Clinical significance of soluble CADM1 as a novel marker for adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/leukemia (ATLL) is an aggressive peripheral T-cell malignancy, caused by infection with the human T-cell leukemia virus type 1 (HTLV-1). We have recently shown that cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is specifically and consistently overexpressed in ATLL cells, and functions as a novel cell surface marker. In this study, we first show that a soluble form of CADM1 (sCADM1) is secreted from ATLL cells by mainly alternative splicing. After developing the Alpha linked immunosorbent assay (AlphaLISA) for sCADM1, we showed that plasma sCADM1 concentrations gradually increased during disease progression from indolent to aggressive ATLL. Although other known biomarkers of tumor burden such as soluble interleukin-2 receptor α (sIL-2Rα) also increased with sCADM1 during ATLL progression, multivariate statistical analysis of biomarkers revealed that only plasma sCADM1 was selected as a specific biomarker for aggressive ATLL, suggesting that plasma sCADM1 may be a potential risk factor for aggressive ATLL. In addition, plasma sCADM1 is a useful marker for monitoring response to chemotherapy as well as for predicting relapse of ATLL. Furthermore, the change in sCADM1 concentration between indolent and aggressive type ATLL was more prominent than the change in the percentage of CD4+CADM1+ ATLL cells. As plasma sCADM1 values fell within normal ranges in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients with higher levels of serum sIL-2Rα, a measurement of sCADM1 may become a useful tool to discriminate between ATLL and other inflammatory diseases, including HAM/TSP.

Acetylation of lysine 564 adjacent to the C-terminal binding protein-binding motif in EVI1 is crucial for transcriptional activation of GATA2.

Ecotropic viral integration site 1 (EVI1) is an important transcription factor for leukemogenesis. EVI1 is a member of a group of transcription factors with C-terminal binding protein (CtBP)-binding motifs that act as transcriptional co-repressors; however, we recently found that EVI1 directly activates GATA2 transcription, which is an important gene for the maintenance of hematopoietic stem cells. We show here that EVI1-activated GATA2 transcripts derive from exon 1S of GATA2, which is specifically activated in neural and hematopoietic cells. EVI1 was acetylated by the histone acetyltransferase p300/CBP association factor (P/CAF) in myeloid leukemia cells and hematopoietic progenitor cells. Acetylation at Lys(564), which is adjacent to the CtBP-binding consensus sequence of EVI1, was found to be important for transcriptional activation of GATA2. Mutation of Lys(564) to alanine (K564A) markedly reduced the ability of EVI1 to bind DNA and activate transcription of GATA2. Furthermore, we confirmed that Lys(564) in EVI1 was specifically acetylated in leukemia and primary hematopoietic cells by using an antibody directed against an acetylated Lys(564) EVI1 peptide. Moreover, co-transfection of P/CAF with EVI1 overcame the suppressive effect of the CtBP co-repressor and resulted in GATA2 transcriptional activation; nonetheless, CtBP2 was still included in the protein complex with EVI1 and P/CAF on the EVI1-binding site in the GATA2 promoter region. Thus, acetylation of EVI1 at Lys(564) by P/CAF enhances the DNA binding capacity of EVI1 and thereby contributes to the activation of GATA2.

Degradation of p47 by autophagy contributes to CADM1 overexpression in ATLL cells through the activation of NF-κB.

Cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is identified as a novel cell surface marker for human T-cell leukemia virus (HTLV-1)-infected T cells. Adult T-cell leukemia/lymphoma (ATLL) is developed in HTLV-1-infected T-cells after a long infection period. To examine the mechanism of CADM1 overexpression in ATLL, we first identified that CADM1 is transcriptionally up-regulated by a transcriptional enhancer element through NF-κB signaling pathway. In HTLV-1-infected T-cells, CADM1 expression is dependent on HTLV-1/Tax through activation of canonical and non-canonical NF-κB; however, in ATLL cells with frequent loss of Tax expression, the activation of canonical NF-κB only enhances the CADM1 expression. Along with active mutations in signaling molecules under T-cell recepor (TCR) signaling, degradation of p47, a negative regulator of NF-κB, was essential for activation of canonical NF-κB through stabilization of NEMO (NF-κB essential modulator). The mechanism of p47 degradation is primarily dependent on activation of lysosomal-autophagy and the autophagy is activated in most of the HTLV-infected and ATLL cells, suggesting that the p47 degradation may be a first key molecular event during HTLV-1 infection to T-cells as a connector of two important signaling pathways, NF-κB and autophagy.

