Arginase inhibition promotes wound healing in mice.

OBJECTIVE: Arginase plays important regulatory roles in polyamine, ornithine, and nitric oxide syntheses. However, its role in the healing process has not been delineated. In this study, we used a highly potent and specific inhibitor of arginase, namely 2(S)-amino-6-boronohexanoic acid NH4 (ABH) to evaluate the role of arginase function in wound healing. MATERIALS AND METHODS: ABH or saline was applied topically to full thickness, dorsal, excisional wounds in C57BL/6 mice every 8 hours for 14 days post surgery and the rate of wound closure was estimated planimetrically. Wound tissue was harvested from mice sacrificed on postoperative days 3 and 7 and examined histologically. The extent of epithelial, connective, and granulation tissue present within the wound area was estimated histomorphometrically. The effect of ABH on wound arginase activity, production of nitric oxide metabolites (NO(x)), and presence of smooth muscle actin positive cells (myofibroblasts) was evaluated. RESULTS: While arginase activity was inhibited in vivo, the rate of wound closure significantly increased 7 days post-surgery, (21 ± 4%: P < .01; Student t test) in ABH treated animals. This was accompanied by an early increase in wound granulation tissue and accumulation of NO(x) followed by enhanced re-epithelialization and localization of myofibroblasts beneath the wound epithelium. CONCLUSION: Arginase inhibition improves excisional wound healing and may be used to develop therapeutics for early wound closure.

Role of notch-1 and E-cadherin in the differential response to calcium in culturing normal versus malignant prostate cells.

A panel of expression markers was validated and used to document that, when radical prostatectomy specimens are cultured in low (i.e., <260 micromol/L)-calcium (Ca2+)-serum-free, growth factor-defined (SFD) medium, what grows out are not prostatic cancer cells but basally derived normal transit-amplifying prostatic epithelial cells. The selective outgrowth of the normal transit-amplifying versus prostatic cancer cells is due to the differential effect of low-Ca2+ medium on the structure of Notch-1 and E-cadherin signaling molecules. In low-Ca2+ medium, Notch-1 receptor is conformationally in a constitutively active, cell autonomous form not requiring reciprocal cell-cell (i.e., ligand) interaction for signaling. Such signaling is required for survival of transit-amplifying cells as shown by the death of transit-amplifying cells induced by treatment with a series of chemically distinct gamma-secretase inhibitors to prevent Notch-1 signaling. Conversely, in low-Ca2+ medium, E-cadherin is conformationally inactive preventing cell-cell homotypic interaction, but low cell density nonaggregated transit-amplifying cells still survived because Notch-1 is able to signal cell autonomously. In contrast, when medium Ca2+ is raised to >400 micromol/L, Notch-1 conformationally is no longer constitutively active but requires cell-cell contact for reciprocal binding of Jagged-1 ligands and Notch-1 receptors between adjacent transit-amplifying cells to activate their survival signaling. Such cell-cell contact is enhanced by the elevated Ca2+ inducing an E-cadherin conformation allowing homotypic interaction between transit-amplifying cells. Such Ca(2+)-dependent, E-cadherin-mediated interaction, however, results in cell aggregation, stratification, and inhibition of proliferation of transit-amplifying cells via contact inhibition-induced up-regulation of p27/kip1 protein. In addition, transit-amplifying cells not contacting other cells undergo squamous differentiation into cornified (i.e., 1% SDS insoluble) envelopes and death in the elevated Ca2+ medium. Stratification and contact inhibition induced by elevated Ca2+ are dependent on E-cadherin-mediated homotypic interaction between transit-amplifying cells as shown by their prevention in the presence of a cell-impermanent, E-cadherin neutralizing antibody. In contrast to growth inhibition of normal transit-amplifying cells, supplementation of low-Ca(2+)-SFD medium with 10% FCS and raising the Ca2+ to >600 micromol/L stimulates the growth of all prostate cancer cell lines tested. Additional results document that, at physiologic levels of Ca2+ (i.e., >600 micromol/L), prostatic cancer cells are not contact inhibited by E-cadherin interactions and Notch-1 signaling is no longer required for survival but instead becomes one of multiple signaling pathways for proliferation of prostatic cancer cells. These characteristic changes are consistent with prostate cancer cells' ability to metastasize to bone, a site of high-Ca2+ levels.

Prostate cancer: potential targets of anti-proliferative and apoptotic signaling pathways.

Prostate cancers are genetically and phenotypically heterogenous. The heterogeneous nature of prostate cancer may be a consequence of mutations in different cell types (basal stem, transit amplifying or luminal cells) resulting in different malignant maturation pathways. One consistent characterization of these cancers, however, is their eventual progression to a hormonal refractory state. The development of effective novel therapeutic strategies requires an understanding of the mechanisms for the development of such a refractory state. Targeting proliferative and survival pathways provides a rationale for drug design and development for hormone refractory prostate cancer. Prostate cancer cells, however, develop an enhanced redundancy in downstream survival signaling. Hence, new combinational therapies must be developed with the understanding that compensatory mechanisms evolve under selective pressure.

Enhanced redundancy in Akt and mitogen-activated protein kinase-induced survival of malignant versus normal prostate epithelial cells.

