Malaysia Stroke Council guide on acute stroke care service during COVID-19 Pandemic.

On the 18th of March 2020, the Malaysia government declared a movement control order (MCO) due to the unprecedented COVID-19 pandemic. Although the majority of patients presented with respiratory-related symptoms, COVID-19 patients may present atypically with neurological manifestations and may even have an increased risk of stroke. The Malaysia Stroke Council is concerned regarding the level of care given to stroke patients during this pandemic. During the recent National Stroke Workflow Steering Committee meeting, a guide was made based on the currently available evidences to assist Malaysian physicians providing acute stroke care in the hospital setting in order to provide the best stroke care while maintaining their own safety. The guide comprises of prehospital stroke awareness, hyperacute stroke care, stroke care unit and intensive care unit admission, post-stroke rehabilitation and secondary prevention practice. We urge continuous initiative to provide the best stroke care possible and ensure adequate safety for both patients and the stroke care team.

Kinship of conditionally immortalized cells derived from fetal bone to human bone-derived mesenchymal stroma cells.

The human fetal osteoblast cell line (hFOB 1.19) has been proposed as an accessible experimental model for study of osteoblast biology relating to drug development and biomaterial engineering. For their multilineage differentiation potential, hFOB has been compared to human mesenchymal progenitor cells and used to investigate bone-metabolism in vitro. Hereby, we studied whether and to what extent the conditionally immortalized cell line hFOB 1.19 can serve as a surrogate model for bone-marrow derived mesenchymal stromal cells (bmMSC). hFOB indeed exhibit specific characteristics reminiscent of bmMSC, as colony formation, migration capacity and the propensity to grow as multicellular aggregates. After prolonged culture, in contrast to the expected effect of immortalization, hFOB acquired a delayed growth rate. In close resemblance to bmMSC at increasing passages, also hFOB showed morphological abnormalities, enlargement and finally reduced proliferation rates together with enhanced expression of the cell cycle inhibitors p21 and p16. hFOB not only have the ability to undergo multilineage differentiation but portray several important aspects of human bone marrow mesenchymal stromal cells. Superior to primary MSC and osteoblasts, hFOB enabled the generation of continuous cell lines. These provide an advanced basis for investigating age-related dysfunctions of MSCs in an in vitro 3D-stem cell microenvironment.

Epithelial-mesenchymal interactions in prostatic development. II. Biochemical observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder.

Adult bladder epithelium (BLE) is induced to differentiate into glandular epithelium after association with urogenital sinus mesenchyme (UGM) and subsequent in vivo growth in syngeneic male hosts. Alteration of epithelial cytodifferentiation is associated with the expression of prostate-specific antigens, histochemical and steroid metabolic activities. These observations suggest that the inductive influence of the UGM has reprogrammed both the morphological and functional characteristics of the urothelium. In this report, differences regarding the mechanisms and effects of androgenic stimulation of prostate and bladder are exploited to determine the extent to which UGM plus BLE recombinants express a prostatelike, androgen-dependent phenotype. Results from cytosolic and autoradiographic binding studies suggest that androgen binding is induced in UGM plus BLE recombinants and that this activity is accounted for by the induced urothelial cells. In UGM plus BLE recombinants, androgen-induced [3H]thymidine or [35S]-methionine uptake analyzed by two-dimensional gel electrophoresis was qualitatively and quantitatively similar to that of prostate as opposed to bladder. These studies indicate that expression within BLE of prostatic phenotype is associated with a loss of urothelial characteristics and that androgen sensitivity is presumably a function of the inductive activities of the stroma.

cAMP-responsive element-binding protein regulates vascular endothelial growth factor expression: implication in human prostate cancer bone metastasis.

Aberrant expression of vascular endothelial growth factor (VEGF) is associated with human prostate cancer (PCa) metastasis and poor clinical outcome. We found that both phosphorylation of cyclic AMP-responsive element-binding protein (CREB) and VEGF levels were significantly elevated in patient bone metastatic PCa specimens. A PCa ARCaP progression model demonstrating epithelial-to-mesenchymal transition exhibited increased CREB phosphorylation and VEGF expression as ARCaP cells became progressively more mesenchymal and bone-metastatic. Activation of CREB induced, whereas inhibition of CREB blocked, VEGF expression in ARCaP cells. CREB may regulate VEGF transcription via a hypoxia-inducible factor-dependent mechanism in normoxic conditions. Activation of CREB signaling is involved in the coordinated regulation of VEGF and may pre-dispose to PCa bone metastasis.