Analysis of hepatitis C virus (HCV) RNA in the lesions of lichen planus in patients with chronic hepatitis C: detection of anti-genomic- as well as genomic-strand HCV RNAs in lichen planus lesions.

BACKGROUND: Hepatitis C virus (HCV) is a single-strand RNA virus. The association of lichen planus with chronic HCV infection has been reported, as has been cryoglobulinemic purpura, psoriasis, urticaria, and porphyria cutanea tarda. However, the cause of lichen planus is unclear. OBJECTIVES: To investigate whether genomic- and/or anti-genomic-strand HCV RNAs are present in the lichen planus lesions of chronic hepatitis C patients and to elucidate the pathogenesis of lichen planus. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) followed by nested-PCR was carried out to detect HCV RNA using RNA samples from lichen planus lesions of three patients with chronic hepatitis C. Since it is well known that commonly there is relatively dense inflammatory cell infiltration mainly in the upper dermis in lichen planus, the same RT-PCR procedure was performed using RNA from peripheral blood leukocytes from the same patients. In addition, in one patient, the same procedure was also performed using an RNA sample from normal skin. RESULTS: Bands of the appropriate size (161 base pairs corresponding to region 98-258 of HCV RNA) in the nested-PCR products for both genomic- and anti-genomic-strands were detected in lichen planus lesions as well as in peripheral blood leukocytes in all the cases. CONCLUSION: To the best of our knowledge, this is the first report showing the presence of anti-genomic- as well as genomic-strand HCV RNAs in lichen planus lesions in patients with chronic hepatitis C; suggesting that HCV-associated lichen planus lesions may be sites of HCV replication.

Increased plasma caveolin-1 levels are associated with progression of prostate cancer among Japanese men.

AIM: Up-regulation of caveolin-1 (CAV1) is associated with aggressive prostate cancer. Among Caucasian and African-American patients, plasma CAV1 levels are elevated in patients with castration-resistant prostate cancer (CRPC), but not in those with hormone-sensitive prostate cancer (non-CRPC), which implies that CAV1 could be a therapeutic target for CRPC. Here, we evaluated associations between plasma CAV1 levels and these types of cancer in Japanese men, and CAV1 expression in PC3 (CRPC) and LNCaP (non-CRPC) cell lines. MATERIALS AND METHODS: Plasma samples were obtained from 58 patients with prostate cancer: 36 with CRPC and 22 with non-CRPC. Enzyme-linked immuno sorbent assay (ELISA) kits were used to determine CAV1 plasma levels; qRT-PCR and western blots were used to evaluate the expression of CAV1 mRNA and protein in cell lines. RESULTS: Plasma CAV1 levels in patients with CRPC were greatly higher than in those with non-CRPC (1.46±1.37 ng/ml in CRPC; 0.56±0.32 ng/ml in non-CRPC, p<0.004). Western blot and real-time qRT-PCR showed CAV1 protein and mRNA in PC3 cells to be significantly overexpressed compared to its expression in LNCaP cells (p<0.0001). CONCLUSION: Our results showed a relationship between CAV1 expression and prostate cancer progression, and support the possibility of CAV1 as a therapeutic target for CRPC.

Oncogenic transcription factor Evi1 regulates hematopoietic stem cell proliferation through GATA-2 expression.

The ecotropic viral integration site-1 (Evi1) is an oncogenic transcription factor in murine and human myeloid leukemia. We herein show that Evi1 is predominantly expressed in hematopoietic stem cells (HSCs) in embryos and adult bone marrows, suggesting a physiological role of Evi1 in HSCs. We therefore investigate the role and authentic target genes of Evi1 in hematopoiesis using Evi1-/- mice, which die at embryonic day 10.5. HSCs in Evi1-/- embryos are markedly decreased in numbers in vivo with defective self-renewing proliferation and repopulating capacity. Notably, expression rate of GATA-2 mRNA, which is essential for proliferation of definitive HSCs, is profoundly reduced in HSCs of Evi1-/- embryos. Restoration of the Evi1 or GATA-2 expression in Evi1-/- HSCs could prevent the failure of in vitro maintenance and proliferation of HSC through upregulation of GATA-2 expression. An analysis of the GATA-2 promoter region revealed that Evi1 directly binds to GATA-2 promoter as an enhancer. Our results reveal that GATA-2 is presumably one of critical targets for Evi1 and that transcription factors regulate the HSC pool hierarchically.

Overexpression of a cell adhesion molecule, TSLC1, as a possible molecular marker for acute-type adult T-cell leukemia.

Adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1) infection, occurs in 2% to 4% of the HTLV-1 carriers with a long latent period, suggesting that additional alterations participate in the development of ATL. To characterize and identify novel markers of ATL, we examined the expression profiles of more than 12 000 genes in 8 cases of acute-type ATL using microarray. One hundred ninety-two genes containing interleukin 2 (IL-2) receptor alpha were up-regulated more than 2-fold compared with CD4(+) and CD4(+)CD45RO(+) T cells, and tumor suppressor in lung cancer 1 (TSLC1), caveolin 1, and prostaglandin D2 synthase showed increased expression of more than 30-fold. TSLC1 is a cell adhesion molecule originally identified as a tumor suppressor in the lung but lacks its expression in normal or activated T cells. We confirmed ectopic expression of the TSLC1 in all acute-type ATL cells and in 7 of 10 ATL- or HTLV-1-infected T-cell lines. Introduction of TSLC1 into a human ATL cell line ED enhanced both self-aggregation and adhesion ability to vascular endothelial cells. These results suggested that the ectopic expression of TSLC1 could provide a novel marker for acute-type ATL and may participate in tissue invasion, a characteristic feature of the malignant ATL cells.

Aberrant expression of the MEL1S gene identified in association with hypomethylation in adult T-cell leukemia cells.

DNA methylation plays critical roles in the development and differentiation of mammalian cells, and its dysregulation has been implicated in oncogenesis. This study was designed to determine whether DNA hypomethylation-associated aberrant gene expression is involved in adult T-cell leukemia (ATL) leukemogenesis. We isolated hypomethylated DNA regions of ATL cells compared with peripheral blood mononuclear cells from a carrier by a methylated CpG-island amplification/representational difference analysis method. The DNA regions identified contained MEL1, CACNA1H, and Nogo receptor genes. Sequencing using sodium bisulfite-treated genomic DNAs revealed the decreased methylated CpG sites, confirming that this method detected hypomethylated DNA regions. Moreover, these hypomethylated genes were aberrantly transcribed. Among them, MEL1S, an alternatively spliced form of MEL1 lacking the PR (positive regulatory domain I binding factor 1 and retinoblastoma-interacting zinc finger protein) domain, was frequently transcribed in ATL cells, and the transcriptional initiation sites were identified upstream from exons 4 and 6. Transfection of MEL1S into CTLL-2 cells conferred resistance against transforming growth factor beta (TGF-beta), suggesting that aberrant expression of MEL1S was associated with dysregulation of TGF-beta-mediated signaling. Although Tax renders cells resistant to TGF-beta, Tax could not be produced in most fresh ATL cells, in which MEL1S might be responsible for TGF-beta resistance. Our results suggest that aberrant gene expression associated with DNA hypomethylation is implicated in leukemogenesis of ATL.

A novel EVI1 gene family, MEL1, lacking a PR domain (MEL1S) is expressed mainly in t(1;3)(p36;q21)-positive AML and blocks G-CSF-induced myeloid differentiation.

We have identified a novel gene MEL1 (MDS1/EVI1-like gene 1) encoding a zinc finger protein near the breakpoint of t(1; 3)(p36;q21)-positive human acute myeloid leukemia (AML) cells. Here, we studied the structure, expression pattern, and function of MEL1 in leukemia cells. In this study, we have identified 3 transcription start sites, 1 in exon 1 and 2 in exon 2, and 2 kinds of translation products, 170 kDa (MEL1) and 150 kDa (MEL1S). Notably, the 150-kDa band of MEL1S was detected mainly in the t(1;3)(p36;q21)-positive AML cells. By immunoblot analysis and proteolytic mapping, it is suggested that the 150-kDa band of MEL1S in the leukemia cells is translated from the internal initiation codon ATG597 in exon 4 and is mostly lacking the amino-terminal PR domain of MEL1. By the cyclic amplification and selection of targets (CASTing) method for identifying consensus sequences, it was shown that the consensus sequences of MEL1 were included in 2 different consensus sequences for DNA-binding domain 1 and 2 (D1-CONS and D2-CONS) of EVI1. In reporter gene assays, MEL1S activated transcription via binding to D2-CONS; however, the fusion of MEL1 or MEL1S to GAL4 DNA-binding domain (DBD) made them GAL4 binding site-dependent transcriptional repressors. Moreover, overexpression of MEL1S blocked granulocytic differentiation induced by granulocyte colony-stimulating factor (G-CSF) in interleukin-3 (IL-3)-dependent murine myeloid L-G3 cells, while MEL1 could not block the differentiation. Thus, it is likely that overexpression of the zinc finger protein lacking the PR domain (EVI1 and MEL1S) in the leukemia cells is one of the causative factors in the pathogenesis of myeloid leukemia.