FGFR3 Down-Regulation is Involved in bacillus Calmette-Guérin Induced Bladder Tumor Growth Inhibition.

PURPOSE: Bacillus Calmette-Guérin is the standard treatment for patients with nonmuscle invasive high histological grade bladder cancer. Previously we found that bacillus Calmette-Guérin induces murine bladder cancer MB49 cell death in vitro and in vivo, generating tissue remodeling, which involves the release of fibroblast growth factor (FGF)-2. MATERIALS AND METHODS: We studied the effect of bacillus Calmette-Guérin treatment on FGF-2 and FGF receptor (FGFR) expression in bladder cancer. RESULTS: In vitro FGF-2 increased MB49 cell proliferation but did not reverse bacillus Calmette-Guérin induced cell death. Increased FGF-2 expression was detected after bacillus Calmette-Guérin treatment. Moreover MB49 cells expressed high FGFR3 levels, which decreased after treatment. Similar results were observed in human T24 bladder cancer cells. In vivo MB49 tumors expressed higher FGFR3 levels than normal urothelium. Tumor FGFR3 decreased after treatment and correlated with tumor growth inhibition in response to bacillus Calmette-Guérin. In a pilot bioassay using 11 human bladder tumors treated ex vivo with bacillus Calmette-Guérin we found a subgroup of 41% of patients in whom FGFR3 was decreased after treatment. CONCLUSIONS: Based on bladder cancer murine model results we infer that down-regulation of FGFR3 is a predictive marker of a good response to bacillus Calmette-Guérin therapy. The decrease in FGFR3 in response to bacillus Calmette-Guérin occurred not only in a murine model but also in a human bladder cancer cell line and in some patient samples. More patients and increased followup are needed to establish the predictive role of FGFR3 as a marker in human bladder cancer.

Cross-desensitization and cointernalization of H1 and H2 histamine receptors reveal new insights into histamine signal integration.

G protein-coupled receptor signaling does not result from sequential activation of a linear pathway of proteins/enzymes, but rather from complex interactions of multiple, branched signaling routes, i.e., signaling networks. In this work we present an exhaustive study of the cross-talk between H1 and H2 histamine receptors (H1R and H2R) in U937 cells and Chinese hamster ovary-transfected cells. By desensitization assays we demonstrated the existence of a crossdesensitization between both receptors independent of protein kinase A or C. H1R-agonist stimulation inhibited cell proliferation and induced apoptosis in U937 cells following treatment of 48 hours. H1R-induced antiproliferative and apoptotic response was inhibited by an H2R agonist suggesting that the cross-talk between both receptors modifies their function. Binding and confocal microscopy studies revealed cointernalization of both receptors upon treatment with the agonists. To evaluate potential heterodimerization of the receptors, sensitized emission fluorescence resonance energy transfer experiments were performed in human embryonic kidney 293T cells using H1R-cyan fluorescent protein and H2R-yellow fluorescent protein. To our knowledge these findings may represent the first demonstration of agonist-induced heterodimerization of the H1R and H2R. In addition, we also show that the inhibition of the internalization process did not prevent receptor crossdesensitization, which was mediated by G protein-coupled receptor kinase 2. Our study provides new insights into the complex signaling network mediated by histamine and further knowledge for the rational use of its ligands.

Multidrug resistance protein 4 (MRP4/ABCC4) regulates cAMP cellular levels and controls human leukemia cell proliferation and differentiation.

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

Structure-anti-leukemic activity relationship study of ortho-dihydroxycoumarins in U-937 cells: key role of the δ-lactone ring in determining differentiation-inducing potency and selective pro-apoptotic action.

Previous studies indicated the need of at least one phenolic hydroxyl group in the coumarin core for induction of cytotoxicity in different cell lines. Herein, we present an exhaustive structure-activity relationship study including ortho-dihydroxycoumarins (o-DHC) derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and 1,2-pyrones (representative of the δ-lactone ring of the coumarin core), carried out to further identify the structural features of o-DHC required to induce leukemic cell differentiation and apoptosis in U-937 cells. Our results show for the first time that the δ-lactone ring positively influences the aforementioned biological effects, by conferring greater potency to compounds with an intact coumarin nucleus. Most tellingly, we reveal herein the crucial role of this molecular portion in determining the selective toxicity that o-DHC show for leukemic cells over normal blood cells. From a pharmacological perspective, our findings point out that o-DHC may be useful prototypes for the development of novel chemotherapeutic agents.

Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways.

It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G(11)-coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changes in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP beta2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or beta2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [(3)H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq-PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.

Mifepristone inhibits MPA-and FGF2-induced mammary tumor growth but not FGF2-induced mammary hyperplasia.

We have previously demonstrated a crosstalk between fibroblast growth factor 2 (FGF2) and progestins inducing experimental breast cancer growth. The aim of the present study was to compare the effects of FGF2 and of medroxyprogesterone acetate (MPA) on the mouse mammary glands and to investigate whether the antiprogestin RU486 was able to reverse the MPA- or FGF2-induced effects on both, mammary gland and tumor growth. We demonstrate that FGF2 administered locally induced an intraductal hyperplasia that was not reverted by RU486, suggesting that FGF2-induced effects are progesterone receptor (PR)-independent. However, MPA-induced paraductal hyperplasia was reverted by RU486 and a partial agonistic effect was observed in RU486-treated glands. Using C4-HD tumors which only grow in the presence of MPA, we showed that FGF2 administered intratumorally was able to stimulate tumor growth as MPA. The histology of FGF2-treated tumors showed different degrees of gland differentiation. RU486 inhibited both, MPA or FGF2 induced tumor growth. However, only complete regression was observed in MPA-treated tumors. Our results support the hypothesis that stromal FGF2 activates PR inducing hormone independent tumor growth.

Histamine H2 receptor trafficking: role of arrestin, dynamin, and clathrin in histamine H2 receptor internalization.

Agonist-induced internalization of G protein-coupled receptors (GPCRs) has been implicated in receptor desensitization, resensitization, and down-regulation. In the present study, we sought to establish whether the histamine H2 receptor (H2r) agonist amthamine, besides promoting receptor desensitization, induced H2r internalization. We further studied the mechanisms involved and its potential role in receptor resensitization. In COS7 transfected cells, amthamine induced H2r time-dependent internalization, showing 70% of receptor endocytosis after 60-min exposure to amthamine. Agonist removal led to the rapid recovery of resensitized receptors to the cell surface. Similar results were obtained in the presence of cycloheximide, an inhibitor of protein synthesis. Treatment with okadaic acid, an inhibitor of the protein phosphatase 2A (PP2A) family of phosphatases, reduced the recovery of both H2r membrane sites and cAMP response. Arrestin 3 but not arrestin 2 overexpression reduced both H2r membrane sites and H2r-evoked cAMP response. Receptor cotransfection with dominant-negative mutants for arrestin, dynamin, Eps15 (a component of the clathrin-mediated endocytosis machinery), or RNA interference against arrestin 3 abolished both H2r internalization and resensitization. Similar results were obtained in U937 cells endogenously expressing H2r. Our findings suggest that amthamine-induced H2r internalization is crucial for H2r resensitization, processes independent of H2r de novo synthesis but dependent on PP2A-mediated dephosphorylation. Although we do not provide direct evidence for H2r interaction with beta-arrestin, dynamin, and/or clathrin, our results support their involvement in H2r endocytosis. The rapid receptor recycling to the cell surface and the specific involvement of arrestin 3 in receptor internalization further suggest that the H2r belongs to class A GPCRs.

Carcinoma-associated fibroblasts activate progesterone receptors and induce hormone independent mammary tumor growth: A role for the FGF-2/FGFR-2 axis.

