Bold R J, Warren R E, Ishizuka J, Cho-Chung Y S, Townsend C M, Thompson J C
Department of Surgery, University of Texas Medical Branch, Galveston 77555-0533.
Surgery. 1994 Aug;116(2):189-95; discussion 195-6.
Gastrin regulates growth of human colon cancer cells by activation of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA). Gastrin and 8-Br-cAMP, a membrane-permeable cAMP analog, inhibit growth of HCT116 cells; both stimulate growth of LoVo cells. This dual effect on growth may be explained by relative amounts of the regulatory subunit (RI alpha or RII beta) of PKA within the cancer cells. Antisense oligodeoxynucleotides (ASO) to either RI alpha or RII beta inhibit protein translation of the target mRNA by sequence-specific binding; subsequently, cellular PKA content and the cAMP-mediated growth may be altered. We determined whether ASO to either the RI alpha or RII beta subunit altered the cAMP-mediated growth of HCT116 and LoVo human colon cancer cells.
HCT116 cells were treated with RII beta ASO (15 mumol/L, 4 days) and then treated with 8-Br-cAMP (25 mumol/L); tritiated thymidine incorporation was measured after 24 hours, and the cell number was determined on alternate days. Protein and mRNA levels of the RII beta subunit were determined by Western and Northern blotting, respectively. Similar studies with an ASO against the RI alpha subunit were performed on LoVo cells.
RII beta ASO reversed the cAMP-mediated inhibition of growth of HCT116 cells, and RII beta ASO decreased the protein level of the RII beta subunit. RII beta ASO did not alter the basal growth of HCT116 cells. RI alpha ASO reversed the cAMP-mediated stimulation of growth of LoVo cells.
The regulatory subunits of PKA are potential targets to alter growth of human colon cancer cells. Gene therapy directed to alter specific steps in signal transduction pathways may provide new therapeutic strategies.
胃泌素通过激活环磷酸腺苷（cAMP）依赖性蛋白激酶A（PKA）来调节人结肠癌细胞的生长。胃泌素和8-溴-cAMP（一种可透过细胞膜的cAMP类似物）可抑制HCT116细胞的生长；二者均可刺激LoVo细胞的生长。对生长的这种双重作用可能由癌细胞内PKA调节亚基（RIα或RIIβ）的相对含量来解释。针对RIα或RIIβ的反义寡脱氧核苷酸（ASO）通过序列特异性结合抑制靶mRNA的蛋白质翻译；随后，细胞内PKA含量及cAMP介导的生长可能会发生改变。我们确定针对RIα或RIIβ亚基的ASO是否会改变HCT116和LoVo人结肠癌细胞的cAMP介导的生长。
用RIIβ ASO（15 μmol/L，处理4天）处理HCT116细胞，然后用8-溴-cAMP（25 μmol/L）处理；24小时后测量氚标记胸腺嘧啶核苷掺入量，并隔天测定细胞数量。分别通过蛋白质免疫印迹法和Northern印迹法测定RIIβ亚基的蛋白质和mRNA水平。对LoVo细胞进行了针对RIα亚基的ASO的类似研究。
RIIβ ASO逆转了cAMP介导的HCT116细胞生长抑制，且RIIβ ASO降低了RIIβ亚基的蛋白质水平。RIIβ ASO未改变HCT116细胞的基础生长。RIα ASO逆转了cAMP介导的LoVo细胞生长刺激。
PKA的调节亚基是改变人结肠癌细胞生长的潜在靶点。针对信号转导通路中特定步骤进行改变的基因治疗可能会提供新的治疗策略。
