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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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Related in: MedlinePlus

Evaluation of the tumorigenic potential of wild type and PP2Cα siRNA-expressing MCF7 cells. A: Tumor growth curves obtained for subcutaneously inoculated MCF7-wt and MCF7-si cells upon the combined implementation of estrogen-containing hormone pellets and Matrigel. The values indicate the individual tumor sizes at day 37. B: Exemplary images of MCF7-wt and MCF7-si tumors at day 37 post inoculation. C: Analysis of the expression of PP2Cα in MCF7-wt and MCF7-si tumors. Magnification 200×. D: Representative images of the cell cycle distribution of MCF7-wt and MCF7-si tumors. Left yellow peak: G0/G1 fraction of the aneuploid tumor cells (i.e. the MCF7 cells). Right yellow peak: G2/M fraction of the aneuploid tumor cells. Dashed blue area: S phase fraction of the aneuploid tumor cells. Red peak: G1 fraction of the diploid host cells (e.g. mouse fibroblasts and endothelial cells). Blue peak: Necrotic cells. E: Multicycle algorithm-based quantification of the cell cycle distribution of MCF7-wt and MCF7-si tumors. Values represent average ± SD (n = 3). F: Analysis of the expression of the proliferation marker Ki-67 in MCF7-wt and MCF7-si tumors. Magnification 200×. G: Representative FACS analyses of the incorporation of the proliferation marker BrdU in MCF7-wt and MCF7-si tumors. H: Quantification of the amount of BrdU-positive cells in MCF7-wt and MCF7-si tumors. Values represent average ± SD (n = 3). * Indicates p < 0.05.
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Figure 4: Evaluation of the tumorigenic potential of wild type and PP2Cα siRNA-expressing MCF7 cells. A: Tumor growth curves obtained for subcutaneously inoculated MCF7-wt and MCF7-si cells upon the combined implementation of estrogen-containing hormone pellets and Matrigel. The values indicate the individual tumor sizes at day 37. B: Exemplary images of MCF7-wt and MCF7-si tumors at day 37 post inoculation. C: Analysis of the expression of PP2Cα in MCF7-wt and MCF7-si tumors. Magnification 200×. D: Representative images of the cell cycle distribution of MCF7-wt and MCF7-si tumors. Left yellow peak: G0/G1 fraction of the aneuploid tumor cells (i.e. the MCF7 cells). Right yellow peak: G2/M fraction of the aneuploid tumor cells. Dashed blue area: S phase fraction of the aneuploid tumor cells. Red peak: G1 fraction of the diploid host cells (e.g. mouse fibroblasts and endothelial cells). Blue peak: Necrotic cells. E: Multicycle algorithm-based quantification of the cell cycle distribution of MCF7-wt and MCF7-si tumors. Values represent average ± SD (n = 3). F: Analysis of the expression of the proliferation marker Ki-67 in MCF7-wt and MCF7-si tumors. Magnification 200×. G: Representative FACS analyses of the incorporation of the proliferation marker BrdU in MCF7-wt and MCF7-si tumors. H: Quantification of the amount of BrdU-positive cells in MCF7-wt and MCF7-si tumors. Values represent average ± SD (n = 3). * Indicates p < 0.05.

Mentions: To address the involvement of PP2Cα in tumorigenesis, 1 * 107 MCF-wt and MCF7-si cells were inoculated subcutaneously (s.c.) into both hind limbs of immunodeficient nude mice. Neither the MCF7-wt cells, however, nor the MCF7-si cells turned out to be able to develop tumors under physiological conditions (Table 4). Based on the notion that MCF7 cells are hormone-dependent breast cancer cells, and on the assumption that their in vivo growth therefore likely depends on the levels of circulating hormones, we next implanted estrogen-containing hormone pellets into 8 nude mice. Ten days after the implantation of the pellets, 1 * 107 MCF7-wt and MCF7-si cells were again inoculated s.c. into both hind limbs of the animals. As shown in Table 4, in one animal injected with MCF7-si cells, a tumor had formed (8 × 11 mm at day 35). In the other three animals injected with MCF7-si cells, and in all four animals injected with MCF7-wt cells, no tumors had developed. In order to increase the number of malignant cells remaining at the site of inoculation, we subsequently suspended 1 * 107 cells in Matrigel and inoculated them s.c. into 10 nude mice. Up to three weeks post inoculation, 2–3 mm sized tumors (or rather, semi-palpable suspensions of tumor cells) could be observed in all animals. After this time point, however, they disappeared one after the other, and at day 50, no tumors had developed (Table 4). Finally, in an ultimate attempt to improve the tumorigenic potential of the two types of MCF7 cells, the estrogen-containing hormone pellets were combined with Matrigel; hereto, 1 * 107 wild type and PP2Cα knockdown cells were suspended in Matrigel, and they were inoculated into mice that had been implanted with pellets 10 days before. Approximately 3 weeks after inoculation, several animals, especially those injected with MCF7-si cells, started to present with signs of tumor growth. As shown in Figs. 4A and 4B, from this day on, several MCF7-si tumors kept on growing steadily, reaching sizes of e.g. 13 × 14 or 10 × 12 mm by day 37. The growth curves in Fig. 4A furthermore demonstrate that a substantially smaller fraction of the animals inoculated with MCF7-wt cells turned out to be able to form tumors (4 out of 8, as compared to 9 out of 10 for MCF7-si), and they also exemplify that those MCF7-wt tumors that did grow, grew much slower than did the MCF7-si tumors; 37 days after inoculation, the largest MCF7-wt tumor was 7 × 10 mm, as compared to 12 × 19 mm for MCF7-si (Figs. 4A and 4B). These findings suggest that, at least under certain conditions, the