A common deletion at chromosomal arm 14q32 in human renal cell carcinoma (RCC) prompted us to explore a tumor suppressor gene (TSG) in this region. We report that imprinted DLK1 at 14q32, a regulator of adipocyte differentiation, is a candidate TSG in RCCs. DLK1 expression was lost in 39 out of 50 (78%) primary RCC tissues, whereas expression of DLK1 was maintained in every normal kidney tissue examined. DLK1 was expressed in only one of 15 (7%) RCC-derived cell lines. In order to see the biological significance of DLK1 inactivation in RCCs, we tested the effect of restoration of DLK1 in RCC cell lines, using a recombinant retrovirus containing the gene. Reintroduction of DLK1 into DLK1-null RCC cell lines markedly increased anchorage-independent cell death, anoikis and suppressed tumor growth in nude mice. We then investigated the underlying mechanisms for DLK1 inactivation in RCCs. We found loss of heterozygosity at this region in 12 out of 50 RCC tissues (24%). To explore the role of epigenetic regulation of DLK1 inactivation in RCCs, we conducted methylation analysis of the upstream region and the gene body of DLK1. We could not find a differentially methylated region in either the upstream region or the gene body of DLK1. However, we found that gain of methylation upstream of GTL2, a reciprocal imprinted gene for DLK1, is a critical epigenetic alteration for the inactivation of DLK1 in RCCs. The present data have shown that gain of methylation upstream of the untranslated GTL2 leads to pathological downregulation of DLK1 in RCCs.

RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200) plays a role in the enhancement of the RB1 pathway through the direct binding to a GC-rich region 201bp upstream (from the initiation ATG) of the RB1 promoter. Here, we identified hSNF5 and p53 as the binding partners of RB1CC1 by immunoprecipitation and immunofluorescence assays. Interaction between these molecules and the RB1 pathway was analyzed by the assays of chromatin immunoprecipitation, luciferase-reporter, reverse transcription-polymerase chain reaction and immunoblot. The tumor growth suppression by RB1CC1 was evaluated by flow cytometry or by a cell growth assay. The nuclear RB1CC1 complex involving hSNF5 and/or p53 activated transcription of RB1, p16 and p21, and suppressed tumor cell growth. Furthermore, nuclear RB1CC1 expression significantly correlated with those of RB1 and p16 in breast cancer tissue in vivo, and the Ki-67 proliferation index was dependent on p53 as well as RB1CC1. The present study indicates that RB1CC1 together with hSNF5 and/or p53 enhances the RB1 pathway through transcriptional activation of RB1, p16 and p21. Evaluation of RB1CC1 expression combined with RB1 and p53 status is expected to provide useful information in clinical practice and future therapeutic strategies in breast cancer.

Purpose: Testicular germ cell tumors (TGCT) have a unique epigenetic profile distinct from that of other types of cancer. Elucidation of these properties has a potential to identify novel markers for TGCTs.Experimental Design: We conducted comprehensive analysis of DNA methyltransferase (DNMT) gene expression in TGCTs. Based on the expression profiles of DNMT genes in TGCTs, we generated a rabbit polyclonal anti-human DNMT3L antibody. We then studied the role of DNMT3L in TGCTs by the treatment of two embryonal carcinoma (EC) cell lines with a small interfering RNA system. Finally, we evaluated the immunohistochemical detection of DNMT3L in TGCT tissues. We also compared the patterns of DNMT3L immunohistochemistry with those of CD30 and SOX2.Results: Among the DNMT genes, we found that mRNA for DNMT3L was specifically expressed in TGCTs, but neither in normal testicular tissues nor in cancer cells of somatic tissue origin. DNMT3L protein was strongly expressed in two EC cell lines, but not in the cell lines of somatic tissue origin. Transfection of small interfering RNA for DNMT3L significantly reduced DNMT3L expression and resulted in growth suppression and apoptosis in EC cells. Immunohistochemical analysis showed that DNMT3L protein was present only in EC cells, but not in the other types of TGCT components and cancer cells of somatic tissue origin. DNMT3L staining was more prominent and specific than CD30 or SOX2 staining for detecting EC cells.Conclusion: DNMT3L is a novel marker and is essential for the growth of human embryonal carcinoma.Clin Cancer Res; 16(10); 2751-9. ©2010 AACR.Testicular germ cell tumors (TGCT) are the most common malignancy in young men, with a peak incidence between 20 and 40 years of age. The incidence of TGCT has increased dramatically over the last century (1). TGCTs are classified into two major histologic subgroups, seminoma and nonseminoma: nonseminomatous TGCTs show embryonal and extraembryonal differentiation patterns, which include primitive zygotic (embryonal carcinoma, EC), embryonal-like somatic differentiated (teratoma), and extraembryonally differentiated (choriocarcinoma, yolk sac tumor) phenotypes (2).We and others have shown that TGCTs have a unique DNA methylation profile distinct from that of somatic tissue-derived neoplasms (3-6). TGCT tissues show less frequent methylation than somatic tissue cancers (3). This unmethylated DNA profile is significant particularly in seminomatous TGCTs (7). We have also shown that X-linked genes on super numerical X chromosomes in seminomatous and nonseminomatous TGCTs were predominantly hypomethylated, regardless of XIST expression (8). These studies suggest that seminoma and EC at least carry epigenetic profiles similar to those of fetal germ cells or embryonic stem (ES) cells.By characterizing the epigenetic profiles of TGCTs, it may be possible to identify novel markers for TGCTs. In the present study, we have performed a comprehensive expression analysis of DNA methyltransferase (DNMT) genes in TGCTs. We found that mR...

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BackgroundCell-based regeneration therapies have great potential for application in new areas in clinical medicine, although some obstacles still remain to be overcome for a wide range of clinical applications. One major impediment is the difficulty in large-scale production of cells of interest with reproducibility. Current protocols of cell therapy require a time-consuming and laborious manual process. To solve this problem, we focused on the robotics of an automated and high-throughput cell culture system. Automated robotic cultivation of stem or progenitor cells in clinical trials has not been reported till date. The system AutoCulture® used in this study can automatically replace the culture medium, centrifuge cells, split cells, and take photographs for morphological assessment. We examined the feasibility of this system in a clinical setting.ResultsWe observed similar characteristics by both the culture methods in terms of the growth rate, gene expression profile, cell surface profile by fluorescence-activated cell sorting, surface glycan profile, and genomic DNA stability. These results indicate that AutoCulture® is a feasible method for the cultivation of human cells for regenerative medicine.ConclusionsAn automated cell-processing machine will play important roles in cell therapy and have widespread use from application in multicenter trials to provision of off-the-shelf cell products.