DDR1 contributes to kidney inflammation and fibrosis by promoting the phosphorylation of BCR and STAT3.

Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase activated by collagen, contributes to chronic kidney disease. However, its role in acute kidney injury and subsequent development of kidney fibrosis is not clear. Thus, we performed a model of severe ischemia/reperfusion-induced acute kidney injury that progressed to kidney fibrosis in WT and Ddr1-null mice. We showed that Ddr1-null mice had reduced acute tubular injury, inflammation, and tubulointerstitial fibrosis with overall decreased renal monocyte chemoattractant protein (MCP-1) levels and STAT3 activation. We identified breakpoint cluster region (BCR) protein as a phosphorylated target of DDR1 that controls MCP-1 production in renal proximal tubule epithelial cells. DDR1-induced BCR phosphorylation or BCR downregulation increased MCP-1 secretion, suggesting that BCR negatively regulates the levels of MCP-1. Mechanistically, phosphorylation or downregulation of BCR increased ß-catenin activity and in turn MCP-1 production. Finally, we showed that DDR1-mediated STAT3 activation was required to stimulate the secretion of TGF-ß. Thus, DDR1 contributes to acute and chronic kidney injury by regulating BCR and STAT3 phosphorylation and in turn the production of MCP-1 and TGF-ß. These findings identify DDR1 an attractive therapeutic target for ameliorating both proinflammatory and profibrotic signaling in kidney disease.

Critical individual roles of the BCR and FGFR1 kinase domains in BCR-FGFR1-driven stem cell leukemia/lymphoma syndrome.

Constitutive activation of FGFR1, as a result of diverse chromosome translocations, is the hallmark of stem cell leukemia/lymphoma syndrome. The BCR-FGFR1 variant is unique in that the BCR component contributes a serine-threonine kinase (STK) to the N-terminal end of the chimeric FGFR1 kinase. We have deleted the STK domain and mutated the critical Y177 residue and demonstrate that the transforming activity of these mutated genes is reduced compared to the BCR-FGFR1 parental kinase. In addition, we demonstrate that deletion of the FGFR1 tyrosine kinase domain abrogates transforming ability, which is not compensated for by BCR STK activity. Unbiased screening for proteins that are inactivated as a result of loss of the BCR STK identified activated S6 kinase and SHP2 kinase. Genetic and pharmacological inhibition of SHP2 function in SCLL cells expressing BCR-FGFR1 in vitro leads to reduced viability and increased apoptosis. In vivo treatment of SCLL in mice with SHP099 leads to suppression of leukemogenesis, supporting an important role for SHP2 in FGFR1-driven leukemogenesis. In combination with the BGJ398 FGFR1 inhibitor, cell viability in vitro is further suppressed and acts synergistically with SHP099 in vivo suggesting a potential combined targeted therapy option in this subtype of SCLL disease.

Virus-directed enzyme prodrug therapy and the assessment of the cytotoxic impact of some benzimidazole derivatives.

Virus-directed enzyme prodrug therapy is one of the major strategy of increasing cytotoxicity of bioreductive agents. This research intended to examine new selected benzimidazole derivatives as a substrate for nitroreductase, the enzyme involved in nitroreduction which is responsible to the production of cytotoxic metabolites. In this way, the selectivity and strength of cytotoxicity can be raised. The effect of benzimidazoles on virus transfected cells and non-virus transfected cells A549 cell line was established by Annexin V + propidium iodide test, western blot, and polymerase chain reaction analysis of specific pro- and anti-apoptotic proteins in the corresponding gene expression and additionally nitroreductase gene expression. Our results proved the pro-apoptotic properties of all tested compounds in normoxia and hypoxia, especially according to virused A549 cells where the time of exposition was reduced from 48 to 4 h. In this shorten period of time, the strongest activity was shown by N-oxide compounds with nitro-groups. The apoptosis was confirmed by generation of BAX gene and protein and reduction of BCL2 gene and protein.

Efficacy and safety of autologous peripheral blood stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia: A study protocol for a multicenter exploratory prospective study (Auto-Ph17 study).

