Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth.

Protein arginine methyltransferase 5 (PRMT5) is an emerging epigenetic enzyme that mainly represses transcription of target genes via symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3. Accumulating evidence suggests that PRMT5 may function as an oncogene to drive cancer cell growth by epigenetic inactivation of several tumor suppressors. Here, we provide evidence that PRMT5 promotes prostate cancer cell growth by epigenetically activating transcription of the androgen receptor (AR) in prostate cancer cells. Knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor reduces the expression of AR and suppresses the growth of multiple AR-positive, but not AR-negative, prostate cancer cells. Significantly, knockdown of PRMT5 in AR-positive LNCaP cells completely suppresses the growth of xenograft tumors in mice. Molecular analysis reveals that PRMT5 binds to the proximal promoter region of the AR gene and contributes mainly to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter by its interaction with Sp1, the major transcription factor responsible for AR transcription, and forms a complex with Brg1, an ATP-dependent chromatin remodeler, on the proximal promoter region of the AR gene. Furthermore, PRMT5 expression in prostate cancer tissues is significantly higher than that in benign prostatic hyperplasia tissues, and PRMT5 expression correlates positively with AR expression at both the protein and mRNA levels. Taken together, our results identify PRMT5 as a novel epigenetic activator of AR in prostate cancer. Given that inhibiting AR transcriptional activity or androgen synthesis remains the major mechanism of action for most existing anti-androgen agents, our findings also raise an interesting possibility that targeting PRMT5 may represent a novel approach for prostate cancer treatment by eliminating AR expression.

YAP activation protects urothelial cell carcinoma from treatment-induced DNA damage.

Current standard of care for muscle-invasive urothelial cell carcinoma (UCC) is surgery along with perioperative platinum-based chemotherapy. UCC is sensitive to cisplatin-based regimens, but acquired resistance eventually occurs, and a subset of tumors is intrinsically resistant. Thus, there is an unmet need for new therapeutic approaches to target chemotherapy-resistant UCC. Yes-associated protein (YAP) is a transcriptional co-activator that has been associated with bladder cancer progression and cisplatin resistance in ovarian cancer. In contrast, YAP has been shown to induce DNA damage associated apoptosis in non-small cell lung carcinoma. However, no data have been reported on the YAP role in UCC chemo-resistance. Thus, we have investigated the potential dichotomous role of YAP in UCC response to chemotherapy utilizing two patient-derived xenograft models recently established. Constitutive expression and activation of YAP inversely correlated with in vitro and in vivo cisplatin sensitivity. YAP overexpression protected while YAP knockdown sensitized UCC cells to chemotherapy and radiation effects via increased accumulation of DNA damage and apoptosis. Furthermore, pharmacological YAP inhibition with verteporfin inhibited tumor cell proliferation and restored sensitivity to cisplatin. In addition, nuclear YAP expression was associated with poor outcome in UCC patients who received perioperative chemotherapy. In conclusion, these results suggest that YAP activation exerts a protective role and represents a pharmacological target to enhance the anti-tumor effects of DNA damaging modalities in the treatment of UCC.

Alterations in chromatin accessibility and DNA methylation in clear cell renal cell carcinoma.

Recent studies have demonstrated that in clear cell renal cell carcinoma (ccRCC) several chromatin remodeling enzymes are genetically inactivated. Although, growing evidence in cancer models has demonstrated the importance of epigenetic changes, currently only changes in DNA methylation can be accurately determined from clinical samples. To address this limitation, we have applied formaldehyde-assisted isolation of regulatory elements (FAIREs) combined with next-generation sequencing (FAIRE-seq) to identify specific changes in chromatin accessibility in clinical samples of ccRCC. We modified the FAIRE procedure to allow us to examine chromatin accessibility for small samples of solid tumors. Our FAIRE results were compared with DNA-methylation analysis and show how chromatin accessibility decreases at many sites where DNA-methylation remains unchanged. In addition, our FAIRE-seq analysis allowed us to identify regulatory elements associated with both normal and tumor tissue. We have identified decreases in chromatin accessibility at key ccRCC-linked genes, including PBRM1, SETD2 and MLL2. Overall, our results demonstrate the power of examining multiple aspects of the epigenome.

