2B4 Cells Research Articles

Non-coding RNAs derived from the foot-and-mouth disease virus genome trigger broad antiviral activity against coronaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a potentially severe respiratory disease, the coronavirus disease 2019 (COVID-19), an ongoing pandemic with limited therapeutic options. Here, we assessed the anti-coronavirus activity of synthetic RNAs mimicking specific domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs). These molecules are known to exert broad-spectrum antiviral activity in cell culture, mice and pigs effectively triggering the host innate immune response. The ncRNAs showed potent antiviral activity against SARS-CoV-2 after transfection in human intestinal Caco-2 and lung epithelium Calu-3 2B4 cells. When the in vivo efficacy of the FMDV ncRNAs was assessed in K18-hACE2 mice, administration of naked ncRNA before intranasal SARS-CoV-2 infection significantly decreased the viral load and the levels of pro-inflammatory cytokines in the lungs compared with untreated infected mice. The ncRNAs were also highly efficacious when assayed against common human HCoV-229E and porcine transmissible gastroenteritis virus (TGEV) in hepatocyte-derived Huh-7 and swine testis ST cells, respectively. These results are a proof of concept of the pan-coronavirus antiviral activity of the FMDV ncRNAs including human and animal divergent coronaviruses and potentially enhance our ability to fight future emerging variants.

VP.83 Ion channels and myogenesis in Duchenne muscular dystrophy: Electrophysiological profile of wild-type and dystrophic myoblasts and myocytes

In skeletal muscle, several ion channels interact with the dystrophin complex, which is disrupted in Duchenne muscular dystrophy (DMD). However, little is known about their involvement in myogenesis in DMD. Here, we performed a pilot study to characterize the electrophysiological asset of myoblasts and myocytes at day 2/4/6/11 by using two immortalized mouse satellite-derived cell lines: the wild-type 2B4 and the dystrophic SF1. We evaluated both inward and outward currents at different time points of differentiation by whole-cell patch clamp. 2B4 cells showed an increment of inward currents as the differentiation program progresses. In SF1 myocytes, inward currents increased up to the 6th day of differentiation, like in 2B4 cells. However, day-11 SF1 myocytes showed 50% lower inward currents compared to day-11 2B4 myocytes (5.2 nA vs 2.8 nA at -20 mV). High concentration of tetrodotoxin blocked these inward currents in both day-6 and day-11 2B4/SF1. Outward currents were clearly detectable in day-11 2B4 cells but very small in day-2/4/6 2B4 cells. Conversely, SF1 outward currents reached the highest value at day-6, being 3-fold higher than 6-day 2B4 cells (733 pA vs 283 pA at +60 mV). However, day-11 SF1 myocytes showed 44% lower outward currents, compared to day-11 2B4 cells (722 pA vs 1283 pA). BaCl₂ decreased outward currents in day-6 2B4/SF1 cells and in day-11 2B4 myocytes. In addition, we assessed resting membrane potentials which became more negative as the days of differentiation increased in both cell-lines. However, day-11 SF1 cells had a more depolarised membrane potential, compared to day-11 2B4 myocytes. This preliminary data suggests that during myogenesis, intrinsic impairments in ion channel development are disclosed by dystrophic conditions, likely in relation to the primary defect. Furthermore, electrophysiological characterization of human wild-type and dystrophic myoblasts/myocytes is on-going.

SARS-CoV-2 induced oxidative stress promotes HMGB1 secretion to induce inflammation

Abstract The coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a major ongoing pandemic, which results in life-threatening pneumonia and multiple-organ failure. Although the molecular mechanisms by which SARS-CoV-2 lead to various disease sequalae remain elusive, exaggerated inflammatory and immune responses can lead to the debilitating and lethal outcomes of this disease. Understanding how immune mechanisms contribute to the development of severe symptoms is essential for designing effective countermeasures against SARS-CoV-2. Our studies show that SARS-CoV-2 induces increased extracellular secretion of high-mobility group box 1 (HMGB1), a prominent damage-associated molecular pattern family member that alerts the immune system to potential damage and triggers immediate response. The goal of this study is to dissect the molecular mechanisms that promotes the release of HMGB1 and its associated cellular signaling in response to SARS-CoV-2 infection. Studies were conducted in human lung epithelial cells including A549-ACE2 and Calu-3/2B4 cells, mice, and COVID-19 patient samples. HMGB1 levels and oxidative stress markers were measured by an immune assay, Western blot, and qRT-PCR. SARS-CoV-2 infection induced severe oxidative stress, leading to enhanced extracellular release of HMGB1 and subsequent activation of signaling cascades to promote inflammation. Therefore, modulation of HMGB1’s proinflammatory signaling may reduce SARS-CoV-2-induced inflammation by controlling oxidative and inflammatory stress in COVID-19 disease. This study was supported by a Young Clinical Scientist Award from the Flight Attendant Medical Research Institute (FAMRI) to YMH (grant I.D. number 123385), NIH/NIAID R21 AI35619 to YMH, and Rapid Response Pilot grant from NIEHS P30 Center Grant ES030285 to YMH. Additional support was from the Institute for Translational Science at UTMB, supported by a Clinical and Translational Science Award Mentored Career Development (KL2 Award; KL2TR001441), and Pilot grant (85393) from Institute for Human Infections & Immunity at UTMB, supported by Sealy & Smith Foundation to YMH.

