George Pan

IFN-γupregulates apoptosis-related molecules and enhances Fas-mediated apoptosis in human cholangiocarcinoma

Human cholangiocarcinoma is a malignancy with no effective therapy and a poor prognosis. Previously, we demonstrated that cultured human cholangiocarcinoma cell lines heterogeneously express Fas on their surface, resulting in 2 subpopulations, Fas‐high and Fas‐low cells. Fas‐low cells are resistant to apoptosis induced by Fas antibody and the calmodulin antagonists tamoxifen and trifluoperazine and are tumorigenic in nude mice (Pan et al., Am J Pathol 1999;155:193–203). Here, we show that IFN‐γ enhances apoptosis in both Fas‐high and Fas‐low cells. IFN‐γ upregulates many apoptosis‐related molecules, including Fas, caspase‐3, caspase‐4, caspase‐7, caspase‐8 and Bak, in both cell lines. Pretreatment with IFN‐γ facilitated Fas‐mediated caspase cleavage, cytochrome c release and Bax translocation. The ability of IFN‐γ to inhibit tumorigenesis of Fas‐low cells was demonstrated in nude mice. Intratumoral injection of IFN‐γ decreased tumor volumes by 78%. These findings indicate that IFN‐γ modulates the apoptotic pathway by upregulating apoptosis‐related genes. This renders tumorigenic Fas‐low cholangiocarcinoma cells nontumorigenic and sensitive to Fas apoptosis, thus representing a possible therapeutic modality.

Dominant inhibition of Fas ligand-mediated apoptosis due to a heterozygous mutation associated with autoimmune lymphoproliferative syndrome (ALPS) Type Ib

Background:Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance due primarily to genetic defects in Fas (CD95/APO-1; TNFRSF6), a cell surface receptor that regulates apoptosis and its signaling apparatus.Methods:Fas ligand gene mutations from ALPS patients were identified through cDNA and genomic DNA sequencing. Molecular and biochemical assessment of these mutant Fas ligand proteins were carried out by expressing the mutant FasL cDNA in mammalian cells and analysis its effects on Fas-mediated programmed cell death.Results:We found an ALPS patient that harbored a heterozygous A530G mutation in the FasL gene that replaced Arg with Gly at position 156 in the proteins extracellular Fas-binding region. This produced a dominant-interfering FasL protein that bound to the wild-type FasL protein and prevented it from effectively inducing apoptosis.Conclusion:Our data explain how a naturally occurring heterozygous human FasL mutation can dominantly interfere with normal FasL apoptotic function and lead to an ALPS phenotype, designated Type Ib.

Stimulatory Role of Substance P on Gonadotropin Release in Ovariectomized Rats

Substance P (SP) has been shown to be present in the hypothalamus and anterior pituitary. To evaluate a possible physiological role of endogenous SP in the control of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release, specific antiserum against SP (anti-SP) was injected intraventricularly (3 microliters into the third ventricle) or intravenously (50 or 200 microliters) into conscious, ovariectomized (OVX) rats. Third ventricular injection of the antiserum induced a significant decrease in both plasma LH and FSH levels when compared to values in control animals injected with normal rabbit serum (p less than 0.01 and p less than 0.025, respectively). The effect was observed within 10 mi and levels remained suppressed for 60 min. In contrast, intravenous injection of large doses of anti-SP had no effect on the release of both hormones. In order to confirm the stimulatory effect of SP itself, synthetic SP was injected intravenously and intraventricularly into estrogen-primed (E-primed), OVX rats. Synthetic SP dramatically stimulated LH release, but not FSH release when injected either intravenously or intraventricularly at doses of 10 and 50 micrograms (p less than 0.001, p less than 0.005 vs. control, respectively). To investigate any direct action of SP on gonadotropin release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats. SP did not affect the release of gonadotropins in vitro. These results indicate that endogenous hypothalamic SP exerts a tonic stimulatory hypothalamic control of basal gonadotropin release in OVX rats.

