Fine structure of pituitary tumors and transplants in mice: effects on the prostate.

In this study, we demonstrate insulin-like growth factor binding protein (IGFBP) acid proteolysis in conditioned media (CM) from normal and malignant primary cultures of prostatic epithelial cells, prostatic cell lines, and in seminal plasma. We further demonstrate the absence of such activity in CM from prostatic stromal cells. Radio-labeled IGFBPs (1–6) were incubated with various acidified CM and seminal plasma. None of these media showed IGFBP proteolytic activity at neutral pH, but all CM from prostatic epithelial cells (PC-E) demonstrated strong IGFBP proteolysis at acidic pH. No acid-activated proteolysis was observed in the CM from stromal cell cultures. In order to ascertain the role of cathepsin D, anti-cathepsin antibodies were used to immunodeplete the media of the selected enzymes prior to incubation with IGFBPs. Depletion of cathepsin D greatly reduced the proteolytic activity of the PC-E CM. Additionally, purified cathepsin D yielded a digestion pattern identical to that produced by prostatic cell CM and seminal plasma, following acidic incubation with IGFBP-3. Remarkably, the proteolytic pattern generated by seminal plasma, when incubated with IGFBP-3 at neutral pH, corresponded to that produced by prostate-specific antigen (PSA), demonstrating the interpolation of both neutral and acid proteases from prostate cells into seminal plasma. In conclusion, prostatic epithelial cells secrete acid-specific IGFBP protease(s) related to cathepsin D. Although no significant statistical difference was observed in the degree of acid-specific proteolysis in the media from normal versus malignant primary epithelial cell cultures, physiologicalcharacteristics of the malignant state might facilitate increased cathepsin D activity. We suspect this proteolysis may play a role in prostatic cell proliferationand invasive tumor growth. J. Cell. Physiol. 171:196–204, 1997. © 1997 Wiley-Liss, Inc.

In this study, we demonstrate insulin-like growth factor binding protein (IGFBP) acid proteolysis in conditioned media (CM) from normal and malignant primary cultures of prostatic epithelial cells, prostatic cell lines, and in seminal plasma. We further demonstrate the absence of such activity in CM from prostatic stromal cells. Radio-labeled IGFBPs (1-6) were incubated with various acidified CM and seminal plasma. None of these media showed IGFBP proteolytic activity at neutral pH, but all CM from prostatic epithelial cells (PC-E) demonstrated strong IGFBP proteolysis at acidic pH. No acid-activated proteolysis was observed in the CM from stromal cell cultures. In order to ascertain the role of cathepsin D, anti-cathepsin antibodies were used to immunodeplete the media of the selected enzymes prior to incubation with IGFBPs. Depletion of cathepsin D greatly reduced the proteolytic activity of the PC-E CM. Additionally, purified cathepsin D yielded a digestion pattern identical to that produced by prostatic cell CM and seminal plasma, following acidic incubation with IGFBP-3. Remarkably, the proteolytic pattern generated by seminal plasma, when incubated with IGFBP-3 at neutral pH, corresponded to that produced by prostate-specific antigen (PSA), demonstrating the interpolation of both neutral and acid proteases from prostate cells into seminal plasma. In conclusion, prostatic epithelial cells secrete acid-specific IGFBP protease(s) related to cathepsin D. Although no significant statistical difference was observed in the degree of acid-specific proteolysis in the media from normal versus malignant primary epithelial cell cultures, physiological characteristics of the malignant state might facilitate increased cathepsin D activity. We suspect this proteolysis may play a role in prostatic cell proliferation and invasive tumor growth.

