C1orf35 contributes to tumorigenesis by activating c-MYC transcription in multiple myeloma.

Multiple myeloma (MM) is a clinically and biologically heterogenous event that accounts for approximately 10% of all hematological malignancies. Chromosome 1 open reading frame 35 (C1orf35) is a gene cloned and identified in our laboratory from a MM cell line (GenBank: AY137773), but little is known about its function. In the current study, we have confirmed that C1orf35 is a candidate oncogene, and it can promote cell cycle progression from G1 to S. Later, we found that C1orf35 is able to affect the cell proliferation by modulating the expression of c-MYC (v-myc myelocytomatosis viral oncogene homolog), and the oncogenic property of C1orf35 can be rescued by c-MYC inhibition. Herein, we found positive association between C1orf35 and c-MYC in MM patients and in MM cell lines. The correlation analysis of the genes coamplified in MM patients from GEO datasets showed a correlation between C1orf35 and c-MYC, and the expression data of different stages of plasma cell neoplasm acquired from GEO datasets showed that the expression of C1orf35 increase with the progression of the disease. This indicates that C1orf35 may play a role in the disease progression. Moreover, C1orf35 can modulate c-MYC expression and rescue c-MYC transcription inhibited by Act D. Finally, we have shown that C1orf35 activates c-MYC transcription by binding to the i-motif of Nuclease hypersensitivity element III1 (NHE III1) in the c-MYC promoter. Not only does our current study advance our knowledge of the pathogenesis and therapeutic landscape of MM, but also of other cancer types and diseases that are initiated with deregulated c-MYC transcription.

Promoter methylation induced epigenetic silencing of DAZAP2, a downstream effector of p38/MAPK pathway, in multiple myeloma cells.

Multiple myeloma (MM) is hematological malignancy characterized by clonal proliferation of malignant plasma cells in the bone marrow environment. Previously, we identified DAZAP2 as a candidate cancer suppressor gene, the downregulation of which is regulated by its own promoter methylation status. In the current study, we analyzed the DAZAP2 promoter in MM cell lines KM3, MM.1S, OPM-2, and ARH77 by bisulfite genomic sequencing assay. We identified the binding site for transcription factor cyclic adenosine monophosphate response element binding (CREB) in the DAZAP2 promoter CpG2, and we found that hypermethylation of the CREB binding motif in the DAZAP2 promoter is responsible for the reduced DAZAP2 expression in MM cells. Later we checked the p38/MAPK signaling cascade, which is reported to regulate expression and function of CREB. Our results showed that the p38/MAPK signaling pathway drives the expression of DAZAP2 by phosphorylation of CREB, and hypermethylation of CREB binding motif in DAZAP2 promoter can inhibit binding of CREB to the latter, thus downregulating DAZAP2 expression. Moreover, treating the MM cells with 5-aza-2' deoxycytidine to demethylate DAZAP2 promoter restored the binding of CREB to its binding motif, and thus upregulated DAZAP2 expression. Our results not only identified DAZAP2 as a new downstream target of p38/MAPK/CREB signaling cascade, but we also clarified that the downregulation of DAZAP2 in MM cells is caused by hypermethylation of CREB binding motif in its own promoter region, which implies that demethylation of DAZAP2 promoter can be a novel therapeutic strategy for MM treatment.

[Resistance of rAAV2-MfE77.43-Transferred Mice to Schistosoma japonicum Infection].