The TMPRSS2 Inhibitor Nafamostat Reduces SARS-CoV-2 Pulmonary Infection in Mouse Models of COVID-19.

ABSTRACTThe coronavirus disease 2019 (COVID-19) pandemic has caused significant morbidity and mortality on a global scale. The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initiates host cell entry when its spike protein (S) binds to its receptor, angiotensin-converting enzyme 2 (ACE2). In airway epithelia, the spike protein is cleaved by the cell surface protease TMPRSS2, facilitating membrane fusion and entry at the cell surface. This dependence on TMPRSS2 and related proteases suggests that protease inhibitors might limit SARS-CoV-2 infection in the respiratory tract. Here, we tested two serine protease inhibitors, camostat mesylate and nafamostat mesylate, for their ability to inhibit entry of SARS-CoV-2 and that of a second pathogenic coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). Both camostat and nafamostat reduced infection in primary human airway epithelia and in the Calu-3 2B4 cell line, with nafamostat exhibiting greater potency. We then assessed whether nafamostat was protective against SARS-CoV-2 in vivo using two mouse models. In mice sensitized to SARS-CoV-2 infection by transduction with human ACE2, intranasal nafamostat treatment prior to or shortly after SARS-CoV-2 infection significantly reduced weight loss and lung tissue titers. Similarly, prophylactic intranasal treatment with nafamostat reduced weight loss, viral burden, and mortality in K18-hACE2 transgenic mice. These findings establish nafamostat as a candidate for the prevention or treatment of SARS-CoV-2 infection and disease pathogenesis.

Expansion of Single Cell Transcriptomics Data of SARS-CoV Infection in Human Bronchial Epithelial Cells to COVID-19

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19) that was emerged as a new member of coronaviruses since December 2019 in Wuhan, China and then after was spread in all continentals. Since SARS-CoV-2 has shown about 77.5% similarity to SARS-CoV, the transcriptome and immunological regulations of SARS-CoV-2 was expected to have high percentage of overlap with SARS-CoV.ResultsIn this study, we applied the single cell transcriptomics data of human bronchial epithelial cells (2B4 cell line) infected with SARS-CoV, which was annotated in the Expression Atlas database to expand this data to COVID-19. In addition, we employed system biology methods including gene ontology (GO) and Reactome pathway analyses to define functional genes and pathways in the infected cells with SARS-CoV. The transcriptomics analysis on the Expression Atlas database revealed that most genes from infected 2B4 cell line with SARS-CoV were downregulated leading to immune system hyperactivation, induction of signaling pathways, and consequently a cytokine storm. In addition, GO:0016192 (vesicle-mediated transport), GO:0006886 (intracellular protein transport), and GO:0006888 (ER to Golgi vesicle-mediated transport) were shown as top three GOs in the ontology network of infected cells with SARS-CoV. Meanwhile, R-HAS-6807070 (phosphatase and tensin homolog or PTEN regulation) showed the highest association with other Reactome pathways in the network of infected cells with SARS-CoV. PTEN plays a critical role in the activation of dendritic cells, B- and T-cells, and secretion of proinflammatory cytokines, which cooperates with downregulated genes in the promotion of cytokine storm in the COVID-19 patients.ConclusionsBased on the high similarity percentage of the transcriptome of SARS-CoV with SARS-CoV-2, the data of immunological regulations, signaling pathways, and proinflammatory cytokines in SARS-CoV infection can be expanded to COVID-19 to have a valid platform for future pharmaceutical and vaccine studies.

Identification of reciprocal microRNA-mRNA pairs associated with metastatic potential disparities in human prostate cancer cells and signaling pathway analysis.