The insulin-like growth factor (IGF) system is involved in the regulation of cell growth. The system involves a network of molecules that includes the IGFs themselves (IGF-I and -II), IGF receptors (types I and II), IGF-binding proteins (IGFBP-1 through -6), and IGFBP proteases. Characterization of this complex system in the prostate has recently been initiated. Prostatic cell lines as well as primary cultures of prostatic epithelial and stromal cells have been analyzed for expression of IGFs, receptors, and IGFBPs. Prostatic epithelial cells and, quite likely, stromal cells as well respond to the mitogenic activity of IGFs via the type I IGF receptor. Prostatic stromal cells synthesize and secrete IGF-II; there is evidence that prostatic cell lines also synthesize IGFs, but this has not been confirmed in primary cultures of prostatic epithelial cells. Prostatic stromal and epithelial cells secrete a number of IGFBPs. The biological impact of some of these IGFBPs on the growth of prostatic cells has been examined, and proteolytic cleavage of IGFBP-3 by prostate-specific antigen (PSA) has been demonstrated. Aberrations in several elements of the IGF system have been observed in stromal cells derived from benign prostatic hyperplasia (BPH). The IGF system may therefore have a part in the etiology of BPH as well as in normal and malignant processes in the prostate.

Mycoplasmal infections alter gene expression in cultured human prostatic and cervical epithelial cells.

Mycoplasmal infections alter gene expression in cultured human prostatic and cervical epithelial cells

Objective To set up the methods of establishing rat primary benign prostatic hyperplasic glandular epithelial cell line.Methods Male spontaneously hypertensive rats were raised to 29 weeks,and then evaluated the situation of BPH with HE staining.The prostate tissue from ventral prostate lobe was aseptically removed,dissected,minced,and then dissociated in collagenase type Ⅰ.Isolated cells were collected,seeded in WAJC-404 and PrEGM medium separately,then cultured and passaged.Specificity of primitive cultured prostatic epithelial cells was identified by cell immunochemistry with CK8/18,and the cell growth curves were drawn.Then the situation of growth of the two prostatic hyperplasic glandular epithelial cell lines were analysed and compared.Results The prostatic hyperplasic glandular epithelial cell lines of the spontaneously hypertensive rats in WAJC-404 and PrEGM medium were successfully primarily cultured,purified and passaged in vitro.Cell immunochemistry proved that the cell lines specifically express cytokeratin 8/18.Cell growth curve showed that prostatic epithelial cells in PrEGM,compared with prostatic epithelial cells in WAJC-404,possessed better cell morphology,more exuberant cell vitality,faster growth rate to enter the logarithmic growth period(4 d vs.7 d)and higher peak of cell growth curve(15.3× 104/ml vs.12.8×104/ml).Conclusions Rat primary benign prostatic hyperplasic glandular epithelial cell line can be established conventionally in vitro.PrEGM medium is more suitable for primary culture of the rat benign prostatic hyperplasic glandular epithelial cell line than WAJC-404 medium. Key words: Prostatic glandular epithelial cell; Benign prostatic hyperplasia; Cell line; Primary culture

Molecular activity of 1,25-dihydroxyvitamin D 3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis

BackgroundSignal transducers and activators of transcription (STATs) are involved in growth regulation of cells. They are usually activated by phosphorylation at specific tyrosine residues. In neoplastic cells, constitutive activation of STATs accompanies growth dysregulation and resistance to apoptosis through changes in gene expression, such as enhanced anti-apoptotic gene expression or reduced pro-apoptotic gene expression. Activated STAT3 is thought to play an important role in prostate cancer (PCA) progression. Because we are interested in how persistently-activated STAT3 changes the cellular phenotype to a malignant one in prostate cancer, we used expression vectors containing a gene for constitutively-activated STAT3, called S3c, into NRP-152 rat and BPH-1 human benign prostatic epithelial cells.ResultsWe observed that prostatic cell lines stably expressing S3c required STAT3 expression for survival, because they became sensitive to antisense oligonucleotide for STAT3. However, S3c-transfected cells were not sensitive to the effects of JAK inhibitors, meaning that STAT3 was constitutively-activated in these transfected cell lines. NRP-152 prostatic epithelial cells lost the requirement for exogenous growth factors. Furthermore, we observed that NRP-152 expressing S3c had enhanced mRNA levels of retinoic acid receptor (RAR)-α, reduced mRNA levels of RAR-β and -γ, while BPH-1 cells transfected with S3c became insensitive to the effects of androgen, and also to the effects of a testosterone antagonist. Both S3c-transfected cell lines grew in soft agar after stable transfection with S3c, however neither S3c-transfected cell line was tumorigenic in severe-combined immunodeficient mice.ConclusionsWe conclude, based on our findings, that persistently-activated STAT3 is an important molecular marker of prostate cancer, which develops in formerly benign prostate cells and changes their phenotype to one more closely resembling transformed prostate cells. That the S3c-transfected cell lines require the continued expression of S3c demonstrates that a significant phenotypic change occurred in the cells. These conclusions are based on our data with respect to loss of growth factor requirement, loss of androgen response, gain of growth in soft agar, and changes in RAR subunit expression, all of which are consistent with a malignant phenotype in prostate cancer. However, an additional genetic change may be required for S3c-transfected prostate cells to become tumorigenic.

