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Role of PP2Calpha in cell growth, in radio- and chemosensitivity, and in tumorigenicity.

Lammers T, Peschke P, Ehemann V, Debus J, Slobodin B, Lavi S, Huber P - Mol. Cancer (2007)

Bottom Line: It was found that knockdown of PP2Calpha did not affect the proliferation, the clonogenic survival and the membrane integrity of MCF7 cells.In addition, it did not alter their radio- and chemosensitivity.Based on these findings, we conclude that PP2Calpha is not involved in controlling cell growth and radio- and chemosensitivity in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Innovative Cancer Diagnosis and Therapy, Clinical Cooperation Unit Radiotherapeutic Oncology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. t.lammers@dkfz.de

ABSTRACT

Background: PP2Calpha is the representative member of the type 2C family of protein phosphatases, and it has recently been implicated in the regulation of p53-, TGFbeta-, cyclin-dependent kinase- and apoptosis-signaling. To investigate the role of PP2Calpha in cell growth and in radio- and chemosensitivity, wild type and PP2Calpha siRNA-expressing MCF7 cells were subjected to several different viability and cell cycle analyses, both under basal conditions and upon treatment with radio- and chemotherapy. By comparing the growth of tumors established from both types of cells, we also evaluated the involvement of PP2Calpha in tumorigenesis.

Results: It was found that knockdown of PP2Calpha did not affect the proliferation, the clonogenic survival and the membrane integrity of MCF7 cells. In addition, it did not alter their radio- and chemosensitivity. For PP2Calpha siRNA-expressing MCF7 cells, the number of cells in the G0/G1 phase of the cell cycle was reduced, the induction of the G1 block was attenuated, the number of cells in G2/M was increased, and the induction of the G2 block was enhanced. The tumorigenic potential of PP2Calpha siRNA-expressing MCF7 cells was found to be higher than that of wild type MCF7 cells, and the in vivo proliferation of these cells was found to be increased.

Conclusion: Based on these findings, we conclude that PP2Calpha is not involved in controlling cell growth and radio- and chemosensitivity in vitro. It does, however, play a role in the regulation of the cell cycle, in the induction of cell cycle checkpoints and in tumorigenesis. The latter notion implies that PP2Calpha may possess tumor-suppressing properties, and it thereby sets the stage for more elaborate analyses on its involvement in the development and progression of cancer.

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Evaluation of the chemosensitivity of wild type and PP2Cα siRNA-expressing MCF7 cells. A and B: Effect of different doses of doxorubicin on the proliferation of MCF7-wt and MCF7-si cells. The cells were either exposed to the drug for 24 h (A) or continuously (B). Values represent average ± SD (n = 3). C: Effect of doxorubicin on the clonogenicity of MCF7-wt and MCF7-si cells. Values represent average ± SD (n = 3). D: Effect of gemcitabine on the clonogenicity of MCF7-wt and MCF7-si cells. Values represent average ± SD (n = 3). E and F: Quantification of the effect of doxorubicin on the viability (i.e. the membrane integrity) of MCF7-wt and MCF7-si cells. The viability was determined by means of FACS analysis. Values represent average ± SD (n = 3). G: Representative images of the cell cycle distribution of MCF7-wt and MCF7-si cells after 48 h of doxorubicin treatment.
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Figure 3: Evaluation of the chemosensitivity of wild type and PP2Cα siRNA-expressing MCF7 cells. A and B: Effect of different doses of doxorubicin on the proliferation of MCF7-wt and MCF7-si cells. The cells were either exposed to the drug for 24 h (A) or continuously (B). Values represent average ± SD (n = 3). C: Effect of doxorubicin on the clonogenicity of MCF7-wt and MCF7-si cells. Values represent average ± SD (n = 3). D: Effect of gemcitabine on the clonogenicity of MCF7-wt and MCF7-si cells. Values represent average ± SD (n = 3). E and F: Quantification of the effect of doxorubicin on the viability (i.e. the membrane integrity) of MCF7-wt and MCF7-si cells. The viability was determined by means of FACS analysis. Values represent average ± SD (n = 3). G: Representative images of the cell cycle distribution of MCF7-wt and MCF7-si cells after 48 h of doxorubicin treatment.

