中国癌症杂志
中國癌癥雜誌
중국암증잡지
CHINA ONCOLOGY
2010年
1期
6-11
,共6页
桑梅香%刘丽华%丁春艳%孟君%单保恩
桑梅香%劉麗華%丁春豔%孟君%單保恩
상매향%류려화%정춘염%맹군%단보은
p1k3%p73%H1299细胞系%荧光素酶报告剂分析
p1k3%p73%H1299細胞繫%熒光素酶報告劑分析
p1k3%p73%H1299세포계%형광소매보고제분석
P1k3%p53%H1299 cell line%luciferase reporter assay
背景与目的:研究表明蛋白激酶P1k3可以增加肿瘤抑制因子p53的转录活性,但对p73转录活性的影响尚不清楚,因此本实验旨在研究蛋白激酶P1k3对p73转录活性的影响.方法:选用p53缺失型人类肺癌细胞系H1299,采用荧光素酶报告剂分析、反转录聚合酶链反应(RT-PCR)及克隆形成实验的方法研究蛋白激酶P1k3及激酶活性缺失的P1k3(K52R)对p73转录活性的影响.结果:荧光素酶报告剂分析结果显示,单独转染p73基因可以增加p21~(WAF1)和Bax启动子介导的荧光素酶的表达(P<0.05);共转染p73基因和P1k3基因,p21~(WAF1)和Bax启动子介导的荧光素酶的表达与单独转染p73基因组相比显著降低(P<0.05),且呈浓度依赖性;而激酶活性缺失的Plk3(K52R)对p21~(WAF1)和Bax启动子介导的荧光素酶的表达没有显著影响(P>0.05).RT-PCR检测结果也显示,p73诱导p2~(WAF1)和Bax的mRNA表达(P<0.05),P1k3降低了p73诱导的p21~(WAF1)和Bax的mRNA表达水平(P<0.05);而激酶活性缺失的P1k3(K52R)对p73诱导的p21~(WAF1)和Bax的mRNA表达水平无显著影响(P>0.05).克隆形成实验结果显示,p73抑制了H1299细胞克隆的形成(P<0.05),P1k3降低了p73对H1299细胞克隆形成的抑制(P<0.05),而激酶活性缺失的P1k3(K52R)对p73抑制H1299细胞克隆形成无显著影响(P>0.05).结论:P1k3可以抑制p73的转录活性,而激酶活性缺失的P1k3(K52R)对p73的转录活性无明显影响.
揹景與目的:研究錶明蛋白激酶P1k3可以增加腫瘤抑製因子p53的轉錄活性,但對p73轉錄活性的影響尚不清楚,因此本實驗旨在研究蛋白激酶P1k3對p73轉錄活性的影響.方法:選用p53缺失型人類肺癌細胞繫H1299,採用熒光素酶報告劑分析、反轉錄聚閤酶鏈反應(RT-PCR)及剋隆形成實驗的方法研究蛋白激酶P1k3及激酶活性缺失的P1k3(K52R)對p73轉錄活性的影響.結果:熒光素酶報告劑分析結果顯示,單獨轉染p73基因可以增加p21~(WAF1)和Bax啟動子介導的熒光素酶的錶達(P<0.05);共轉染p73基因和P1k3基因,p21~(WAF1)和Bax啟動子介導的熒光素酶的錶達與單獨轉染p73基因組相比顯著降低(P<0.05),且呈濃度依賴性;而激酶活性缺失的Plk3(K52R)對p21~(WAF1)和Bax啟動子介導的熒光素酶的錶達沒有顯著影響(P>0.05).RT-PCR檢測結果也顯示,p73誘導p2~(WAF1)和Bax的mRNA錶達(P<0.05),P1k3降低瞭p73誘導的p21~(WAF1)和Bax的mRNA錶達水平(P<0.05);而激酶活性缺失的P1k3(K52R)對p73誘導的p21~(WAF1)和Bax的mRNA錶達水平無顯著影響(P>0.05).剋隆形成實驗結果顯示,p73抑製瞭H1299細胞剋隆的形成(P<0.05),P1k3降低瞭p73對H1299細胞剋隆形成的抑製(P<0.05),而激酶活性缺失的P1k3(K52R)對p73抑製H1299細胞剋隆形成無顯著影響(P>0.05).結論:P1k3可以抑製p73的轉錄活性,而激酶活性缺失的P1k3(K52R)對p73的轉錄活性無明顯影響.