This article will review the different modes of action of soluble growth factors in the growth of benign and malignant prostatic cells. Cellular proliferation, growth arrest or even apoptosis requires the participation of appropriate growth factors [1]. Proliferative activities of prostatic epithelial cells, like other cells, are governed by the action of a variety of growth factors. Prostatic growth is traditionally considered to be regulated by androgen, as the prostate is an androgen-sensitive organ, in that the growth and maintenance of the structure and functional integrity are dependent upon the presence of circulating androgen [2,3]. A depletion of this androgenic support, e.g. by bilateral orchidectomy in the host, results in massive apoptosis in prostatic epithelial cells, leading to a rapid rate of tissue involution [4]. Subsequent androgen replacement therapy reactivates prostatic growth. Therefore, androgen is the most potent mitogen to the prostate. It is now apparent that this mitogenic effect of androgen on the prostate is mediated through the action of various growth factors as a consequence of an intricate cell-to-cell interaction, a characteristic feature of benign prostatic growth [5,6]. On the other hand, malignant prostatic growth is characterized by additional mechanisms of cellular proliferation that provide a distinct growth advantage over that of their benign counterparts. Cancer develops as a result of a series of genetic mutations [7]. Prostatic cancer is no exception; however, its progression seems to follow a relatively predictable course, from an androgen-sensitive state to an autonomous state [8]. Sensitivity to androgen in prostatic cancer is mainly due to the presence of androgen receptors in malignant cells, which retain some properties of the benign prostatic cells. However, the manner in which androgen interacts with prostatic cancer cells can be drastically different from that in which it reacts with benign cells. Despite androgen playing an important role in the progression of prostatic cancer, eventually these cancer cells are able to convert from an androgen-responsive growth mode to an androgen-independent mode. Often, a conversion from one state to another is associated with a poorer prognosis and is preceded by the acquisition of new growth advantages within the cancer cells. Again, soluble growth factors are the underlying mechanisms of androgen-sensitive and androgen-insensitive malignant growth. The abnormal growth behaviour in prostatic cancer cells can be manifested by an over-expression of growth-stimulating factor(s) and/or a loss of expression (or function) of growth-suppressive factor(s). The production and action of growth factors in the context of benign and malignant growth of prostatic cells will be the focus of the present discussion.

The attachment and spreading of canine prostatic epithelial cells in primary monolayers and their subsequent proliferation were studied in Primaria and in polystyrene dishes either uncoated or coated with collagen type I, fibronectin, laminin, poly-D-lysine, or natural extracellular matrices (nECM) produced by canine prostatic epithelial or fibroblastic cells. Cells were inoculated in serum-free medium or in medium supplemented with either dialyzed fetal bovine serum (dFBS) or charcoal-treated dog serum at 10%. Dihydrotestosterone (DHT) and 5 alpha-androstane 3 alpha, 17 beta-diol (3 alpha, 17 beta-diol) at 10(-6) M or a mixture of steroids (androstenedione, testosterone, DHT, 3 alpha, 17 beta-diol, 5 alpha-androstane 3 beta, 17 beta-diol, estrone, and estradiol) were also added. Of all components and dishes tested, only dFBS and the nECM produced by prostatic epithelial cells increased cell attachment (850% and 450%, respectively). When the latter preparations were used in combination, an additive effect (1,500%) was observed, and the subsequent addition of dog serum after the attachment period yielded the highest number of growing prostatic epithelial cells. When prostatic epithelial cells were inoculated either in dishes coated with a nECM derived from prostatic fibroblasts or in presence of dog serum, a 50% inhibition in their plating efficiency was observed. The presence of collagen type I, fibronectin, laminin, or an nECM in the cultures, together with various steroids, had no effect on the cell unresponsiveness to steroids nor did it alter the mitogenic effect of dog serum. Thus, the attachment and spreading of canine prostatic epithelial cells in monolayers are mediated by nonsteroidal factors present in dFBS and in their nECM. Their presence, together with steroids, does not elicit a proliferative response to steroids nor did it increase the effect of growth-promoting factors in dog serum.

