The Immune Inhibitor A1 Protease of Bacillus anthracis

Bacillus anthracis, an organism ubiquitous in the soil and the causative agent of anthrax, utilizes multiple mechanisms to regulate secreted factors; one example is the activity of secreted proteases. One of the most abundant proteins in the culture supernates of B. anthracis is the Immune Inhibitor A1 (InhA1) protease. Here, I demonstrate that InhA1 modulates the abundance of approximately half of the proteins secreted into the culture supernates, including substrates that are known to contribute to the ability of the organism to cause virulence. For example, InhA1 cleaves the anthrax toxin proteins, PA, LF, and EF. InhA1 also targets a number of additional proteases, including Npr599, contributing to a complex proteolytic regulatory cascade with far-reaching affects on the secretome. Using an intra-tracheal mouse model of infection, I found that an inhA-null strain is attenuated in relation to the parent strain. The data indicate that reduced virulence of the inhA mutant strain may be the result of toxin protein deregulation, decreased association with macrophages, and/or the inability to degrade host antimicrobial peptides. Given the significant modulation of the secretome by InhA1, it is likely that expression of the protease is tightly regulated. To test this I examined inhA1 transcript and protein levels in the parent and various isogenic mutant strains and found that InhA1 expression is regulated by several mechanisms. First, the steady state levels of inhA1 transcript are controlled by the regulatory protein SinR, which inhibits inhA1 expression. Second, InhA1 abundance is inversely proportional to the SinR-regulated protease camelysin, indicating the post-transcriptional regulation of InhA1 by camelysin. Third, InhA1 activity is dependent on a conserved zinc binding motif, suggesting that zinc availability regulates InhA1 activity. The convergence of these regulatory mechanisms signifies the importance of tight regulation of InhA1 activity, activity that substantially affects how B. anthracis interacts with its environment.

LY2109761, a novel transforming growth factor beta receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis.

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

THE HISTONE DEACETYLASE INHIBITOR, MS-275, SENSITIZES METASTATIC OSTEOSARCOMA TO FASL-INDUCED CELL DEATH: A ROLE FOR C-FLIP

The purpose of this study was to determine the effects of the histone deacetylase inhibitor, MS-275, on the Fas signaling pathway and susceptibility of osteosarcoma (OS) to Fas ligand (FasL)-induced cell death. OS metastasizes almost exclusively to the lungs. We have shown that Fas expression in OS cells is inversely correlated with their metastatic potential. Fas+ cells are rapidly eliminated when they enter the lungs via interaction with FasL, which is constitutively expressed in the lungs. Fas- OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Moreover, upregulation of Fas in established OS lung metastases results in tumor regression. Therefore, agents that upregulate Fas expression or activate the Fas signaling pathway may have therapeutic potential. Treatment of Fas- metastatic OS cell lines with 2 μM MS-275 sensitized cells to FasL-induced cell death in vitro. We found that MS-275 did not alter the expression of Fas on the cell surface; rather it resulted in increased levels of Fas within the membrane lipid rafts, as demonstrated by an increase in Fas expression in detergent insoluble lipid raft fractions. We further demonstrated that following MS-275 treatment, Fas colocalized with GM1+ lipid rafts and that there was a decrease in c-FLIP (cellular FLICE-inhibitory protein) mRNA and protein. Downregulation of c-FLIP correlated with caspase activation and apoptosis induction. Transfection of cells with shRNA to c-FLIP also resulted in the localization of Fas to lipid rafts. These studies indicate that MS-275 sensitizes OS cells to FasL by upregulating the expression of Fas in membrane lipid rafts, which correlated with the downregulation of c-FLIP. Treatment of nu/nu-mice with established OS lung metastases with oral MS-275 resulted in increased apoptosis, a significant inhibition of c-FLIP expression in tumors and tumor regression. Histopathological examination of mice showed no significant organ toxicity. Overall, these results suggest that the mechanism by which MS-275 sensitizes OS cells and lung metastases to FasL-induced cell death may be by a reduction in the expression of c-FLIP.

