SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.

Matrix metalloproteinase expression during experimental autoimmune encephalomyelitis and effects of a combined matrix metalloproteinase and tumour necrosis factor-alpha inhibitor.

Matrix metalloproteinases (MMPs) are a large family of Zn2+ endopeptidases that are expressed in inflammatory conditions and are capable of degrading connective tissue macromolecules. MMP-like enzymes are also involved in the processing of a variety of cell surface molecules including the pro-inflammatory cytokine TNF-alpha. MMPs and TNF-alpha have both been implicated in the pathology associated with neuro-inflammatory diseases (NIDs), particularly multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have shown that BB-1101, a broad spectrum hydroxamic acid-based combined inhibitor of MMP activity and TNF processing, reduces the clinical signs and weight loss in an acute EAE model in Lewis rats. However, little is known about which MMPs are involved in the neuroinflammatory process. In order to determine the optimum inhibitory profile for an MMP inhibitor in the treatment of NID, we investigated the profile of MMP expression and activity during EAE. The development of disease symptoms was associated with a 3-fold increase in MMP activity in the cerebrospinal fluid (CSF), which could be inhibited by treatment with BB-1101, and an increase in 92 kDa gelatinase activity detected by gelatin substrate zymography. Quantitative PCR analysis of normal and EAE spinal cord revealed the expression of at least seven MMPs. Of these, matrilysin showed the most significant change, being elevated over 500 fold with onset of clinical symptoms and peaking at maximum disease severity. Of the other six MMPs detected, 92 kDa gelatinase showed a modest 5 fold increase which peaked at the onset of clinical signs and then declined during the most severe phase of the disease. Matrilysin was localised by immunohistochemistry to the invading macrophages within the inflammatory lesions of the spinal cord. Matrilysin's potent broad spectrum proteolytic activity and its localisation to inflammatory lesions in the CNS suggest this enzyme could be particularly involved in the pathological processes associated with neuro-inflammatory disease.

Quantitation of matrix metalloproteinases in cultured rat astrocytes using the polymerase chain reaction with a multi-competitor cDNA standard.

Matrix metalloproteinases (MMPs) are a family of Zn2+ endopeptidases that are expressed in many inflammatory conditions and that contribute to connective tissue breakdown and the release of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha). There is emerging evidence that MMPs have a role in inflammatory disorders of the central nervous system (CNS) such as multiple sclerosis. However, little is known about the expression of MMPs by inflamed tissue within the CNS or by the glia, neurones, and leucocytes which participate in the inflammatory response. To address this issue we have developed a polymerase chain reaction (PCR)-based method for the quantitation of rat MMP mRNA levels, which we have applied to astrocyte cultures with and without inflammatory stimulation. The technique relies on a competition reaction in which a synthetic standard cDNA is co-amplified with the target cDNA in the same PCR reaction. Standard multi-competitor cDNAs, containing priming sites for nine MMPs, and two housekeeping genes were constructed. We have shown that MMP activity is increased over three-fold in neonatal rat astrocyte cultures following stimulation with lipopolysaccharide (LPS). At the mRNA level, MT-MMP-1, 72 kDa gelatinase, and stromelysin-3 were constitutively expressed and unaffected by LPS treatment, whereas 92 kDa gelatinase, and stromelysin-1 were strongly induced (1,000-fold). Stromelysin-2, rat collagenase, and macrophage metalloelastase were modestly upregulated by LPS treatment. Matrilysin was not expressed. This technique is suitable for quantifying MMP expression in the cells which contribute to inflammation in the CNS and could also be applied directly to tissue samples from animal models of disease.

Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells.

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.

Instability of expression of major histocompatibility antigens in fibroblasts expressing activated ras oncogene: constitutive and interferon-gamma induced class I and class II antigens in a series of clonal isolates of murine fibroblasts transformed by v-Ki-ras.

We have examined the expression of major histocompatibility complex (MHC) antigens, constitutive or induced with interferon gamma (IFN-gamma), in a line of C3H mouse embryo fibroblasts (C3H 201) transformed with a helper-virus-free preparation of the Kirsten strain of murine sarcoma virus. C3H 201 cells expressed some class I antigen (H-2Kk) in the absence of added interferon, unlike the parental C3H 10T1/2 cells from which they were derived. However, this declined with (in vitro) passage level after transformation. Treatment with IFN-gamma induced very high expression of H-2Kk at all passage levels. There was no constitutive expression of class II antigen (I-Ak); however, this could be induced by IFN-gamma. Inducibility of I-Ak was found also to be related to the number of passages after transformation; at early passage levels after transformation more I-Ak was induced than after the cells had been allowed to grow for several passages, until at high passage levels little or no I-Ak was induced. This was not due to the presence of a subpopulation of untransformed cells since when the cells were cloned shortly after infection all the resulting clones were transformed. In addition, IFN-gamma at any passage level induced clearly less I-Ak than was found in C3H 10T1/2 cells, in which I-Ak inducibility was high and stable. Twenty-one clones were derived from C3H 201 cells at early passage (less than 8) either from soft agar or from liquid culture. These clones showed a wide variation in MHC antigen phenotype. Many expressed H-2Kk in the absence of IFN-gamma, and all were strongly inducible for H-2Kk. None showed I-Ak in the absence of IFN-gamma. All but two expressed I-Ak after IFN-gamma treatment but, with four exceptions, clearly less than the untransformed line. Four clones derived at late passage (40) resembled the late passage line. The expression of the ras oncogene and tumorigenicity was studied in representative clones; there was no obvious correlation with MHC phenotype, nor with the method of cloning. We conclude from these studies that the expression of MHC antigens by fibroblasts expressing the v-Ki-ras oncogene, either with or without exposure to interferon gamma, is unstable, varying with the number of cell generations from transformation and from clone to clone.

