The Inhibition Of Retroviral Vector Containing DPC4/Samd4 Gene On Human Pancreatic Cacinoma Cells

1. Expression of DPC4/Smad4 in The Malignant and Nonmalignant Specimens of Human Pancreas The tumor suppressor DPC4 (deleted in pancreatic carcinoma, locus 4), which was identified by Scott Kern in 1996, is frequently lost in many tumor cells, especially in pancreatic cells. DPC4, also named as Smad4, belongs to the evolutionarily conserved family of Smad proteins that are crucial intracellular mediators of signals from the transforming growth factor β(TGF-β). Here, we determined the expression of DPC4 in 20 pancreatic adenocarcinoma and 18 nonmalignant pancreatic specimins by reverse transcription-polymerase chain reaction (RT-PCR) assay and immunohistochemistry respectively. The RT-PCR assay showed that positive rate of DPC4 mRNA accounted for 100% (18/18) in all normal specimens, whereas 40% (8/20) in adenocarcinoma specimens. The regional and intense positive case of the expression of DPC4 protein revealed by immunohistochemistry assay were 5 and 2 respectively and all positive rate accounted for 35%(7/20), whereas it was all positive expression in normal tissues. It was a significant difference of DPC4 expression between the normal and malignant pancreatic specimens, and suggested that loss of DPC4 maybe be involved in the development of the pancreatic carcinoma and a putative clinical diagnostic and prognostic indicator for pancreatic cancer. 2. Construction of The Retroviral Vectors Containing DPC4/Smad4. The human DPC4/Smad4 complementary DNA was amplified from Smad4/DPC4-pBluescript plasmid by PCR and subcloned to the retroviral vector pLXSN to obtain pLXSN/ DPC4/Smad4+ recombinant with direct inserting, and then packaged with GP+E86 and PA317 amphotropic packaging cells respectively. AntiG418 clones were acquired and named as PA317/ pLXSN DPC4+ cells. As a control, pLXSN also was packaged with GP+E86 and PA317 cells and the antiG418 clones were named as PA317/pLXSN. The virus titer was elevated through the cross infection from GP+E86 to PA317 cells and reached 6.0×105 pfu/L. The PCR assay confirmed there was DPC4/Smad4 gene integration in PA317/pLXSN DPC4+ cells, but PA317/pLXSN. This suggested that introduction of DPC4 gene to retroviral vectors was an effective way, which maybe can provide theoretical basis for the gene therapy of pancreatic cancer and other malignancy tumor. 3. Inhibition of DPC4/Smad4 on The Pancreatic Cancer Cells The retroviral vector pLXSN containing DPC4 which could be used to transfect the pancreatic carcinoma cells BxPC-3 was constructed, with the empty vector pLXSN as a positive control. AntiG418 clones were acquired and the daughter cells were named as BxPC-3/DPC4 and BxPC-3/pLXSN respectively. As a negative control, the parent BxPC-3 cells were named as BxPC-3/-. All the cells were identified by PCR and Western assay. The cell in vitro proliferation was assessed by MTT assay and colon-forming unit assay, the apoptosis and the cell cycle were assessed by flow cytometry, and the mRNA level of Chk1 and VEGF were detected by semi-quantitative PCR assay. We found the transfection and stable expression of DPC4 in the BxPC-3 cells could result in increase of the pancreatic cells in G1 phase, apoptosis induction, and inhibition of the cells growth. Rather, The semi-quantitative PCR showed that DPC4 could decrease the Chk1 and VEGF mRNA transcription levels. Our study suggested that the retroviral vector pLXSN contaiming DPC4 could inhibit the proliferation of the pancreatic cacinoma cells, arrest the cell cycle, and induce apoptosis dependant of down-regulation of Chk1, even without the presentation of TGF-β. It also down-regulated the expression of VEGF, which indicated that DPC4 could mediate tumor suppression through suppression of angiogenesis. 4. Combination Inhibition of DPC4 Gene Transfection and 5-Fu, Gemcitabine on Pancreatic Carcinoma Cells The daughter cell BxPC-3/DPC4 which had DPC4 stable expression was acquired after the pancreatic carcinoma cells BxPC-3 had been transfected with pLXSN/DPC4.The sensitivity of all cells for 5-Fu and gemcitabine were observed, and the 50% inhibiting concentrations of 5-Fu and gemcitabine for BxPC-3/DPC4 (culturing for 72h), where were calculated using curve-fitting parameters, were rather lower than the ones of BxPC-3/pLXSN and BxPC-3/-cells. Moreover, the semi-quantity PCR assay revealed that the mRNA level of Mdr-1 and Chk1 were down-regulated. Our experiment suggested that pLXSN/DPC4 vector, 5-Fu and gemcitabine could inhibit the growth of pancreatic cancer cells respectively. But the combination therapy of pLXSN/DPC4 vector and chemotherapy drugs could more inhibit the growth of cancer cells. The DPC4 gene transfection could enhance the sensitivity of pancreatic cells to chemotherapy drugs, which maybe be through the down-regulation of Mdr-1 and chk1 gene expression.