Analysis of pp60$\sp{{\rm c}{-}src}$ and pp62$\sp{{\rm c}{-}yes}$ intracellular protein tyrosine kinase function in colon tumor cell growth regulation using herbimycin A, a tyrosine kinase specific inhibitor

Two approaches were utilized to investigate the role of pp60c-src activation in growth control of model colon tumor cell lines. The first approach involved analysis of pp60c-src activity in response to growth factor treatment to determine if transient activation of the protein was associated with ligand induced mitogenic signal transduction as occurs in non-colonic cell types. Activation of pp60c-src was detected using colon tumor cell lysates after treatment with platelet derived growth factor (PDGF). Activation of pp60c-src was also detected in response to epidermal growth factor (EGF) treatment using cellular lysates and intact cells. In contrast, down-regulation of purified pp60c-src occurred after incubation with EGF-treated EGFr immune complexes in vitro suggesting additional cellular events were potentially required for the stimulatory response observed in intact cells. The results demonstrated activation of pp60c-src in colon tumor cells in response to PDGF and EGF which is consistent with the role of the protein in mitogenic signal transduction in non-colonic cell types.^ The second approach used to study the role of pp60c-src activation in colonic cell growth control focused on analysis of the role of constitutive activation of the protein, which occurs in approximately 80% of colon tumors and cell lines, in growth control. These studies involved analysis of the effects of the tyrosine kinase specific inhibitor Herbimycin A (HA) on monolayer growth and pp60c-src enzymatic activity using model colon tumor cell lines. HA induced dose-dependent growth inhibition of all colon tumor cell lines examined possessing elevated pp60c-src activity. In HT29 cells the dose-dependent growth inhibition induced by HA correlated with dose-dependent pp60c-src inactivation. Inactivation of pp60c-src was shown to be an early event in response to treatment with HA which preceded induction of HT29 colon tumor cell growth inhibition. The growth effects of HA towards the colon tumor cells examined did not appear to be associated with induction of differentiation or a cytotoxic mechanism of action as changes in morphology were not detected in treated cells and growth inhibition (and pp60c-src inactivation) were reversible upon release from treatment with the compound. The results suggested the constitutive activation of pp60c-src functioned as a proliferative signal in colon tumor cells. Correlation between pp60c-src inactivation and growth inhibition was also observed using HA chemical derivatives confirming the role of tyrosine kinase inactivation by these compounds in inhibition of mitogenic signalling. In contrast, in AS15 cells possessing specific antisense mRNA mediated inactivation of pp60c-src, HA-induced inactivation of the related pp62c-yes tyrosine kinase, which is also activated during colon tumor progression, was not associated with induction of monolayer growth inhibition. These results suggested a function for the constitutively activated pp62c-yes protein in colon tumor cell proliferation which was different from that of activated pp60c-src. (Abstract shortened by UMI.) ^

Regulation of sex hormone dependent growth by a serum borne inhibitor

This thesis project focused on understanding the basic process controlling cell proliferation in sex-steroid hormone dependent cancers. The availability of inculture models using cloned cell lines offers the greatest advantage for studying the control of this event. Incubation of cloned sex-hormone sensitive cells in medium containing increasing concentrations of sex-hormone stripped serum, results in a dose dependent growth inhibition; this inhibition is reversed by the addition of physiological concentrations of steroid hormones. The mechanisms explaining this phenomenon are not yet fully understood, but different theories propose the existence in serum of a sex hormone binding protein with growth inhibitory properties. We were able to identify a protein that specifically binds sex hormones in rat and horse serum with affinities 10-fold lower to the ones observed with the classic sex-hormone binding globulin (SHBG) in humans. Purification of this protein on a large scale Lowed a more detailed analysis. The putative sex-hormone binding protein has an apparent molecular weight of 386 KDa. SDS-PAGE with commassie staining of the purified product, displayed a pattern non-characteristic of SMG, but all bands cross-reacted with a commercial anti-SMG antibody in western analysis. Titrations of the purified product on cell proliferation assays using sex-hormone dependent lines, resulted in a dose dependent growth inhibition. This inhibition was reversed by the addition of sex hormones. Our results indicate that we have identified and purified a sex-hormone binding protein in serum with characteristics similar to SHBG and with cell growth inhibitory properties. ^