The mechanisms by which mammary carcinomas acquire hormone independence are still unknown. To study the role of cancer-associated fibroblasts (CAF) in the acquisition of hormone-independence we used a hormone-dependent (HD) mouse mammary tumor and its hormone-independent (HI) variant, which grows in vivo without hormone supply. HI tumors express higher levels of FGFR-2 than HD tumors. In spite of their in vivo differences, both tumors have the same hormone requirement in primary cultures. We demonstrated that CAF from HI tumors (CAF-HI) growing in vitro, express higher levels of FGF-2 than HD counterparts (CAF-HD). FGF-2 activated the progesterone receptors (PR) in the tumor cells, thus increasing cell proliferation in both HI and HD tumors. CAF-HI induced a higher proliferative rate on the tumor cells and in PR activation than CAF-HD. The blockage of FGF-2 in the co-cultures or the genetic or pharmacological inhibition of FGFR-2 inhibited PR activation and tumor cell proliferation. Moreover, in vivo, the FGFR inhibitor decreased C4-HI tumor growth, whereas FGF-2 was able to stimulate C4-HD tumor growth as MPA. T47D human breast cancer cells were also stimulated by progestins, FGF-2 or CAF-HI, and this stimulation was abrogated by antiprogestins, suggesting that the murine C4-HI cells respond as the human T47D cells. In summary, this is the first study reporting differences between CAF from HD and HI tumors suggesting that CAF-HI actively participate in driving HI tumor growth.

Biochemical mechanisms underlying the pro-apoptotic activity of 7,8-dihydroxy-4-methylcoumarin in human leukemic cells.

The search for new drugs requires a deep understanding of the molecular basis of drug action, being necessary the elucidation of the mechanism of action with the understanding of the relationship between structure and activity. In the present study, we evaluated the pro-apoptotic activity of 7,8-dihydroxy-4-methylcoumarin (DHMC) and its underlying mechanisms in human leukemic cells. Here, we present evidence that DHMC induced selective and concentration-dependent apoptosis in human leukemic cells. The pro-apoptotic effect of DHMC was mediated by activation of the JNKs and inhibition of the ERK1/2 and PI3K/Akt pathways, with no participation of the p38 cascade after 24h of treatment. Indeed, down-regulation of the proto-oncogene c-myc as well as induction of the cell cycle inhibitor p21(WAF1/CIP1) through a p53 independent mechanism were observed in U-937 cells. These findings suggest that DHCM may have a potential therapeutic role in the future treatment of hematological malignancies.

In Vitro Human Umbilical Vein Endothelial Cells Response to Ionic Dissolution Products from Lithium-Containing 45S5 Bioactive Glass.

Since lithium (Li⁺) plays roles in angiogenesis, the localized and controlled release of Li⁺ ions from bioactive glasses (BGs) represents a promising alternative therapy for the regeneration and repair of tissues with a high degree of vascularization. Here, microparticles from a base 45S5 BG composition containing (wt %) 45% SiO2, 24.5% Na2O, 24.5% CaO, and 6% P2O5, in which Na2O was partially substituted by 5% Li2O (45S5.5Li), were obtained. The results demonstrate that human umbilical vein endothelial cells (HUVECs) have greater migratory and proliferative response and ability to form tubules in vitro after stimulation with the ionic dissolution products (IDPs) of the 45S5.5Li BG. The results also show the activation of the canonical Wnt/ß-catenin pathway and the increase in expression of proangiogenic cytokines insulin like growth factor 1 (IGF1) and transforming growth factor beta (TGFß). We conclude that the IDPs of 45S5.5Li BG would act as useful inorganic agents to improve tissue repair and regeneration, ultimately stimulating HUVECs behavior in the absence of exogenous growth factors.

Histamine H2 receptor overexpression induces U937 cell differentiation despite triggered mechanisms to attenuate cAMP signalling.

Knowing that cell-surface receptors that recognize and respond to extracellular stimuli are key components for the regular communication between individual cells required for the survival of any living organism, the aim of the present work was to investigate the effect of H2R overexpression on the U937 signal transduction pathway and its consequences on cell proliferation and differentiation. The overexpression of H2R led to an increase in cAMP basal levels, a leftward shift of agonist concentration-response curves, and similar maximal response to agonist treatment, suggesting that overexpressed H2Rs act as functional spare receptors. In this system cells triggered several mechanisms tending to restore cAMP basal levels to those of the naïve cells. H2R overexpression induced PDE activity stimulation and GRK2 overexpression. In spite of the onset of these regulatory mechanisms, H2 agonist and rolipram treatments induced the terminal differentiation of the H2R overexpressed clone, conversely to the naïve cells. Present findings show that stably H2R overexpression alters cAMP signalling as the result of not only the amounts of second messenger generated but also the activation or upregulation of various components of signalling cascade, leading to an adapted biologically unique system. This adaptation may represent an advantage or a disadvantage, depending on the biological system, but in any case, the existence of compensatory mechanisms should be considered when a clinical treatment is designed.