The major cause of mortality for prostate cancer (PCa) is metastasis; however, the metastatic mechanism remains unclear. MicroRNAs (miRNAs) alter the expression patterns of essential genes through posttranscriptional regulation during cancer development. The study was mainly aimed at identifying specific miRNA-messenger RNA (mRNA) interactions and signaling pathways associated with PCa distant metastasis. New analytical approaches were applied, combining miRNA and gene expression microarray, to screen differentially expressed miRNA-mRNA pairs in the normal prostate epithelial cell line RWPE-1, the highly-metastatic human PCa cell line PC-3M-1E8 (H-1E8 or 1E8) and the lowly metastatic cell line PC-3M-2B4 (L-2B4 or 2B4). Eight differentially expressed candidate miRNAs and their targets closely related to PCa metastasis were identified and validated in patients by using the Gene Expression Omnibus database. Among them, overexpression of hsa-miR-92b-3p and hsa-let-7a-5p and underexpression of their targets, such as glutathione-S-transferase M3 (GSTM3), baculoviral IAP repeat-containing 3, and cyclin-dependent kinase inhibitor 1 (CDKN1A), were also validated in H-1E8 cells compared with L-2B4 cells. Bioinformatics suggested that hsa-miR-92b-3p and hsa-let-7a-5p and their targets might promote PCa metastasis through platinum-based drug resistance and the JAK-STAT signaling pathway. H-1E8 and L-2B4 cells treated by cisplatin showed the greatly decreased levels of hsa-miR-92b-3p and hsa-let-7a-5p; however, in contrast to 2B4 cells, 1E8 cells did not negatively regulate the increase in the expression levels of the targets GSTM3 and CDKN1A. This finding suggests that the dysregulation between hsa-let-7a-5p/CDKN1A and hsa-miR-92b-3p/GSTM3 pairs is associated with platinum-based chemoresistance of metastatic cancer cells.

Modulation of Human Leukocyte Antigen-C by Human Cytomegalovirus Stimulates KIR2DS1 Recognition by Natural Killer Cells.

The interaction of inhibitory killer cell Ig-like receptors (KIRs) with human leukocyte antigen (HLA) class I molecules has been characterized in detail. By contrast, activating members of the KIR family, although closely related to inhibitory KIRs, appear to interact weakly, if at all, with HLA class I. KIR2DS1 is the best studied activating KIR and it interacts with C2 group HLA-C (C2-HLA-C) in some assays, but not as strongly as KIR2DL1. We used a mouse 2B4 cell reporter system, which carries NFAT-green fluorescent protein with KIR2DS1 and a modified DAP12 adaptor protein. KIR2DS1 reporter cells were not activated upon coculture with 721.221 cells transfected with different HLA-C molecules, or with interferon-γ stimulated primary dermal fibroblasts. However, KIR2DS1 reporter cells and KIR2DS1+ primary natural killer (NK) cells were activated by C2-HLA-C homozygous human fetal foreskin fibroblasts (HFFFs) but only after infection with specific clones of a clinical strain of human cytomegalovirus (HCMV). Active viral gene expression was required for activation of both cell types. Primary NKG2A−KIR2DS1+ NK cell subsets degranulated after coculture with HCMV-infected HFFFs. The W6/32 antibody to HLA class I blocked the KIR2DS1 reporter cell interaction with its ligand on HCMV-infected HFFFs but did not block interaction with KIR2DL1. This implies a differential recognition of HLA-C by KIR2DL1 and KIR2DS1. The data suggest that modulation of HLA-C by HCMV is required for a potent KIR2DS1-mediated NK cell activation.

Id3 Controls Cell Death of 2B4+ Virus-Specific CD8+ T Cells in Chronic Viral Infection.

Sustained Ag persistence in chronic infection results in a deregulated CD8(+) T cell response that is characterized by T cell exhaustion and cell death of Ag-specific CD8(+) T cells. Yet, the underlying transcriptional mechanisms regulating CD8(+) T cell exhaustion and cell death are poorly defined. Using the experimental mouse model of lymphocytic choriomeningitis virus infection, we demonstrate that the transcriptional regulator Id3 controls cell death of virus-specific CD8(+) T cells in chronic infection. By comparing acute and chronic infection, we showed that Id3 (-) virus-specific CD8(+) T cells were less abundant, whereas the absolute numbers of Id3 (+) virus-specific CD8(+) T cells were equal in chronic and acute infection. Phenotypically, Id3 (-) and Id3 (+) cells most prominently differed with regard to expression of the surface receptor 2B4; although Id3 (-) cells were 2B4(+), almost all Id3 (+) cells lacked expression of 2B4. Lineage-tracing experiments showed that cells initially expressing Id3 differentiated into Id3 (-)2B4(+) cells; in turn, these cells were terminally differentiated and highly susceptible to cell death under conditions of persisting Ag. Enforced Id3 expression specifically increased the persistence of 2B4(+) virus-specific CD8(+) T cells by decreasing susceptibility to Fas/Fas ligand-mediated cell death. Thus, our findings reveal that the transcriptional regulator Id3 promotes the survival of virus-specific CD8(+) T cells in chronic infection and suggest that targeting Id3 might be beneficial for preventing cell death of CD8(+) T cells in chronic infection or for promoting cell death of uncontrolled, hyperactive CD8(+) T cells to prevent immunopathology.