Quantum dots-based multiplexed immunohistochemistry of protein expression in human prostate cancer cells.

Semiconductor quantum dots (QDs) are bright fluorescent nanoparticles that have been successfully used for the detection of biomarker expression in cells. The objective of the present study is to use this technology in a multiplexing manner to determine at a single cell level the expression of a cell-specific bio-marker, prostate-specific antigen (PSA) expressed by human prostate cancer LNCaP and ARCaP cell lines. Here we compared the sensitivity of immunohistochemistry (IHC) and QD-based detection of AR and PSA expression in these cell lines. Further, we conducted multiplexing QD-based detection of PSA and androgen receptor (AR) expression in LNCaP cells subjecting to androgen (R1881) stimulation. The involvement of AR in PSA regulation in LNCaP cells, at a single cell level, was confirmed by the co-incubation of LNCaP cells in the presence of both R1881 and its receptor antagonist, bicalutamide (Casodex). We showed here the superior quality of QDs, in comparison to IHC, for the detection of AR and PSA in cultured LNCaP and ARCaP cells. Multiplexing QDs technique can be used to detect simultaneously AR and PSA expression induced by R1881 which promoted AR translocation from its cytosolic to the nuclear compartment. We observed AR antagonist, bicalutamide, inhibited AR nuclear translocation and PSA, but not AR expression in LNCaP cells.

Suramin-induced decrease in prostate-specific antigen expression with no effect on tumor growth in the LNCaP model of human prostate cancer.

BACKGROUND: Suramin, a polysulfonated naphthylurea and a recognized antitrypanosomal agent, has shown some promise in phase II clinical trials in the management of hormone-refractory human prostate cancer. Reduction of serum prostate-specific antigen (PSA) levels has been proposed as an end point for evaluating the antitumor efficacy of treatments for hormone-refractory prostate cancer. PURPOSE: We examined the antitumor effect of suramin in an in vivo mouse model of hormone-refractory human prostate cancer to determine whether a decrease in PSA levels reflects a reduction in tumor growth (volume). The tumors were induced in castrated, athymic nude mice by use of the androgen-independent, tumorigenic human prostate cancer cell line C4-2, which is a subline of the androgen-dependent, parental nontumorigenic cell line LNCaP. We also evaluated the effects of suramin in vitro on cell growth and the expression of PSA messenger RNA (mRNA) in both LNCaP and C4-2 cells. METHODS: For the in vivo studies, 24 mice were given a subcutaneous injection of 5 x 10(6) C4-2 cells at each of four sites. Animals (n = 20) with tumor volumes greater than 1 mm3 or less than 5 mm3 were divided equally into two groups. Drug treatment was initiated in one group by administration of 1 mg suramin intraperitoneally, followed by 0.1 mg suramin at 10-day intervals to maintain constant serum levels. Tumor growth and PSA expression levels were monitored. For the in vitro studies, both LNCaP and C4-2 cells were exposed to 100-400 microgram/mL suramin, and cell growth was monitored by a quantitative crystal violet assay. PSA mRNA expression was assessed by northern blot analysis in cells treated with either 250 microgram/mL suramin, 400 ng/mL dihydrotestosterone (DHT) (positive control), or 0.5-75 microgram/mL hydrocortisone (to mimic the clinical use of hydrocortisone during suramin treatment to compensate for the loss of adrenocortical function). In some studies, the combined effect of DHT and suramin on PSA mRNA expression was also evaluated. A two-way analysis of variance was performed to evaluate the treatment differences, and P values were obtained from two-sided tests for statistical significance. RESULTS: In vivo, suramin did not significantly affect the growth of androgen-independent C4-2 tumors (relative to the growth of tumors in 5% glucose-treated control animals; P = .76). However, suramin significantly decreased the ratio of PSA level to tumor volume (ng/mL PSA per mm(3) of tumor) (P<.001). Mice developed bone metastases in both treatment arms. Suramin affected the in vitro growth of LNCaP cells but not of C4-2 cells. Suramin diminished PSA mRNA expression in both LNCaP and C4-2 cells grown in vitro. Hydrocortisone had no effect on PSA mRNA levels. CONCLUSIONS: Although suramin inhibited the growth of androgen-dependent LNCaP cells, it did not inhibit the growth of androgen-independent C4-2 cells either in vitro or in vivo. Suramin significantly decreased PSA mRNA expression in both cell lines in vitro and depressed serum PSA levels in mice bearing androgen-independent C4-2 tumors. IMPLICATIONS: PSA level should be used with caution as an end point in clinical trials using suramin therapy for hormone-refractory prostate cancer.