INTRODUCTION: The prognosis of Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL) has been dramatically improved since the introduction of tyrosine kinase inhibitors (TKIs). Although allogeneic hematopoietic cell transplantation (allo-HCT) is a major treatment option, the role of autologous peripheral blood stem cell transplantation (auto-PBSCT) has been reconsidered, especially in patients who achieved early molecular remission. METHODS AND ANALYSIS: This is a multicenter exploratory study for Ph + ALL patients aged between 55 and 70 years who achieved complete molecular remission within 3 cycles of chemotherapy. The target sample size is 5, and the registration period is 2 years. The primary endpoint is Day100- mortality after transplantation, and the secondary endpoints are survival, relapse rate, nonrelapse mortality, and adverse events.This study is divided into 3 phases: peripheral blood stem cell harvest, transplantation, and maintenance. Chemomobilization is performed using a combination of cyclophosphamide (CPM), doxorubicin, vincristine (VCR), and prednisolone (PSL). As a preparative regimen, the LEED regimen is used, which consists of melphalan, CPM, etoposide, and dexamethasone. Twelve cycles of maintenance therapy using a combination of VCR, PSL, and dasatinib are performed.In association with relapse, the minimal residual disease (MRD) of BCR-ABL chimeric gene and T-cell subsets are analyzed both before and after auto-PBSCT. ETHICS AND DISSEMINATION: The protocol was approved by the institutional review board of Nagoya University Hospital and all the participating hospitals. Written informed consent was obtained from all patients before registration, in accordance with the Declaration of Helsinki. Results of the study will be disseminated via publications in peer-reviewed journals. TRIAL REGISTRATION: Trial registration number UMIN000026445.

Novel fusion between the breakpoint cluster region and platelet-derived growth factor receptor-alpha genes in a patient with chronic myeloid leukemia-like neoplasm: undetectable residual disease after imatinib therapy.

Rare patients suffering from myeloid neoplasms share clinical and cytological features indistinguishable from chronic myeloid leukemia (CML) but lack the BCR-ABL1 fusion gene. Several studies provide evidence that alterations in genes encoding tyrosine kinase receptors such as the platelet-derived growth factor receptor (PDGFR) may be involved in the pathogenesis of these disorders. Here we describe a patient with a rare CML-like disease in whom we identified a novel in-frame BCR-PDGFRA rearrangement joining BCR exon 17 to PDGFRA exon 13, resulting in overexpression of PDGFRA. The design of a specific quantitative PCR assay to monitor the molecular response during treatment with imatinib, a multitargeted tyrosine kinase inhibitor (TKI) with activity against ABL, c-Kit, and PDGFRA revealed an outstanding disease control with durably undetectable BCR-PDGFRA transcripts. Multiple TKIs are currently available yet with distinct target profiles; thus, accurate molecular diagnosis and monitoring tools are essential to establish tailored treatments and assess response to therapy in this type of rare hematological malignancy.

Curcumin and quercetin trigger apoptosis during benzo(a)pyrene-induced lung carcinogenesis.

The aim of the present study was to assess the cumulative effects of curcumin and quercetin in inducing apoptosis during benzo(a)pyrene (BP) (100 mg/Kg body weight)-induced lung carcinogenesis in mice. BP treatment resulted in a significant increase in the protein expression of Bcl-2 whereas expression of Bax was significantly decreased. Further, BP treatment brought about a significant decrease in the activities of caspase 3, caspase 9 as well as the number of apoptotic cells. Interestingly, separate as well as combined supplementation of curcumin (60 mg/kg body weight) and quercetin (40 mg/kg body weight) to BP-treated animals resulted in a significant decrease in the protein expression of Bcl-2 but caused a significant increase in the protein expression of Bax along with a noticeable improvement in the number of apoptotic cells. Also, supplementation with curcumin and quercetin separately to BP-treated mice brought a significant improvement in the enzyme activities of caspase 9 as well as caspase 3 but the improvement was more pronounced following combined treatment. Therefore, curcumin and quercetin, if given in combination shall exhibit enhanced chemopreventive potential against development of lung carcinogenesis by stimulating the apoptotic machinery.

The expression of sphingosine-1 phosphate receptor-1 in chronic lymphocytic leukemia cells is impaired by tumor microenvironmental signals and enhanced by piceatannol and R406.

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon interaction of leukemic cells with a supportive microenvironment. Therefore, the mobilization of tissue-resident CLL cells into the circulation is a useful therapeutic strategy to minimize the reservoir of tumor cells within survival niches. Because the exit of normal lymphocytes from lymphoid tissues depends on the presence of sphingosine-1 phosphate (S1P) and the regulated expression of S1P receptor-1 (S1PR1), we investigated whether the expression and function of S1PR1 can be modulated by key microenvironment signals. We found that activation of CLL cells with CXCL12, fibroblast CD40L(+), BCR cross-linking, or autologous nurse-like cells reduces their S1PR1 expression and the migratory response toward S1P. Moreover, we found that S1PR1 expression was reduced in the proliferative/activated subset of leukemic cells compared with the quiescent subset from the same patient. Similarly, bone marrow-resident CLL cells expressing high levels of the activation marker CD38 showed a lower expression of S1PR1 compared with CD38(low) counterparts. Finally, given that treatment with BCR-associated kinase inhibitors induces a transient redistribution of leukemic cells from lymphoid tissues to circulation, we studied the effect of the Syk inhibitors piceatannol and R406 on S1PR1 expression and function. We found that they enhance S1PR1 expression in CLL cells and their migratory response toward S1P. Based on our results, we suggest that the regulated expression of S1PR1 might modulate the egress of the leukemic clone from lymphoid tissues.