Silencing of a novel tumor metastasis suppressor gene LASS2/TMSG1 promotes invasion of prostate cancer cell in vitro through increase of vacuolar ATPase activity

Homo sapiens longevity assurance homologue 2 of yeast LAG1 (LASS2), also known as tumor metastasis suppressor gene 1 (TMSG1), is a newly found tumor metastasis suppressor gene in 1999. Preliminary studies showed that it not only suppressed tumor growth but also closely related to tumor metastasis, however, its molecular mechanisms is still unclear. There have been reported that protein encoded by LASS2/TMSG-1 could directly interact with the C subunit of Vacuolar ATPase (V-ATPase), which suggested that LASS2/TMSG1 might inhibit the invasion and metastasis through regulating the function of V-ATPase. Thus, in this study, we explored the effect of small interference RNA (siRNA) targeting LASS2/TMSG1 on the invasion of human prostate carcinoma cell line PC-3M-2B4 and its molecular mechanisms associated with the V-ATPase. Real-time fluorogentic quantitative PCR (RFQ-PCR) and Western blot revealed dramatic reduction of 84.5% and 60% in the levels of LASS2/TMSG1 mRNA and protein after transfection of siRNA in PC-3M-2B4 cells. The V-ATPase activity and extracellular hydrogen ion concentration were significantly increased in 2B4 cells transfected with the LASS2/TMSG1-siRNA compared with the controls. The activity of secreted MMP-2 was up-regulated in LASS2/TMSG1-siRNA treated cells compared with the controls; and the capacity for migration and invasion in LASS2/TMSG1-siRNA treated cells was significantly higher than the controls. Thus, we concluded that silencing of LASS2/TMSG1 may promote invasion of prostate cancer cell in vitro through increase of V-ATPase activity and extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2.

Loss of E-cadherin promotes prostate cancer metastasis via upregulation of metastasis-associated gene 1 expression

E-cadherin is a key cell-to-cell adhesion molecule associated with the invasion and metastasis of tumor cells; however, the molecular mechanisms are not entirely understood. In this study, we investigated whether downregulation of E-cadherin by E-cadherin-specific small intefering RNA (siRNA) was able to promote malignant phenotypes of prostate cancer cells through upregulating the metastasis-associated gene 1 (MTA1) in vitro. The expression levels of E-cadherin in human paired prostate adenocarcinoma cell lines, PC-3M-2B4 (2B4) and PC-3M-1E8 (1E8), were investigated using western blot analysis. The alteration of malignant phenotypes associated with decreasing E-cadherin expression were assessed in 2B4 cells using wound-healing assays, solid-phase adhesion assays, invasion assays and cytoskeletal staining. The expression of E-cadherin and MTA1 in normal, localized and metastatic prostate cancer cells was analyzed using immunohistochemistry. Downregulation of E-cadherin using an RNA interference approach led to the upregulation of MTA1 expression, decreased tumor cell adhesion ability as well as enhanced cell mobility, invasion and cellular polarity compared with the controls (P<0.05). E-cadherin regulated MTA1 in a time-dependent manner. The correlation between E-cadherin and MTA1 was inversed in the prostate cancer group (P<0.05; r(s)=-0.434). The data suggest that E-cadherin plays an important role in prostate cancer metastasis, which is likely to be due to the regulation of MTA1 expression. E-cadherin may combine with MTA1 and alter the malignant phenotype of prostate cancer cells. A combined testing strategy for detecting MTA1 and E-cadherin may be sufficient for selecting high-risk patients with metastasis. Therefore, E-cadherin and MTA1 may be potential powerful factors for the treatment of various types of cancer.