Mechanisms of action of tasquinimod on the tumour microenvironment.

Tasquinimod is a small molecule with pleiotropic effects on the tumour microenvironment. Tasquinimod inhibits the growth and metastasis of tumour cells in vitro and in vivo. It targets the tumour microenvironment, enhancing the host immune response and inhibiting the angiogenic response. Tasquinimod influences infiltrating myeloid cells in the tumour milieu shifting the balance towards a less immunosuppressive phenotype. Myeloid-derived suppressor cells and tumour-associated macrophages are major components of the immunosuppressive microenvironment and as a result promote tumour growth and favour angiogenesis and metastasis formation. Growing evidence indicates that tasquinimod targets these myeloid cells and modulates local tumour immunity by blocking the interaction between the multifunctional protein S100A9 and its ligands receptor of advanced glycation end products and Toll-like receptor 4. Its anti-angiogenic effects are achieved at least in part through these effects on regulatory myeloid cells and also potentially through inactivating histone deacetylase-4 and reducing expression of hypoxia-inducible factor 1-controlled genes. The aim is to comprehensively review the mode of action of tasquinimod as a novel oral anti-cancer agent. Based on its unique combination of effects, tasquinimod is a novel agent with clinical therapeutic potential in various solid tumours, both alone and as part of rational combination therapy.

Long-term survival and biomarker correlates of tasquinimod efficacy in a multicenter randomized study of men with minimally symptomatic metastatic castration-resistant prostate cancer.

PURPOSE: Tasquinimod (Active Biotech) is an oral immunomodulatory, anti-angiogenic, and anti-metastatic agent that delayed metastatic disease progression in a randomized placebo-controlled phase II trial in men with metastatic castration-resistant prostate cancer (mCRPC). Here, we report long-term survival with biomarker correlates from this trial. EXPERIMENTAL DESIGN: Two hundred and one (134 tasquinimod and 67 placebo) men with mCRPC were evaluated. Forty-one men randomized to placebo crossed over to tasquinimod. Survival data were collected with a median follow-up time of 37 months. Exploratory biomarker studies at baseline and over time were collected to evaluate potential mechanism-based correlates with tasquinimod efficacy including progression-free survival (PFS) and overall survival (OS). RESULTS: With 111 mortality events, median OS was 33.4 months for tasquinimod versus 30.4 months for placebo overall, and 34.2 versus 27.1 months in men with bone metastases (n = 136), respectively. Multivariable analysis demonstrated an adjusted HR of 0.52 [95% confidence interval (CI), 0.35-0.78; P = 0.001] for PFS and 0.64 (95% CI, 0.42-0.97; P = 0.034) for OS, favoring tasquinimod. Time-to-symptomatic progression was improved with tasquinimod (P = 0.039, HR = 0.42). Toxicities tended to be mild in nature and improved over time. Biomarker analyses suggested a favorable impact on bone alkaline phosphatase and lactate dehydrogenase (LDH) over time and a transient induction of inflammatory biomarkers, VEGF-A, and thrombospondin-1 levels with tasquinimod. Baseline levels of thrombospondin-1 less than the median were predictive of treatment benefit. CONCLUSIONS: The survival observed in this trial of men with minimally symptomatic mCRPC suggests that the prolongation in PFS with tasquinimod may lead to a survival advantage in this setting, particularly among men with skeletal metastases, and has a favorable risk:benefit ratio.

Effect of abiraterone acetate plus prednisone on the QT interval in patients with metastatic castration-resistant prostate cancer.

