Objective To explore the expression and activation of transcription factor E2F1 in cultured human retinal pigment epithelium (RPE) cells. Methods Cultured human RPE cells were divided into two groups after synchronization: one was cultured in Dulbecco′s modified Eagle′s medium (DMEM) without serum; the other was cultured in DMEM supplemented with 20% serum of newborn calf. The expressions of E2F1 protein in two groups were detected by Western blot analysis. The E2F1-DNA binding activities were measured by gel mobility-shift assay(EMSA). Results E2F1 protein of 60 000 molecular weight was detected in the nuclear extract of human RPE cells, and serum stimulation could increase its expression（P＜0.001）. EMSA exhibited the increased binding activity of E2F1 in the serum-stimulated RPE cells with DNA. Conclusions E2F1 is expressed in the nuclei of human RPE cells. Serum stimulation can increase its protein expression as well as binding activity, so as to play a regulation role of gene transcription. (Chin J Ocul Fundus Dis, 2002, 18: 224-226)
Objective To investigate the expression of tumor suppressor gene Lumican mRNA in gastric cancer and its role in the development of gastric cancer. Methods The expressions of Lumican mRNA in gastric cancer tissues, tissues near tumor and normal tissues were detected by using reverse transcription polymerase chain reaction (RT-PCR), clinical pathology for those tissues was studied as well. Results The expression absence rates of Lumican mRNA were 42.4% (28/66), 15.2% (10/66) and 0 in gastric cancer tissues, tissues near tumor and normal tissues respectively. The expression absence rate of Lumican mRNA in patients with lymph node metastasis was 61.1%, which was significantly higher than that of the patients without lymph node metastasis (20.0%, χ2=11.323, P=0.001). The expression absence rate of Lumican mRNA in the gastric cancer at the advanced stages (stages Ⅲ, Ⅳ) was 61.5%, which was significantly higher than that in the gastric cancer at the early stages (stages Ⅰ, Ⅱ, 30.0%, χ2=6.417， P=0.011). The expression absence rates of Lumican mRNA in the high, moderate and poor differentiation tumors were 38.5% (10/26), 38.5% (10/26) and 57.1% (8/14) respectively, the expression absence rate of Lumican mRNA was no significant association with the differentiation degree (χ2=1.576, P=0.455). Conclusion The expression absence of tumor suppressor gene Lumican mRNA may play an important role in the tumorgenesis and influences the prognosis of gastric cancer.
To study the significance of T-lymphocytes rDNA transcription activity in diagnosis, differential diagnosis, therapeutical effect and evaluation of treatment for colorectal carcinoma, 59 cases of colorectal carcinoma, 20 cases of colorectal inflammatory disease and 9 volunteers were choosen to detect the T-lymphocyte rDNA transcription activity of peripheral blood T-lymphocyte by cell culture and CMIAS008 image analysis system of Ag-NOR. Results: T-lymphocytes rDNA transcription activity was decreased obviously in colorectal inflammatory patients. Compared with control group, both group showed markedly statistical difference (Ｐ＜0.01). Tlymphocytes rDNA transcription activity increased gradually to normal groups after operation and chemical treatment for colorectal carcinoma patients; but it decreased for recurrent patients three years after operation. Conclusions: The detection of T-lymphocytes transcription activity can be used as a differential criterion for colorectal carcinoma and colorectal inflammatory disease, meanwhile it also can be used as a reference criterion for evaluation of treatment and supervision of tumor recurrence.
Objective To screen the possible regulatory proteins showing the ability for interaction with serum response factor ( SRF) in the progress of myofibroblast activation, and to see if the proteinprotein interaction is contributing to induce the expression of smooth muscle αactin ( α-SMA) . Methods Phage display cDNA libraries were constructed from the transdifferentiated airway epithelial cells and parental cells. Phage clones were then selectively amplified during the biopanning procedure by using SRF as a bait protein for the two cDNA libraries. Following four rounds of biopanning, recovered cDNAs were sequenced and the obtained sequences were aligned by BLAST tool to select the candidate gene. PAI-RBP1 of the candidate gene was cloned and sub-cloned into pcDNA3. 0 plasmid. Transient transfection and RT-PCR analysis were performed for investigation of the expression of α-SMA. Results Three candidate proteinbinding partners, PAI-RBP1, Nucleolin, and HF1OO, were identified. Among them, PAI-RBP1 pcDNA3. 0 plasmid was subjected to transient co-transfection with SRF, showing up-regulation of α-SMA expression. Conclusions Combined with phage display technique, through protein-protein interaction between core transcription factor and unknown proteins to find a newtranscriptional regulator may serve as an effective strategy. Three novel SRF binding proteins were found from transdifferentiated cells. This study indicates that PAI-RBP1 involves in the activation of myofibroblast by induction of α-SMA expression.