Human breast cell lines exhibit functional alpha2-adrenoceptors.

Adrenergic compounds (epinephrine and norepinephrine) are the most important hormones released during stress. Several different receptors are associated with their action in different tissues. However, alpha(2)-adrenoceptors have not yet been described in either normal or tumour human breast tissue. The aim of this work was to describe and characterize these receptors in several tumour and non-tumour human cell lines. The expression of alpha(2)-adrenoceptors was analyzed at the RNA (RT-PCR) and protein ([(3)H]-rauwolscine binding and immunocytochemistry) levels in different human breast cell lines, and the biological activity assessed by [(3)H]-thymidine incorporation. The cancer IBH-6, IBH-7 and MCF-7 and the non-tumour HBL-100 cells line, expressed both alpha(2B)- and alpha(2C)-adrenoceptor-subtypes. A single subtype was expressed in malignant HS-578T (alpha(2A)) and MDA-MB-231 and non-tumour MCF-10A cells (alpha(2B)). All cell lines exhibited significant binding for the specific antagonist [(3)H]-rauwolscine. The alpha-, alpha(2)-, and the alpha(1)-compounds with known affinity for alpha(2)-adrenoceptors, including epinephrine, norepinephrine, yohimbine, clonidine, rauwolscine and prazosin, competed significantly with binding in MCF-7 cells. In addition, IBH-6, IBH-7 and MCF-7 cells showed significant staining with specific antibodies against alpha(2B)- and alpha(2C)-adrenoceptor-subtypes, when tested by immunocytochemistry. In all cell lines, the specific agonist clonidine or oxymetazoline stimulated [(3)H]-thymidine incorporation. EC(50) values were in the range of 20-50 fM for IBH-6, IBH-7, and HS-578T; 0.14 pM for MCF-7; 2-82 pM for HBL-100 and MCF-10A cells, and a biphasic behaviour with a maximum value at 38.0 pM, was observed for MDA-MB-231 cells. The specific alpha(2)-adrenergic antagonist rauwolscine always reversed this stimulation at 0.1 nM. In conclusion, this study describes for the first time, the presence of alpha(2)-adrenoceptors in human epithelial breast cell lines. Moreover, activation of these receptors was associated with an enhancement of cell proliferation.

Angiogenic effects of ionic dissolution products released from a boron-doped 45S5 bioactive glass.

In regenerative medicine of vascularized tissues, there is a great interest in the use of biomaterials that are able to stimulate angiogenesis, a process necessary for rapid revascularization to allow the transport and exchange of oxygen, nutrients, growth factors and cells that take part in tissue repair and/or regeneration. An increasing number of publications have shown that bioactive glasses stimulate angiogenesis. Because it has been established that boron (B) may play a role in angiogenesis, the aim of this study was to assess the in vivo angiogenic effects of the ionic dissolution products that from a bioactive glass (BG) in the 45S5 system doped with 2 wt% B2O3 (45S5.2B). The pro-angiogenic capacity of 45S5.2B BG was assessed on the vasculature of the embryonic quail chorioallantoic membrane (CAM). Ionic dissolution products from 45S5.2B BG increased angiogenesis. This is quantitatively evidenced by the greater expression of integrin αvß3 and higher vascular density in the embryonic quail CAM. The response observed at 2 and 5 days post-treatment was equivalent to that achieved by applying 10 µg mL-1 of basic fibroblast growth factor. These results show that the ionic dissolution products released from the bioactive glass 45S5.2B stimulate angiogenesis in vivo. The effects observed are attributed to the presence the ionic dissolution products, which contained 160 ± 10 µM borate.

Induction of cell differentiation in human leukemia U-937 cells by 5-oxygenated-6,7-methylenedioxycoumarins from Pterocaulon polystachyum.

The present study focused on the effect of a series of extracts and two 5,6,7-trioxygenated coumarins isolated from Pterocaulon polystachyum on the proliferation and differentiation of human promonocytic U-937 cells. The petroleum ether extract was the only extract that significantly reduced cell proliferation and induced cell differentiation. Treatment with pure 5-methoxy-6,7-methylenedioxycoumarin (C1) and 5-(3-methyl-2-butenyloxy)-6,7-methylenedioxycoumarin (C2), present in the petroleum ether extract, showed a time and concentration-dependent inhibition on cell proliferation. In addition, the coumarin derivatives were also able to induce CD88 functionality and NBT reduction, markers of monocytic cell differentiation. These results suggest that C1 and C2 might have a potential therapeutic role in the management of leukemia.