Dissimilar expression of multidrug resistance mdr1 and bcrp by the replication of hepatitis C virus: role of the nonstructural 5A protein.

Multidrug resistance associated with the overexpression of ATP-dependent binding cassette (ABC) proteins is widely accepted as an important cause of treatment failure in patients with neoplastic or infectious diseases. Some of them play also a pivotal role in detoxification processes. Herein, we investigated the effect of hepatitis C virus (HCV) replication and nonstructural 5A (NS5A) protein on the expression and functional activity of two ABC transport proteins: MDR1 and BCRP. RT-quantitative real-time polymerase chain reaction (qPCR) was carried out for mdr1 and bcrp mRNAs in both Huh7 cells expressing NS5A and Huh7.5 cells containing either full-length- or subgenomic-HCV replicon systems. The functional activity of these pumps was studied by performing a dye efflux assay with DiOC2 and Rhodamine 123. A dose-dependent down-regulation of mdr1 expression was documented in Huh7 cells expressing the NS5A protein, as well as in both replicon systems. In contrast, a significant increase of bcrp expression in both systems was recorded, which were in full agreement with the dye efflux assay results. These results warrant further in vivo studies in HCV patients with cholestasis and/or patients that are refractive to the pharmacotherapy due to the activity of these pumps.

Selection of individual VH genes occurs at the pro-B to pre-B cell transition.

B cells are subjected to selection at multiple checkpoints during their development. The selection of Ab H chains is difficult to study because of the large diversity of the CDR3. To study the selection of individual Ab H chain V region genes (V(H)), we performed CDR3 spectratyping of ∼ 75-300 rearrangements per individual V(H) in C57BL6/J mice. We measured the fraction of rearrangements that were in-frame in B cell DNA. We demonstrate that individual V(H)s have different fractions of in-frame rearrangements (IF fractions) ranging from 10 to 90% and that these IF fractions are reproducible in different mice. For most V(H)s, the IF fraction in pro-B cells approximated 33% and then shifted to the nearly final (mature) B cell value by the cycling pre-B cell stage. The frequency of high in-frame (IF) V(H) usage increased in cycling pre-B cells compared with that in pro-B cells, whereas this did not occur for low IF V(H)s. The IF fraction did not shift as much in BCR-expressing B cells and was minimally affected by L chain usage for most V(H). High IF clan II/III V(H)s share more positively charged CDR2 sequences, whereas high IF clan I J558 CDR2 sequences are diverse. These data indicate that individual V(H)s are subjected to differential selection, that V(H) IF fraction is mainly established through pre-BCR-mediated selection, that it may operate differently in clan I versus II/III V(H)s, and that it has a lasting influence on the Ab repertoire.

[Relationship between thymus output function in CML patients and their bcr-abl mRNA levels].

The study was purposed to analyze the relationship between the content of T-cell receptor excision DNA circles (TREC) and bcr-abl mRNA levels in CML patients and to evaluate the prognostic significance of recent thymic output function detection in patients with chronic myelogenous leukemia (CML). Quantitative detection of TREC and bcr-abl fusion gene transcripts in peripheral blood from 15 CML patients were preformed by real-time PCR. The change of bcr-abl levels in 6 patients was followed-up for two years. The results showed that there was no significant correlation between TREC and bcr-abl mRNA levels in peripheral blood from CML patients for the first attack. Patients who had higher TREC at diagnosis had a larger reduction of bcr-abl after 2 years of follow-up. While out of 2 patients who underwent haemopoietic stem cell transplantation (HSCT), one patient with higher level of TREC before transplantation was confirmed to express undetectable level of TREC by three consecutive detections after transplantation, other one patient was identified to express low level of bcr-abl. It is concluded that high thymic output function in CML patients can be beneficial for killing the residual CML cells.

[Experimental advance of targeted medicines for chronic myeloid leukemia--review].