Objective: To investigate the resistance of E77.43 gene of Microtus fortis（MfE77.43） to Schistosoma japonicum infection. Methods: MfE77.43 was constructed into the recombinant Adeno-associated virus AAV2. The AAV2-MfE77.43 was transfected into HEK293 cells by the calcium phosphate DNA coprecipitation method. The recombinant rAAV2-MfE77.43 was purified and total RNA was extracted from the transfected cells. The expression of E77.43 was examined by RT-PCR and the purity of rAAV2-MfE77.43 was analyzed by SDS-PAGE. Eighteen KM mice were divided into three groups （n=6 in each group）. Mice in the experiment group were intramuscularly injected on days 0, 3 and 7 with 400 µl recombinant AAV2-MfE77.43 virus which was 5-fold diluted in normal saline. Mice in negative control and blank control groups received same volume of pAAV or normal saline. Venous blood was collected through the tail before each injection, and E77.43 expression in plasma was detected by dot-ELISA method. After the last injection, each mouse was infected with 40 S. japonicum cercariae and sacrificed on day 41 after infection. Adult worms and liver eggs per gram（LEPG） were counted. Worm and egg reduction rate was calculated respectively. Egg granulomas were observed by HE staining. Results: RT-PCR resulted in a 330 bp specific band. SDS-PAGE of virus shell protein revealed three protein bands with M(r) of 87 000, 72 000 and 62 000, respectively. Dot-ELISA showed that E77.43 protein began to be expressed on day 3 after rAAV2-MfE77.43 injection, remaining stable till day 41. The adult worm number and LEPG were 20.16±3.93 and 19 800±2 715, respectively, with a worm and egg reduction rate of 27.3% and 26.2% in the experiment group. While the worm number and LEPG in the negative control group were 29.16±2.44 and 28 000±2 192（P<0.01）, respectively. HE staining and observation revealed fewer eosinophils and inflammatory cells around the liver eggs in the therapy group. Conclusion: The E77.43 gene shows protective effects against S. japonicum infection, indicating that E77.43 may participate in the natural resistance of Microtus fortis to S. japonicum infection.

The effects of promoter methylation on downregulation of DAZAP2 in multiple myeloma cell lines.

Our previous studies had shown that DAZAP2 was profoundly downregulated in bone marrow mononuclear cells from multiple myeloma patients. In this report, we analyzed epigenetic changes in multiple myeloma cell lines to understand the molecular mechanisms underlying the downregulation of DAZAP2. Four multiple myeloma cell lines, KM3, MM.1S, OPM-2 and ARH-77, were studied. The results of methylation specific PCR (MSP) showed that the promoter of DAZAP2 was methylated for KM3, MM.1S, OPM-2 and unmethylated for ARH-77. The DAZAP2 promoter region was amplified to obtain a series of different length sequences. All of the amplified sequences were inserted to luciferase reporter vector. The constructs were transfected into COS-7 cells and the luciferase activities were measured to search for the core region of DAZAP2 promoter. Two CpG islands were found in DAZAP2 promoter region. The results of luciferase assay showed that CpG island 1 displayed weak transcriptional activity, whereas CpG island 2 exhibited strong transcriptional activity (273 folds) compared to the control. The sequence that covered both CpG islands 1 and 2 showed higher activity (1,734 folds) compared to the control, suggesting that the two islands had synergistic effect on regulating DAZAP2 expression. We also found that M. Sss I methylase could inhibit the luciferase activity, whereas demethylation using 5-aza-2'-deoxycytidine treatment rescued the expression of DAZAP2 for multiple myeloma cell lines. These data revealed that methylation of DAZAP2 promoter was involved in downregulation of DAZAP2 in multiple myeloma cells.

The association between non-Hodgkin lymphoma and methylation of p73.

To investigate the effects of methylation of the p73 gene on the pathogenesis of non-Hodgkin lymphoma (NHL), the methylation status of the p73 gene promoter and the expression of p73 mRNA were examined in NHLs by methylation-specific polymerase chain reaction (MSP) and reverse transcription-polymerase chain reaction, respectively; p73 protein was detected by Western blotting analysis. Furthermore, the expression of p73 mRNA in NHL cells treated with 5-Aza-2'-deoxycytidine was analyzed. MSP results revealed that the promoter of p73 was methylated in 87.5% of NHLs but was not methylated in reactive hyperplasia lymph node samples. The expression of p73 mRNA was not detected in 83.33% of NHLs but was detected in all of the reactive hyperplasia lymph node samples. The p73 protein was not detected in 91.67% of NHLs but was detected in all of the reactive hyperplasia lymph node samples. The expression of p73 mRNA was detected in NHL cells treated with 5-Aza-2'-deoxycytidine. The inactivation of p73, predominantly by methylation, may be involved in the pathogenesis of NHLs.