A New Extensively Characterised Conditionally Immortal Muscle Cell-Line for Investigating Therapeutic Strategies in Muscular Dystrophies

A new conditionally immortal satellite cell-derived cell-line, H2K 2B4, was generated from the H2Kb-tsA58 immortomouse. Under permissive conditions H2K 2B4 cells terminally differentiate in vitro to form uniform myotubes with a myogenic protein profile comparable with freshly isolated satellite cells. Following engraftment into immunodeficient dystrophin-deficient mice, H2K 2B4 cells regenerated host muscle with donor derived myofibres that persisted for at least 24 weeks, without forming tumours. These cells were readily transfectable using both retrovirus and the non-viral transfection methods and importantly upon transplantation, were able to reconstitute the satellite cell niche with functional donor derived satellite cells. Finally using the Class II DNA transposon, Sleeping Beauty, we successfully integrated a reporter plasmid into the genome of H2K 2B4 cells without hindering the myogenic differentiation. Overall, these data suggest that H2K 2B4 cells represent a readily transfectable stable cell-line in which to investigate future stem cell based therapies for muscle disease.

Dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection.

Human lung epithelial cells are likely among the first targets to encounter invading severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Not only can these cells support the growth of SARS-CoV infection, but they are also capable of secreting inflammatory cytokines to initiate and, eventually, aggravate host innate inflammatory responses, causing detrimental immune-mediated pathology within the lungs. Thus, a comprehensive evaluation of the complex epithelial signaling to SARS-CoV is crucial for paving the way to better understand SARS pathogenesis. Based on microarray-based functional genomics, we report here the global gene response of 2B4 cells, a cloned bronchial epithelial cell line derived from Calu-3 cells. Specifically, we found a temporal and spatial activation of nuclear factor (NF)κB, activator protein (AP)-1, and interferon regulatory factor (IRF)-3/7 in infected 2B4 cells at 12-, 24-, and 48-hrs post infection (p.i.), resulting in the activation of many antiviral genes, including interferon (IFN)-β, -λs, inflammatory mediators, and many IFN-stimulated genes (ISGs). We also showed, for the first time, that IFN-β and IFN-λs were capable of exerting previously unrecognized, non-redundant, and complementary abilities to limit SARS-CoV replication, even though their expression could not be detected in infected 2B4 bronchial epithelial cells until 48 hrs p.i. Collectively, our results highlight the mechanics of the sequential events of antiviral signaling pathway/s triggered by SARS-CoV in bronchial epithelial cells and identify novel cellular targets for future studies, aiming at advancing strategies against SARS.

Simultaneous Use of Histone Deacetylase Inhibitor Valproic Acid Potentiates Natural Killer Cell Cytotoxicity against T-Lineage Acute Lymphoblastic Leukemia

Leukemic cells could survive after aggressive therapies including mega-dose chemotherapy. Natural killer (NK) cells have great promise for treatment of such refractory disease because many kinds of hematopoietic malignancies and solid tumors are reported to be intrinsically sensitive to NK cell lysis. Indeed, NK cell allo-reactivity has been shown to reduce leukemia relapse after haploidentical stem cell transplantation (SCT) and cord blood transplantation (CBT) in patients with myeloid leukemia (Ruggeri et al. Science 2002; Willemze, et al. EBMT 2008 abstract). However, NK cell allo-reactivity against acute lymphoblastic leukemia (ALL) has been reported to be generally weak regardless of the presence of killer immunoglobulin receptor (KIR) ligand mismatch. We previously showed that genetic modification of primary NK cells with anti-CD19 chimeric receptor could overcome the refractoriness of B-lineage ALL cells to NK cell lysis (Imai et al, Blood 2005). For T-lineage ALL, however, development of antigen-specific artificial receptors against T-ALL cells has been hampered by the similarity in immunophenotype between NK cells and T cells. Novel strategy to augment anti-T-ALL activity of NK cells, such as combined use of a kind of agents which has anti-leukemic activity and does not inhibit NK cell activity, is needed. Valproic acid (VPA) has been widely used as an anticonvulsant and a mood stabilizer for a long time, and its safety has been established. Recently, a number of studies have shown that VPA could act as a histone deacetylase (HDAC) inhibitor and possess anti-cancer activity. We thus studied the effect of VPA on NK cell cytotoxicity against T-ALL cell lines and asked whether VPA could be incorporated into NK cell therapy. NK cells were activated and expanded by coculture of peripheral blood mononuclear cells with K562 cells transfected with 4-1BB ligand and membrane-bound interleukin-15. After 7 days, cells were subjected to CD3-depletion procedure to earn highly pure (>99%) NK cells, and then cultured in the presence of high dose interleukin (IL)-2 (1000 U/ml). Efficient killing of T-ALL cell lines MOLT4, Jurkat and CEM-C7 by activated NK cells were observed in 4-hour cytotoxicity assay, even if KIR ligand mismatch were not present. VPA suppressed leukemic cell proliferation and induced apoptosis in MOLT4, Jurkat and CEM-C7 at clinically achievable concentrations and in a dose-dependent fashion. Exposure of activated NK cells to VPA at 100 mg/ml for 48 hours in the presence of IL-2 resulted in mild inhibition of NK cell proliferation by approximately 20% and decreased surface expression of activating receptors such as NKG2D, 2B4, and natural cell cytotoxicity receptors p30, p44, p46 (10–40% decrease in mean fluorescence intensity). However, the addition of VPA to coculture of NK cells with MOLT-4, Jurkat and CEM-C7 did not result in interfering the anti-leukemia activity of NK cells. When anti-leukemic activity was evaluated in more prolonged culture experiments at very low effector: target (E/T) ratio, the number of residual leukemic cells was significantly lower in culture with VPA compared to those without VPA (MOLT-4: residual leukemia cells 12.2 ± 1.9% vs 46.0 ± 2.3% of control culture at E/T ratio of 0.13:1; CEM-C7: 21.0 ± 0.4% vs 40.1 ± 1.1% of control culture at E/T ratio of 0.13:1, 72-hour incubation). Similar results were also obtained when VPA was substituted by another HDAC inhibitor Suberoylanilide hydroxamic acid (SAHA). We found that upregulation of NK-activating receptor ligands MICA/B, ULBP1 and ULBP2 occurred in MOLT-4, Jurkat and CEM-C7 after 24–48 hour exposure to VPA at 100 mg/ml. On the contrary, T-ALL cells did not show increased sensitivity to NK cell lysis after pretreatment by VPA for 48 hours. Therefore, we thought that the synergistic effect of VPA observed in prolonged culture experiments could be mainly attributed to growth suppression of continuously growing leukemic cells, not to its immunomodulatory activity. These data suggest that simultaneous use of VPA would not abrogate NK cell cytotoxic activity and VPA might be used in combination with infusion of NK cells for patients bearing VPA-sensitive tumors. In patients after haploidentical SCT and CBT, NK cell allo-reactivity might be potentiated by the use of VPA.