Mentions: To investigate the impact of PP2Cα knockdown on chemosensitivity, wild type and siRNA-expressing MCF7 cells were exposed to doxorubicin and subjected to the three abovementioned viability assays. Figs. 3A and 3B demonstrate that at doxorubicin concentrations of 0.1–10 μM, no difference in proliferation could be observed between the two types of cells, neither when they were pulse-incubated (i.e. for 24 h; Fig. 3A), nor when they were incubated continuously (i.e. for 120 h; Fig. 3B). Next, the impact of doxorubicin on the clonogenic survival of wild type and PP2Cα knockdown cells was evaluated. As shown in Fig. 3C, also in this case, no difference in response to chemotherapeutic treatment could be observed between the two types of cells. To exclude the possibility that the observed lack of an altered chemosensitivity was drug-specific, we also evaluated the clonogenicity of the cells in response to gemcitabine. As shown in Fig. 3D, however, also upon treatment with gemcitabine, no difference in clonogenic survival could be detected between wild type and PP2Cα knockdown cells. This finding indicates that the observed lack of a differential chemosensitivity was not specific for doxorubicin, and it suggests that MCF7-wt and MCF-si cells are equally sensitive towards chemotherapeutic treatment. Subsequently, as for radiotherapy, we then also investigated the impact of chemotherapy on the membrane integrity of MCF7-wt and MCF7-si cells. Figs. 3E and 3F present the pooled results of three independent analyses, and they demonstrate that there was hardly any difference in chemosensitivity between the two types of cells; at 24 h, the uptake of PI was slightly higher for MCF7-si, at 48 h, the viability of the cells was identical, and at 72 h, the degree of membrane-damage was slightly higher for MCF7-wt. In line with information obtained in the SRB and the CFA analyses, these findings demonstrate that the knockdown of PP2Cα does not alter the chemosensitivity of MCF7 cells.


Role of PP2Calpha in cell growth, in radio- and chemosensitivity, and in tumorigenicity.

Lammers T, Peschke P, Ehemann V, Debus J, Slobodin B, Lavi S, Huber P - Mol. Cancer (2007)

Evaluation of the chemosensitivity of wild type and PP2Cα siRNA-expressing MCF7 cells. A and B: Effect of different doses of doxorubicin on the proliferation of MCF7-wt and MCF7-si cells. The cells were either exposed to the drug for 24 h (A) or continuously (B). Values represent average ± SD (n = 3). C: Effect of doxorubicin on the clonogenicity of MCF7-wt and MCF7-si cells. Values represent average ± SD (n = 3). D: Effect of gemcitabine on the clonogenicity of MCF7-wt and MCF7-si cells. Values represent average ± SD (n = 3). E and F: Quantification of the effect of doxorubicin on the viability (i.e. the membrane integrity) of MCF7-wt and MCF7-si cells. The viability was determined by means of FACS analysis. Values represent average ± SD (n = 3). G: Representative images of the cell cycle distribution of MCF7-wt and MCF7-si cells after 48 h of doxorubicin treatment.
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Related In: Results  -  Collection

