma cell lines (PC-3 and LNCaP) by a mechanism independent of de novo HSP70 synthesis or cell death. This pathway is similar to one used by the leaderless protein IL-1beta. Our studies show that HSP70 release involves transit though an endolysosomal compartment and is inhibited by lysosomotropic compounds. In addition, the rate of HSP70 secretion correlates well with the appearance of the lysosomal marker LAMP1 on the cell surface, further suggesting the role for endolysosomes. The entry of HSP70 into this secretory compartment appears to involve the ABC family transporter proteins and ABC transporter inhibitor glibenclamide antagonizes secretion. Although the cell signals involved in triggering stress induced HSP70 release though this lysosomal pathway are largely unknown, our experiments suggest a regulatory role for extracellular ATP. These mechanisms appear to be shared by IL-1beta secretion. Following release, we observed the binding of extracellular HSP70 to the cell surface of the prostate carcinoma cells. These findings suggest that secreted HSP70 can take part in paracrine or autocrine interactions with adjacent cell surfaces. Our experiments therefore suggest a mechanism for HSP70 secretion and binding to the surface of other cells that may be involved in recognition of the tumor cells by the immune system.     

Immunohistological detection of BRAF25 in human prostate tumor and cancer specimens

BRAF25 is an alternatively spliced protein of BRAF35 (see associated paper). We have mapped the BRAF25 gene to chromosome sub-band 19p13.3, a region where loss of chromosomal heterozygosity has been reported in about 50% of ovarian cancers. Because of the high incidence of genetic links of prostate cancer to breast and ovarian cancers, we investigated the BRAF25 expression in the prostate specimens. Immunohistochemical analysis using antibodies specific for BRAF25 revealed a strong immunostaining in sections of the benign prostatic hyperplasia (BPH). The staining was concentrated on the nuclei of cells facing the lumen of prostatic glands, even though the sporadic nuclei of cells in stromas were also stained. However, the expression of BRAF25 was dramatically reduced in intermediate prostate cancer and absent in advanced prostate cancer. Preincubation of the antibody with the immunizing peptide abolished immunostaining in BPH specimens. Therefore, the expression of BRAF25 was gradually lost in prostate cancer.     

Molecular cloning and characterization of a novel 3'-phosphoadenosine 5'-phosphosulfate transporter, PAPST2

Sulfation is an important posttranslational modification associated with a variety of molecules. It requires the involvement of the high energy form of the universal sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Recently, we identified a PAPS transporter gene in both humans and Drosophila. Although human colonic epithelial tissues express many sulfated glycoconjugates, PAPST1 expression in the colon is trace. In the present study, we identified a novel human PAPS transporter gene that is closely related to human PAPST1. This gene, called PAPST2, is predominantly expressed in human colon tissues. The PAPST2 protein is localized on the Golgi apparatus in a manner similar to the PAPST1 protein. By using yeast expression studies, PAPST2 protein was shown to have PAPS transport activity with an apparent Km value of 2.2 microM, which is comparable with that of PAPST1 (0.8 microM). Overexpression of either the PAPST1 or PAPST2 gene increased PAPS transport activity in human colon cancer HCT116 cells. The RNA interference of the PAPST2 gene in the HCT116 cells significantly reduced the reactivity of G72 antibody directed against the sialyl 6-sulfo N-acetyllactosamine epitope and total sulfate incorporation into cellular proteins. These findings indicate that PAPST2 is a PAPS transporter gene involved in the synthesis of sulfated glycoconjugates in the colon.     

Not all ALMT1-type transporters mediate aluminum-activated organic acid responses: the case of ZmALMT1 – an anion-selective transporter

The phytotoxic effects of aluminum (Al) on root systems of crop plants constitute a major agricultural problem in many areas of the world. Root exudation of Al-chelating molecules such as low-molecular-weight organic acids has been shown to be an important mechanism of plant Al tolerance/resistance. Differences observed in the physiology and electrophysiology of root function for two maize genotypes with contrasting Al tolerance revealed an association between rates of Al-activated root organic acid release and Al tolerance. Using these genotypes, we cloned ZmALMT1 , a maize gene homologous to the wheat ALMT1 and Arabidopsis AtALMT1 genes that have recently been described as encoding functional, Al-activated transporters that play a role in tolerance by mediating Al-activated organic acid exudation in roots. The ZmALMT1 cDNA encodes a 451 amino acid protein containing six transmembrane helices. Transient expression of a ZmALMT1 ::GFP chimera confirmed that the protein is targeted to the plant cell plasma membrane. We addressed whether ZmALMT1 might underlie the Al-resistance response (i.e. Al-activated citrate exudation) observed in the roots of the Al-tolerant genotype. The physiological, gene expression and functional data from this study confirm that ZmALMT1 is a plasma membrane transporter that is capable of mediating elective anion efflux and influx. However, gene expression data as well as biophysical transport characteristics obtained from Xenopus oocytes expressing ZmALMT1 indicate that this transporter is implicated in the selective transport of anions involved in mineral nutrition and ion homeostasis processes, rather than mediating a specific Al-activated citrate exudation response at the rhizosphere of maize roots.     