As men age, a growing number develop benign prostatic hyperplasia (BPH). According to previous research, diabetes may be a risk factor. Pyruvate dehydrogenase kinase 4 (PDK4) is closely related to glucose metabolism and plays a role in the onset and progression of numerous illnesses. This study aimed to determine the direct effects of high glucose environment on prostate epithelial cells, in particular by altering PDK4 expression levels. In this investigation, normal prostatic epithelial cells (RWPE-1) and human benign prostatic hyperplasia epithelial cells (BPH-1) were treated with 50 mM glucose to show the alteration of high glucose in prostate cells. PDK4-target siRNA, PDK4-expression plasmid were used to investigate the effects of PDK4. Rosiglitazone (RG), a PPARγ agonist, with the potential to up-regulate PDK4 expression was also used for treating prostate cells. The expression of PDK4 in human prostate samples was also analyzed. The effects of high glucose therapy on BPH-1 and RWPE-1 cells were demonstrated to enhance proliferation, epithelial-mesenchymal transition (EMT), suppress apoptosis, and down-regulate PDK4 expression. Additionally, diabetes-related BPH patients had reduced PDK4 expression. Following the application of PDK4-target siRNA, a comparable outcome was seen. The PDK4-expression plasmid therapy, however, produced the opposite results. RG with the ability to elevate PDK4 expression might be used to treat BPH. Changes in the metabolism of lipids and glucose may be the cause of these consequences. These findings showed that high glucose treatment might facilitate BPH development, and may be related to the down-regulation of PDK4. PDK4 might be a potential therapeutic target of BPH.

We report on two cases of hepatocellular carcinoma (HCC) with metastasis to the cavernous sinus and sphenoid sinus. Both cases presented with diplopia and retro-orbital headache and both underwent surgery for a primary pituitary gland tumor. After surgery, both cases were diagnosed with metastases from HCC. Case 1 was a 67-year-old male with a history of HCC who was referred to our hospital for pituitary tumor surgery. The tumor appeared to be in the sella turcica and to invade the sphenoid sinus and right cavernous sinus. Transnasal transsphenoidal surgery (TSS) was performed. The tumor was postoperatively diagnosed by histology to be a metastatic pituitary tumor from HCC. Radiotherapy was administered to the metastatic site. Case 2 was a 58-year-old male with a history of TSS for a pituitary tumor 16years previously. He was referred to our hospital for TSS for a recurrent pituitary adenoma. TSS was performed twice in 3months. During a preoperative general examination, HCC and chronic hepatitis B were revealed. TSS was performed initially, followed by arterial infusion chemotherapy. After TSS, the pituitary tumor was diagnosed by histology to be a metastasis from HCC. As with Case 1, radiotherapy was administered to the metastasis. Most tumors in the sella turcica are pituitary adenomas, although some cases of metastatic pituitary tumors and skull base metastases have been reported. Distant metastases generally have a poor prognosis; however, surgery to the metastatic site can effectively control symptoms caused by the metastatic tumor.