AZT Enhances Osteoclastogenesis and Bone Loss

A variety of metabolic complications have been reported to be associated with highly active antiretroviral therapy (HAART), including osteopenia and osteoporosis. In this study, we determine the effects of zidovudine (AZT), a nucleoside reverse transcriptase inhibitor, on osteoclastogenesis in a cultured mouse macrophage preosteoclast cell line (RAW264.7), in mouse primary bone marrow macrophage-monocyte precursors, and on bone mineral density in mice. The results indicate that AZT induces an increase in osteoclastogenesis in the mouse preosteoclast cell line and in mouse bone marrow osteoclast precursors in the presence of RANKL. This increased osteoclastogenesis is dependent upon the concentration of AZT. AZT increases the promoter activity of tartrate-resistant acid phosphatase (TRAP) and the binding and function of the nuclear transcription protein, NF-kappaB, in RAW264.7 cells. Therefore, the effect of AZT is mediated, at least in part, by enhancing RANKL-mediated osteoclastogenesis. Bone mineral density (BMD) in AZT-treated mice is decreased and histopathology shows marked osteopenia. These results support an important role of AZT-stimulated osteoclastogenesis in HAART-induced osteopenia.

Pathogenesis of Osteopenia/Osteoporosis Induced by Highly Active Anti-Retroviral Therapy for AIDS

Abstract:  The advent of highly active anti‐retroviral therapy (HAART) has dramatically decreased the rate of AIDS‐related mortality and significantly extended the life span of patients with AIDS. A variety of metabolic side effects are associated with these therapies, one of which is metabolic bone disease. A higher prevalence of osteopenia and osteoporosis in HIV‐infected patients receiving anti‐retroviral therapy than in patients not on therapy has now been reported in several studies. Several factors have been demonstrated to influence HIV‐associated decreases in bone mineral density (BMD), including administration of nucleoside reverse transcriptase inhibitors (NRTIs). In this article, discussion will focus on the molecular pathogenesis and treatment of HAART‐associated osteopenia and osteoporosis.

RANKL Regulates Fas Expression and Fas-Mediated Apoptosis in Osteoclasts†

Osteoclast apoptosis is an influential determinant of osteoclast bone‐resorbing activity. RANKL, a critical factor for osteoclastogenesis, is also important in osteoclast survival. However, the mechanisms by which RANKL prevents osteoclast apoptosis remain largely unknown.

Requirement of Calmodulin Binding by HIV-1 gp160 for Enhanced FAS-mediated Apoptosis*

Accelerated apoptosis is one mechanism proposed for the loss of CD4+ T-lymphocytes in human immunodeficiency virus type 1 (HIV-1) infection. The HIV-1 envelope glycoprotein, gp160, contains two C-terminal calmodulin-binding domains. Expression of gp160 in Jurkat T-cells results in increased sensitivity to FAS- and ceramide-mediated apoptosis. The pro-apoptotic effect of gp160 expression is blocked by two calmodulin antagonists, tamoxifen and trifluoperazine. This enhanced apoptosis in response to FAS antibody or C2-ceramide is associated with activation of caspase 3, a critical mediator of apoptosis. A point mutation in the C-terminal calmodulin-binding domain of gp160 (alanine 835 to tryptophan, A835W) eliminates gp160-dependent enhanced FAS-mediated apoptosis in transiently transfected cells, as well asin vitro calmodulin binding to a peptide corresponding to the C-terminal calmodulin-binding domain of gp160. Stable Tet-off Jurkat cell lines were developed that inducibly express wild type gp160 or gp160A835W. Increasing expression of wild type gp160, but not gp160A835W, correlates with increased calmodulin levels, increased apoptosis, and caspase 3 activation in response to anti-FAS treatment. The data indicate that gp160-enhanced apoptosis is dependent upon calmodulin up-regulation, involves the activation of caspase 3, and requires calmodulin binding to the C-terminal binding domain of gp160.