Objective To elucidate the role of the transcription factor liver activator protein (LAP, a member of the C/EBP family) in the expression of α1(I) collagen gene in activated hepatic stellate cells (HSCs). Methods Rat HSCs were prepared from SD rats by in situ perfusion and singlestep density Nycodenz gradient. Two chimeric luciferase reporter gene plasmids containing the human collagen α1(I) gene promoter fragments (-804～+1 452 or -804～+222) were constructed. Culture-activated HSCs were co-transfected with the reporter gene contructs and mammalian vector expressing LAP using the cationic-liposome mediated method, and the promoter activity was determined by measuring luciferase activity. Results The luciferase reporter gene construct containing the first intron of α1(I) collagen gene (-804～+1 452, was called as PGL3-col) had a higher level of gene expression, as compared with the construct lacking the first intron 〔was called as PGL3-col (△intron)-in activated HSCs (315±45 U/mg protein vs 220±70 U/mg protein, Ｐ＜0.05). Transient transfection of the vector expressing LAP significantly increased basal transcription from PGL3-col and PGL3-col (△intron) reporter gene vectors (587±62 U/mg protein vs 315±45 U/mg protein and 326±52 U/mg protein vs 220±70 U/mg protein respectively, both Ｐ＜0.05). Conclusion The transcription factor LAP transactivates collagen α1(I) gene in activated HSCs, and the first intron is important for α1(I) collagen gene transcription activity in activated HSCs.
ObjectiveTo summarize the role of epithelial-mesenchymal-transition （EMT） in occurrence and development of gastrointestinal cancer. MethodsDomestic and international publications online involving EMT of gastrointestinal cancer in recent years were collected and reviewed. ResultsEMT was a highly conserved process that has been well characterised in embryogenesis. Studies had shown that the aberrant activation of EMT in adult epithelia could promote tumour metastasis by repressing cell adhesion molecules. E-cadherin， one of the epithelial cell markers, maybe involved in the process of the EMT, especially of the Ecadherin transcriptional repressors， these transcriptional repressors significantly increased in the gastrointestinal cancer. Further more， EMT might involve in the process of gastrointestinal cancer stem cells formation. ConclusionsEMT and it’s regulators play a very important role in gastrointestinal cancer， and may provide a newsight into the gastrointestinal cancer. It also can provide a novel clinical targets to treat the gastrointestinal cancer.
Objective To investigate the inhibitive effect of E2F decoy oligodeoxynucleotides (E2F decoy ODNs) on cultured human retinal pigment epithelial (HRPE) cells.Methods E2F decoy ODNs or scramble decoy ODNs at varied concentrations were put into the HRPE cells mediated by lipofectamineTM2000. The proliferative activity of HRPE was detected by methythiazolyl-terazollium assay, and the competitive combinative activity of E2F decoy ODNs and transcription factor E2F was detected by electrophoresis mobility-shift assay. Results The proliferation of HRPE was inhibited markedly by E2F decoy ODNs at the concentration of 0.2 μmol/L (P=0.002) in a dose-dependent manner but not by scrambled decoy. The results of electrophoresis mobility-shift assay showed that the combinative activity of transcription factor E2F was abolished completely by E2F decoy ODNs. Conclusions E2F decoy ODNs may sequence-specifically inhibit the combinative activity of transcripti on factor E2F,and inhibit the proliferation of HRPE cells.(Chin J Ocul Fundus Dis，2004，20：182-185)
ObjectiveTo determine the optimizing parameters in transfecting the SV-40-PED cells mediated by oligofectamine. Methods With a change of Decoy oligodeoxynucleotides(ODNs)/oligofectamine in ratio and the transfection time, the uptake rate and the mean fluorescence intensity of SP1 ODNs in the SV-40-PED cells were measured by flow cytometry to evaluate the transfection efficiencies. 4 μl oligofectamine with different concentrations of ODNs(2.5，5.0，7.5，10.0 and 12.5 μl) were put into 100 μl of DMEM without serum and antibiotics. the (SV-40-PED) cells were transfected after 20 min at room temperature. the final concentration of SP1 decay ODNs were 50，100，150，200 and 250 nmol/L. Transfection effieiency was detected at 26 h after transfection. The intracellular distribution ofSP1 ODNs was determined with a fluorescence microscope. The lactate dehydrogenase (LDH) activity in the supernatant was measured to assess the cytotoxicity.Results The uptake of SP1 ODNs into the SV-40-PED cells was significantly improved by oligofectamine. The cell appearance did not change much in the groups of 50, 100 and 150 nmol/L. In the groups of 200 and 250 nmol/L, the cell reverted after being shrinked and altered to round. At 26 h after the transfection, there was no marked change in the cell form at the concentration of 250 nmol/L. There was floatation at 48 and 72 h after the transfection. Under the fluorescence microscope, we observed fluorescent materials distributed in the cell nucleus in the successfully-transferred groups. We could see the nucleoli clearly in the groups of 200 nmol/L and 250 nmol/L. There was a ber fluorescence intensitywith a higher concentration and the fluorescent materials gathered at the cell nucleus. At the final concentration of 250 nmol/L, the LDH level was 137.12±3.92 U/L in the 72hgroup, which was significantly higher those that in the 26h group（49.61±17.13 U/L）and the 48h group（120.26±8.42 U/L）(Plt;0.01). At 26 h after the transfection, there were no statistical differences at the above LDHlevels in the different-concentration groups（Pgt;0.05）. Conclusion Transfection efficiency is the highest when the final concentration of the SP1 decoy ODNs is 250 nmol/L during the incubation of for 24 h in transfecting the SV-40-PED cells.