Acquisition of a tumorigenic phenotype by a rat ventral prostate epithelial cell line expressing a transfected activated neu oncogene.

The neu oncogene has been demonstrated to be a potent transforming gene in rodent fibroblasts. The overexpression of the human erbB-2/neu oncogene has been implicated in the development and/or prognosis of several human carcinomas including that of the prostate. To assess the transforming potential of the activated rat neu oncogene in prostatic epithelial carcinogenesis, this laboratory has transfected a cloned non-tumorigenic, rat ventral prostate epithelial cell line, NbE-1.4, with an activated, point-mutated neu oncogene. Transfection of NbE-1.4 cells with the activated neu oncogene expression vector, pSV-neu-T (neu-T), resulted in an altered cell morphology, an increase in soft agar colony-forming efficiency, and conversion to a tumorigenic phenotype. Although the parental NbE-1.4 cells expressed endogenous c-neu mRNA, a reverse transcriptase polymerase chain reaction assay determined that the neu-T-transfected clones expressed only the point-mutated neu-T mRNA. The suppression of the c-neu transcripts occurred regardless of the neu-T mRNA level expressed in these cell clones. These data provide evidence to show that low-level expression of an activated neu oncogene alone was insufficient to transform rat prostate epithelial cells. Rather, overexpression of an activated neu oncogene correlated well with the acquisition of a tumorigenic phenotype by the NbE-1.4 epithelial cell line.

Extraction of nuclear androgen receptors by sodium molybdate from normal rat prostates and prostatic tumors.

Because sodium molybdate stabilizes steroid receptors, this compound has been included in the homogenizing medium in order to maximize the recovery of measurable steroid receptors in normal and neoplastic tissues. This study demonstrates that sodium molybdate extracts additional androgen receptors from prostatic nuclei in a concentration-dependent manner. Nuclei previously washed with Triton X-100 to remove the outer nuclear membranes released similar numbers of androgen receptors with sodium molybdate as the unwashed nuclei, suggesting that the extracted nuclear androgen receptors are associated with intranuclear matrices. Sucrose density gradient analyses revealed that sodium molybdate-extractable nuclear androgen receptors sedimented similarly to the 0.4 M KCl extract as 4S receptor complexes under high-salt conditions. We have compared the amount of nuclear androgen receptors extracted from normal prostates (ventral prostate and dorsolateral prostate) and Noble hooded rat and Dunning prostatic tumors by a sensitive translocation-extraction procedure. This procedure involves the incubation of minced prostatic tissues, isolated from castrated rats, with [3H]R1881 ( [3H]methyltrienolone; [6,7-3H]-17 beta-hydroxy-17 alpha-methylestra-4,9,11-trien-3-one) at 37 degrees for 2 hr. Crude nuclear pellets were prepared from the minced tissues, and nuclear androgen receptors were extracted with 40 mM Na2MoO4 or 0.4 M KCl. Results showed that the amount of nuclear androgen receptors present in the prostatic tumor nuclei is lower than that found in the normal. Although the percentage of nuclear androgen receptors extracted by sodium molybdate or KCl is similar between androgen-dependent and androgen-independent prostatic tumors, the absolute amounts of nuclear androgen receptors per mg DNA extracted from the former are 2- to 8-fold higher than those found in the latter.

Comparison of soluble protein kinases from normal rat prostates and prostatic Dunning tumors.

