Tissue factor over-expression by human pancreatic cancer cells BXPC3 is related to higher prothrombotic potential as compared to breast cancer cells MCF7

Cancer histology influences the risk of venous thromboembolism and tissue factor (TF) is the key molecule in cancer-induced hypercoagulability. We investigated the relation between TF expression by pancreatic and breast cancer cells (BXPC3 and MCF7 respectively) and their capacity to trigger in vitro thrombin generation in normal human plasma. Flow cytometry and Western blot analysis for TF expression were performed using murine IgG1 monoclonal antibody against human TF. Real-time PCR for TFmRNA was also performed. Activity of TF expressed by cancer cells was measured with a specific chromogenic assay. Thrombin generation in PPP was assessed using calibrated automated thrombogram. Cancer cells were added to platelet poor plasma from healthy volunteers. In separate experiments cells were incubated with the anti-TF antibody at concentration that completely neutralized the activity of recombinant human TF on thrombin generation. BXPC3 cells expressed significantly higher amounts of functional TF as compared to MCF7 cells. Incubation of BXPC3 and MCF7 cells with PPP resulted in acceleration of the initiation phase of thrombin generation. BXPC3 cells manifested higher procoagulant potential than MCF7 cells. The incubation of BXPC3 or MCF7 cells with the anti-TF monoclonal antibody which resulted in reversal of their effect on thrombin generation.The present study establishes a link between the amount of TF expressed by cancer cells with their procoagulant activity. Both studied types of cancer cells trigger thrombin generation but they have different procoagulant potential. The procoagulant activity of BXPC3 and MCF7 cells is related to the amount of TF expressed. Kinetic parameters of thrombogram are the most relevant for the detection of the TF-dependent procoagulant activity of cancer cells. TF expression is one of the mechanisms by which cancer cells manifest their procoagulant potential but it is not the unique one. The present experimental model will allow the characterization the procoagulant fingerprint of cell lines from the same or different histological types of cancer.     

Endogenous competition against binding of [18F]DMFP and [18F]fallypride to dopamine D2/3 receptors in brain of living mouse

Aim . Molecular imaging studies with benzamide radioligands can reveal competition from endogenous binding at D_2/3‐receptors in living brain. However, single photon emission computed tomography (SPECT) methods suffer from limited spatial resolution, and [¹¹C]‐labeled ligands are only available at positron emission tomography (PET) research sites with cyclotron‐radiochemistry facilities, whereas [¹⁸F] can be transported, due to its longer physical half‐life. Therefore, we endeavored to characterize the vulnerabilities of the benzamide antagonist [¹⁸F]desmethoxyfallypride (DMFP) and its high‐affinity congener [¹⁸F]fallypride (FP) to competition from endogenous dopamine in living mouse brain. Methods . Groups of awake mice were pretreated with saline, amphetamine (10 mg/kg), or reserpine (5 mg/kg), followed by i.v. tracer injections. Mice were killed at 2.5–90 min (DMFP) or 2.5–180 min (FP) circulation times. Brains were dissected and regional radioactivity concentration measured by gamma counting. Other groups of mice were anesthetized for dynamic microPET recordings with DMFP or FP. Binding potentials (BP_ND) were calculated using cerebellum as reference region. Results . With 90‐min circulation, DMFP BP_ND in striatum was 2.4 by dissection and 2.2 by microPET, which showed a 62% decrease in response to amphetamine‐evoked dopamine release and a 33% increase after reserpine‐evoked dopamine depletion. With 120‐min circulation, FP BP_ND in striatum was 24.1 by dissection and 9.2 by microPET, which showed a 31% decrease in the amphetamine group, but no effect of reserpine. Dissection showed similar sensitivities for FP binding, but only a 29% amphetamine‐evoked reduction for DMFP. Conclusions . Relative to gold standard ex vivo results, microPET estimates of DMFP BP_ND were unbiased, whereas FP BP_ND in striatum was substantially underestimated. Both tracers proved suitable for revealing pharmacologically evoked changes in competition at D_2/3‐receptors in striatum of living mice. Synapse 64:313–322, 2010. © 2009 Wiley‐Liss, Inc.     