Emodin, a tyrosine kinase inhibitor, and human cancer

Many human diseases, including cancers, result from aberrations of signal transduction pathways. The recent understanding of the molecular biochemistry of signal transduction in normal and transformed cells enable us to have a better insight about cancer and design new drugs to target this abnormal signaling in the cancer cells. Tyrosine kinase pathway plays a very important role in normal and cancer cells. Enhanced activity of tyrosine kinases has been associated with many human cancer types. Therefore, identifying the type of tyrosine kinases involved in a particular cancer type and blocking these tyrosine kinase pathways may provide a way to treat cancer. Receptor tyrosine kinase expression, namely epidermal growth factor receptor (EGFR) family, was examined in the oral squamous cell carcinoma patients. The expression levels of different members of the EGFR family were found to be significantly associated with shorter patients' survival. Combining EGFR, HER-2/neu, and HER-3 expression can significantly improve the predicting power. The effect of emodin, a tyrosine kinase inhibitor, on these receptors in head and neck squamous cell carcinoma cell lines was examined. Emodin was found to suppress the tyrosine phosphorylation of HER-2/neu and EGF-induced tyrosine phosphorylation of EGFR. Emodin also induced apoptosis and downregulated the expression of anti-apoptotic protein bcl-2 in oral squamous cell carcinoma cells. It is known that tyrosine kinase pathways are involved in estrogen receptor signaling pathway. Therefore, the effects of inhibiting the tyrosine kinase pathway in estrogen receptor-positive breast cancers was studied. Emodin was found to act similarly to antiestrogens, capable of inhibiting estrogen-stimulated growth and DNA synthesis, and the phosphorylation of Rb protein. Interestingly, emodin, and other tyrosine kinase inhibitors, such as RG 13022 and genistein, depleted cellular levels of estrogen receptor protein. Emodin-induced depletion of estrogen receptor was mediated by the proteasome degradation pathway. In summary, we have demonstrated that tyrosine kinase pathways play an important role in oral squamous cell carcinoma and estrogen receptor-positive breast cancer. Targeting the tyrosine kinases by inhibitors, such as emodin, may provide a potential way to treat the cancer patients. ^

Mechanism of IFN-induced apoptosis and sensitization by the proteasome inhibitor bortezomib in bladder cancer cells

Bladder cancer is the fifth most common cancer with more than 50,000 cases diagnosed each year. Interferon-α (IFNα) is mostly used in combination with BCG for the treatment of transitional cell carcinoma (TCC). To examine the effects of IFNα on bladder cancer cells, I analyzed a panel of 20 bladder cancer cell lines in terms of their sensitivity to IFNα-induced apoptosis and the underlying mechanisms. I identified three categories: cells that die after 48hr, after 72h, and cells resistant even after 72hr of IFNα treatment. Examination of the IFN-signal transduction pathway revealed that the defect was not due to abrogation of IFN signaling. Further analysis demonstrated dependency of IFN-induced apoptosis on caspase-8, implicating the role of death receptors in IFN-induced cell death. Of the six most-IFN-sensitive cell lines, the majority upregulated Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) at the mRNA and protein level and IFN-induced cell death was mediated through TRAIL, while a minority of the most IFN-sensitive cells undergo apoptosis through a TNFα-dependent mechanism. IFNα resistance was due to either absence of TRAIL upregulation at the mRNA or protein level, resistance to exogenous rhTRAIL itself or lack of sensitization to IFN-induced cell death. Downregulation of XIAP, or XIAP inactivation through its regulator NFκB has been reported to sensitize tumor cells to death receptor-induced cell death. Baseline and IFN-inducible XIAP levels were examined in the most and least IFN-sensitive cells, knocking down XIAP and the p65 subunit of NFκB enhanced IFN-induced cell death, implicating XIAP downregulation as a mechanism through which bladder cancer cells are sensitized to IFN-induced apoptosis. To determine whether or not the proteasome inhibitor Bortezomib (BZ) sensitizes bladder cancer cells to IFN-induced cell death, the combined effects of IFN+BZ and the underlying molecular mechanisms were examined both in vitro and in vivo using two bladder xenograft models. In both models, tumor growth inhibition was the result of either increased cell death of tumor cells exerted by the two agents and/or inhibition of angiogenesis. In vitro, MAP downregulation in response to the combined treatment of IFN+BZ accounts for one of the mechanisms mediating IFN+BZ cell death in bladder cancer cells. ^