The photoaffinity labeling of the regulatory subunits of adenosine cyclic 3':5'-monophosphate-dependent soluble protein kinases with an adenosine cyclic 3':5' monophosphate analogue, 8-azidoadenosine cyclic 3':5'-monophosphate (8-N3-cAMP) was compared in normal prostates and prostatic (Dunning) tumors. Marked differences in the proportions of photoincorporation of 8-N3-[32P]cAMP into the regulatory subunit of type I (RI) and type II (RII) protein kinases and the proteolytic fragment of these regulatory subunits (Rpf) were observed in normal prostate (ventral prostate versus dorsolateral prostate) and Dunning prostatic tumors [hormone-dependent (R-3327-H) versus hormone-independent (R-3327-HI) or anaplastic (R-3327-AT) tumors]. The proportion of 8-N3-[32P]cAMP photoincorporation into RII versus RI was 2-fold higher in the dorsolateral prostate than that observed in the ventral prostate, whereas the ratio of photoincorporation into RI or Rpf was comparable between these two tissues. Comparison between tumor tissues revealed that the proportion of photoincorporation of 8-N3-[32P]cAMP into RII versus RI was 4.2-fold higher in hormone-dependent than in hormone-independent or anaplastic tumors, whereas the ratio of photoincorporation into RI versus Rpf in the former was only 6 to 7% that found in the latter. Since Dunning hormone-dependent and hormone-independent tumors have similar morphology, the present photoaffinity labeling of these regulatory subunits of soluble protein kinases provides a sensitive method (4 mg tissue per assay) to distinguish these prostatic tumors of different hormonal dependency.

Aberrant expression of epidermal growth factor receptor and HER-2 (erbB-2) messenger RNAs in human renal cancers.

Amplification, rearrangement, or overexpression of the gene for the epidermal growth factor receptor (EGFR) occurs in certain types of human neoplasia. We investigated EGFR gene structure and measured EGFR mRNA levels in human renal tumor biopsies. Seventeen renal tumors [13 renal cell carcinomas (RCCs), two Wilms' tumors, one oncocytoma, and one metastatic ganglioneuroblastoma] and their corresponding normal kidney tissues were examined for EGFR gene structural integrity by Southern blot hybridization. Twelve of these tumors (including 11 RCCs) were examined for EGFR mRNA expression levels by RNA blot hybridization. The EGFR gene was rearranged in one of 13 (8%) of the RCC specimens examined and was highly amplified in the ganglioneuroblastoma. The overall frequency of EGFR gene structure alterations in this series of renal tumors was 12%. Nine of 11 RCC specimens (82%) exhibited markedly elevated EGFR mRNA levels (approximately 2- to 6-fold). In contrast, expression of the EGFR-related protooncogene HER-2 (erbB-2) was found to be decreased in 11 RCCs and one Wilms' tumor; HER-2 gene structure, however, appeared normal in all specimens. These results indicate that overexpression of EGFR mRNA, probably due to changes in gene regulation, and underexpression of HER-2 mRNA are characteristic features of human RCC.

Extracellular matrix and androgen receptor expression associated with spontaneous transformation of rat prostate fibroblasts.

Spontaneous transformation in continuous culture of the androgen-sensitive rat prostate fibroblast cell line, NbF-1, resulted in an aggressively tumorigenic nonmetastatic phenotype that coincided with few gross chromosome abnormalities. This study identified transformation-associated alterations in extracellular matrix and androgen receptor expression in the NbF-1 cell line. Substantial levels of procollagens I, III, and IV and fibronectin mRNAs were detected in nontumorigenic NbF-1 cells. Laminin B1 and B2 mRNAs were also detectable, but at lower levels. Expression of all six extracellular matrix mRNAs was nonuniformly lower in tumorigenic NbF-1 cells. This decrease in expression was greatest for alpha 2 procollagen IV mRNA, which was reduced 17-fold. Proteoglycans and glycosaminoglycans synthesized by the NbF-1 cultures were also characterized. The NbF-1 cell line expressed chondroitin sulfate proteoglycans predominantly, and expression was reduced 5- to 10-fold in tumorigenic cultures. In contrast to the extensive alterations in the extracellular matrix, measurement of high-affinity androgen binding and androgen receptor mRNA levels showed substantial expression of androgen receptors in both NbF-1 cultures. Cultures of early and late passage NbF-1 cells demonstrated a mitogenic response to dihydrotestosterone. These data indicate (a) that alterations in expression of extracellular matrix components may represent early markers for tumorigenic transformation in prostatic mesenchymal cells and (b) that these changes can occur without disrupting androgen receptor expression and androgen sensitivity.