PURPOSE: Abiraterone is the active metabolite of the pro-drug abiraterone acetate (AA) and a selective inhibitor of CYP17, a key enzyme in testosterone synthesis, and improves overall survival in postdocetaxel metastatic castration-resistant prostate cancer (mCRPC). This open-label, single-arm phase 1b study was conducted to assess the effect of AA and abiraterone on the QT interval. METHODS: The study was conducted in 33 patients with mCRPC. Patients received AA 1,000 mg orally once daily + prednisone 5 mg orally twice daily. Electrocardiograms (ECGs) were collected in triplicate using 12-lead Holter monitoring. Baseline ECGs were obtained on Cycle 1 Day-1. Serial ECG recordings and time-matched pharmacokinetic (PK) blood samples were collected over 24 h on Cycle 1 Day 1 and Cycle 2 Day 1. Serial PK blood samples were also collected over 24 h on Cycle 1 Day 8. RESULTS: After AA administration, the upper bound of the 2-sided 90 % confidence interval (CI) for the mean baseline-adjusted QTcF change was <10 ms; no patients discontinued due to QTc prolongation or adverse events. No apparent relationship between change in QTcF and abiraterone plasma concentrations was observed [estimated slope (90 % CI): 0.0031 (-0.0040, 0.0102)]. CONCLUSIONS: There is no significant effect of AA plus prednisone on the QT/QTc interval in patients with mCRPC.

A phase I/IIA study of AGS-PSCA for castration-resistant prostate cancer.

BACKGROUND: This first-in-human phase I/IIA study was designed to evaluate the safety and pharmacokinetics (PKs) of AGS-PSCA a fully human monoclonal antibody directed to prostate stem cell antigen (PSCA) in progressive castration-resistant prostate cancer. PATIENTS AND METHODS: Twenty-nine patients were administered infusions of AGS-PSCA (1-40 mg/kg) every 3 weeks for 12 weeks; 18 final patients received a 40-mg/kg loading dose followed by 20-mg/kg repeat doses. Primary end points were safety and PK. Immunogenicity, antitumor activity and circulating tumor cells were also evaluated. RESULTS: No drug-related serious adverse events were noted. Dose escalation stopped before reaching the maximum tolerated dose as target concentrations were achieved. Drug levels accumulated linearly with dose and the mean terminal half-life was 2-3 weeks across dose levels. The 40-mg/kg loading dose followed by repeated 20-mg/kg doses yielded serum drug concentrations above the projected minimum therapeutic threshold after two to three doses without excessive drug accumulation or toxicity. Significant antitumor effects were not seen. CONCLUSIONS: A 40-mg/kg loading dose followed by 20-mg/kg infusions every 3 weeks is the recommended phase II dose of AGS-PSCA. PSCA is a promising drug target and studies in prostate and other relevant solid tumors are planned.

Phase I study of the histone deacetylase inhibitor entinostat in combination with 13-cis retinoic acid in patients with solid tumours.

BACKGROUND: Preclinical studies suggest that histone deacetylase (HDAC) inhibitors may restore tumour sensitivity to retinoids. The objective of this study was to determine the safety, tolerability, and the pharmacokinetic (PK)/pharmacodynamic (PD) profiles of the HDAC inhibitor entinostat in combination with 13-cis retinoic acid (CRA) in patients with solid tumours. METHODS: Patients with advanced solid tumours were treated with entinostat orally once weekly and with CRA orally twice daily × 3 weeks every 4 weeks. The starting dose for entinostat was 4 mg m(-2) with a fixed dose of CRA at 1 mg kg(-1) per day. Entinostat dose was escalated by 1 mg m(-2) increments. Pharmacokinetic concentrations of entinostat and CRA were determined by LC/MS/MS. Western blot analysis of peripheral blood mononuclear cells and tumour samples were performed to evaluate target inhibition. RESULTS: A total of 19 patients were enroled. The maximum tolerated dose (MTD) was exceeded at the entinostat 5 mg m(-2) dose level (G3 hyponatremia, neutropenia, and anaemia). Fatigue (G1 or G2) was a common side effect. Entinostat exhibited substantial variability in clearance (147%) and exposure. CRA trough concentrations were consistent with prior reports. No objective responses were observed, however, prolonged stable disease occurred in patients with prostate, pancreatic, and kidney cancer. Data further showed increased tumour histone acetylation and decreased phosphorylated ERK protein expression. CONCLUSION: The combination of entinostat with CRA was reasonably well tolerated. The recommended phase II doses are entinostat 4 mg m(-2) once weekly and CRA 1 mg kg(-1) per day. Although no tumour responses were seen, further evaluation of this combination is warranted.