배경여목적:연구표명단백격매P1k3가이증가종류억제인자p53적전록활성,단대p73전록활성적영향상불청초,인차본실험지재연구단백격매P1k3대p73전록활성적영향.방법:선용p53결실형인류폐암세포계H1299,채용형광소매보고제분석、반전록취합매련반응(RT-PCR)급극륭형성실험적방법연구단백격매P1k3급격매활성결실적P1k3(K52R)대p73전록활성적영향.결과:형광소매보고제분석결과현시,단독전염p73기인가이증가p21~(WAF1)화Bax계동자개도적형광소매적표체(P<0.05);공전염p73기인화P1k3기인,p21~(WAF1)화Bax계동자개도적형광소매적표체여단독전염p73기인조상비현저강저(P<0.05),차정농도의뢰성;이격매활성결실적Plk3(K52R)대p21~(WAF1)화Bax계동자개도적형광소매적표체몰유현저영향(P>0.05).RT-PCR검측결과야현시,p73유도p2~(WAF1)화Bax적mRNA표체(P<0.05),P1k3강저료p73유도적p21~(WAF1)화Bax적mRNA표체수평(P<0.05);이격매활성결실적P1k3(K52R)대p73유도적p21~(WAF1)화Bax적mRNA표체수평무현저영향(P>0.05).극륭형성실험결과현시,p73억제료H1299세포극륭적형성(P<0.05),P1k3강저료p73대H1299세포극륭형성적억제(P<0.05),이격매활성결실적P1k3(K52R)대p73억제H1299세포극륭형성무현저영향(P>0.05).결론:P1k3가이억제p73적전록활성,이격매활성결실적P1k3(K52R)대p73적전록활성무명현영향.
Background and purpose: Protein kinase P1k3 could increase the transcriptional activity of p53. However, the effect of P1k3 on the transcriptional activity of p73 is still unknown. Our study was to investigate the effect of P1k3 on the transcriptional activity of p73. Methods: Luciferase reporter assay, RT-PCR and colony formation assay were adopted to study the effect of P1k3 and kinase-deficient P1k3 (K52R) on the transcriptional activity of p73 in p53-deficient human lung carcinoma H1299 cells. Results: Luciferase reporter assay showed that p73 increased the luciferase activities induced by p21~(WAF1) and Bax promoters (P<0.05). After co-transfection with p73 and P1k3, the luciferase activities induced by p21~(WAF1) and Bax promotors were significantly decreased in a dose-dependent manner as compared with the group that transfected p73 only (P<0.05). However, kinase-deficient Plk3 (K52R) had no significant effect on the luciferase activities induced by p21~(WAF1) and Bax promoters (P>0.05). RT-PCR showed that p73 increased the mRNA expressions of p21~(WAF1) and BAX (P<0.05). P1k3 decreased the expressions of p21~(WAF1) and Bax induced by p73 (P<0.05). Kinase-deficient P1k3 (K52R) had no significant effect on the expressions of p21~(WAF1) and Bax induced by p73 (P>0.05). Colony formation assay revealed that p73 decreased the colony formation of H1299 cells (P<0.05). P1k3 decreased the inhibitory effect of p73 on the colony formation of H1299 cells (P<0.05). Kinase-deficient P1k3 (K52R) had no significant effect on the inhibitory effect of p73 on the colony formation of H1299 cells (P>0.05).Conclusion: P1k3 can inhibit the transcriptional activity of p73, where as kinase-deficient P1k3 has no effect on the transcriptional activity of p73.