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder from hematopoietic stem cell disorder characterized by the consecutive expression of bcr-abl gene, and the translation product of which has enhanced tyrosine kinase activity and can activate a series of downstream signal transduction proteins and results in the occurence of CML. Although the application of imatinib (IM) makes nearly all patients with CML in chronic phase achieve a complete hematologic remission, and 90%of those treated in the early chronic phase achieve a complete cytogenetic remission, but the development of resistance to IM in the course of treatment and even in the beginning of the treatment forced people to develop new agents and to combine the new agents with IM in order to achieve better therapeutic result. This article reviews the experimental advances of targeted therapeutics in CML recent years.

Different target range and cytotoxic specificity of adaphostin and 17-allylamino-17-demethoxygeldanamycin in imatinib-resistant and sensitive cell lines.

Imatinib mesylate is a selective inhibitor of the oncogenic tyrosine kinase, Bcr-Abl, and is widely used as a first-line treatment for chronic myeloid leukaemia (CML). Prolonged monotherapy is frequently associated with patients becoming refractory to imatinib. Therefore, there is considerable interest in small molecule inhibitors which may be used either as replacements or as adjuncts to existing imatinib therapy. For this purpose, it is most likely that drugs which do not share imatinib's mechanism of action will be most valuable. We compared two such compounds with different modes of action, adaphostin and 17-allylamino-17-demethoxygeldanamycin (17-AAG), for their cytotoxic effect and ability to induce the downregulation of cellular proteins in a murine haemopoietic cell line transformed with human p210(Bcr-Abl), and two subclones resistant to imatinib owing to an Abl-kinase domain mutation (E255K) or amplification of the BCR-ABL gene, respectively. We found that, whereas 17-AAG selectively killed Bcr-Abl-positive cells and inhibited proteins dependent on heat-shock protein 90 for their stability (p210(Bcr-Abl) and Akt), adaphostin induced the downregulation of multiple cell-signalling proteins (p210(Bcr-Abl), Akt, Bcr, Abl and STAT5a) and was cytotoxic to both Bcr-Abl-positive and -negative cells. We suggest that both compounds may prove useful in the treatment of CML but caution that undesirable side-effects may result from the inhibition of multiple cell signalling proteins.

Analysis of immunoglobulin heavy and light chain variable genes in post-transplant lymphoproliferative disorders.

Post-transplant lymphoproliferative disorders (PTLD) derive from antigen-experienced B-cells and represent a major complication of solid organ transplantation. We characterized usage, mutation frequency and mutation pattern of immunoglobulin variable (IGV) gene rearrangements in 50 PTLD (polymorphic PTLD, n=10; diffuse large B-cell lymphoma, n=35; and Burkitt/Burkitt-like lymphoma, n=5). Among PTLD yielding clonal IGV amplimers, a functional IGV heavy chain (IGHV) rearrangement was found in 40/50 (80.0%) cases, whereas a potentially functional IGV light chain rearrangement was identified in 36/46 (78.3%) PTLD. By combining IGHV and IGV light chain rearrangements, 10/50 (20.0%) PTLD carried crippling mutations, precluding expression of a functional B-cell receptor (BCR). Immunohistochemistry showed detectable expression of IG light chains in only 18/43 (41.9%) PTLD. Failure to detect a functional IGV rearrangement associated with lack of IGV expression. Our data suggest that a large fraction of PTLD arise from germinal centre (GC)-experienced B-cells that display impaired BCR. Since a functional BCR is required for normal B-cell survival during GC transit, PTLD development may implicate rescue from apoptosis and expansion of B-cells that have failed the GC reaction. The high frequency of IGV loci inactivation appears to be a peculiar feature of PTLD among immunodeficiency-associated lymphoproliferations.

[Purification of a new phospholipase A2 homologue from Agkistrodon blomhoffii siniticus and its effects on gene expression profile of Hep3B cells].

OBJECTIVE: To isolate and purify a new phospholipase A2 (PLA2) homologue from Agkistrodon blomhoffii siniticus and investigate its effects on the gene expression profile of Hep3B cells. METHODS: The PLA2 homologue was isolated and purified by reverse-phase high-performance liquid chromatography (HPLC) and its purity was determined also by HPLC. The relative molecular mass of the homologue was measured by electrospray ionization mass spectrum. The gene expression profile of Hep3B cells was detected with gene chip after exposure of the cells to 139 microg/ml PLA2 homologue for 12 h. RESULTS: The purity of the PLA2 homologue was 97.2%, whose relative molecular mass was 13,900. After exposure of Hep3B cells to 139 microg/ml PLA2 homologue for 12 h, 19 genes were down-regulated and 20 up-regulated in the cells. The genes showing altered expressions in response to the exposure were mainly involved in cell cycle control and DNA damage repair, cell apoptosis and senescence, production of signal transduction molecules and transcription factors, cell adhesion, angiogenesis, and tumor invasion and metastasis. CONCLUSIONS: The PLA2 homologue induces alterations in the expression of a wide variety of genes involved in the growth and metastasis of tumor cells. The results of this study provide clues for further study of the possible mechanism for the action of PLA2 homologue on Hep3B cells.