The transcriptional repressor Delta EF1 controls resistance to the EGFR inhibitor erlotinib in human HNSCC lines

Epidermal Growth Factor Receptor (EGFR) overexpression occurs in about 90% of Head and Neck Squamous Cell Carcinoma (HNSCC) cases. Aberrant EGFR signaling has been implicated in the malignant features of HNSCC. Thus, EGFR appears to be a logical therapeutic target with increased tumor specificity for the treatment of HNSCC. Erlotinib, a small molecule tyrosine kinase inhibitor, specifically inhibits aberrant EGFR signaling in HNSCC. Only a minority of HNSCC patients were able to derive a substantial clinical benefit from erlotinib. ^ This dissertation identifies Epithelial to Mesenchymal Transition (EMT) as the biological marker that distinguishes EGFR-dependent (erlotinib-sensitive) tumors from the EGFR-independent (erlotinib-resistant) tumors. This will allow us to prospectively identify the patients who are most likely to benefit from EGFR-directed therapy. More importantly, our data identifies the transcriptional repressor DeltaEF1 as the mesenchymal marker that controls EMT phenotype and resistance to erlotinib in human HNSCC lines. si-RNA mediated knockdown of DeltaEF1 in the erlotinib-resistant lines resulted in reversal of the mesenchymal phenotype to an epithelial phenotype and significant increase in sensitivity to erlotinib. ^ DeltaEF1 represses the expression of the epithelial markers by recruiting HDACs to chromatin. This observation allows us to translate our findings into clinical application. To test whether the transcriptional repression by DeltaEF1 underlines the mechanism responsible for erlotinib resistance, erlotinib-resistant lines were treated with an HDAC inhibitor (SAHA) followed by erlotinib. This resulted in a synergistic effect and substantial increase in sensitivity to erlotinib in the resistant cell lines. Thus, combining an HDAC inhibitor with erlotinib represents a novel promising pharmacologic strategy for reversing resistance to erlotinib in HNSCC patients. ^

Caspase 8: Mediating the effects of a novel proteasome inhibitor, NPI-0052

Targeting the proteasome with the sole FDA approved proteasome inhibitor (PI), bortezomib, has been fruitful in specific cancers. Its success has generated an interest in next-generation PIs that might have a therapeutic advantage in cancers, such as leukemia, where bortezomib monotherapy was less effective. This study focuses on a novel, clinically relevant PI, NPI-0052. Experiments show that NPI-0052 targets chymotrypsin- and caspase-like activities more potently than the trypsin-like activity in leukemia cells. NPI-0052 induced apoptosis, as determined by caspase-3 activation and DNA fragmentation. Using caspase inhibitors and caspase-8 (I9.2) or FADD (I2.1) deficient cells revealed that caspase-8 was essential for NPI-0052-induced apoptosis. NPI-0052 killed cells via a caspase-8-tBid-mitochondrial pathway, relying on caspase-8, whereas bortezomib relies on several caspases. NPI-0052 increased reactive oxygen species (ROS) levels, which contributed towards cytotoxicity since an antioxidant conferred protection. To improve the clinical efficacy of PIs, NPI-0052 was combined with epigenetic anti-cancer agents, histone deacetylase inhibitors (HDACi). NPI-0052 with MS-275 or vorinostat (FDA approved HDACi), synergistically induced apoptosis more effectively than an HDACi/bortezomib regimen in Jurkat cells. Caspase-8 and ROS contributed towards NPI-0052/HDACi cytotoxicity and caspase-8 mediated superoxide production by NPI-0052 or NPI-0052/HDACi. The proximal targets of these agents: proteasome activity and histone acetylation were examined to determine if they contributed towards synergistic effects. HDACi targeted proteasomal β subunits and corresponding catalytic activities responsible for degrading proteins. Immunoblotting showed increases in histone-H3 expression and its acetylation with NPI-0052 or NPI-0052/HDACi in Jurkat and primary cells. Importantly, the hyper-acetylation by NPI-0052 was not detected with bortezomib, suggesting that this effect may be unique to NPI-0052. An antioxidant attenuated histone-H3 expression and acetylation induced by NPI-0052 alone or with HDACi. Furthermore, the hyper-acetylation by NPI-0052 relied on caspase-8. These novel results show that a PI is eliciting classical epigenetic alterations, demonstrated by hyper-acetylation of histone-H3. This alteration was oxidant and caspase-8 dependent. Overall, results reveal that caspase-8 mediates many effects induced by NPI-0052. Data show overlapping activities by NPI-0052 and HDACi which are contributing, along with caspase-8 activation and oxidative stress, to cytotoxic interactions in leukemia cells, reinforcing the potential clinical utility of combining these two compounds. ^