: The purpose of this project is to determine the biological basis of the many unique features of prostate cancer, including high incidence, multifocal origin, zonal specificity, and resistance to chemotherapy. We proposed that these characteristics are the result of dysregulated, albeit wild-type, p53 in prostatic epithelial cells. We determined that p53 is not induced in cells derived from either the central zone (resistant to cancer) or the peripheral zone (high frequency of cancer) in response to ionizing radiation. Since the frequency of premalignant lesions is equivalent in these zones, this finding suggests that genomic instability resulting from dysfunctional p53 predisposses both zones to cancer, but that development of invasive cancer in the central zone is limited by unknown factors. We also determined that p53 is not irreversibly nonfunctional in prostatic cells. Inhibition of RNA transcription or of nuclear export led to increased levels of p53 and increased levels of its transcriptional targets, p2l and mdm2. Therefore, mechanisms responsible for upregulation and activation of p53 protein are intact in prostatic epithelial cells. The deficiency, then, apparently lies in the inability of these cells to respond to signals sent by certain DNA-damaging agents, such as ionizing radiation. We recently found that ultraviolet irradiation, in contrast to ionizing radiation, upregulates and activates p53. This finding demonstrates that the pathway leading to induction of p53 is in fact intact in prostatic epithelial cells, but is not triggered by ionizing radiation. Determining the molecular pathways by which these cells recognize and respond to DNA damage will be extremely relevant to prevention of prostate cancer as well as to treatment.

Benign prostatic hyperplasia (BPH) is an extremely common disease of older men characterized by increased growth of prostatic epithelial and stromal cells. Previously we showed that senescent epithelial cells accumulate in the prostate of aging men and secrete interleukin-1 alpha (IL-1 alpha). IL-8 is also present at increased levels in BPH tissues and induces expression of FGF2, a potent stromal growth factor. Therefore, we sought to determine if IL-8 is also expressed at increased levels by senescent epithelial cells and if this secreted IL-8 plays a role in the pathogenesis of BPH. Expression of IL-8 in human BPH tissue and primary cultures of prostatic epithelial cells was analyzed using an enzyme-linked immunoabsorption assay (ELISA). Tissue senescence was assessed by a quantitative assay for senescence-associated beta galactosidase (SA-beta gal). Proliferation of primary and immortalized prostatic epithelial cells in response to IL-8 was determined by counting of cells at intervals after addition of IL-8. Expression of IL-8 is significantly increased in vitro when cultured prostatic epithelial cells undergo senescence. Quantitative assay of BPH tissue extracts revealed that tissue IL-8 levels are correlated with both SA-beta gal activity and prostate weight. IL-8 promotes proliferation of primary and immortalized prostatic epithelial cells in culture. Senescence of prostatic epithelial cells results in increased expression of IL-8, which can promote proliferation of non-senescent epithelial and stromal cells by direct and indirect mechanisms, and in this manner contributes to the increased tissue growth seen in BPH.

To identify the sequence of inflammation-driven signaling cascades and other molecular events that might cause tumor-like transformation of prostatic cells. Cytokine array analysis, Reactome and STRING analysis, immunoblotting, and immunocytochemistry were used to investigate the molecular mechanisms governing inflammation-driven adverse changes in human prostatic cells caused by the sexually transmitted infection, Trichomonas vaginalis, resulting in prostatitis, benign prostatic hyperplasia and prostate cancer. Array analysis showed upregulation of 23 cytokines within 24h of infection of human prostatic epithelial RWPE-1 cells with the parasite, invitro. Reactome and STRING analysis of array data identified interleukin-6, interleukin-8, nuclear factor kappa B, signal transducer and activator of transcription3 and cyclooxygenase2 as chief instigators of prostatic anomaly, which were found to be significantly upregulated by immunofluorescence and western blotting analyses. STRING further connected these instigators with macrophage migration inhibitory factor, PIM-1 and prostate-specific antigen; which was confirmed by their marked stimulation in infected prostatic cells by immunoblotting and immunocytochemistry. Upregulated proliferation markers, such as Ki67, proliferating cell nuclear antigen and B-cell lymphoma2, suggested tumor-like signaling in infected RWPE-1 cells, which was further supported by downregulation of E-cadherin, upregulation of vimentin and activation of focal adhesion kinase. Prostate tumor DU145 cells were more sensitive to parasite invasion, and showed rapid upregulation with nuclear translocation of sensitive parameters, such as nuclear factor kappa B, signal transducer and activator of transcription3, and macrophage migration inhibitory factor. The migration of DU145 cells augmented when incubated in spent media from parasite-infected RWPE-1 cells. The initiation of inflammation driven tumor-like cell signaling in parasite-infected human prostatic epithelial cells is apparent, with the prostate tumor (DU145) cells being more sensitive to T. vaginalis than normal (RWPE-1) prostatic cells.