Objectives To investigate the expressions and significance of E2F1, ID1, and Bax protein in gallbladder adenocarcinoma tissues. MethodsThe expressions of E2F1, ID1, and Bax protein in 70 cases of gallbladder adenocarcinoma, 20 cases of high level intraepithelial neoplasia, 30 cases of low level intraepithelial neoplasia, and 20 cases of cholecystitis tissues were tested by using immunohistochemical method. ResultsThe positive expression rates of E2F1, ID1, and Bax protein in gallbladder adenocarcinoma was 84.3%, 70.0%, and 25.7%, respectively; the positive expression rates in high level intraepithelial neoplasia was 75.0%, 65.0%, and 55.0%, respectively; the positive expression rates in low level intraepithelial neoplasia was 16.7%, 23.3%, and 56.7%, respectively; and the positive expression rates in cholecystitis tissues was 10.0%, 20.0%, and 75%, respectively.The positive expression rates of E2F1 and ID1 protein in gallbladder adenocarcinoma were significantly higher than those intraepithelial neoplasia and cholecystitis tissues (P < 0.05), but the positive expression rate of Bax protein in gallbladder adenocarcinoma was lower (P < 0.05).The expressions of E2F1 and ID1 protein were significantly correlated with clinical Nevin staging of gallbladder adenocarcinoma (P < 0.05), but not correlated with the gallbladder adenocarcinoma differentiation degree (P > 0.05).The expression of Bax protein was related to the gallbladder adenocarcinoma differentiation degree (P < 0.05), but not correlated with clinical Nevin staging (P > 0.05).The expression of E2F1 protein was negatively correlated with expression of Bax protein (r=-0.375, P < 0.05), ID1 protein expression has nothing to do with the protein expression of Bax protein (P > 0.05).The expression of E2F1 protein was positively correlated with ID1 protein (r=7.031, P < 0.05). ConclusionsThe E2F1, ID1, and Bax may play an important role in the generation and development of the gallbladder adenocarcinoma.The combined detection of E2F1, ID1, and Bax have important guiding significance for auxiliary diagnosis and clinical staging of gallbladder adenocarcinoma.
Objective To investigate the expression of transcription factor e2f-1 in the different development stages of gastric cancer, the relationships between clinicopathologic characteristics and e2f-1 expression status, as well as its influences on the prognosis. Methods The operative samples from primary lesion of 121 patients who underwent radical resection for gastric cancer were detected by SABC immunohistochemical staining. The relationships of e2f-1 expression with clinicopathologic characteristics and with the prognosis were observed by univariate, multivariate and relative analyses. Results The total positive expression rate of e2f-1 in all patients was 38.8% （47/121）. With the advancement of gastric cancer, the level of e2f-1 expression in TNMⅠ-Ⅳ stage was gradually decreased (r=－0.320， Plt;0.05)： Ⅰa stage with 62.5% （10/16）， Ⅰb with 47.1% （8/17）， Ⅱwith 55.0% （11/20）， Ⅲa with 40.0% （8/20）， Ⅲb with 27.3% （6/22）， Ⅳ with 15.4% （4/26）. The expression of e2f-1 was significantly negative correlated with tumor diameter, depth of infiltration, lymph node metastasis ratio, and N stage （Plt;0.05）. The multivariate analysis revealed that either histology type, or survival time was respectively an independent factor for e2f-1 expression （Plt;0.05）. Log-Rank test showed the relative factors to survival included N stage, tumor diameter, tumor position, lymph node metastasis ratio, depth of infiltration, and TNM stage （Plt;0.05）. Cox survival analysis found that both of later N stage and e2f-1 higher expression were independent prognostic factors （Plt;0.05）. The higher e2f-1 expression was related to a poor survival in TNM stageⅠand Ⅱ patients （r=－0.304, Plt;0.05）, the prognosis of patients with e2f-1 positive expression was worse than that of patients with negative expression （χ2=13.437， Plt;0.05）, and there was no statistic relationship between the expression of e2f-1 and prognosis in stage Ⅲ and Ⅳ patients （Pgt;0.05）. Conclusions e2f-1, as a useful marker, seems to be an indication for the malignant behavior in relatively earlier gastric cancer, in which the e2f-1 positive expression shares a significantly poor survival. And the lower expression of e2f-1 has been identified in later advanced gastric cancer, the more malignances in advanced gastric cancer might associate with a lower expression of e2f-1.