A Gene Expression Connectivity Map Identifies Rapamycin as a Glucocorticoid Resistance Reversal Agent in Lymphoblastic Leukemia.

Glucocorticoids (GC) remain a cornerstone of treatment for acute lymphoblastic leukemia (ALL), and failure of GC to induce apoptosis in vitro is predictive of a poor prognosis. Strategies to pharmacologically overcome drug resistance in general, and GC resistance in particular, are lacking. We hypothesized that gene expression signatures of disease states (e.g. GC resistant ALL) and gene expression signatures of drug action could be computationally connected by systematically performing gene expression profiling on cells treated with known drugs. As a pilot for the Connectivity Map concept, we profiled ~300 drugs on Affymetrix U113A arrays following 6 hour treatment of MCF7, HL60 or PC3 cells. This signature database was queried with a signature of GC-sensitive ALL obtained by comparing pre-treatment marrow blasts from patients with GC-sensitive or resistant ALL. Using Kolmogorov-Smirnov based computational analysis, the Connectivity Map indicated that the signature of the mTOR inhibitor rapamycin matched the signature of GC-sensitivity. We thus tested the hypothesis that rapamycin would induce GC sensitivity in GC resistant lymphoid cells. Because rapamycin is known to act downstream of AKT, we used a constitutively active AKT (myr-AKT) to activate the mTOR pathway in the glucocorticoid-sensitive murine T-cell line 2B4. Myr-AKT induced glucocorticoid resistance that was reversible by rapamycin treatment. Rapamycin also reversed the GC-resistance signature, and sensitized glucocorticoid resistant human CEM-c1 T-ALL cells and 2B4 cells to GC-induced apoptosis at a rapamycin dose that alone had no effect on cell proliferation or survival. To determine the mechanism of rapamycin induction of GC sensitivity, we examined the components of the GC-resistance signature and noted overexpression of the anti-apoptotic gene MCL-1. MCL-1 expression decreased in rapamycin-treated CEM-c1 and 2B4 cells whereas expression of other multi-domain antiapoptotic molecules BCL-2 and BCL-xL was unaffected. We over-expressed Mcl-1 in 2B4 cells and developed a transgenic mouse that expresses mcl-1 under the control of the H2-K promoter. Over-expression of MCL-1 conferred glucocorticoid resistance to 2B4 cells, primary splenic B and CD8+ T cells. Furthermore MCL-1 over-expression protected against the combination of rapamycin and glucocorticoids, suggesting that a decrease in MCL-1 expression is necessary for the rapamycin-mediated increase in GC sensitivity. Conversely, shRNA-mediated knockdown of MCL-1 rendered GC-resistant CEM-c1 cells sensitive to GC. Finally, MCL-1 over expression sequestered the majority of proapoptotic BIM, even after GC-induced BIM expression. Thus, rapamycin sensitizes ALL cells to GC through down-regulation of MCL-1. These data indicate that (1) rapamycin/GC combination clinical trials are warranted for GC-resistant ALL, (2) rapamycin reverses GC resistance via down-regulation of MCL-1, and (3) the Connectivity Map approach represents a novel strategy for in silico identification of promising combination therapies for cancer. A public-access Connectivity Map database containing profiles of all FDA-approved drugs in therefore warranted.