Activation of the downstream akt and mitogen-activated protein kinases is associated with development and progression of prostate cancer to the lethal androgen-independent state. However, the causal role of these downstream kinases in androgen-independent prostate cancers is unknown. In this study, activation and requirements of akt and mitogen-activated protein kinase (erk, p38, and jnk) signaling for the survival and proliferation of five malignant human cell lines encompassing the spectrum of androgen-independent prostate cancers was compared with the activation and requirements in normal prostate epithelial cells. Using Western blotting with phospho-antibodies, we detected differential activation in exponentially growing, growth factor-deprived, and restimulated cultures of malignant versus normal cells. The inhibition of erk, p38, jnk, and akt with U0126, SB203580, SP600125, and Akt inhibitor, respectively, document that normal cells require simultaneous erk and jnk signaling for survival, plus akt signaling for proliferation. In malignant cells, however, only jnk inhibition as monotherapy produces a consistent apoptotic response, although the combinatorial inhibition of jnk, erk, p38 plus akt results in statistically enhanced apoptosis. These results demonstrate that prostate cancer progression to a lethal androgen-independent state involves the acquisition of an enhanced redundancy in downstream survival signaling.

In vitro culturing and characteristics of transit amplifying epithelial cells from human prostate tissue.

The prostatic epithelium is functionally organized in stem cell units. This unit consists of a slow turn over stem cell within the basal epithelial layer which can replenish itself and provide progeny which differentiate down either a neuroendocrine or exocrine pathway. The maturation along the exocrine pathway initially involves transit amplifying cells within the basal layer proliferating and subsequently the progeny maturing into intermediate cells. These intermediate cells migrate into the luminal layer where they terminally differentiate into non-proliferative secretory luminal cells which express prostate specific differentiation markers, like PSA. A growing body of experimental evidence has identified the proliferating transit amplifying/intermediate cells as the cells of origin for the common prostatic adenocarcinomas. Using a series of growth characteristics, and mRNA and protein markers, we have validated that primary cultures can be established in serum free defined media from surgically resected human prostates which are composed of essentially pure population of transit amplifying cells. At each serial passage, the subsequent cultures undergo enhanced maturation into intermediate cells and by the 7-10th passage these cells eventually lose their proliferative ability. This study validates that these cells are a useful and relevant system for the determination of molecular events involved in prostatic carcinogenesis.

Differential expression and/or activation of P38MAPK, erk1/2, and jnk during the initiation and progression of prostate cancer.

BACKGROUND: Members of the mitogen-activated protein kinase (MAPK) family are capable of transducing signals from a wide variety of stimuli, including growth factors, G-protein coupled receptors, and cytokines that are likely to play a role in the initiation and/or progression of prostate cancer. METHODS: The expression and activation of three members of the MAPK family, namely, erk, jnk, and p38MAPK was examined using Western blotting and immunohistochemistry during tumor progression in a transgenic mouse model for prostate cancer. RESULTS: Activation of p38MAPK was significantly elevated (2.3-fold) in well-differentiated prostatic tumors compared to normal controls. Furthermore, prostatic intraepithelial neoplastic (PIN) lesions expressing activated p38MAPK were observed to be proliferative rather than apoptotic. Expression of activated erk1/2 also preferentially co-located to a sub-population of epithelial cells within PIN lesions that correlated with Ki67 expression. In dramatic contrast, activated forms of erk1/2, jnk, and p38MAPK were reduced or absent in late stage adenocarcinomas and metastatic deposits. CONCLUSIONS: Erk1/2, jnk, and p38MAPKs are differentially expressed and/or activated during prostate cancer progression. Activation of both erk1/2 and p38MAPK occurs concomitant with prostatic epithelial cell proliferation and the initiation of prostate cancer while inactivation is contemporaneous with the emergence of the poorly differentiated metastatic and androgen-independent phenotype.

Trk receptor inhibition induces apoptosis of proliferating but not quiescent human osteoblasts.

Prostate cancer frequently metastasizes to the skeleton, producing painful osteoblastic lesions, which are associated with significant morbidity and mortality. This bone tropism involves the bidirectional paracrine interactions between prostate cancer cells and osteoblasts. These interactions enhance prostate cancer cell survival and proliferation of osteoblasts. Therefore, agents that can induce apoptosis of prostate cancer cells and proliferating osteoblasts would be highly advantageous. Previously, we have documented that the unique survival pathway for prostate cancer cells involves a neurotrophin/Trk receptor autocrine pathway. The indocarbazole compounds, CEP-701 and CEP-751, are potent inhibitor of this Trk receptor survival signaling and thus selectively induces apoptosis of prostate cancer cells in various in vitro and in vivo models. In this study, we documented the effects of CEP-751 on the conditionally immortalized osteoblastic cell line, hFOB, in vitro. At the permissive temperature of 34 degrees C, these cells express large T antigen, inducing their continuous proliferation, whereas at 39 degrees C, T antigen is degraded and the cells stop proliferating without undergoing apoptosis. Trk receptors are expressed in hFOB cells, as determined both by reverse transcription-PCR and Western blots. These osteoblasts were shown to produce nerve growth factor and brain-derived neurotrophic factor but not neurotrophin-3, as measured by ELISA. hFOB osteoblasts, cultured at 34 degrees C, secreted significantly (P < 0.01) more brain-derived neurotrophic factor and nerve growth factor into the medium than hFOB cells cultured at 39 degrees C. Because the Trk/neurotrophin axis is present in both proliferating and quiescent (i.e., nonproliferating) osteoblasts, the effects of 48 h of exposure to various doses of CEP-751 on cell viability and apoptosis of hFOB cells were assessed by trypan blue exclusion assays and 4',6-diamidino-2-phenylindole nuclear staining. Cell viability and apoptosis of hFOB cells at 34 degrees C were significantly and dose-dependently decreased compared with untreated proliferating cells. In contrast, even the highest concentration of CEP-751 (200 nM) did not affect cell viability and apoptosis of quiescent hFOB cells cultured at 39 degrees C. This trk inhibition-induced cytotoxicity was confirmed using early-passage, proliferating normal (i.e., non-SV40-transformed) human osteoblasts, which also express Trk receptor protein. These combined results demonstrate that proliferating osteoblasts acquire a sensitivity to trk inhibition- induced apoptosis not shared with normally quiescent osteoblasts.