Mental disorders in obese patients with and without metabolic syndrome.

OBJECTIVE: The authors sought to evaluate lifetime prevalence of mental disorders in patients affected by metabolic syndrome compared with patients affected by central obesity alone. METHODS: One hundred eighty-six (63.5%) patients affected by central obesity and 107 (36.5%) affected by metabolic syndrome according to ICF criteria were interviewed by means of SCID I and SCID II. RESULTS: Axis I and axis II lifetime prevalence were respectively 53.8% and 30.1% among patients with central obesity, 50.5% and 28% among patients with metabolic syndrome, differences which were not significant. No statistically significant differences were found between groups as far as each single axis I and II diagnostic category was considered. CONCLUSION: Metabolic syndrome is not associated with a higher risk of mental disorders compared to central obesity alone.

Modified synthesis and antiangiogenic activity of linomide.

A modified procedure for the synthesis of Linomide is described. The synthesized drug was characterized and assessed for its in vivo antiangiogenic activity. In a murine angiogenesis assay Linomide treatment inhibited new blood vessel formation as documented by reduced microvessel area and blood volume.

Combination of phenylbutyrate and 13-cis retinoic acid inhibits prostate tumor growth and angiogenesis.

Differentiation-inducing agents, such as retinoids and short-chain fatty acids, have an inhibitory effect on tumor cell proliferation and tumor growth in preclinical studies. Clinical trials involving these compounds as single agents have been suboptimal in terms of clinical benefit. Our study evaluated the combination of phenylbutyrate (PB) and 13-cis retinoic acid (CRA) as a differentiation and antiangiogenesis strategy for prostate cancer. On the basis of previous evidence, common signal transduction pathways and possible modulation of retinoid receptors and retinoid response elements by PB could be responsible for such activities. We assessed the effect of the combination of PB and CRA on human and rodent prostate carcinoma cell lines. The combination of PB and CRA inhibited cell proliferation and increased apoptosis in vitro in an additive fashion as compared with single agents (P < 0.014). Prostate tumor cells treated with both PB and CRA revealed an increased expression of a subtype of retinoic acid receptor (retinoic acid receptor-beta), suggesting a molecular mechanism for the biological additive effect. The combination of PB and CRA also inhibited prostate tumor growth in vivo (up to 82-92%) as compared with single agents (P < 0.025). Histological examination of tumor xenografts revealed decreased in vivo tumor cell proliferation, an increased apoptosis rate, and a reduced microvessel density in the animals treated with combined drugs, suggesting an antiangiogenesis effect of this combination. Thus, endothelial cell treatment with both PB and CRA resulted in reduced in vitro cell proliferation. In vivo testing using the Matrigel angiogenesis assay showed an additive inhibitory effect in the animals treated with a combination of PB + CRA (P < 0.004 versus single agents). In summary, this study showed an additive inhibitory effect of combination of differentiation agents PB and CRA on prostate tumor growth through a direct effect on both tumor and endothelial cells.

Adenovirus-mediated gene transfer of fibroblast growth factor-1: angiogenesis and tumorigenicity in nude mice.