Immunohistochemical expression pattern of MMR protein can specifically identify patients with colorectal cancer microsatellite instability

The microsatellite instability (MSI) pathway is found in most cases of hereditary nonpolyposis colorectal cancer (HNPCC) and in 12 % of sporadic colorectal cancer (CRC). It involves inactivation of deoxyribonucleic acid mismatch repair (MMR) genes MLH1, MSH2, PMS2, and MSH6. MMR germline mutation detections are an important supplement to HNPCC clinical diagnosis. It enables at-risk and mutation-positive relatives to be informed about their cancer risks and to benefit from intensive surveillance programs that have been proven to reduce the incidence of CRC. In this study, we analyzed for the first time in Tunisia the potential value of immunohistochemical assessment of MMR protein to identify microsatellite instability in CRC. We evaluate by immunohistochemistry MMR protein expression loss in tumoral tissue compared to positive expression in normal mucosa. Immunohistochemistry revealed loss of expression for MLH1, MSH2, MSH6, and PMS2 in 15, 21, 13, and 15 % of cases, respectively. Here, we report a more elevated frequency of MSI compared to data of the literature. In fact, by immunohistochemistry, 70 % of cases were shown to be MSS phenotype, whereas 30 % of cases, in our set, were instable. Moreover, according to molecular investigation, 71 % of cases were instable (MSI-H) and remaining cases were stable (29 %). Thus, we found a perfect association between MMR immunohistochemical analyses and MSI molecular investigation. Immunohistochemical analysis of MMR gene product expression may allow one to specifically identify MSI phenotype of patients with colorectal carcinomas.     

Modulation of type 1 diabetes susceptibility by tumor necrosis factor alpha -308 G/A and lymphotoxin alpha +249 A/G haplotypes and lack of linkage disequilibrium with predisposing DQB1-DRB1 haplotypes in Bahraini patients

Tumor necrosis factor alpha (TNF-α) −308 G/A and lymphotoxin alpha (LTα) +249 A/G single-nucleotide polymorphisms were investigated in 228 type 1 diabetes mellitus (T1DM) patients and 240 controls. Only LTα +249G allele and +249G/+249G genotype frequencies were higher among patients, and no linkage disequilibrium was found between TNF-α/LTα alleles and susceptible/protective DRB1-DQB1 haplotypes. TNF-α/LTα T1DM-susceptible (−308G/+249G) and protective (−308G/+249A) haplotypes were identified.     

