Experimental Study On Adenovirus Mediated P27mt Gene Therapy In Extrahepatic Cholangiocarcinoma

Objective Skp2 overexpression may be involved in carcinogenesis andprogression of in primary breast cancer , human gastric carcinoma, prostate cancer in vivo, possibly via P27kipl proteolysis. To investigate the significance of S phase kinase-associated protein 2 (Skp2) and cell cycle regulatory proteins P27kipl expression in human cholangiocarcinoma and the relationship with clinicopathologic features. The abundance of P27kip1, an inhibitor of cell proliferation, is controlled by Skp2-dependent proteolysis.Methods The expressions of Skp2 and P27^kip1 were examined in 43 samples ofcholangiocarcinoma tissues with pathological confirmation and 10 specimens of bile duct with chronic inflammation were determined with immunohistochemical.Results The expression rates of SKP2 and P27^kip1 were 34.88%(15/43) and30.23%(13/43) respectively in cancerous tissues, but 10%(0/10) and 80.00%(8/10) in normal bile duct tissues with chronic inflammation. The expressions of SKP2 and P27^KIP1 had a significantly correlation with high tumor grade and clinical stage of cholangiocardinoma.Conclusions1. Skp2 may regulate the expression of P27^kip1 by negatively regulating Skp2expression.2. Our results suggest that aberrant expressions of Skp2 and P27^kip1 may be associated with the incidence and development of cholangiocarcinoma.3. The Skp2 and P27^kip1 protein level can be regarded as prognostic markers for cholangiocarcinoma.Part II: P27^kip1 Interaction with the SCF Component Skp2in QBC939 cellsThesis 3: Expression of SKP2 by transfecting adenovirus vectormediated P27^kip1 into cholangiocarcinoma QBC939 Cell Line Thesis 4: The Effects of Skp2 Antisense Oligodeoxynucleotides (ASODN) on the Expression of P27^Kip1 in Cholangiocarcinoma QBC939 Cell LineObjective To investigate the effect on the expression of P27^kip1 and Skp2 geneby transfecting SKP2 antisense oligodeoxynucleotides and mutated P27^kip1 into cholangiocarcinoma cells, which has a mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) respectively.Skp2 and P27^kip1 may be involved in carcinogenesis and progression of human cholangiocarcinoma in vivo, possibly via P27^krp1 proteolysis. Ubiquitin proteasome pathway may regulate the expression of P27^kip1 by Skp2 expression.Methods Expression of SKP2 and P27^kip1 and was analyzed and cell growthwas observed QBC939 cell. The expression levels of mRNA and protein were determined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively.Results1.Western blot and RT-PCR assay for Skp2 and P27^kip1 in proteins extracted from Ad-P27mt transduced cholangiocarcinoma cells showed overexpression of Skp2 and P27^kip1 protein than control groups(P<0.05)2.By treatment with SKP2 ASODN, the level of expression both Skp2 mRNA and protein were down-regulated. There was a lower expression of Skp2 mRNA and protein in ASODN treatment group than control groups(P<0.05).3.Although P27^kip1 mRNA remained unchanged, its protein level was up-regulated than control groups(P<0.05).Conclusion1. There is enhanced ubiquitination and P27^kip1 proteolysis by the proteasome in the QBC939 cell line. We speculate that may be an abnormality in the lysosomal degradation pathway or related components.2. The enhanced ubiquitin-proteosome pathway is important for selective degradation of short-lived protein P27^kip1 in cholangiocarcinoma cells. Targeting of the cyclin-dependent kinase inhibitor P27^kip1 for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex.The level of expression of P27^kip1 mRNA and protein expression predominantly increase in QBC939 cell lines by blocking ubiquitin proteosome pathway.3. Skp2 ASODN can inhibit the level of expression of Skp2 mRNA and protein expression predominantly in QBC939 cell lines by blocking ubiquitin proteosome pathway.Although P27^kip1 mRNA level remaine unchanged, its protein level was up-regulated. From these results, we conclude that P27^kip1 is regulated predominantly by posttranslational mechanisms mediated by the ubiquitin-proteasome pathway.4. By transfecting adenovirus vector mediated P27^kip1 into cholangiocarcinoma cells, the mRNA and protein level of expression of Skp2 and P27^kip1 gene increase predominantly in QBC939 cell lines.It shows that overexpression of P27^kip1 can regulate Skp2 by positive feedback. We conclude that Skp2 is regulated by both transcription and posttranscription mechanisms.5. P27^Kip1 proteolysis, which is triggered by its phosphorylation on threonine (Thr)187. The P27^kip1 gene mutates in this point would decrease dramaticly the expression of Skp2.6. From these results, we P27^kip1 and Skp2 gene may be potentially useful for gene therapy against cholangiocarcinoma.Part III: Adenovirus Expressing Mutant P27^kip1 on Gene Therapy for Cholangiocarcinoma.Thesis 5: The effect of Adenovirus Expressing Mutant P27^kip1 on the proliferation of Cholangiocarcinoma Cell line QBC939 in vivo andvitroThesis 6: Sensitivities of Cholangiocarcinoma Cells Transfected by Exogenous P27^kip1 Gene to ChemotherapyObjective To investigate the effect on cell cycle and apoptosis and chemosensitivity in vivo and vitro by transfecting mutated P27^kip1 intocholangiocarcinoma cells QBC939 line, which has a mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188(ATGATC).Methods The recombinant vector was constructed and the mutated P27^kip1 genewas transfected into human cholangiocarcinoma cell line (QBC₉₃₉). Expression of P27^kip1 was analyzed by RT-PCR, Western blot. And the effects of 5-fluorouracil (5-FU), mitomycin (MMC) and cyclophosphamide(CTX) on the transfected cells were detected by assaying the rate of apoptosis and growth inhibition by methabenzthiazuron (MTT) assay and flow cytometry (FCM).Results1. Expression of P27^kip1 in protein and mRNA increased significantly in QBC939 cell line transfected with Ad-P27mt.2. P27^kip1 over expression caused the cell growth suppression in vivo and in vitro and cell cycle arrest, apoptosis.3. The overexpression P27^kip1can remarkably increase the drug sensibility of the cholangiocarcinoma cells. Which enhanced the growth inhibition of QBC₉₃₉ inducted by 5-FU, MMC and CTX. and the ratio of growth from 41.89%(5-FU) , 45.59%(MMC) 38.91%(CTX) to 56.15%(5-FU) , 55.65%(MMC) , 51.69%(CTX) and apoptosis index from 16.76%(5-FU), 13.63%(MMC), 10.46%(CTX) to 44.39%(5-FU), 37.46%(MMC), 36.94% (CTX) (P<0.01).Conclusions1. P27^kip1 may cause cell cycle arrest in G0/G1 phase and subsequently lead to apoptosis. Thus recombinant adenovirus expressing mutant P27^kip1 may be potentially useful for gene therapy against cholangiocarcinoma.2. The exogenous P27^kip1 gene transfer can remarkably increase the drug sensibility of the cholangiocarcinoma cells. The combination of P27^kip1 gene therapy with chemotherapy may be more effective in cancer treatment.