Recombinant AAV-mediated HSVtk gene transfer with direct intratumoral injections and Tet-On regulation for implanted human breast cancer.

BACKGROUND: HSVtk/ganciclovir (GCV) gene therapy has been extensively studied in tumors and relies largely on the gene expression of HSVtk. Most studies, however, have failed to demonstrate any significant benefit of a controlled gene expression strategy in cancer treatment. The Tet-On system is commonly used to regulate gene expression following Dox induction. We have evaluated the antitumor effect of HSVtk/ganciclovir gene therapy under Tet-On regulation by means of adeno-associated virus-2 (AAV-2)-mediated HSVtk gene transfer with direct intratumoral injections in mice bearing breast cancer tumors. METHODS: Recombinant adeno-associated virus-2 (rAAV) was constructed and transduced into MCF-7 cell line. GCV treatment to the rAAV infected MCF-7 cells was performed by MTT assay under the doxycycline (Dox) induction or without Dox induction at a vp (viral particle) number of > or =10(4)/cell. The virus was administered intratumorally to nude mice that had also received GCV intraperitoneally. The antitumor effects were evaluated by measuring tumor regression and histological analysis. RESULTS: We have demonstrated that GCV treatment to the infected MCF-7 cells under the Dox induction was of more inhibited effects than those without Dox induction at > or =10(4) vp/cell. In ex vivo experiments, tumor growth of BALB/C nude mice breast cancer was retarded after rAAV-2/HSVtk/Tet-On was injected into the tumors under the Dox induction. Infiltrating cells were also observed in tumors after Dox induction followed by GCV treatment and cells were profoundly damaged. The expression of HSVtk gene in MCF-7 cells and BALB/C nude mice tumors was up-regulated by Tet-On under Dox induction with reverse transcription-PCR (RT-PCR) analysis. CONCLUSION: The antitumor effect of rAAV-mediated HSVtk/GCV gene therapy under the Dox induction with direct intratumoral injections may be a useful treatment for breast cancer and other solid tumors.

[The construction of recombinant AAV vector expressing HSVtk gene controlled by Tet-On and the detection of its activity].

In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.

[In vitro killing activity of the fractional proteins from microtus fortis serum to Schistosoma japonicum juveniles].

OBJECTIVE: To explore the killing effect of different fractional proteins from Microtus fortis (Mf) serum to S. japonicum juveniles, and to find possible association of the proteins with the natural resistance to schistosome infection. METHODS: The proteins in Mf serum were separated by means of ion-exchange column chromatography and molecular sieve column chromatography. After desalted by dialysis and lyophilized, the proteins were dissolved in DMEM medium which contained 300 U/ml penicillin and 300 microg/ml streptomycin, and the two-day old schistosomula were added in for in vitro cultivation (100 +/- 20/well). The killing activity of the fractional proteins to the juvenile worms was defined by mortality rate. RESULTS: 58 fractional proteins were separated from Mf serum, in which six proteins were confirmed to have a significant killing activity to schistosomula. The mortality of schistosomula all reached 37% and above, and the highest mortality (87.5%) was observed in the fraction 18.1, while the negative control was 25.09% (P < 0.01). CONCLUSION: Some fractional proteins in Microtus fortis serum show an effect in the natural resistance to schistosome infection.

Suicide gene therapy of human breast cancer in SCID mice model by the regulation of Tet-On.