Serum prostate specific antigen levels in mice bearing human prostate LNCaP tumors are determined by tumor volume and endocrine and growth factors.

The ability of prostate-specific antigen (PSA) to predict tumor volume and stage in patients with prostate cancer would be improved if factors regulating its production and clearance were better defined. A thorough understanding of the pharmacokinetics (regulation of production, metabolism, and excretion) of PSA has been precluded, however, by the absence of an in vivo animal model. The purposes of this study are to develop a murine model for evaluating PSA pharmacokinetics in vivo and to assess factors that influence PSA production in vitro. The human prostate cancer cell line, LNCaP, was chosen because it is androgen sensitive and PSA positive. Although LNCaP cells are usually nontumorigenic when inoculated s.c. in athymic mice, coinoculation of 1 x 10(6) LNCaP cells with 1 x 10(6) human bone fibroblasts reliably produces PSA-secreting carcinomas. This LNCaP model provides accurate correlation between tumor volume and serum PSA levels (r = 0.94) and demonstrates that tumor volume and androgens are codeterminants of circulating PSA levels. Following castration, serum PSA levels decrease rapidly up to 8-fold and increase up to 20-fold following androgen supplementation, without detectable castration-induced tumor cell death or concomitant changes in tumor volume. Serum PSA levels increase 0.24 ng/ml/mm3 of tumor, which is approximately 5-fold less than that estimated for humans. Most likely this reduced PSA index (PSA:tumor volume ratio) results from a 7-fold faster clearance of PSA in athymic mice than in humans; other than this shorter half-life, PSA elimination in the murine model appears similar to that in humans, with both following first-order kinetics characteristic of a two-compartment model. Interestingly, following prolonged growth (greater than 21 days) in castrate hosts, LNCaP tumors are capable of adapting to an androgen-deprived environment whereby LNCaP tumors regain the ability to secrete PSA in amounts similar to the precastrate state. In LNCaP cells, androgens increase PSA mRNA levels 4-fold in vivo and in vitro. PSA mRNA expression is also altered by various growth factors. Changes in PSA production induced by androgens and growth factors do not always parallel changes in LNCaP cell growth rate induced by these factors, suggesting that PSA production occurs independently of cell growth rate and may be influenced by various interrelated factors, including hormonal and stromal milieu. Observations from this murine model suggest that androgens and tumor volume are independent determinants of serum PSA levels and imply that decreases in circulating PSA following antiandrogen therapy may not always reflect a corresponding reduction in tumor volume.

Autocrine regulation of prostate-specific antigen gene expression in a human prostatic cancer (LNCaP) subline.

Prostate-specific antigen (PSA), a M(r) 34,000 serine protease, is recognized as a useful marker for the detection and prognosis of patients with prostate cancer. Although serum PSA is an excellent prognostic indicator, an increasing number of factors were found to regulate the PSA expression of prostatic cancer cells, which include androgenic steroids, the growth factors (GFs) and the extracellular matrix. The purpose of this study is to define a novel protein factor that may be responsible for regulating PSA expression by androgen-independent (AI) human prostate cancer cells. We have established a LNCaP subline (C4) from a parental LNCaP tumor grown in a castrated host. The C4 subline overexpressed PSA mRNA and protein. Serum-free conditioned medium (CM) isolated from the C4 subline is able to stimulate PSA gene expression in parental LNCaP cells in a concentration-dependent manner. This autocrine PSA-inducing activity was found to be organ specific because CMs from other fibroblast cell lines (such as bone, prostate, kidney, and lung fibroblasts) and the CMs from several prostatic carcinoma cell lines (such as parental LNCaP, PC-3, DU-145) and a bladder transitional carcinoma cell line (WH) fail to exhibit similar activity. The activity of the CM from the C4 subline cannot be substituted by GFs such as TGF-alpha, TGF-beta, bFGF, HGF, KGF, or NGF; neuropeptide (bombesin/GRP); secondary messenger analogue (dibutyryl cAMP); beta 2-adrenergic agonist (isoproterenol); or alpha 1-adrenergic agonist (phenylephrine), indicating that the factor(s) may be a novel prostate-specific autocrine factor (PSAF). Both androgen and PSAF exhibit an additive effect on up-regulating PSA gene expression, suggesting that the signal transduction pathway elicited by PSAF may differ from that mediated by the androgen receptor. Further characterization of PSAF by heat, acid, and trypsin digestion revealed that the PSAF may be a protein factor with a unique amino acid composition. These observations suggest that a novel autocrine pathway mediated by PSAF may be responsible for the overexpression of PSA mRNA and protein in a human prostatic cancer cell line. The potential clinical significance of this factor will be discussed.