Interferon-gamma release assays for latent tuberculosis infection in child and young adult candidates or recipients of tumor necrosis factor inhibitor therapy

Background. Inhibition of tumor necrosis factor (TNF) is associated with progression of latent tuberculosis infection (LTBI) to active disease. LTBI screening prior to starting TNF inhibitor therapy is recommended. Blood tests, collectively known as interferon-gamma release assays (IGRAs), offer a means other than the tuberculin skin test (TST) of screening for LTBI. However, in the setting of immune compromise, anergy may limit the clinical utility of IGRAs. ^ Methods. A cross-sectional study was conducted in children and young adults ≤ 21 years of age who were cared for at Texas Children's Hospital in Houston, TX, during 2011 and who were candidates for, or were receiving, tumor necrosis factor (TNF)-inhibitor therapy. All subjects answered a risk factor questionnaire and were tested for LTBI by two commercially available IGRAs (QuantiFERON-Gold In-Tube assay and the T-SPOT.TB assay), along with the TST. T-cell phenotypes were evaluated through flow cytometry, both at baseline and after antigen stimulation. ^ Results. Twenty-eight subjects were enrolled. All were TST negative and none were IGRA positive. Results were negative for the 27 subjects who were tested with QuantiFERON-Gold In-Tube. However, 26% of subjects demonstrated anergy in the T-SPOT.T. Patients with T-SPOT. TB anergy had lower quantitative IFN-γ responses to mitogen in the QFT assay—the mean IFN-γ level to mitogen in patients without T-SPOT.TB anergy was 9.84 IU/ml compared to 6.91 IU/ml in patients with T-SPOT.TB anergy (P = 0.046). Age and use of TNF inhibitors, corticosteroids, or methotrexate use were not significantly associated with T-SPOT.TB anergy. Antigen stimulation revealed depressed expression of intracellular IFN-γ in subjects with T-SPOT. TB anergy. ^ Conclusions. The frequency of anergy in this population is higher than would be expected from studies in adults. There appears to be inappropriate IFN-γ responses to antigen in subjects with T-SPOT. TB anergy. This immune defect was detected by the T-SPOT. TB assay but not by the QuantiFERON-Gold In-Tube assay. Further data are needed to clarify the utility of IGRAs in this population.^

