No abstract

DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b -/-lymphomas, but not in Dnmt3b -/-pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b -/-lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b +/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by an accumulation of neoplastic B cells due to their resistance to apoptosis and increased survival. Among various factors, the tumor microenvironment is known to play a role in the regulation of cell proliferation and survival of many cancers. However, it remains unclear how the tumor microenvironment contributes to the increased survival of B-CLL cells. Therefore, we studied the influence of bone marrow stromal cell -induced hedgehog (Hh) signaling on the survival of B-CLL cells. Our results show that a Hh signaling inhibitor, cyclopamine, inhibits bone marrow stromal cell -induced survival of B-CLL cells, suggesting a role for Hh signaling in the survival of B-CLL cells. Furthermore, gene expression profiling of primary B-CLL cells (n = 48) indicates that the expression of Hh signaling molecules, such as GLI1, GLI2, SUFU, and BCL2, is significantly increased and correlates with disease progression of B-CLL patients with clinical outcome. In addition, SUFU and GLI1 transcripts, as determined by real-time PCR, are significantly overexpressed and correlate with adverse indicators of clinical outcome in B-CLL patients, such as cytogenetics or CD38 expression. Furthermore, selective downregulation of GLI1 by antisense oligodeoxynucleotides (GLI1-ASO) results in decreased BCL2 expression and cell survival, suggesting that GLI1 may regulate BCL2 and, thereby, modulate cell survival in B-CLL. In addition, there was significantly increased apoptosis of B-CLL cells when cultured in the presence of GLI1-ASO and fludarabine. Together, these results reveal that Hh signaling is important in the pathogenesis of B-CLL and, hence, may be a potential therapeutic target.

No abstract

Summary:Forty-one patients were studied at set times after allogeneic blood stem cell transplantation (alloBSCT) for recovery of lymphocyte numbers and function. Cells were mobilized with G-CSF from HLA-matched related donors and cryopreserved. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate; G-CSF was administered post-transplant. Median time to absolute lymphocyte count (ALC) у500/l was 17 days vs 41 and 49 days in historical alloBMT patients with G-CSF (n = 23) or no cytokine (n = 29) post-transplant, respectively (P Ͻ 0.0001). CD4/CD8+ ratio was 1.9 on day 28 after alloBSCT, then gradually declined to 0.8 at 1 year due to more rapid CD8 + cell recovery. Mean phytohemagglutinin-induced T cell responses were lower than normal on day +28 (P Ͻ 0.05), then tended to recover towards normal values. Natural-killer cytotoxicity remained low from day +28 to 1 year post-alloBSCT, but considerable lymphokineactivated killer cytotoxicity was induced from cells already obtained on day +28. Faster lymphocyte recovery correlated with better survival in alloBSCT patients (median follow-up 287 days, P = 0.002), ALC recovery was not affected by acute GVHD, CMV infections or doses of infused cells. ALC recovery did not correlate with survival in either historical alloBMT group. These data suggest that after alloBSCT lymphocyte reconstitution is faster than after alloBMT, and that quicker lymphocyte recovery predicts better survival in the alloBSCT setting.

Purpose: In B-cell chronic lymphocytic leukemia (CLL), high CD38 expression has been associated with unfavorable clinical course, advanced disease, resistance to therapy, shorter time to first treatment, and shorter survival. However, the genes associated with CLL patient subgroups with high and low CD38 expression and their potential role in disease progression is not known. Experimental Design: To identify the genes associated with the clinical disparity in CLL patients with high versus low CD38 expression, transcriptional profiles were obtained from CLL cells from 39 different patients using oligonucleotide microarray. Gene expression was also compared between CLL cells and B cells from healthy individuals. Results: Gene expression analysis identified 76 differentially expressed genes in CD38 high versus low groups. Out of these genes, HEM1, CTLA4, and MNDA were selected for further studies and their differential expression was confirmed by real-time PCR. HEM1 overexpression was associated with poor outcome, whereas the overexpression of CTLA4 and MNDA was associated with good outcome. Down-regulation of HEM1 expression in patient CLL cells resulted in a significant increase in their susceptibility to fludarabine-mediated killing. In addition, when gene expression patterns in CD38 high and low CLL cells were compared with normal B-cell profiles, ATM expression was found to be significantly lower in CD38 high compared with CD38 low CLL as confirmed by real-time reverse transcription-PCR. Conclusions: These results identify the possible genes that may be involved in cell proliferation and survival and, thus, determining the clinical behavior of CLL patients expressing high or low CD38.

Mantle cell lymphoma (MCL) has one of the worst clinical outcomes among the B-cell lymphomas, with a median survival of only 3 to 4 years. Therefore, a better understanding of the underlying mechanisms that regulate MCL proliferation/survival is needed to develop an effective therapy. Because sonic hedgehog (Shh)-GLI signaling has been shown to be important in the proliferation and survival of several cancers, and no such information is available for MCL, this study was undertaken. Our results show that the molecules associated with Shh-GLI signaling, such as PTCH and SMO receptors, and GLI1 and GLI2 target transcription factors were expressed in the human MCL cell lines and primary MCL cells from patients. Perturbation of this signaling in the presence of exogenous Shh/cyclopamine significantly (P < 0.001) influenced the proliferation of JVM2 MCL cells. Furthermore, down-regulation of GLI transcription factors using antisense oligonucleotides not only resulted in significantly (P < 0.001) decreased proliferation of the MCL cells but also significantly (P < 0.05) increased their susceptibility to chemotherapeutic drug, doxorubicin. Also, downregulation of GLI decreased cyclin D1 and BCL2 transcript levels, which suggests that these key molecules might be regulated by GLI in MCL. Thus, our results indicate a significant role for Shh-GLI signaling in the proliferation of MCL, and molecular targeting of GLI is a potential therapeutic approach to improve the treatment for MCL.

The Hoechst dye efflux assay represents an effective method for direct identification of retinal stem cells/progenitors. These results demonstrate that the prospectively isolated retinal stem cells/progenitors and those enriched as SP cells from mitogen-exposed retinal cell culture may be similar in their properties and potential.