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Figure 3: Evaluation of the chemosensitivity of wild type and PP2Cα siRNA-expressing MCF7 cells. A and B: Effect of different doses of doxorubicin on the proliferation of MCF7-wt and MCF7-si cells. The cells were either exposed to the drug for 24 h (A) or continuously (B). Values represent average ± SD (n = 3). C: Effect of doxorubicin on the clonogenicity of MCF7-wt and MCF7-si cells. Values represent average ± SD (n = 3). D: Effect of gemcitabine on the clonogenicity of MCF7-wt and MCF7-si cells. Values represent average ± SD (n = 3). E and F: Quantification of the effect of doxorubicin on the viability (i.e. the membrane integrity) of MCF7-wt and MCF7-si cells. The viability was determined by means of FACS analysis. Values represent average ± SD (n = 3). G: Representative images of the cell cycle distribution of MCF7-wt and MCF7-si cells after 48 h of doxorubicin treatment.
Mentions: To investigate the impact of PP2Cα knockdown on chemosensitivity, wild type and siRNA-expressing MCF7 cells were exposed to doxorubicin and subjected to the three abovementioned viability assays. Figs. 3A and 3B demonstrate that at doxorubicin concentrations of 0.1–10 μM, no difference in proliferation could be observed between the two types of cells, neither when they were pulse-incubated (i.e. for 24 h; Fig. 3A), nor when they were incubated continuously (i.e. for 120 h; Fig. 3B). Next, the impact of doxorubicin on the clonogenic survival of wild type and PP2Cα knockdown cells was evaluated. As shown in Fig. 3C, also in this case, no difference in response to chemotherapeutic treatment could be observed between the two types of cells. To exclude the possibility that the observed lack of an altered chemosensitivity was drug-specific, we also evaluated the clonogenicity of the cells in response to gemcitabine. As shown in Fig. 3D, however, also upon treatment with gemcitabine, no difference in clonogenic survival could be detected between wild type and PP2Cα knockdown cells. This finding indicates that the observed lack of a differential chemosensitivity was not specific for doxorubicin, and it suggests that MCF7-wt and MCF-si cells are equally sensitive towards chemotherapeutic treatment. Subsequently, as for radiotherapy, we then also investigated the impact of chemotherapy on the membrane integrity of MCF7-wt and MCF7-si cells. Figs. 3E and 3F present the pooled results of three independent analyses, and they demonstrate that there was hardly any difference in chemosensitivity between the two types of cells; at 24 h, the uptake of PI was slightly higher for MCF7-si, at 48 h, the viability of the cells was identical, and at 72 h, the degree of membrane-damage was slightly higher for MCF7-wt. In line with information obtained in the SRB and the CFA analyses, these findings demonstrate that the knockdown of PP2Cα does not alter the chemosensitivity of MCF7 cells.

Bottom Line: It was found that knockdown of PP2Calpha did not affect the proliferation, the clonogenic survival and the membrane integrity of MCF7 cells.In addition, it did not alter their radio- and chemosensitivity.Based on these findings, we conclude that PP2Calpha is not involved in controlling cell growth and radio- and chemosensitivity in vitro.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Innovative Cancer Diagnosis and Therapy, Clinical Cooperation Unit Radiotherapeutic Oncology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. t.lammers@dkfz.de

ABSTRACT

Background: PP2Calpha is the representative member of the type 2C family of protein phosphatases, and it has recently been implicated in the regulation of p53-, TGFbeta-, cyclin-dependent kinase- and apoptosis-signaling. To investigate the role of PP2Calpha in cell growth and in radio- and chemosensitivity, wild type and PP2Calpha siRNA-expressing MCF7 cells were subjected to several different viability and cell cycle analyses, both under basal conditions and upon treatment with radio- and chemotherapy. By comparing the growth of tumors established from both types of cells, we also evaluated the involvement of PP2Calpha in tumorigenesis.

Results: It was found that knockdown of PP2Calpha did not affect the proliferation, the clonogenic survival and the membrane integrity of MCF7 cells. In addition, it did not alter their radio- and chemosensitivity. For PP2Calpha siRNA-expressing MCF7 cells, the number of cells in the G0/G1 phase of the cell cycle was reduced, the induction of the G1 block was attenuated, the number of cells in G2/M was increased, and the induction of the G2 block was enhanced. The tumorigenic potential of PP2Calpha siRNA-expressing MCF7 cells was found to be higher than that of wild type MCF7 cells, and the in vivo proliferation of these cells was found to be increased.

Conclusion: Based on these findings, we conclude that PP2Calpha is not involved in controlling cell growth and radio- and chemosensitivity in vitro. It does, however, play a role in the regulation of the cell cycle, in the induction of cell cycle checkpoints and in tumorigenesis. The latter notion implies that PP2Calpha may possess tumor-suppressing properties, and it thereby sets the stage for more elaborate analyses on its involvement in the development and progression of cancer.

Show MeSH
Related in: MedlinePlus