SHP2 and MKP5 in P2Y purinergic receptor-mediated prostate cancer invasion

To investigate the effects of protein tyrosine phosphatase-SHP2 and dual-specificity MAPK phosphatase-MKP5 on the activation of MAPKs and cell invasion induced by P2Y purinergic receptor in human prostate cancer cell lines with different metastatic potentials. The wide type (-wt) SHP2, mutant type (-cs) SHP2 and wide type (-wt) MKP5 cDNA expression vectors were constructed and stably transfected into 1E8 cells (highly metastatic) and/or 2B4 cells (non-metastatic). The tyrosine phosphorylation of SHP2 was examined by immunoprecipitation. The activation of ERK1/2 and p38 induced by P2Y receptor agonist ATP was analyzed by Western blot with phospho-specific antibodies against the dually phosphorylated, active forms of ERK1/2 and p38. The in-vitro invasive ability through Matrigel was measured by boyden-chamber assay. ATP induced significant SHP2 phosphorylation, which was stronger and lasted longer in 1E8 than in 2B4. SHP2-wt enhanced the ERK1/2 activation induced by ATP in 2B4 cells, while SHP2-cs delayed and decreased this effect in 1E8 cells. Both SHP2-wt and SHP2-cs had no obvious influence on p38 activation. ATP stimulated cell invasion of both 1E8 and 2B4, while transfection of SHP2-wt into 2B4 cells further increased the invasive-stimulating ability of ATP (18.7% increase compared with ATP treatment alone). Transfection of SHP2-cs into 1E8 cells, however, antagonized the invasive-stimulating ability of ATP (40.9% decrease compared with ATP treated group). Up-regulation of MKP5-wt inhibited phosphorylation of p38 by ATP and reduced cell invasion stimulated by ATP (22.4% and 28.7% decrease compared with ATP treated group of 1E8 and 2B4, respectively). Both SHP2 and MKP5 play some roles in P2Y receptor-mediated activation of MEK/ERK, p38 signaling pathways and prostate cancer invasion. SHP2 positively regulates ERK activation and prostate cancer invasion, whereas MKP5 inhibits the invasion by suppressing p38 activation.

Monoclonal Antibodies Against Human Tumor Metastasis Suppressor Gene–1 (TMSG-1): Preparation, Characterization, and Application

To better understand the molecular mechanism of metastasis, the monoclonal antibody against tumor metastasis suppressor gene-1 (TMSG-1, one novel gene) was prepared, characterized, and applied in estimating the metastatic potential of human tumors. A dominant epitope, TMSG-1(15)--derived from TMSG-1--was automatically synthesized based on the Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen solution was used to immunize BALB/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and their characterization was performed by Western blotting and immunohistochemical staining. The hybridoma cell line secreting anti-TMSG-1 antibody, designated C8, was established after primary ELISA screening and consequent rapid limited dilution. C8 was IgM in isotyping. The competitive inhibition assay showed that the antibody was TMSG-1 specific. Furthermore, in Western blotting, a protein band of about 45 kD was detected with nonmetastatic variant PC-3M-2B4 and PG-LH7 cells, but not with the isogenetic metastatic variant PC-3M-1E8 and PG-BE1 cells. Immunohistochemistry method showed that positive staining presented in the cytomembrane and cytoplasm of 2B4 and LH7 cells, while 1E8 and BE1 cells were non-reactive. The sections from paraffin-embedded blocks of human cancer (52 cases of breast carcinoma and 41 cases of colon cancer) were stained, showing strongly positive in non-metastatic tumor and weakly positive or negative in metastatic tumor; the strongly positive rate of TMSG-1 expression in human cancers were, respectively, 36%, 7.4%, 52.4%, and 35% in non-metastatic and metastatic breast cancer and non-metastatic and metastatic colon cancer (p = 0.02). The monoclonal antibody developed against synthetic peptide was TMSG-1 specific, and it has promise in Western blot and immunohistochemistry for detecting the TMSG-1 expression of cancer cells and tissues. It may provide an important tool in the study of TMSG-1 expression and function in both experimental and clinical studies. Furthermore, our tests confirmed that TMSG-1 protein has excellent inverse correlation to tumor metastasis potential, with the molecular weight of 45 kD (supporting the encoded protein containing 380 amino acids) and localization in cytomembrane and cytoplasm of tumor cell.