Human prostate cancer expresses the low affinity insulin-like growth factor binding protein IGFBP-rP1.

Many of the alterations in the insulin-like growth factor (IGF) axis in prostatic disease have been associated with changes in the insulin-like growth factor binding proteins (IGFBPs), a multigene family of proteins that are thought to mediate the action of IGFs on target tissues. IGFBP-related protein 1 (rP1), also known as IGFBP-7 or mac25, is a recently described member of the IGFBP family, the biological function of which has yet to be completely ascertained. In this study, we analyzed the localization of IGFBP-rP1 in prostate cancer and benign prostate tissues using immunohistochemistry and a polyclonal antibody, T1A12, that is specific for IGFBP-rP1. The most intense staining was observed in nerves, whereas smooth muscle cells in the prostate stained weakly. Lymphocytes were always negative. When normal prostatic secretory epithelium was present, staining was usually absent. The lining secretory epithelium stained positively in 0 of 12 (0%) cases of benign prostatic hyperplasia, 57 of 63 (90.5%) primary adenocarcinomas, and 7 of 7 (100%) prostate cancer metastases. Prostatic intraepithelial neoplasia showed a similar pattern of staining to that observed for the invasive tumors. Analysis of Northern blots showed that none of the prostate cancer cell lines (LNCaP, C4, C4-2, C4-2B4, 9069E3, DU145, and PC3) expressed IGFBP-rP1 mRNA. This lack of expression was confirmed by immunohistochemistry of s.c.-generated tumor xenografts of LNCaP and C4-2 and by immunoblot on serum-free-conditioned media from all prostatic cell lines. In contrast to these results, tumor xenografts generated by direct intraosseous injection of LNCaP or C4-2 to bone marrow space resulted in tumors that stained positively for IGFBP-rP1. Our results show that IGFBP-rP1 is expressed in both in situ and invasive prostate neoplasms, but not typically in normal secretory or BPH epithelium; furthermore, the expression of IGFBP-rP1 can be induced in human prostate cancer cell lines in vivo on interaction with an appropriate host environment.

Acceleration of human prostate cancer growth in vivo by factors produced by prostate and bone fibroblasts.

Prostate cancer, the most prevalent cancer affecting men, frequently metastasizes to the axial skeleton where it produces osteoblastic lesions with growth rates often exceeding that of the primary tumor. To evaluate the role of tumor cell-host stromal interaction and stromal specific growth factors (GFs) in prostate cancer growth and progression, we coinoculated athymic mice with human prostate cancer cells (LNCaP) and various nontumorigenic fibroblasts s.c. LNCaP tumor formation was most consistently induced by human bone (MS) fibroblasts (62%), followed by embryonic rat urogenital sinus mesenchymal (rUGM) cells (31%) and Noble rat prostatic fibroblasts (17%), but not by NIH-3T3, normal rat kidney, or human lung CCD16 fibroblasts. Carcinomas formed preferentially in male hosts, demonstrating in vivo androgen sensitivity. The human prostate component of these tumors was confirmed with immunohistochemical staining for prostate-specific antigen (PSA), Northern analysis for PSA expression, and Southern analysis for human repetitive Alu sequences. Elevations in serum PSA paralleled the histomorphological and biochemical findings. LNCaP and fibroblast cell-conditioned media (CM) was used to determine whether autocrine and paracrine mitogenic pathways exist between LNCaP and fibroblast cells in vitro, and various defined GFs were tested to identify possible active factors. Mitogenic assays revealed a 200-300% bidirectional stimulation between LNCaP and bone or prostate fibroblast-derived CM. Lung, normal rat kidney, and 3T3 fibroblast CM were not mitogenic for LNCaP cells. Among the purified GFs tested basic fibroblast growth factor (bFGF) was the most potent mitogen, stimulating LNCaP growth 180% in a concentration-dependent manner. Transforming growth factor alpha and epidermal growth factor were both minimally mitogenic. Coinoculation of LNCaP cells with a slowly absorbed matrix (Gelfoam) absorbed with bFGF or dialyzed and concentrated rUGM or MS CM was also capable of inducing LNCaP tumor formation in vivo. These observations illustrate that fibroblasts differentially modulate prostate cancer growth through the release of paracrine-mediated GFs, possibly including bFGF, and that tumor-stromal cell interactions play an important role in prostate cancer growth and progression.

