1.THE EFFECTS OF db-cAMP ON CaM LEVEL,PK Ⅱ ACTIVITY AND INHIBITION OF TRANSFORMED CELLS PROLIFERATION
Acta Anatomica Sinica 1953;0(01):-
Objective To study the mechanism of the effects of db cAMP on the inhibition of cell proliferation and phenotypic changes in transformed cells. Methods The progression of cell cycle was analyzed by the flow cytometry.The transforming phenotype of C 3H 10 T 1/2 cells was observed by the soft agar colony formation.Northern blotting and radioimmunoassay were used to analyze the level of CaM expression.The activity of PKⅡ was determined through ? 32 p ATP incorporation assay. Results The proliferation of transformed C 3H 10 T 1/2 cells was inhibited markedly by 1mmol/L db cAMP.The db cAMP treated transformed cells were accumulated at G 1 phase.After treatment with db cAMP,the soft agar colony formation efficient in transformed cells was decreased evidently,simultaneously,cells altered in morphology,from rounded to flattened shape.In contrast to normal cells,CaM level and PKⅡ activity increased in transformed cells.However,both CaM level and PKⅡ activity in transformed cells treated by db cAMP decreased obviously.Conclusion\ The results indicate that not only alteration of CaM expression,but also the changes of the PKⅡ activity are involved in account for the cell transformation and induced differentiation of transformed cells.It is possible that the role for CaM in the cell transformation and induced differentiation may be mediatied by the effect of PKⅡ. \;[
2.EFFECT OF THE CHANGES IN MICROTUBULAR-MICROFILAMENTOUS SYSTEM ON THE MUCUS SECRETION OF CULTURED MGc8e-3 HUMAN STOMACH CANCER CELLS
Duanshun WANG ; Dongliang GU ; Xianghuan QIU ; Kunren WANG
Acta Anatomica Sinica 1954;0(02):-
cells was not influenced if they were treated with 1(T~(-5)M taxol and 0.1?g/ml colcemid for 2 hrs. either simultaneously or in accession. The secretory activity of these cells was evidently inhibited after treatment with 4?g/ml cytochalasin B for 6 hrs. Our results indicate that the mucus secretion of MGc80-3 human stomach cancer cells closely depended on the presence of both microtubules and microfilaments. We also found that the mucus secretion of MGc80-3 cells was enhanced by treatment with l?g/ml pilocarpine for 6 hrs. The proliferation of MGc80-3 cell population was inhibited by thiazolidine-4 carboxylic acid. However, the secretory activity of these cells was evidently enhanced after treatment with 0.1?g/ml thiazolidine-4 carboxylic acid for 10 hrs. The stimulating effect of pilocarpine and thiazolidine-4 carboxylic acid disappeared after the microtubules and microfilaments were disassembled by treatment with colcemid and cytochalasin B. In addition, our experiments also show that the mucus secretion of these cells was increased after treatment with ImM db-cAMP. These findings convincingly support the assumption that motile events placed under the control of the microtubular-microfilamentous system are intimately involved in the mucus secretion of MGc80-3 human stomach cancer cells. cells was not influenced if they were treated with 1(T~(-5)M taxol and 0.1?g/ml colcemid for 2 hrs. either simultaneously or in accession. The secretory activity of these cells was evidently inhibited after treatment with 4?g/ml cytochalasin B for 6 hrs. Our results indicate that the mucus secretion of MGc80-3 human stomach cancer cells closely depended on the presence of both microtubules and microfilaments. We also found that the mucus secretion of MGc80-3 cells was enhanced by treatment with l?g/ml pilocarpine for 6 hrs. The proliferation of MGc80-3 cell population was inhibited by thiazolidine-4 carboxylic acid. However, the secretory activity of these cells was evidently enhanced after treatment with 0.1?g/ml thiazolidine-4 carboxylic acid for 10 hrs. The stimulating effect of pilocarpine and thiazolidine-4 carboxylic acid disappeared after the microtubules and microfilaments were disassembled by treatment with colcemid and cytochalasin B. In addition, our experiments also show that the mucus secretion of these cells was increased after treatment with ImM db-cAMP. These findings convincingly support the assumption that motile events placed under the control of the microtubular-microfilamentous system are intimately involved in the mucus secretion of MGc80-3 human stomach cancer cells.
3.Primary Studies on the Progression of S Phase and its Molecular Mechanism by Protein Kinase A Inhibitor in the HeLa Cell
Xia SUN ; Huitu LIU ; Yingkai TONG ; Duanshun WANG
Progress in Biochemistry and Biophysics 2001;28(2):232-235
The synchronized HeLa cells were used to study the effect of protein kinase A(PKA) inhibitor on the progression of S phase. Synchronized cell s in S phase were obtained by the method of TdR double block through 3H-T dR incorporation assay. The PKA inhibitor typeⅢ obviously increased the level of 3H-TdR incorporation of S phase in HeLa cells. In contrast with contro l, the activity of thymidine kinase (TK) in S phase increased, too. It indicated that PKA played an inhibitory role in S phase progression of HeLa cells. With t he method of Western blotting, the PKA inhibitor typeⅢ enhanced the level of C yclinA and PCNA, inhibited the expression of p21, which is a negative regulator of cell cycle, but had no effect on the expression of CDK2. The results showed t hat PKA could negatively regulate the S phase progression by affecting the level of CyclinA, PCNA and influencing the expression of p21 protein. This may be one of the molecular mechanisms which is involved in the negative regulation of S p hase progression by PKA in HeLa cells.