CHARACTERIZATION OF SELECTIVE INHIBITION OF ADENYLYL CYCLASE ACTIVITY BY SMALL MOLECULE INHIBITOR NKY80

The nine membrane-bound isoforms of adenylyl cyclase (AC), via synthesis of the signaling molecule cyclic AMP (cAMP), are involved in many isoform specific physiological functions. Decreasing AC5 activity has been shown to have potential therapeutic benefit, including reduced stress on the heart, pain relief, and attenuation of morphine dependence and withdrawal behaviors. However, AC structure is well conserved, and there are currently no isoform selective AC inhibitors in clinical use. P-site inhibitors inhibit AC directly at the catalytic site, but with an uncompetitive or noncompetitive mechanism. Due to this mechanism and nanomolar potency in cell-free systems, attempts at ligand-based drug design of novel AC inhibitors frequently use P-site inhibitors as a starting template. One small molecule inhibitor designed through this process, NKY80, is described as an AC5 selective inhibitor with low micromolar potency in vitro. P-site inhibitors reveal important ligand binding “pockets” in the AC catalytic site, but specific interactions that give NKY80 selectivity are unclear. Identifying and characterizing unique interactions between NKY80 and AC isoforms would significantly aid the development of isoform selective AC inhibitors. I hypothesized that NKY80’s selective inhibition is conferred by AC isoform specific interactions with the compound within the catalytic site. A structure-based virtual screen of the AC catalytic site was used to identify novel small molecule AC inhibitors. Identified novel inhibitors are isoform selective, supporting the catalytic site as a region capable of more potent isoform selective inhibition. Although NKY80 is touted commercially as an AC5 selective inhibitor, its characterization suggests strong inhibition of both AC5 and the closely related AC6. NKY80 was also virtually docked to AC to determine how NKY80 binds to the catalytic site. My results show a difference between NKY80 binding and the conformation of classic P-site inhibitors. The selectivity and notable differences in NKY80 binding to the AC catalytic site suggest a catalytic subregion more flexible in AC5 and AC6 that can be targeted by selective small molecule inhibitors.

STIMULATION THROUGH TLR4 INCREASES FVIII INHIBITOR FORMATION IN A MOUSE MODEL OF HEMOPHILIA A

Hemophilia A is a clotting disorder caused by functional factor VIII (FVIII) deficiency. About 25% of patients treated with therapeutic recombinant FVIII develop antibodies (inhibitors) that render subsequent FVIII treatments ineffective. The immune mechanisms of inhibitor formation are not entirely understood, but circumstantial evidence indicates a role for increased inflammatory response, possibly via stimulation of Toll-like receptors (TLRs), at the time of FVIII immunization. I hypothesized that stimulation through TLR4 in conjunction with FVIII treatments would increase the formation of FVIII inhibitors. To test this hypothesis, FVIII K.O. mice were injected with recombinant human FVIII with or without concomitant doses of TLR4 agonist (lipopoysaccharide; LPS). The addition of LPS combined with FVIII significantly increased the rate and the production of anti-FVIII IgG antibodies and neutralizing FVIII inhibitors. In the spleen, repeated in vivo TLR4 stimulation with LPS increased the relative percentage of macrophages and dendritic cells (DCs) over the course of 4 injections. However, repeated in vivo FVIII stimulation significantly increased the density of TLR4 expressed on the surface of all spleen antigen presenting cells (APCs). Culture of splenocytes isolated from mice revealed that the combined stimulation of LPS and FVIII also synergistically increased early secretion of the inflammatory cytokines IL-6, TNF-α, and IL-10, which was not maintained throughout the course of the repeated injections. While cytokine secretion was relatively unchanged in response to FVIII re-stimulation in culture, LPS re-stimulation in culture induced increased and prolonged inflammatory cytokine secretion. Re-stimulation with both LPS and FVIII induced cytokine secretion similar to LPS stimulation alone. Interestingly, long term treatment of mice with LPS alone resulted in splenocytes that showed reduced response to FVIII in culture. Together these results indicated that creating a pro-inflammatory environment through the combined stimulation of chronic, low-dose LPS and FVIII changed not only the populations but also the repertoire of APCs in the spleen, triggering the increased production of FVIII inhibitors. These results suggested an anti-inflammatory regimen should be instituted for all hemophilia A patients to reduce or delay the formation of FVIII inhibitors during replacement therapy.

Study on the effect of metformin in patients of metastatic renal cell cancer with diabetes receiving tyrosine kinase inhibitor therapy