Gene transfer of angiogenic growth factors with replication-deficient recombinant adenovirus (Ad) vectors may provide a new approach to the treatment of ischemic diseases. To determine if Ad-infected cells could stimulate angiogenesis in vivo and to assess the tumorigenicity of cells infected with these vectors, NIH3T3 fibroblasts infected with Ad vectors coding for human acidic fibroblast growth factor (aFGF-1) were used in angiogenic and tumorigenic assays. Infected cells induced a strong angiogenic response in vivo, while cells infected with control virus did not. Stable 3T3 transfectants expressing the FGF-1 gene were also highly angiogenic and exhibited growth in soft agar, while Ad-infected cells did not. Ad-infected cells grew transiently in nude mice, whereas 3T3 transfectants formed large tumors which grew exponentially. Extrapolation of cell dose-response curves showed that a minimum of 1.5 x 10(4) infected cells were required for transient tumor cell growth in vivo. Ad-infected cells cultured in vitro for 30 days lost their invasive phenotype and the ability for transient cell growth in nude mice. Thus, phenotypic changes induced by Ad-mediated gene transfer of FGF-1 are transient both in vitro and in vivo, suggesting that these Ad vectors do not have tumorigenic potential. Stimulation of angiogenesis by Ad-infected cells may be useful for the evaluation of anti-angiogenic and anti-tumor agents.

VEGF165 expressed by a replication-deficient recombinant adenovirus vector induces angiogenesis in vivo.

To evaluate the concept that localized delivery of angiogenic factors via virus-mediated gene transfer may be useful in the treatment of ischemic disorders, the replication-deficient adenovirus (Ad) vector AdCMV.VEGF165 (where CMV is cytomegalovirus and VEGF is vascular endothelial growth factor) containing the cDNA for human VEGF165, a secreted endothelial cell-specific angiogenic growth factor, was constructed. Human umbilical vein endothelial cells (HUVECs) and rat aorta smooth muscle cells (RASMCs) infected with AdCMV.VEGF165 (5 and 20 plaque-forming units [pfu] per cell) demonstrated VEGF mRNA expression and protein secretion into the supernatant. Furthermore, the conditioned medium from these cells enhanced vascular permeability in vivo. In contrast, neither VEGF mRNA nor secreted protein was found in uninfected HUVECs or RASMCs or in cells infected with the control vector AdCMV.beta gal (where beta gal is beta-galactosidase). Assessment of starved HUVECs at 14 days demonstrated sixfold more cells for AdCMV.VEGF165-infected HUVECs (20 pfu per cell) than for either infected or uninfected control cells. RASMC proliferation was unaffected by infection with AdCMV.VEGF165. When plated in 2% serum on dishes precoated with reconstituted basement membrane (Matrigel), HUVECs infected with AdCMV.VEGF165 (20 pfu per cell) differentiated into capillary-like structures. Under similar conditions, both uninfected HUVECs and HUVECs infected with AdCMV.beta gal did not differentiate. To evaluate the ability of AdCMV.VEGF165 to function in vivo, either AdCMV. VEGF165 or AdCMV.beta gal (2 x 10(10) pfu) was resuspended in 0.5 mL Matrigel and injected subcutaneously into mice. Immunohistochemical staining demonstrated VEGF in the tissues surrounding the Matrigel plugs containing AdCMV.VEGF165 up to 3 weeks after injection, whereas no VEGF was found in the control plugs with AdCMV.beta gal. Two weeks after injection, there was histological evidence of neovascularization in the tissues surrounding the Matrigel containing AdCMV.VEGF165, whereas no significant angiogenesis was observed in response to AdCMV.beta gal. Furthermore, the Matrigel plugs with AdCMV.VEGF165 demonstrated hemoglobin content fourfold higher than the plugs with AdCMV.beta gal. Together, these in vitro and in vivo studies are consistent with the concept that Ad vectors may provide a useful strategy for efficient local delivery of VEGF165 in the treatment of ischemic diseases.

Adenovirus-mediated gene transfer of wild-type p53 results in melanoma cell apoptosis in vitro and in vivo.