Androgen receptor mediates the reduced tumor growth, enhanced androgen responsiveness, and selected target gene transactivation in a human prostate cancer cell line.

The growth and development of the prostate gland are regulated by the androgen and the androgen receptor (AR). Despite our molecular understanding of the roles of the AR regulating; a downstream target gene transcription, the direct or indirect (stromally mediated) actions of the androgen in controlling prostate cell growth and differentiation are still unclear. In this report, an invasive; and metastatic human prostate tumor cell line, androgen-repressed human prostate cancer cell line (ARCaP), either transduced with wild-type human AR (hAR) or a control neomycin-resistant plasmid DNA, was used to evaluate the direct role of AR in regulating prostate tumor cell growth and gene transcription. Results showed that: (a) introduction of wild-type hAR to ARCaP cells restored positive androgen regulation of prostate tumor cell growth in vitro through an enhanced cell-cycle progression from G(0)/G(1) to S and G(2)-M phases; (b) hAR was shown to transactivate glucocorticoid-responsive element but not prostate-specific antigen promoter-directed reporter gene expression; and (c) hAR-transduced ARCaP cells exhibited reduced growth, invasion, and migratory behavior in vitro and tumor growth in vivo. These results suggest that the introduction of hAR into the invasive human prostate cancer ARCaP cell line restored its androgen-regulated cell growth, decreased the rate of tumor growth, and selectively activated AR target gene expression. These cellular functions in response to androgen are commonly associated with increased differentiation of prostate epithelial cells.

Epidermal growth factor receptor-mediated autocrine and paracrine stimulation of human transitional cell carcinoma.

Stromal-epithelial interactions may play a key role in tumor growth and metastasis. We have established a model to study the cellular and molecular basis of this paracrine interaction both in vivo and in vitro using a human transitional cell carcinoma cell line (WH). s.c. coinoculation of 1 x 10(6) WH cells with 1 x 10(6) nontumorigenic fetal rat urogenital sinus mesenchymal (rUGM) cells in athymic mice accelerated carcinoma growth 20 times faster than isolated WH cell inoculations and 4 times faster than coinoculations of the same number of NIH-3T3 or human bladder fibroblasts. Characterization of these chimeric tumors with immunohistochemical and DNA dot-blot analyses documented their predominantly human component. To evaluate the underlying mechanisms involved in this paracrine-mediated in vivo tumor growth acceleration, Northern analyses for growth factors (GFs) and extracellular matrix (ECM) expression in the different cell lines, as well as in vitro mitogenic assays, were performed. Northern analysis revealed basic fibroblast growth factor, transforming growth factor alpha, and epidermal growth factor receptor expression by WH cells but not rUGM cells; ECM components (fibronectin and collagens I and IV) were expressed only in the fibroblast cell lines. Cell type-specific paracrine growth factors are produced by cultured stromal and epithelial cells with a 2-3-fold bidirectional increase in WH and rUGM cell growth when cultured with reciprocal cell-type conditioned medium. An autocrine growth loop was observed for WH but not rUGM cells. WH cell growth is stimulated in vitro by low concentrations of transforming growth factor alpha and epidermal growth factor, while rUGM cell growth is stimulated 3-fold by basic fibroblast growth factor. Antiepidermal growth factor receptor antibodies completely inhibited autocrine and paracrine pathways stimulating WH cell growth, while anti-basic fibroblast growth factor antibodies had no inhibitory effect. These observations suggest that autocrine and paracrine growth factor stimulation of WH bladder carcinoma cell growth is most likely mediated by an epidermal growth factor receptor-related pathway. The predominant expression of ECM by fibroblasts in this model suggests that stromal cell ECM components may modulate tumor cell growth and angiogenesis possibly through mechanisms involving cellular adhesion, chemotaxis, or growth factor action.