Middle cerebral artery occlusion in rats studied by magnetic resonance imaging.

Ischemia due to middle cerebral artery occlusion was studied in 29 rats from 1 to 24 hours after occlusion using magnetic resonance imaging. Images were made before and after the injection of a superparamagnetic iron oxide compound, AMI-25. Subtraction images demonstrated the region of perfusion deficit as early as 1 hour after occlusion, earlier than conventional T2-weighted images. The area of altered perfusion detected by this technique (subtraction imaging after AMI-25 administration) correlated with that demonstrated by iodoantipyrine autoradiography. Since this magnetic resonance technique can be used to serially estimate the location and size of the ischemic area, the technique can be an important adjunct to metabolic studies of focal ischemia using magnetic resonance spectroscopy. The technique may have clinical applications as well.

Interaction of v-Ki-ras oncogene and interferon-gamma in the control of histocompatibility antigen expression in mouse fibroblasts.

C3H10T1/2 fibroblasts when transformed with Kirsten murine sarcoma virus lose the ability to be induced to express class II major histocompatibility complex antigens when induced with interferon-gamma (IFN-gamma). Sublines were derived from transformed lines by cell sorting after treatment with IFN-gamma, sorting for low or high expression of H-2Ak. These sublines remained stably noninducible or inducible for class II antigen for several passages after sorting. In all other respects tested, viz, sensitivity to IFN-gamma for the generation of an antiviral state or the induction of class I antigen, content of ras gene products, the sorted sublines were very similar. We conclude that ras oncogene expression in these cells can influence the induction of class II antigen but that because ras expression in the sorted lines is similar the effect of ras expression is indirect and presumably involves interaction with other cellular factors.

Role of interferon-gamma in T-cell responses to Semliki Forest virus-infected murine brain cells.

Primary brain cell cultures prepared from newborn C3H mice were infected with Semliki Forest virus (SFV) or treated with a beta-propiolactone-inactivated preparation of SFV (BPL-SFV). The effects of recombinant interferon-gamma (IFN-gamma) treatment on SFV replication, SFV antigen display, major histocompatibility complex (MHC) class I and class II antigen expression, susceptibility to lysis by SFV-specific cytotoxic T lymphocytes (CTL) and the ability to stimulate SFV-specific T lymphocytes to release IFN-gamma were determined. The IFN-gamma treatment prevented replication of SFV, as determined by incorporation of [3H]uridine into SFV-RNA, and reduced expression of SFV antigens on the cell surface, as determined by lysis with antibody and complement or indirect immunofluorescence. BPL-SFV-treated brain cells expressed no SFV antigen detectable by lysis with antibody and complement or indirect immunofluorescence. IFN-gamma increased expression of MHC class I and class II antigens, measured by indirect immunofluorescence, susceptibility to killing by alloreactive T-cell lines and ability to stimulate an allogeneic mixed lymphocyte reaction (MLR). Brain cells infected with SFV or treated with BPL-SFV were susceptible to killing by the CTL. The killing was MHC restricted and neither uninfected nor untreated cells were killed. IFN-gamma treatment prior to SFV infection or BPL-SFV treatment resulted in an augmentation of lysis by the CTL, indicating that even where SFV antigen expression is reduced or present at very low levels, in the context of enhanced MHC class I expression cells remain susceptible to CTL killing. Brain cells treated with BPL-SFV stimulated SFV-specific T cells to release IFN-gamma. Pretreatment of brain cells with IFN-alpha beta or IFN-gamma prior to BPL-SFV treatment markedly increased the ability of the cells to stimulate the SFV-specific T cells to release IFN-gamma. Release of IFN-gamma was MHC restricted and brain cells untreated with BPL-SFV did not stimulate IFN-gamma release. IFN-gamma released by T cells stimulated with BPL-SFV-treated brain cells increased class II MHC expression by brain cells as assessed by indirect immunofluorescence.