Gene transfer techniques may provide efficient treatment for a variety of malignant neoplasms. A replication-deficient adenovirus (Ad) vector which carries the cDNA for wild-type p53 (AdCMV.p53) was tested for its in vitro and in vivo effects on the growth of murine melanoma cell line B16-G3.26 and human melanoma cell line SK-MEL-24. The growth of B16-G3.26 cells infected with AdCMV.p53 was inhibited when compared to the uninfected cells or cells infected with the control vector AdCMV.NLS beta gal. Similarly, the growth of SK-MEL-24 cells infected with AdCMV.p53 was also below that of AdCMV.NLS beta gal-infected and uninfected controls. DNA laddering using agarose gel electrophoresis and in situ labeling of DNA fragmentation (TUNEL) showed that AdCMV.p53-infected murine and human melanoma cells underwent apoptosis. Nude mice injected s.c. either with B16-G3.26 cells or with SK-MEL-24 cells developed localized tumors. These tumors were subsequently infiltrated with either AdCMV.p53, AdCMV.NLS beta gal or saline alone. One week after infection, B16-G3.26 tumors exposed to AdCMV.p53 were 2.5 times smaller than control tumors and exhibited DNA fragmentation. A similar growth-inhibitory effect of AdCMV.p53 was observed with SK-MEL-24 tumors. Thus, Ad-mediated wild-type p53 overexpression resulted in melanoma cell apoptosis and inhibition of melanoma growth in vitro and in vivo. These gene therapy approaches may be useful in targeting rapidly growing, malignant melanomas in a clinical setting.

In vivo angiogenesis induced by recombinant adenovirus vectors coding either for secreted or nonsecreted forms of acidic fibroblast growth factor.

In vivo gene transfer of angiogenic growth factors represents a potential approach to the treatment of ischemic diseases. The present study examined the in vitro and in vivo effects of two replication-deficient recombinant adenovirus (Ad) vectors coding for human acidic fibroblast growth factor (aFGF1-154). One vector codes for the nonsecreted form of the peptide (AdCMV.aFGF1-154), and the other vector codes for a recombinant, secreted form (AdCMV.sp+aFGF1-154). AdCMV.NLS beta gal, an adenovirus vector coding for beta-galactosidase (beta-Gal), was used as a control. Assessment of proliferation of starved human umbilical vein endothelial cells infected with AdCMV.aFGF1-154 and AdCMV.sp+aFGF1-154 (20 pfu/cell) showed approximately 6- and 10-fold increase in cell number over control, respectively. Infection with AdCMV.sp+aFGF1-154 and with AdCMV.aFGF1-154 enhanced endothelial cell differentiation into capillary-like structures in vitro. However, this effect was significantly more pronounced with AdCMV.sp+aFGF1-154 than with AdCMV.aFGF1-154. Angiogenesis in vivo was assessed by injecting subcutaneously into mice 750 microliters of reconstituted basement membrane proteins (Matrigel) and the Ad vectors (2 x 10(8) pfu). After 14 days, there was histologic evidence of neovascularization in the animal's tissue surrounding the Matrigel plugs with AdCMV.aFGF1-154 and AdCMV.sp+aFGF1-154. Further, the hemoglobin content of the Matrigel plugs with AdCMV.aFGF1-154 and with AdCMV.sp+aFGF1-154 was, respectively, 2.3- and 2.6-fold higher than with AdCMV.NLS beta gal. Together, these observations support the concept that adenovirus vectors coding for various forms of acidic FGF1-154 may be used to induce angiogenesis in vivo and may provide a new therapeutic approach to ischemic diseases.

The alpha-glucosidase I inhibitor castanospermine alters endothelial cell glycosylation, prevents angiogenesis, and inhibits tumor growth.