Activation of mTORC1 Signaling Pathway in AIDS-Related Lymphomas

Using immunohistochemistry with antibodies against the phosphoserine residues in both S6rp and 4E binding protein 1, we identified the activation of the mammalian target of rapamycin (mTORC)1 pathway in 29 cases of AIDS-related lymphoma. These cases represented a diverse spectrum of histological types of non-Hodgkin lymphoma (24 cases) and classic Hodgkin lymphoma (five cases). mTORC1 was also activated in the hyperplastic but not involuted follicles of HIV-associated lymphadenopathy in eight cases, supporting the notion that mTORC1 activation is a common feature of transformed lymphocytes irrespective of either their reactive or malignant phenotype. We also found that in B-cell lines that represent diffuse large B-cell lymphoma, Burkitt lymphoma, Epstein-Barr virus-infected lymphocytes, and human herpesvirus 8-positive primary effusion lymphoma, inhibitors of Syk, MEK, and, seemingly, phosphoinositide 3 kinases suppressed mTORC1 activation, in particular when these inhibitors were used in combination. These findings indicate that AIDS-related lymphoma and other histologically similar types of lymphomas that are derived from transformed B lymphocytes may display clinical responses to inhibitors that directly target mTORC1 or, possibly, upstream activators of the mTORC1 pathway.The incidence of lymphomas in HIV-positive patients is nearly 200 times higher than in those uninfected by the virus. Lymphomas accounts for an increasing percentage of AIDS-defining illness, particularly from the advent of highly active antiretroviral therapy therapy.^1,2 These AIDS-related lymphomas (ARLs) typically represent the proliferation of enlarged, transformed B lymphocytes, which usually fall into the category of diffuse large cell lymphoma (DLBCL), with morphology ranging from centroblasts to immunoblasts. Other histological types such as Burkitt lymphoma (BL), Hodgkin lymphoma, and T/null cell anaplastic large cell lymphoma are also overrepresented among ARLs.^1,2,3,4 The pathogenesis of ARL is poorly understood. It has been postulated that cell proliferation occurring in the setting of severe immunosuppression and driven by chronic antigenemia resulting at first in the polyclonal and ultimately in the monoclonal lymphoproliferation plays a key role in lymphomagenesis in HIV patients. In addition, cell infection by the Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV8) most likely contributes to the malignant cell phenotype in some subtypes of ARL, with the association of primary effusion lymphoma with HHV8 being essentially universal.^1,2,3,4 Regardless of the histological type of ARL, chemotherapy is typically ineffective and new treatment approaches are clearly needed to combat this group of lymphomas. In addition to ARL, HIV patients develop a benign reactive lymphadenopathy, particularly early after the infection as an overall ineffective response to the virus. This immune response is characterized by florid follicular hyperplasia that over time may lead to follicular involution and lymphocyte depletion.Mammalian target of rapamycin (mTOR) is a ubiquitously expressed serine/threonine kinase involved in key cellular functions including protein synthesis and proliferation.^5,6 mTOR associates with several proteins including either raptor or rictor to form the mTORC1 and mTORC2 complexes, respectively, with the signaling pathways activated by mTORC1 being much better characterized.^4,5,6,7 Accordingly, it is well established that mTORC1 activates p70S6 kinase 1 and inhibits 4E binding protein 1 (4E-BP1). In turn, p70S6 kinase 1 phosphorylates an S6 protein of the 40S ribosomal subunit (S6rp) at several sites including serines 235 and 236. The exact mechanisms of mTORC1 activation are less understood but both phosphoinositide 3 kinases (PI3K)/Akt^8,9,10 and extracellular regulated (ERK)/mitogen-activated kinase (MEK) kinases^11,12 signaling pathways have been found to activate mTORC1 with members of the insulin growth factor family providing the primary signal, at least in some instances. The highly potent and specific inhibitors from the rapamycin family can functionally inactivate mTORC1. In addition to being used as immunosuppressants, mTORC1 inhibitors are evaluated as therapeutic agents in various types of cancer,^5,6 with high efficacy already documented in renal cell carcinoma.¹³Syk is a protein tyrosine kinase expressed in B lymphocytes,¹⁴ monocytes/macrophages,¹⁵ mast cells,¹⁶ and other cell types. Syk has been found to be involved in signal transduction through several types of receptors including the antigen B-cell receptor¹⁷ and at least three different receptors for the Fc component of immunoglobulins G and E.^{14,15,16,18,19,20} A recent report suggests that Syk may be involved in mTORC1 activation in a follicular and possibly other types of B-cell lymphoma.²¹ Although inhibitors of either PI3K/Akt or MEK/ERK signaling pathways did fully inhibit mTORC1 activation in transformed B lymphocytes, at least when applied alone,²² these pathways have been found to contribute to mTORC1 stimulation in two types of T-cell lymphoma.^23,24In this study, we identified the activation of the mTORC1 pathway in all ARL cases examined, regardless of their specific histological classification and immunophenotype. mTORC1 was also activated in the hyperplastic follicles of the HIV-associated lymphadenopathy. Furthermore, we found that in the different types of transformed B-lymphocytes cell lines, inhibition of Syk, MEK, and, seemingly, PI3K resulted in suppression of the mTORC1 activation, in particular when the combination of the inhibitors was used. These findings indicate that ARL and histologically similar types of lymphoma may benefit from targeted therapy with inhibitors of mTORC1 or, possibly, its upstream activators.