Bioinformatic analysis of the human DHRS4 gene cluster and a proposed mechanism for its transcriptional regulation.

BACKGROUND: The human DHRS4 gene cluster consists of three genes, DHRS4, DHRS4L2 and DHRS4L1. Among them, DHRS4 encodes NADP(H)-dependent retinol dehydrogenase/reductase. In a previous study, we investigated the alternative splicing of DHRS4 and DHRS4L2. DHRS4L1 was added to the gene cluster recently, but little is known about its structure and expression. To reveal the regulatory mechanism of the DHRS4 gene cluster expression, we studied the structure and transcription of DHRS4L1 in the context of the transcriptional behaviors of the human DHRS4 gene cluster. Based on the results of bioinformatics analysis, we propose a possible mechanism for the transcriptional regulation of the human DHRS4 gene cluster. RESULTS: The homologous comparison analysis suggests that DHRS4, DHRS4L2 and DHRS4L1 are three homologous genes in human. DHRS4L1 and DHRS4L2 are paralogues of DHRS4, and DHRS4L2 is the most recent member of the DHRS4 gene cluster. In the minus strand of the human DHRS4 gene cluster, a gene transcribed in an antisense direction was found containing a 5' sequence overlapping the region of exon 1 and promoter of DHRS4. By cloning the full length of RNA variants through 5'RACE and 3'RACE, we identified two transcription start sites, within exon a2 and exon 1, of this newly named gene DHRS4L1 using neuroblastoma cell line BE(2)-M17. Analysis of exon composition in the transcripts of DHRS4 gene cluster revealed that exon 1 was absent in all the transcripts initiated from exon a1 of DHRS4L2 and exon a2 of DHRS4L1. CONCLUSIONS: Alternatively spliced RNA variants are prevalent in the human DHRS4 gene cluster. Based on the analysis of gene transcripts and bioinformatic prediction, we propose here that antisense transcription may be involved in the transcriptional initiation regulation of DHRS4 and in the posttranscriptional splicing of DHRS4L2 and DRHS4L1 for the homologous identity of DHRS4 gene cluster. Beside the alternative transcriptional start sites, the antisense RNA is novel possible factor serving to remove exon 1 from the transcripts initiated from exon a1 and exon a2.

In vivo anti-tumor effect of hybrid vaccine of dendritic cells and esophageal carcinoma cells on esophageal carcinoma cell line 109 in mice with severe combined immune deficiency.

AIM: To develop a fusion vaccine of esophageal carcinoma cells and dendritic cells (DC) and observe its protective and therapeutic effect against esophageal carcinoma cell line 109 (EC109). METHODS: The fusion vaccine was produced by fusing traditional polyethyleneglycol (PEG), inducing cytokine, sorting CD34+ magnetic microbead marker and magnetic cell system (MACS). The liver, spleen and lung were pathologically tested after injection of the fusion vaccine. To study the therapeutic and protective effect of the fusion vaccine against tumor EC109, mice were divided immune group and therapeutic group. The immune group was divided into P, E, D and ED subgroups, immunized by phosphate buffered solution (PBS), inactivated EC109, DC and the fusion vaccine respectively, and attacked by EC109 cells. The tumor size, weight, latent period and mouse survival period were recorded and statistically analyzed. The therapeutic group was divided into four subgroups: P, inactivated EC109, D and ED subgroups, which were attacked by EC109 and then treated with PBS, inactivated EC109, DC, and EC109-DC respectively. Pathology and flow cytometry were also used to study the therapeutic effect of the fusion vaccine against EC109 cells. RESULTS: Flow cytometry showed that the expression of folate receptor (FR), EC109 (C), DCs (D) in human nasopharyngeal carcinoma cell line (HNE1) (B) was 78.21%, 89.50%, and 0.18%, respectively. The fusion cells (C) were highly expressed. No tumor was found in the spleen, lung and liver after injection of the fusion vaccine. Human IgG was tested in peripheral blood lymphocytes (PBL). In the immune group, the latent period was longer in EC109-DC subgroup than in other subgroups, while the tumor size and weight were also smaller than those in ED subgroup. In the therapeutic group, the tumor size and weight were smaller in ED subgroup than in P, inactivated EC109 and DC subgroups. CONCLUSION: Fusion cells are highly expressed not only in FR but also in CD80. The fusion vaccine has a distinctive protective effect against tumor EC109 and can inhibit the growth of tumor in mice, and its immune protection against tumor attack is more significant.

Effects of dendritic cells from cord blood CD34+ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice.

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in severe combined immunodeficiency (SCID) mice. METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocarcinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect. RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%, 47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80% vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11 d vs 7 d, P<0.01). CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.

Comparative proteomic analysis reveals differentially expressed proteins regulated by a potential tumor promoter, BRE, in human esophageal carcinoma cells.

Esophageal tumorigenesis is a complex and cascading process, involving the interaction of many genes and proteins. In this study, we have used the comparative proteomic approach to identify tumor-associated proteins and explore the carcinogenic mechanisms. Two-dimensional electrophoresis (2-DE) and MALDI-TOF MS analysis of esophageal carcinoma and control cells revealed 10 proteins that were upregulated. A further 10 proteins were downregulated. Among these 20 differentially expressed proteins, brain and reproductive organ-expressed (BRE) protein was identified as a potential tumor promoter. It was high expressed by the esophageal carcinoma cells, as confirmed by RT-PCR and immunoblotting. BRE has been reported to be a stress-responsive protein. To gain further insight into its function, BRE expression was silenced in esophageal carcinoma cells using BRE-specific small interference RNA. It was discovered that silencing BRE expression downregulated prohibitin expression, but upregulated tumor-suppressor p53 expression. Furthermore, cyclin A and CDK2 expressions were suppressed suggesting that BRE inhibited cell proliferation. These results implied that BRE plays a significant role in mediating antiapoptotic and proliferative responses in esophageal carcinoma cells.