In vitro endothelial cell response to ionic dissolution products from boron-doped bioactive glass in the SiO2-CaO-P2O5-Na2O system.

As it has been established that boron (B) may perform functions in angiogenesis and osteogenesis, the controlled and localized release of B ions from bioactive glasses (BGs) is expected to provide a promising therapeutic alternative for regenerative medicine of vascularized tissues, such as bone. The aim of this study was to assess the in vitro angiogenic effects of the ionic dissolution products (IDPs) from BGs in the SiO2-CaO-Na2O-P2O5 (45S5) system and of those from 45S5 BG doped with 2 wt% B2O3 (45S5.2B). The results show, for the first time, the IDPs from 45S5.2B BG stimulated human umbilical vein endothelial cell (HUVEC) proliferation and migration that were associated with phosphorylation of extracellular signal-related kinase (ERK) 1/2, focal adhesion kinase (FAK) and p38 protein. It was also shown that IDPs from 45S5.2B BG could enhance in vitro HUVEC tubule formation and secretion of interleukin 6 (IL6) and the basic fibroblast growth factor (bFGF). The effects observed are attributed to the presence of B in the IDPs. These findings are relevant to bone tissue engineering and regeneration because the IDPs from 45S5.2B BG may act as inexpensive inorganic angiogenic agents providing a convenient alternative to the application of conventional angiogenic growth factors.

Pharmacodynamic study of the 7,8-dihydroxy-4-methylcoumarin-induced selective cytotoxicity toward U-937 leukemic cells versus mature monocytes: cytoplasmic p21(Cip1/WAF1) as resistance factor.

The development of tumor-selective drugs with low systemic toxicity has always been a major challenge in cancer treatment. Our group previously identified the 7,8-dihydroxy-4-methylcoumarin (DHMC) as a potential chemotherapeutic agent due to its potent, selective anti-proliferative and apoptosis-inducing effects on several cancer cell lines over peripheral blood mononuclear cells. However, there are still no published reports that can explain such selectivity of action. Herein, we addressed this question by using the U-937 promonocytic leukemia cell line, which can be forced to differentiate into a monocyte-like phenotype in vitro. U-937 cells differentiation is dependent on the nuclear expression of p21(Cip1/WAF1), a protein that is absent in immature U-937 cells but present in both the nucleus and the cytoplasm of normal DHMC-resistant monocytes. Considering that induction of differentiation rendered U-937 cells resistant to DHMC, we evaluated the possible causal role of cytoplasmic p21(Cip1/WAF1) in the onset of such resistance by employing U-937 cells stably transfected with a ZnCl2-inducible p21(Cip1/WAF1) variant lacking the nuclear localization signal (U-937/CB6-ΔNLS-p21 cells). Expression of cytoplasmic p21(Cip1/WAF1) did not induce differentiation of the cells but turned them resistant to DHMC through inhibition of JNK, a crucial mediator of DHMC-induced apoptosis in U-937 cells. Sub-acute toxicity evaluation of DHMC in Balb/c mice indicated that DHMC administered intraperitoneally at doses up to 100mg/kg induced no systemic damage. Collectively, our results explain for the first time the selective cytotoxicity of DHMC for tumor cells over normal monocytes, and encourage further in vivo studies on this compound as potential anti-leukemic agent.

Toddaculin, a natural coumarin from Toddalia asiatica, induces differentiation and apoptosis in U-937 leukemic cells.