Apoptosis and tumorigenesis in human cholangiocarcinoma cells : Involvement of Fas/APO-1 (CD95) and calmodulin

We have previously demonstrated that tamoxifen inhibits the growth of human cholangiocarcinoma cells in culture and inhibits tumor growth when cells are injected into nude mice. However, the mechanism of action of tamoxifen remains unknown. Here we demonstrate that tamoxifen and trifluoperazine, both potent calmodulin antagonists, induce apoptosis in vitro, probably acting via the Fas system, in human cholangiocarcinoma cells. Human cholangiocarcinoma cell lines heterogeneously express Fas antigen on their surface. Fas-negative and Fas-positive surface-expressing cells were isolated, cloned, and cultured. Fas antibody, tamoxifen, and trifluoperazine induced dose-dependent apoptosis only in Fas-positive cells; Fas-negative cells were unaffected. Furthermore, apoptosis induced by tamoxifen in Fas-positive cells was blocked by an inhibitory Fas antibody. Tamoxifen was not acting through an anti-estrogenic mechanism, because neither Fas-negative nor Fas-positive cells expressed estrogen receptors and the pure anti-estrogen compound, ICI 182780, did not induce apoptosis in either cell line. Fas-negative cells, but not Fas-positive cells, were able to produce tumors when subcutaneously injected into nude mice. These findings suggest Fas may be a candidate oncogene involved in the pathogenesis of cholangiocarcinoma. Furthermore, the similarity between the pro-apoptotic effects of tamoxifen and trifluoperazine support an underlying molecular mechanism for Fas-mediated apoptosis that involves calmodulin.

Osteoblast and osteoclast differentiation in modeled microgravity.

Abstract:  Prolonged microgravity experienced by astronauts is associated with a decrease in bone mineral density. To investigate the effect of microgravity on differentiation of osteoclasts and osteoblasts, we used a NASA‐recommended, ground‐based, microgravity‐simulating system, the Rotary Cell Culture System (RCCS). Using the RCCS, we demonstrated that modeled microgravity (MMG) inhibited osteoblastogenesis and increased adipocyte differentiation in human mesenchymal stem cells incubated under osteogenic conditions. This transformation involves reduced RhoA activity and cofilin phosphorylation, disruption of F‐actin stress fibers, and decreased integrin signaling through focal adhesion kinase. We have used the system to show that MMG also stimulates osteoclastogenesis. These systems provide the opportunity to develop pharmacological agents that will stimulate osteoblastogenesis and inhibit osteoclastogenesis.

The Combination of Calmodulin Antagonists and Interferon-γ Induces Apoptosis through Caspase-Dependent and -Independent Pathways in Cholangiocarcinoma Cells

Calmodulin (CaM) antagonists have been shown to inhibit tumor cell invasion and metastasis and to induce apoptosis in various tumor models, but the molecular mechanism of CaM antagonist-mediated apoptosis is poorly understood. Here, we demonstrate that interferon (IFN)-gamma induces susceptibility to CaM antagonist-mediated apoptosis in human cholangiocarcinoma cells weakly expressing Fas (Fas-low cells). During CaM antagonist-mediated apoptosis in IFN-gamma-pretreated Fas-low cells, cleavage of caspases-8, -9, and -3 and Bid, release of cytochrome c from the mitochondria and an increase in the free cytosolic calcium concentration were observed. CaM antagonists also caused depolarization of the mitochondrial membrane independent of caspase activation. Although a broad-range caspase inhibitor partially blocked CaM antagonist-mediated apoptosis, the neutralizing Fas antibody had no effect, suggesting that CaM antagonist-mediated apoptosis does not require interaction between CaM antagonists and surface Fas. CaM antagonists induce apoptosis via mechanisms other than inhibition of CaM-dependent protein kinase II and calcineurin, as their inhibitors, KN93 and cyclosporine A, had no effect on apoptosis. Taken together, these results indicate that CaM antagonists induce apoptosis in both caspase-dependent and -independent manners, and that susceptibility to CaM antagonists is modulated by IFN-gamma. The combination of IFN-gamma and CaM antagonists, including tamoxifen, may be a potential therapeutic modality for cholangiocarcinoma and possibly other malignancies.