knockdown of PP2Cα increases the tumorigenicity 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 tumorigenic potential of wild type and PP2Cα siRNA-expressing MCF7 cells. A: Tumor growth curves obtained for subcutaneously inoculated MCF7-wt and MCF7-si cells upon the combined implementation of estrogen-containing hormone pellets and Matrigel. The values indicate the individual tumor sizes at day 37. B: Exemplary images of MCF7-wt and MCF7-si tumors at day 37 post inoculation. C: Analysis of the expression of PP2Cα in MCF7-wt and MCF7-si tumors. Magnification 200×. D: Representative images of the cell cycle distribution of MCF7-wt and MCF7-si tumors. Left yellow peak: G0/G1 fraction of the aneuploid tumor cells (i.e. the MCF7 cells). Right yellow peak: G2/M fraction of the aneuploid tumor cells. Dashed blue area: S phase fraction of the aneuploid tumor cells. Red peak: G1 fraction of the diploid host cells (e.g. mouse fibroblasts and endothelial cells). Blue peak: Necrotic cells. E: Multicycle algorithm-based quantification of the cell cycle distribution of MCF7-wt and MCF7-si tumors. Values represent average ± SD (n = 3). F: Analysis of the expression of the proliferation marker Ki-67 in MCF7-wt and MCF7-si tumors. Magnification 200×. G: Representative FACS analyses of the incorporation of the proliferation marker BrdU in MCF7-wt and MCF7-si tumors. H: Quantification of the amount of BrdU-positive cells in MCF7-wt and MCF7-si tumors. Values represent average ± SD (n = 3). * Indicates p < 0.05.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Evaluation of the tumorigenic potential of wild type and PP2Cα siRNA-expressing MCF7 cells. A: Tumor growth curves obtained for subcutaneously inoculated MCF7-wt and MCF7-si cells upon the combined implementation of estrogen-containing hormone pellets and Matrigel. The values indicate the individual tumor sizes at day 37. B: Exemplary images of MCF7-wt and MCF7-si tumors at day 37 post inoculation. C: Analysis of the expression of PP2Cα in MCF7-wt and MCF7-si tumors. Magnification 200×. D: Representative images of the cell cycle distribution of MCF7-wt and MCF7-si tumors. Left yellow peak: G0/G1 fraction of the aneuploid tumor cells (i.e. the MCF7 cells). Right yellow peak: G2/M fraction of the aneuploid tumor cells. Dashed blue area: S phase fraction of the aneuploid tumor cells. Red peak: G1 fraction of the diploid host cells (e.g. mouse fibroblasts and endothelial cells). Blue peak: Necrotic cells. E: Multicycle algorithm-based quantification of the cell cycle distribution of MCF7-wt and MCF7-si tumors. Values represent average ± SD (n = 3). F: Analysis of the expression of the proliferation marker Ki-67 in MCF7-wt and MCF7-si tumors. Magnification 200×. G: Representative FACS analyses of the incorporation of the proliferation marker BrdU in MCF7-wt and MCF7-si tumors. H: Quantification of the amount of BrdU-positive cells in MCF7-wt and MCF7-si tumors. Values represent average ± SD (n = 3). * Indicates p < 0.05.
Mentions: To address the involvement of PP2Cα in tumorigenesis, 1 * 107 MCF-wt and MCF7-si cells were inoculated subcutaneously (s.c.) into both hind limbs of immunodeficient nude mice. Neither the MCF7-wt cells, however, nor the MCF7-si cells turned out to be able to develop tumors under physiological conditions (Table 4). Based on the notion that MCF7 cells are hormone-dependent breast cancer cells, and on the assumption that their in vivo growth therefore likely depends on the levels of circulating hormones, we next implanted estrogen-containing hormone pellets into 8 nude mice. Ten days after the implantation of the pellets, 1 * 107 MCF7-wt and MCF7-si cells were again inoculated s.c. into both hind limbs of the animals. As shown in Table 4, in one animal injected with MCF7-si cells, a tumor had formed (8 × 11 mm at day 35). In the other three animals injected with MCF7-si cells, and in all four animals injected with MCF7-wt cells, no tumors had developed. In order to increase the number of malignant cells remaining at the site of inoculation, we subsequently suspended 1 * 107 cells in Matrigel and inoculated them s.c. into 10 nude mice. Up to three weeks post inoculation, 2–3 mm sized tumors (or rather, semi-palpable suspensions of tumor cells) could be observed in all animals. After this time point, however, they disappeared one after the other, and at day 50, no tumors had developed (Table 4). Finally, in an ultimate attempt to improve the tumorigenic potential of the two types of MCF7 cells, the estrogen-containing hormone pellets were combined with Matrigel; hereto, 1 * 107 wild type and PP2Cα knockdown cells were suspended in Matrigel, and they were inoculated into mice that had been implanted with pellets 10 days before. Approximately 3 weeks after inoculation, several animals, especially those injected with MCF7-si cells, started to present with signs of tumor growth. As shown in Figs. 4A and 4B, from this day on, several MCF7-si tumors kept on growing steadily, reaching sizes of e.g. 13 × 14 or 10 × 12 mm by day 37. The growth curves in Fig. 4A furthermore demonstrate that a substantially smaller fraction of the animals inoculated with MCF7-wt cells turned out to be able to form tumors (4 out of 8, as compared to 9 out of 10 for MCF7-si), and they also exemplify that those MCF7-wt tumors that did grow, grew much slower than did the MCF7-si tumors; 37 days after inoculation, the largest MCF7-wt tumor was 7 × 10 mm, as compared to 12 × 19 mm for MCF7-si (Figs. 4A and 4B). These findings suggest that, at least under certain conditions, the knockdown of PP2Cα increases the tumorigenicity 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