Chemotherapeutics represent the main approach for the treatment of leukemia. However, the occurrence of adverse side effects and the complete lack of effectiveness in some cases make it necessary to develop new drugs. As part of our screening program to evaluate the potential chemotherapeutic effect of natural coumarins, we investigated the anti-leukemic activities of a series of six prenylated coumarins isolated from the stem bark of Toddalia asiatica (Rutaceae). Among these, 6-(3-methyl-2-butenyl)-5,7-dimethoxycoumarin (toddaculin) displayed the most potent cytotoxic and anti-proliferative effects in U-937 cells. To determine whether these effects resulted from induction of cell death or differentiation, we further evaluated the expression of several apoptosis and maturation markers. Interestingly, while toddaculin at 250 µM was able to induce apoptosis in U-937 cells, involving decreased phosphorylation levels of ERK and Akt, 50 µM toddaculin exerted differentiating effects, inducing both the capacity of U-937 cells to reduce NBT and the expression of differentiation markers CD88 and CD11b, but no change in p-Akt or p-ERK levels. Taken together, these findings indicate that toddaculin displays a dual effect as a cell differentiating agent and apoptosis inducer in U-937 cells, suggesting it may serve as a pharmacological prototype for the development of novel anti-leukemic agents.

Bacillus Calmette Guerin induces fibroblast activation both directly and through macrophages in a mouse bladder cancer model.

BACKGROUND: Bacillus Calmette-Guerin (BCG) is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA) expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy.

Fibroblast growth factor-2 in hyperplastic pituitaries of D2R knockout female mice.

Dopamine D2 receptor (D2R) knockout (KO) female mice develop chronic hyperprolactinemia and pituitary hyperplasia. Our objective was to study the expression of the mitogen fibroblast growth factor (FGF2) and its receptor, FGFR1, comparatively in pituitaries from KO and wild-type (WT) female mice. We also evaluated FGF2 subcellular localization and FGF2 effects on pituitary function. FGF2-induced prolactin release showed a similar response pattern in both genotypes, even though basal and FGF2-stimulated release was higher in KO. FGF2 stimulated pituitary cellular proliferation (MTS assay and [(3)H]thymidine incorporation), with no differences between genotypes. FGF2 concentration (measured by ELISA) in whole pituitaries or cultured cells was lower in KO (P < 0.00001 and 0.00014). Immunofluorescence histochemistry showed less FGF2 in pituitaries from KO females and revealed a distinct FGF2 localization pattern between genotypes, being predominantly nuclear in KO and cytosolic in WT pituitaries. Finally, FGF2 could not be detected in the conditioned media from pituitary cultures of both genotypes. FGFR1 levels (Western blot and immunohistochemistry) were higher in pituitaries of KO. Basal concentration of phosphorylated ERKs was lower in KO cells (P = 0.018). However, when stimulated with FGF2, a significantly higher increment of ERK phosphorylation was evidenced in KO cells (P < or = 0.02). We conclude that disruption of the D2R caused an overall decrease in pituitary FGF2 levels, with an increased distribution in the nucleus, and increased FGFR1 levels. These results are important in the search for reliable prognostic indicators for patients with pituitary dopamine-resistant prolactinomas, which will make tumor-specific therapy possible.

Reduction of G protein-coupled receptor kinase 2 expression in U-937 cells attenuates H2 histamine receptor desensitization and induces cell maturation.

Histamine and H2 agonists transiently induce an important cAMP response in promonocytic U-937 cells but fail to induce monocytic differentiation because of a rapid receptor desensitization mediated by G protein-coupled receptor kinases (GRKs). The aims of the present study were to investigate the participation of GRK2 in the desensitization mechanism of the H2 receptor in U-937 cells by reducing GRK2 levels through antisense technology and to evaluate the differentiating capacity of cells expressing lower GRK2 level, stimulated by H2 agonists. By stable U-937 cell transfection with a GRK2-antisense cDNA, we obtained D5 and A2 cell clones exhibiting a reduction in GRK2 expression and an H3 clone with no significant difference in GRK2 expression from control cells. The cAMP response induced by the H2 agonist in D5 and A2 but not in H3 cells was higher than in U-937 and persisted for a longer period of time, although the number of H2 receptors in D5 and A2 cells was lower than in U-937. Furthermore, D5 and A2 cells treated with H2 agonist showed patterns of c-Fos and CD88 expression consistent with monocytic differentiated cells. Overall, these results indicate a direct correlation between the expression of GRK2 and the desensitization of natively expressed H2 receptors in U-937 cells, suggesting that GRK2 plays a major role in the regulation of these receptors' response. In turn, desensitization process is a key component of H2 receptor signaling, determining the differentiation capability of promonocytic cells.