The chemosensitivity to therapy of childhood early B acute lymphoblastic leukemia could be determined by the combined expression of CD34, SPI-B and BCR genes.

We have identified genes differentially expressed in childhood early B acute lymphoblastic leukemia at diagnosis, according to chemosensitivity. Chemosensitive (M1) and chemoresistant (M3) patients present <5% and >25% of residual leukemic blasts at 21 days of treatment, respectively. The expression profiles of 4205 genes for 32 patients included in the FRALLE93 protocol have been determined using microarray. From differential analysis, CD34, SPI-B and BCR distinguished M1 from M3 patients using microarray and RT-PCR data. Linear discriminant analysis (LDA) and cross-validation show that the combined expression of these three genes classify and predict correctly around 90% and 80% of patients, respectively.

Over-representation of bcr3 subtype of PML/RARalpha fusion gene in APL in Indian patients.

Thirty six patients with acute promyelocytic leukemia were studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR. There was concordance between the results achieved by both the methods except in one case, which was negative by RT-PCR but positive by real-time PCR. The prevalence of bcr3 (short isoform) was found to be significantly higher than that of bcr1 (long isoform) (64 vs. 36%, P=0.03). No correlation was found between age, sex, and white blood cell (WBC) count at diagnosis. Molecular remission was achieved in 66.6% of patients with bcr3 isoform. Median WBC count at presentation was found to be higher than that in the West.

[Effects of berbamine on K562 cells and its mechanisms in vitro and in vivo].

To elucidate the antileukemi effects of berbamine and the possible molecular mechanisms in vitro and in vivo, MTT method was used to examine the effect of berbamine on K562 cell growth. The apoptosis rate was measured by flow cytometry. The mRNA expression level of BCR/ABL gene (semiquantity value) was determined by RT-PCR and the BCR/ABL protein (P210) level was detected by Western blot. The K562-bearing mice were used to reveal the therapeutic effect in vivo. The results showed that a significant time- and concentration-dependent inhibition of cell growth was found in the cells treated with berbamine. After the cells were exposed to 8.0 microg/ml berbamine for 24, 48 and 72 hours, the percentage of growth inhibition of K562 cells progressively increased by (26.63 +/- 3.57)%, (61.84 +/- 4.74)%, (75.32 +/- 1.95)%, respectively (compared with control, P < 0.01). The IC(50) (72 hours) value was 5.227 +/- 1.307 microg/ml. The apoptosis rate of K562 cells treated with 8.0 microg/ml berbamine for 24 and 72 hours increased from (29.20 +/- 3.82)% to (61.77 +/- 4.35)% (P < 0.01). Berbamine down-regulated the expression levels of bcr/abl gene and P210 in K562 cells in a time- and concentration-dependent manner. The bcr/abl expression decreased from (1.38 +/- 0.02) to (0.97 +/- 0.01) after exposure of the cells to 8.0 microg/ml berbamine for 0 and 72 hours (P < 0.01). When the cells were treated with 4.0 - 16.0 microg/ml berbamine for 24 hours, the level of P210 decreased from (0.95 +/- 0.03) to (0.63 +/- 0.01) (P < 0.01). In vivo, after treatment for 4 weeks, the tumor weight of berbamine-treated group was also lower than that of untreated group [(1.46 +/- 0.43) g vs (2.90 +/- 0.94) g, P < 0.01] and the inhibition rate was 49.66%, moreover, berbamine down-regulated the expression level of bcr/abl gene of tumor cells. It is concluded that berbamine can obviously inhibit the cell proliferation and induce apoptosis in K562 cell lines in a time- and concentration-dependent manner in vitro. The mechanisms of berbamine-induced apoptosis may be involved in down-regulation of bcr/abl gene expression and P210 level. In vivo, berbamine can aslo display a better antileukemic effect and down-regulate expression of bcr/abl gene. Berbamine extracted from Chinese herb may be a promising candidate of new drug for clinical anticancer treatment, especially for bcr-abl(+) diseases.