BACKGROUND: RevTet-On gene expression system was used to deliver the suicide gene tk to human breast cancer cell line MCF-7 and control the tk gene expression level. The animal model of human breast cancer on severe combined immune deficiency (SCID) mice was set up to explore the suicide gene therapy by the regulation of Tet-On. METHODS: Herpes simplex virus-thymidine kinase (HSVtk) gene was inserted into the plasmid pRevTRE and the recombinant retroviral vector pRevTRE/HSVtk was constructed. Using modified calcium phosphate co-precipitation method, two transfections, pRevTRE/HSVtk and pRevTet-On were performed for MCF-7 cell line and selected by hygromycin B and G418. MCF-7 cell line that stably expressed Tet-regulated tk gene was established. HSVtk gene expression in the MCF/TRE/tk/Tet-On cell line was under the control of Doxycycline (Dox). Cell viability was also determined by MTT assay, whereas HSVtk gene expression was analyzed by reverse transcription-PCR (RT-PCR). RESULTS: MCF/TRE/tk/Tet-On cell survival rate was decreased from 100% to less than 20% when ganciclovir (GCV) concentration was increased from 0 to 1000 microg/ml at 1 microg/ml of Dox after 72 hours of GCV administration. At 1 microg/ml of GCV concentration, the cell numbers decreased from 7 x 10(4) cells/ml to 2 x 10(4) cells/ml when Dox concentration was increased from 0 to 1500 ng/ml after 72 hours culture. In addition, bystander effects were generated in vitro when 10% - 25% of transduced MCF-7 cells were mixed in untransduced MCF-7 cells. On the other hand, the human breast cancer models in SCID mice were set up. The tk gene was expressed with the regulated character after MCF/TRE/tk/Tet-On cells were implanted into the female SCID mice 7 days after Dox induction followed by intraperitoneally administration of GCV for 23 days. Subcutaneous tumors in SCID mice that were implanted with MCF/TRE/tk/Tet-On cells shrank remarkably after Dox and GCV administration as compared with the control. CONCLUSION: The human breast tumor cells (MCF-7) expressing HSVtk gene can be eradicated by administration of GCV and induced with tetracycline or its derivative Dox in vitro and in vivo.

[Retroviral vector-mediated HSVtk gene expression and acquisition of high titer recombinant virus].

OBJECTIVE: To explore the HSVtk gene expression mediated by the retroviral vector and to obtain high titer recombinant retroviral virus. METHODS: The recombinant vector pRevTRE/HSVtk was constructed by inserting HSVtk gene into pRevTRE. The recombinant retrovirus, which was produced from cloned PA317 cells screened by hygromycin B after "micro-pingpong" technique transferring with pRevTRE/HSVtk plasmids DNA by using modified calcium phosphate precipitation method. HSVtk gene expression was performed on target cells and virus titers were detected in different cultured temper, time and sodium butyrate concentration. RESULTS: The recombinant retroviral vector pRevTRE/HSVtk was constructed and HSVtk gene expression was detected on target cells after they were infected with the recombinant retrovirus. CONCLUSION: High titer of retroviruses could be obtained in the culture medium of PA317 cell line through "micro-pingpong" technique at 30 hours and 10 mmol/L sodium butyrate concentration followed by frozen ultrafiltration.

Identification of Novel Regulatory Elements of Human Stem Cell Factor Gene in MCF Cell.

Human stem cell factor(hSCF)is a pluripotent growth factor that regulates proliferation, differentiation and migration of certain mammalian stem cells, such as primordial germ cells etc. It is shown that hSCF and its receptor are commonly co-expressed in human breast cancer cells. Up to now, the definite regulatory mechanism of hSCF gene in breast cancer cells is unclear, except that its 5'flanking sequence contains essential elements for regulating transcription. To localize the regulatory elements responsible for the regulation of the hSCF gene, we performed transient transfection study in MCF cells, with a series of luciferase reporter gene constructs, containing different 5x end deletions of hSCF gene. This study indicates that the region of -1190 -853 significantly enhanced the luc gene expression, while the region of -339 -162 inhibited the expression. Eletrophoretic mobility shift assay confirmed that MCF nuclear extract proteins bound to both -1190 -853 and -339 -273 regions, forming specific DNA-protein complexes, indicating that there were nuclear protein binding sites in these regions. The results suggest that both -1190 -853 and -339 -273 DNA fragments of the hSCF 5'flanking sequence may be novel regulatory elements, and may play a role in the regulation of hSCF gene expression in MCF cells.