The development of drugs that target the tumor neovasculature may hold promise in inhibiting tumor growth. Experiments in vivo with castanospermine, an inhibitor of the glucosidases that convert protein N-linked high mannose carbohydrates to complex oligosaccharides, resulted in significant inhibition of tumor growth in nude mice. Angiogenesis to basic fibroblast growth factor in castanospermine-treated C57/BL mice was similarly reduced. Endothelial cell proliferation, invasion of basement membrane, and differentiation are crucial steps during neovascularization. In vitro differentiation models using Matrigel and postconfluent cultures of endothelial cells were used to study the effects of glycosidase inhibitors on endothelial cell behavior. FACS analysis of cell surface oligosaccharides using either Concanavalin A or L-phytohemagglutinin lectins confirmed an increase in high mannose groups and a decrease in tri- and tetra antennary beta-linked galactose-N-acetylglucosamine on mannose residues of Asn-linked oligosaccharides upon drug treatment. Castanospermine and the glucosidase inhibitor N-methyldeoxynojirimycin prevented the morphological differentiation of endothelial cells in vitro. These compounds did not alter the proliferation of cultured endothelial cells or their ability to attach to various extracellular matrix molecules. However, the cells showed a reduced ability to migrate and to invade basement membrane gels in vitro and an increased tendency to form aggregates that was inhibitable by D-mannose. These studies suggest that certain cell surface oligosaccharides are required for angiogenesis and that glucosidase inhibitors that alter these structures on endothelial cells are able to inhibit tumor growth.

Initial contact and subsequent adhesion of human neutrophils or monocytes to human aortic endothelial cells releases an endothelial intracellular calcium store.

BACKGROUND: Increases in both leukocyte and endothelial cytosolic free [Ca2+] may be involved in intercellular adhesion by regulating the affinity of surface adhesion molecules or by facilitating transendothelial leukocyte migration. The purpose of this study was to examine the effect of initial contact and subsequent adhesion of human neutrophils or monocytes on human aortic endothelial [Ca2+]. METHODS AND RESULTS: Endothelial monolayers were loaded with the fluorescent Ca2+ indicator indo 1 and exposed to isolated human peripheral blood neutrophils or to a cultured human monocyte cell line. A rapid, fourfold to fivefold increase in endothelial cytosolic [Ca2+] occurred within seconds of leukocyte contact. No increase in endothelial [Ca2+] occurred on contact of 18.25-microns inert microspheres, isolated red blood cells, or suspensions of cultured human aortic endothelial cells. In experiments performed on monolayers grown in 1-mm2 capillary flow tubes, the increase in endothelial cytosolic [Ca2+] on initial leukocyte contact was found to be related to the subsequent resistance to leukocyte detachment during exposure to arterial levels of shear stress (13.4 dyne.cm-2). The increase in endothelial cytosolic [Ca2+] during leukocyte contact was not inhibited in Ca(2+)-free buffer but was abolished by prior depletion of an endoplasmic reticulum Ca2+ store by thapsigargin. Pretreatment of neutrophils with R15.7, a specific monoclonal antibody to the adhesion protein CD-18, inhibited the increase in endothelial cytosolic [Ca2+] on neutrophil contact. CONCLUSIONS: Initial contact leading to subsequent adhesion of human leukocytes to human aortic endothelial cells releases an endothelial intracellular Ca2+ store. This may, in part, be mediated by specific adhesion proteins and may in turn regulate the affinity of surface adhesion molecules or facilitate transendothelial migration of leukocytes.

Altered angiogenesis underlying age-dependent changes in tumor growth.