Regulation of the maintenance of peripheral T-cell anergy by TAB1-mediated p38 alpha activation.

In anergic T cells, T-cell receptor (TCR)-mediated responses are functionally inactivated by negative regulatory signals whose mechanisms are poorly understood. Here, we show that CD4(+) T cells anergized in vivo by superantigen Mls-1(a) express a scaffolding protein, transforming growth factor beta-activated protein kinase 1-binding protein 1 (TAB1), that negatively regulates TCR signaling through the activation of mitogen-activated protein kinase p38 alpha. TAB1 was not expressed in naive and activated CD4(+) T cells. Inhibition of p38 activity in anergic T cells by a chemical inhibitor resulted in the recovery of interleukin 2 (IL-2) and the inhibition of IL-10 secretion. T-cell hybridoma 2B4 cells transduced with TAB1-containing retrovirus (TAB1-2B4 cells) showed activated p38 alpha, inhibited extracellular signal-regulated kinase (ERK) activity, culminating in reduced IL-2 levels and increased IL-10 production. The use of a p38 inhibitor or cotransfection of a dominant-negative form of p38 in TAB1-2B4 cells resulted in the recovery of ERK activity and IL-2 production. These results imply that TAB1-mediated activation of p38 alpha in anergic T cells regulates the maintenance of T-cell unresponsiveness both by inhibiting IL-2 production and by promoting IL-10 production.

SAP mediates specific cytotoxic T-cell functions in X-linked lymphoproliferative disease

AbstractCytotoxic T cells (CTLs) and natural killer cells play a major role in the immune response to Epstein-Barr virus (EBV) infection. In X-linked lymphoproliferative (XLP) disease, a severe immunodeficiency, immunodysregulatory phenomena are observed following EBV infection, suggesting that defects exist in these effector populations. The gene defective in XLP is SAP (signaling lymphocytic activation molecule [SLAM]–associated protein), an adaptor protein that mediates signals through SLAM and other immunoglobulin superfamily receptors including 2B4. We generated EBV-specific T-cell lines from controls and XLP patients and examined CTL function in response to different stimuli. We show that XLP patients can generate EBV–T-cell lines that are phenotypically similar to those from controls. XLP patient EBV–T-cell lines showed a significant decrease in interferon-gamma (IFN-γ) production in response to 2B4 and autologous EBV-transformed lymphoblastoid cell line (LCL) stimulation but not in response to SLAM. Furthermore, XLP EBV–T-cell lines demonstrated markedly decreased cytotoxic activity against autologous LCLs. By retroviral gene transfer of the SAP gene into XLP EBV–T-cell lines, we show reconstitution of IFN-γ production and of cytotoxic activity confirming SAP-dependent defects. These studies demonstrate that in XLP the lack of SAP affects specific signaling pathways resulting in severe disruption of CTL function.

Methotrexate differentially affects growth of suspension and adherent cells

The effects of low concentrations of methotrexate (MTX) on the growth of suspension (FM3A, 2B4 and THP-1) and adherent (NIH3T3 and V79) cells were compared. The concentration of methotrexate to cause the inhibition of cell growth was lower in suspension cells than in adherent cells. The IC 50 for FM3A, 2B4, THP-1, NIH3T3 and V79 cells were 3.5, 5, 9, 30 and 50 nM, respectively. The inhibition of cell growth was reversed completely by tetrahydrofolate and was fully or significantly reversed by adenosine and thymidine, suggesting that the effects of low concentrations of methotrexate result from the inhibition of biosynthesis of purines and pyrimidines. In suspension cells but not in adherent cells there was a decrease in the levels of S-adenosylmethionine and polyamines after methotrexate treatment. Growth of suspension but not adherent cells was significantly recovered by treatment with S-adenosylmethionine. However, treatment with spermidine did not reverse the effects of methotrexate in any of the cell lines. The preferential inhibitory effect of methotrexate in suspension cells versus adherent cells was due mainly to a more rapid uptake of methotrexate. This may be relevant to the in vivo effects of low doses of methotrexate, which have immunosuppressive and anti-inflammatory effects, because lymphocytes are suspension cells.

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22.

A unique metabolite with a molecular mass of 119 Da (C(2)H(5)N(3)O(3)) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.