LIV-1 promotes prostate cancer epithelial-to-mesenchymal transition and metastasis through HB-EGF shedding and EGFR-mediated ERK signaling

LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there was no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In the present communication, we found increased LIV-1 expression in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives human prostate cancer EMT in an androgen-refractory prostate cancer cells (ARCaP) prostate cancer bone metastasis model. LIV-1, when overexpressed in ARCaP(E) (derivative cells of ARCaP with epithelial phenotype) cells, promoted EMT irreversibly. LIV-1 overexpressed ARCaP(E) cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaP(E) cells that elicited constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promoted EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases.     

Not all ALMT1-type transporters mediate aluminum-activated organic acid responses: the case of ZmALMT1 - an anion-selective transporter.

The phytotoxic effects of aluminum (Al) on root systems of crop plants constitute a major agricultural problem in many areas of the world. Root exudation of Al-chelating molecules such as low-molecular-weight organic acids has been shown to be an important mechanism of plant Al tolerance/resistance. Differences observed in the physiology and electrophysiology of root function for two maize genotypes with contrasting Al tolerance revealed an association between rates of Al-activated root organic acid release and Al tolerance. Using these genotypes, we cloned ZmALMT1, a maize gene homologous to the wheat ALMT1 and Arabidopsis AtALMT1 genes that have recently been described as encoding functional, Al-activated transporters that play a role in tolerance by mediating Al-activated organic acid exudation in roots. The ZmALMT1 cDNA encodes a 451 amino acid protein containing six transmembrane helices. Transient expression of a ZmALMT1::GFP chimera confirmed that the protein is targeted to the plant cell plasma membrane. We addressed whether ZmALMT1 might underlie the Al-resistance response (i.e. Al-activated citrate exudation) observed in the roots of the Al-tolerant genotype. The physiological, gene expression and functional data from this study confirm that ZmALMT1 is a plasma membrane transporter that is capable of mediating elective anion efflux and influx. However, gene expression data as well as biophysical transport characteristics obtained from Xenopus oocytes expressing ZmALMT1 indicate that this transporter is implicated in the selective transport of anions involved in mineral nutrition and ion homeostasis processes, rather than mediating a specific Al-activated citrate exudation response at the rhizosphere of maize roots.     

Human Na+ -coupled citrate transporter: primary structure,genomic organization,and transport function

This paper describes the cloning and functional characterization of the human Na(+)-coupled citrate transporter (NaCT). The cloned human NaCT shows 77% sequence identity with rat NaCT. The nact gene is located on human chromosome 17 at p12-13. NaCT mRNA is expressed most predominantly in the liver, with moderate expression detectable in the brain and testis. When functionally expressed in mammalian cells, human NaCT mediates the Na(+)-coupled transport of citrate. Studies with several monocarboxylates, dicarboxylates, and tricarboxylates show that the transporter is selective for citrate with comparatively several-fold lower affinity for other intermediates of citric acid cycle. The Michelis-Menten constant for citrate is approximately 650 microM. The activation of citrate transport by Na(+) is sigmoidal, suggesting involvement of multiple Na(+) ions in the activation process. The transport process is electrogenic. This represents the first plasma membrane transporter in humans that mediates the preferential entry of citrate into cells. Citrate occupies a pivotal position in many important biochemical pathways. Among various citric acid cycle intermediates, citrate is present at the highest concentrations in human blood. The selectivity of NaCT towards citrate and its predominant expression in the liver suggest that this transporter may facilitate the utilization of circulating citrate for the generation of metabolic energy and for the synthesis of fatty acids and cholesterol.     

Conditional expression of the androgen receptor induces oncogenic transformation of the mouse prostate

The androgen signaling pathway, mediated through the androgen receptor (AR), is critical in prostate tumorigenesis. However, the precise role of AR in prostate cancer development and progression still remains largely unknown. Specifically, it is unclear whether overexpression of AR is sufficient to induce prostate tumor formation in vivo. Here, we inserted the human AR transgene with a LoxP-stop-loxP (LSL) cassette into the mouse ROSA26 locus, permitting "conditionally" activated AR transgene expression through Cre recombinase-mediated removal of the LSL cassette. By crossing this AR floxed strain with Osr1-Cre (odd skipped related) mice, in which the Osr1 promoter activates at embryonic day 11.5 in urogenital sinus epithelium, we generated a conditional transgenic line, R26hAR(loxP):Osr1-Cre+. Expression of transgenic AR was detected in both prostatic luminal and basal epithelial cells and is resistant to castration. Approximately one-half of the transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) lesions. Intriguingly, four mice (10%) developed prostatic adenocarcinomas, with two demonstrating invasive diseases. Positive immunostaining of transgenic AR protein was observed in the majority of atypical and tumor cells in the mPIN and prostatic adenocarcinomas, providing a link between transgenic AR expression and oncogenic transformation. An increase in Ki67-positive cells appeared in all mPIN and prostatic adenocarcinoma lesions of the mice. Thus, we demonstrated for the first time that conditional activation of transgenic AR expression by Osr1 promoter induces prostate tumor formation in mice. This new AR transgenic mouse line mimics the human disease and can be used for study of prostate tumorigenesis and drug development.