Interaction Between P16 Gene Promoter And DNA-binding Protein In Eca-109 Cells Induced By Differentiation Agents

8-Br-cAMP (8-bromo-cyclic 3', 5'-adenosine monophosphate, 8-Br-cAMP) may induce cell differentiation and suppress maliganant tumor growth through selective binding site with cAMP receptor bound to DNA . It has been reported that 8-Br-cAMP could up-regulate expression of wP53, iNOS and P21WAF1 and down-regulate that of EGFR, H-ras, c-myc and bcl-2. Quercetin is one kind of plant fiavonoid. It has been reported that quercetin could inhibit the tumor cell growth through repressing activity of proteinkinase C (PKC) and tyrosin kinase (TPK) and could up-regulate expression of Caspase-3, wP53 and iNOS and down-regulate VEGF, bcl-2, and c-myc. The DNA-binging proteins (DBP) play an important role in activity of oncogene and tumor suppressor gene. The changes of the nuclear matrix protein in human esophageal cancer have been reported, but the interaction between p16 gene promoter and nuclear matrix DBP in human esophageal cancer Eca-109 cells induced by 8-Br-cAMP and quercetin has not been reported.The cultured Eca-109 cells were divided randomly into three groups: (1) the control group was cultured only with DMEM (10% fetal bovine serum) ; (2) 8-Br-cAMP ( Br ) group was cocultured with a final concentration ,2 1 0-5mol/L of8-Br-cAMP for 48h; (3) quercetin ( Q ) group was cocultured with 43Hmol/L final concentration of quercetin for 48 h. The cell suspension with concentration of 2X 106/ml was droped onto the treated slids for each group. The nuclear matrix DNA-binging proteins and extranuclear DNA-binging proteins were extracted from Eca-109 cells respectivly for SDS-PAGE and Southwestern blot.P16-promoter-luc-pGL2-Basic plasmid DNA was prepared after plasmid amplification, and digested by EcoR I and Hind III. The 890 bp fragment was purified and labelled by random priming with Biotin. The probe sensitivity was detected by DNA dot blot.The nuclear matrix proteins and extranuclear DBFs were separated by SDS-PAGE and electrotransferred onto the nitrocellulose membrane for Southwestern blot. The bands were analyzed by Genetools software.Modified in situ hybridization was used for DBF localization in cells; The immunoreactivity(IR) of PCNA was detected by immunocytochemistry; The expression of pi 6 mRNA was detected by in situ hybridization.Results:1. The sensitivity of biotin-labelled probe was up to Ipg and the specificity qualified.2. SDS-PAGE: The molecular weight of nuclear matrix proteins(NMP) were 66-31KD and lower than 30KD; Compared with the control group, 58KD, 64KD and 66KD NMPs appeared in Br and Q groups, and the expression level of 14KD, 17KD and 18KD NMPs in Br and Q groups increased, while that of 92KD NMP in Br and Q groupes decreased. The bands of extranuclear proteins were much more than that of NMPs.3. Southwestern blot: The hybridization signals were stronger in Br and Q groups than that in the control group (P<0.05), and the hybridization signals were stronger in Br group than that in Q group (P<0.05). On the NCM there were three major bands ( 66KD, 58KD and 20KD ) in the NMP lanes, while 33KD, 28KD, 20KD and 14KD bands exhibited in the extranuclear DBF lanes.4. Modified in situ hybridization technique (DBF localization): The signals of blueviolet color were located mainly in nuclei, signals also could be observed in the cytoplasm and cytomembrane of some cells. The difference between the control and Br or Q group was significant.5. The PCNA-IR in brownish color was located in the nuclei, the PCNA-IR was weaker in Br and Q groups than that in the control group ( PO.05 ).6. The PCNA-IR in protein dot blot was weaker in Br and Q groups than that in the control group ( PO.05 ).7. The in situ hybridization signals localized in the cytoplasm appeared as blue violet granules. The hybridization signals were stronger in Br and Q groups than that in the control group (P<0.05 ).Conclusion:1. 8-Br-cAMP and quercetin could induce changes in the DBF of Eca-109 cells, and the 869bp fragment in the upstream of exonl a of pi 6 gene may play an impor...