BACKGROUND: Cancer incidence increases with age, but the growth and spread of tumors is often slow and prolonged in the elderly. Transplantable murine tumors grow and spread less readily in older mice and can be used as models to study the effect of host age. Neovascularization is crucial for the growth of solid tumors, and alterations in the host vascular response may underlie the changes in tumor growth occurring with age. PURPOSE: We have used transplantable tumor cells and tumors, and tumor extracts, to better understand differences in the biology of tumor growth and vascularization as a function of host age. METHODS: Englebreth-Holm-Swarm (EHS) carcinoma and B16-F10 melanoma cells were injected into C57BL mice of different ages. Tumor growth, histology, and cellular DNA synthesis were compared. Vascularization was determined using basic fibroblast growth factor and an in vivo angiogenesis assay. EHS tumor extracts were assayed for biologic activity in vitro using human endothelial cells and in vivo using mouse EHS-BAM carcinoma and human TSU-Pr1 prostate carcinoma cells. RESULTS: EHS tumors formed larger tumors in young than in old C57BL mice. Rapid tumor growth resumed upon transfer of tumor tissue from old animals into young animals. The rate of DNA synthesis of tumor tissue from old animals in organ culture was lower than in tissue from young animals. Histologically, tumors grown in old animals exhibited a threefold higher ratio of extracellular matrix to tumor cells than those grown in young animals. Tumors from adult animals exhibited numerous small blood vessels; those from old animals contained fewer, much larger vessels. Similar results were observed in young mice fed a reduced-calorie diet. Young animals elicited a greater and more rapid angiogenic response than old animals. Extracts of tumors grown in old animals failed to support endothelial cell differentiation in culture. Tumor cells injected together with such old extracts showed reduced tumor growth in nude mice. CONCLUSIONS: The rate of growth and morphology of the EHS tumor were altered with age, partly due to a reduced capacity to vascularize the tumors because of a lack of angiogenic factors or the presence of host inhibitors. IMPLICATIONS: Alterations in host factors detected in this tumor model may underlie a variety of age-dependent changes that could influence tumor growth and the repair and regeneration of normal tissue. Reducing the vascularization of tumors represents a potential target to reduce their growth and progression.

Regulation of prostate-specific antigen gene expression in LNCaP human prostatic carcinoma cells by growth, dihydrotestosterone, and extracellular matrix.

We have examined the role of extracellular matrix (ECM), cell growth, and dihydrotestosterone on the expression of prostate-specific antigen (PSA) by human prostatic carcinoma cells LNCaP. ECM induced a transient decrease in PSA mRNA even in the presence of growth factors. PSA mRNA, but not actin mRNA, was down-regulated on ECM in a biphasic manner and was not detected up to 48 hr after culture, but was re-expressed after 3 days. Cycloheximide and actinomycin D pretreatment did not prevent ECM-induced down-regulation of PSA mRNA, while actinomycin D-treated cells on plastic maintained stable PSA mRNA levels. DNA synthesis and PSA glycoprotein secretion were also transiently suppressed on ECM. LNCaP growth inhibition correlated with decreased glyceraldehyde phosphate dehydrogenase mRNA levels. However, the transient growth suppression induced by ECM was not observed with primary endothelial cells on Matrigel. Down-regulation of PSA mRNA by culture on Matrigel was reversible upon transfer to a different matrix substrate. Re-expression was highest on heparan sulfate proteoglycan (4-fold) and fibronectin or collagen I (2-fold) compared to plastic or laminin. Our results indicate that the morphology and proliferation of LNCaP cells may be regulated by the ability of ECM to control cellular differentiation and proliferation.

A functional ryanodine-sensitive intracellular Ca2+ store is present in vascular endothelial cells.

The presence of the ryanodine receptor was recently demonstrated in vascular and endocardial endothelium, but its function has not been established. We investigated whether functional ryanodine-sensitive Ca2+ stores are present in cultured endothelial cells from rat aorta (RAECs), human aorta (HAECs), human umbilical vein (HUVECs), and bovine pulmonary artery (BPAECs) and what role these may play in intracellular Ca2+ regulation. Under resting conditions, HAECs, BPAECs, and HUVECs demonstrated a slow increase in intracellular Ca2+ (indexed by indo 1 fluorescence) on exposure to 5 mumol/L ryanodine, whereas RAECs did not. However, after an initial bradykinin exposure in RAECs, ryanodine markedly blunted the rapid increase in Ca2+ on a second exposure to bradykinin. In HUVECs, ryanodine in buffer with 1.5 mmol/L Ca2+ did not inhibit the agonist-sensitive Ca2+ increase, whereas it blunted the rapid increase in Ca2+ on histamine exposure in buffer with 5 mmol/L Ca2+, suggesting that increasing [Ca2+] enhances the binding of ryanodine to its receptor. Thus, functional ryanodine-sensitive Ca2+ stores are present in vascular endothelial cells. These appear to be involved in regulation of Ca2+ storage and release from agonist-sensitive intracellular compartments.