Objective: The primary objective of our study was to study the effect of metformin in patients of metastatic renal cell cancer (mRCC) and diabetes who are on treatment with frontline therapy of tyrosine kinase inhibitors. The effect of therapy was described in terms of overall survival and progression free survival. Comparisons were made between group of patients receiving metformin versus group of patients receiving insulin in diabetic patients of metastatic renal cancer on frontline therapy. Exploratory analyses were also done comparing non-diabetic patients of metastatic renal cell cancer receiving frontline therapy compared to diabetic patients of metastatic renal cell cancer receiving metformin therapy. ^ Methods: The study design is a retrospective case series to elaborate the response rate of frontline therapy in combination with metformin for mRCC patients with type 2 diabetes mellitus. The cohort was selected from a database, which was generated for assessing the effect of tyrosine kinase inhibitor therapy associated hypertension in metastatic renal cell cancer at MD Anderson Cancer Center. Patients who had been started on frontline therapy for metastatic renal cell carcinoma from all ethnic and racial backgrounds were selected for the study. The exclusion criteria would be of patients who took frontline therapy for less than 3 months or were lost to follow-up. Our exposure variable was treatment with metformin, which comprised of patients who took metformin for the treatment of type 2 diabetes at any time of diagnosis of metastatic renal cell carcinoma. The outcomes assessed were last available follow-up or date of death for the overall survival and date of progression of disease from their radiological reports for time to progression. The response rates were compared by covariates that are known to be strongly associated with renal cell cancer. ^ Results: For our primary analyses between the insulin and metformin group, there were 82 patients, out of which 50 took insulin therapy and 32 took metformin therapy for type 2 diabetes. For our exploratory analysis, we compared 32 diabetic patients on metformin to 146 non-diabetic patients, not on metformin. Baseline characteristics were compared among the population. The time from the start of treatment until the date of progression of renal cell cancer and date of death or last follow-up were estimated for survival analysis. ^ In our primary analyses, there was a significant difference in the time to progression of patients receiving metformin therapy vs insulin therapy, which was also seen in our exploratory analyses. The median time to progression in primary analyses was 1259 days (95% CI: 659-1832 days) in patients on metformin therapy compared to 540 days (95% CI: 350-894) in patients who were receiving insulin therapy (p=0.024). The median time to progression in exploratory analyses was 1259 days (95% CI: 659-1832 days) in patients on metformin therapy compared to 279 days (95% CI: 202-372 days) in non-diabetic group (p-value <0.0001). ^ The median overall survival was 1004 days in metformin group (95% CI: 761-1212 days) compared to 816 days (95%CI: 558-1405 days) in insulin group (p-value<0.91). For the exploratory analyses, the median overall survival was 1004 days in metformin group (95% CI: 761-1212 days) compared to 766 days (95%CI: 649-965 days) in the non-diabetic group (p-value<0.78). Metformin was observed to increase the progression free survival in both the primary and exploratory analyses (HR=0.52 in metformin Vs insulin group and HR=0.36 in metformin Vs non-diabetic group, respectively). ^ Conclusion: In laboratory studies and a few clinical studies metformin has been proven to have dual benefits in patients suffering from cancer and type 2-diabetes via its action on the mammalian target of Rapamycin pathway and effect in decreasing blood sugar by increasing the sensitivity of the insulin receptors to insulin. Several studies in breast cancer patients have documented a beneficial effect (quantified by pathological remission of cancer) of metformin use in patients taking treatment for breast cancer therapy. Combination of metformin therapy in patients taking frontline therapy for renal cell cancer may provide a significant benefit in prolonging the overall survival in patients with metastatic renal cell cancer and diabetes. ^

Inhibitor of cytochrome c messenger RNA -protein interaction in stimulated rat skeletal muscle

The purpose of this work was to examine the possible mechanisms for the regulation of cytochrome c gene expression in response to increased contractile activity in rat skeletal muscle. The working hypothesis was that increased contractile activity enhances cytochrome c gene expression through a cis-element. A 110% increase in cytochrome c mRNA concentration was observed in tibialis anterior (TA) muscle after 9 days of chronic stimulation. Similar difference (120%) exists between soleus (SO) muscle of higher contractile activity and white vastus lateralis (WV) muscle of lower contractile activity. These results suggest that the endogenous cytochrome c gene expression is regulated by contractile activity. Cytochrome c-reporter genes were injected into skeletal muscles to identify the cis-element that is responsible for the regulation. Although the data was inconclusive, part of it suggested the importance of the 3$\sp\prime$-untranslated region (3$\sp\prime$-UTR) in mediating the response to increased contractile activity.^ RNA gel mobility shift (GMSA) and ultraviolet (UV) cross-linking assays revealed specific RNA-protein interaction in a 50-nucleotide region of the 3$\sp\prime$-UTR in unstimulated TA muscle. Computer analysis predicted a stem-loop structure of 17 nucleotides, which provides a structural basis for RNA-protein interaction. These 17 nucleotides are 100% conserved among rat, mouse and human cytochrome c genes and their 13 pseudogenes, suggesting a functional role for this region. The RNA-protein interaction was significantly less in highly active SO muscle than in inactive WV muscle and was dramatically decreased in stimulated TA muscle due to a protein inhibitor(s) associated with ribosome. It is possible that cytochrome c mRNAs undergoing translation are subject to a compartmentalized regulatory influence.^ The conclusion from these results is that increases in contractile activity induce or activate a protein inhibitor(s) associated with ribosome in rat skeletal muscle. The inhibitor decreases RNA-protein interaction in the 3$\sp\prime$-UTR of cytochrome c mRNA, which may result in increased mRNA stability and/or translation. ^