Expression of the protooncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer.

The significance of apoptosis in relation to the development and progression of prostate cancer remains largely undefined. bcl-2 is an oncogene that functions by overriding apoptosis. bcl-2 expression was localized to the basal epithelial cells in the normal human prostate with the use of immunohistochemistry. Androgen-dependent and androgen-independent prostate carcinomas were evaluated immunohistochemically for bcl-2 expression. bcl-2 was undetectable in 13 of 19 cases of androgen-dependent cancers. In contrast, androgen-independent cancers displayed diffuse, high levels of bcl-2 staining (P < 0.01). In rats, steady-state levels of bcl-2 mRNA, assessed by S1 assays, reached maximum levels 10 days following castration. Addition of exogenous testosterone with, or without, flutamide demonstrated that the increased bcl-2 mRNA resulted from androgen ablation. Our findings indicate that bcl-2 expression is augmented following androgen ablation and is correlated with the progression of prostate cancer from androgen dependence to androgen independence.

Adenovirus-mediated expression of PML suppresses growth and tumorigenicity of prostate cancer cells.

Our previous studies demonstrated that the promyelocytic leukemia gene, PML, encodes a growth and transformation suppressor. Overexpression of PML inhibits cancer cell growth in vitro and in vivo. In this study, we further explored the possibility of applying PML as a potential agent for developing prostate cancer gene therapy using an adenovirus delivery system. We have constructed and produced the recombinant PML-adenovirus, Ad-PML, in which the full-length PML cDNA is driven by the strong cytomegalovirus promoter. In LNCaP, DU145, and PC-3 prostate cancer cell lines, an infection efficiency of 90% can be achieved at a concentration of 2, 10, and 100 multiplicity of infection (MOI), respectively. Western blotting and immunofluorescence staining demonstrated that the AD-PML-infected cells expressed a high level of PML protein. The protein expression peaked at days 3-4 postinfection, and a detectable level of PML was found at day 18 after viral infection. To test the effect of Ad-PML on the growth of prostate cancer cells, the DU145 and LNCaP cells were infected with 10 and 2 MOI of Ad-PML. We found that the growth rate of the Ad-PML-infected DU145 and LNCaP cells were significantly inhibited. A tumorigenicity test in nude mice showed that the Ad-PML-treated DU145 cells failed to form tumors. Most importantly, direct injection of Ad-PML into DU145-induced tumors was able to repress tumor growth in nude mice by 64%. Taken together, these data indicate that PML is a tumor growth suppressor in prostate cancer and that Ad-PML may be a potential candidate for human prostate cancer therapy.

Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models.

Osteocalcin (OC), a noncollagenous bone matrix protein, is expressed in high levels by osteoblasts. To determine whether the OC promoter mediates cell-specific gene expression in cells of osteoblast lineage, we constructed a recombinant adenovirus, Ad-OC-TK, which contains the OC promoter that drives the expression of herpes simplex virus thymidine kinase (TK). We tested the expression of TK by this virus in osteoblast cell lines as well as in non-osteoblastic cell lines by assessing the enzyme activity of TK in vitro. Whereas the OC promoter failed to drive the expression of the TK gene in several non-osteoblastic cell lines such as WH, a human bladder transitional carcinoma, and NIH 3T3, an embryonic mouse fibroblast cell line, the OC promoter mediated high levels of expression in osteoblast cell lines including murine ROS and human MG-63 cells. The addition of acyclovir (ACV), a pro-drug for the inhibition of cell proliferation, resulted in the induction of osteoblast-specific cell death in vitro. Intratumoral injection of Ad-OC-TK into murine ROS osteosarcoma abolished tumor growth in a host treated with subsequent i.p. ACV injection in vivo. The Ad-OC-TK virus plus ACV treatment appears to be highly selective in blocking the growth of both murine and human osteosarcoma cell lines in vitro and murine osteosarcoma in vivo.