Bcr: A conditional inhibitor of Bcr -Abl oncoprotein

Chronic myelogenous leukemia (CML) is characterized cytogenetically by the presence of the Philadelphia chromosome and clinically by the clonal expansion of the hematopoietic stem cells and the accumulation of large numbers of myeloid cells. Philadelphia chromosome results from the reciprocal translocation between chromosomes 9 and 22 [t(9;22)(324;q11)], which fuses parts of the ABL proto-oncogene to 5′ portions of the BCR gene. The product of the fused gene is Bcr-Abl oncoprotein. Bcr-Abl oncoprotein has elevated protein tyrosine kinase activity, and is the cause of Philadelphia chromosome associated leukemias. The Bcr sequence in the fusion protein is crucial for the activation of Abl kinase activity and transforming phenotype of Bcr-Abl oncoprotein. Although the Bcr-Abl oncoprotein has been studied extensively, its normal counterpart, the Bcr protein, has been less studied and its function is not well understood. At this point, Bcr is known to encode a novel serine/threonine protein kinase. In Bcr-Abl positive leukemia cells, we found that the serine kinase activity of Bcr is impaired by tyrosine phosphorylation. Both the Bcr protein sequences within Bcr-Abl and the normal cellular Bcr protein lack serine/threonine kinase activity when they become phosphorylated on tyrosine residues by Bcr-Abl. Therefore, the goal of this study was to investigate the role of Bcr in Bcr-Abl positive leukemia cells. We found that overexpression of Bcr can inhibit Bcr-Abl tyrosine kinase activity, and the inhibition is dependent on its intact serine/threonine kinase function. Using the tet repressible promoter system, we demonstrated that Bcr when induced in Bcr-Abl positive leukemia cells inhibited the Bcr-Abl oncoprotein tyrosine kinase. Furthermore, induction of Bcr also increased the number of cells undergoing apoptosis and inhibited the transforming ability of Bcr-Abl. In contrast to the wild-type Bcr, the kinase-inactive mutant of Bcr (Y328F/Y360F) had no effects on Bcr-Abl tyrosine kinase in cells. Results from other experiments indicated that phosphoserine-containing Bcr sequences within the first exon, which are known to bind to the Abl SH2 domain, are responsible for observed inhibition of the Bcr-Abl tyrosine kinase. Several lines of evidence suggest that the phosphoserine form of Bcr, which binds to the Abl SH2 domain, strongly inhibits the Abl tyrosine kinase domain of Bcr-Abl Previously published findings from our laboratory have also shown that Bcr is phosphorylated on tyrosine residue 177 in Bcr-Abl positive cells and that this form of Bcr recruits the Grb2 adaptor protein, which is known to activate the Ras pathway. These findings implicate Bcr as an effector of Bcr-Abl's oncogenic activity. Therefore based on the findings presented above, we propose a model for dual Function of Bcr in Bcr-Abl positive leukemia cells. Bcr, when active as a serine/threonine kinase and thus autophosphorylating its own serine residues, inhibits Bcr-Abl's oncogenic functions. However, when Ber is tyrosine phosphorylated, its Bcr-Abl inhibitory function is neutralized thus allowing Bcr-Abl to exert its full oncogenic potential. Moreover, tyrosine phosphorylated Bcr would compliment Bcr-Abl's neoplastic effects by the activation of the Ras signaling pathway. ^