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A critical role of CDKN3 in Bcr-Abl-mediated tumorigenesis.

Chen Q, Chen K, Guo G, Li F, Chen C, Wang S, Nalepa G, Huang S, Chen JL - PLoS ONE (2014)

Bottom Line: Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence.Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression.Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China.

ABSTRACT
CDKN3 (cyclin-dependent kinase inhibitor 3), a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs) and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML) remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

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CDKN3 negatively regulates K562 cell survival.(A) Expression of CDKN3 in K562 cells stably overexpressing FLAG-tagged wild type CDKN3 (CDKN3-WT) or empty vector (EV) was detected by Western blotting using indicated antibodies. (B) K562 cells stably overexpressing CDKN3-WT or EV were treated with 10 µM of imatinib for the indicated time. Samples were stained with Annexin V-APC and PI, examined by flow cytometry and analyzed by FCS Express V3. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05. (C) Shown is an immunoblot examining FLAG-tagged CDKN3-C140S in K562 cells ectopically expressing CDKN3 mutant (CDKN3-C140S) or empty vector (EV). (D) Cell viability of K562 cells expressing CDKN3-C140S or EV was assessed by flow cytometry after treatment with 10 µM of imatinib for 36 h. Samples were stained with Annexin V-APC and PI. Plotted are results from three independent experiments. Error bars represent SEM, n = 3. (E) Shown is an immunoblot examining shRNA-based knockdown of CDKN3. (F) K562 cells stably expressing sh-luc or sh-CDKN3 were treated with 10 µM imatinib for the indicated time. Samples were then stained with Annexin V-APC and PI, followed by flow cytometry analysis. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05.
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pone-0111611-g001: CDKN3 negatively regulates K562 cell survival.(A) Expression of CDKN3 in K562 cells stably overexpressing FLAG-tagged wild type CDKN3 (CDKN3-WT) or empty vector (EV) was detected by Western blotting using indicated antibodies. (B) K562 cells stably overexpressing CDKN3-WT or EV were treated with 10 µM of imatinib for the indicated time. Samples were stained with Annexin V-APC and PI, examined by flow cytometry and analyzed by FCS Express V3. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05. (C) Shown is an immunoblot examining FLAG-tagged CDKN3-C140S in K562 cells ectopically expressing CDKN3 mutant (CDKN3-C140S) or empty vector (EV). (D) Cell viability of K562 cells expressing CDKN3-C140S or EV was assessed by flow cytometry after treatment with 10 µM of imatinib for 36 h. Samples were stained with Annexin V-APC and PI. Plotted are results from three independent experiments. Error bars represent SEM, n = 3. (E) Shown is an immunoblot examining shRNA-based knockdown of CDKN3. (F) K562 cells stably expressing sh-luc or sh-CDKN3 were treated with 10 µM imatinib for the indicated time. Samples were then stained with Annexin V-APC and PI, followed by flow cytometry analysis. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05.

Mentions: Deregulation or mutation of CDKN3 has been associated with a variety of human cancers [17], [20], [22], [23], [32], but it is unknown whether it plays a role in Bcr-Abl-induced tumorigenesis. To address this issue, we generated K562 leukemic cells stably expressing wild type CDKN3 (CDKN3-WT), or empty vector control (EV) (Figure 1A, Figure S1). These cells were treated with 10 µM of imatinib for up to 36 h, followed by Annexin V and PI staining and flow cytometry analysis. We found that under these culture conditions, approximately 50% of the control cells remained viable after incubation with imatinib for 36 h. In contrast, only approximately 31% of CDKN3-WT overexpressing cells were viable under the same imatinib treatment (Figure 1B), although overexpression of CDKN3 had no significant effect on the cell survival in the absence of imatinib (Figure S2A). To rule out the possibility of off-target responses caused by the imatinib dose at 10 µM, we also treated K562 cells with 5 µM imatinib for 36 h. Similarly, overexpression of CDKN3 significantly reduced the cell viability under this condition as compared with the control (Figure S2B). Importantly, there was no significant difference in cell survival between the control cells and cells expressing CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity, in response to imatinib treatment (Figures 1C and 1D). These data suggest that CDKN3 overexpression promotes imatinib-induced apoptosis in K562 cells, and that the phosphatase activity of CDKN3 is required for its function in regulating leukemic cell survival in the presence of imatinib.


A critical role of CDKN3 in Bcr-Abl-mediated tumorigenesis.

Chen Q, Chen K, Guo G, Li F, Chen C, Wang S, Nalepa G, Huang S, Chen JL - PLoS ONE (2014)

CDKN3 negatively regulates K562 cell survival.(A) Expression of CDKN3 in K562 cells stably overexpressing FLAG-tagged wild type CDKN3 (CDKN3-WT) or empty vector (EV) was detected by Western blotting using indicated antibodies. (B) K562 cells stably overexpressing CDKN3-WT or EV were treated with 10 µM of imatinib for the indicated time. Samples were stained with Annexin V-APC and PI, examined by flow cytometry and analyzed by FCS Express V3. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05. (C) Shown is an immunoblot examining FLAG-tagged CDKN3-C140S in K562 cells ectopically expressing CDKN3 mutant (CDKN3-C140S) or empty vector (EV). (D) Cell viability of K562 cells expressing CDKN3-C140S or EV was assessed by flow cytometry after treatment with 10 µM of imatinib for 36 h. Samples were stained with Annexin V-APC and PI. Plotted are results from three independent experiments. Error bars represent SEM, n = 3. (E) Shown is an immunoblot examining shRNA-based knockdown of CDKN3. (F) K562 cells stably expressing sh-luc or sh-CDKN3 were treated with 10 µM imatinib for the indicated time. Samples were then stained with Annexin V-APC and PI, followed by flow cytometry analysis. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05.
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pone-0111611-g001: CDKN3 negatively regulates K562 cell survival.(A) Expression of CDKN3 in K562 cells stably overexpressing FLAG-tagged wild type CDKN3 (CDKN3-WT) or empty vector (EV) was detected by Western blotting using indicated antibodies. (B) K562 cells stably overexpressing CDKN3-WT or EV were treated with 10 µM of imatinib for the indicated time. Samples were stained with Annexin V-APC and PI, examined by flow cytometry and analyzed by FCS Express V3. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05. (C) Shown is an immunoblot examining FLAG-tagged CDKN3-C140S in K562 cells ectopically expressing CDKN3 mutant (CDKN3-C140S) or empty vector (EV). (D) Cell viability of K562 cells expressing CDKN3-C140S or EV was assessed by flow cytometry after treatment with 10 µM of imatinib for 36 h. Samples were stained with Annexin V-APC and PI. Plotted are results from three independent experiments. Error bars represent SEM, n = 3. (E) Shown is an immunoblot examining shRNA-based knockdown of CDKN3. (F) K562 cells stably expressing sh-luc or sh-CDKN3 were treated with 10 µM imatinib for the indicated time. Samples were then stained with Annexin V-APC and PI, followed by flow cytometry analysis. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05.
Mentions: Deregulation or mutation of CDKN3 has been associated with a variety of human cancers [17], [20], [22], [23], [32], but it is unknown whether it plays a role in Bcr-Abl-induced tumorigenesis. To address this issue, we generated K562 leukemic cells stably expressing wild type CDKN3 (CDKN3-WT), or empty vector control (EV) (Figure 1A, Figure S1). These cells were treated with 10 µM of imatinib for up to 36 h, followed by Annexin V and PI staining and flow cytometry analysis. We found that under these culture conditions, approximately 50% of the control cells remained viable after incubation with imatinib for 36 h. In contrast, only approximately 31% of CDKN3-WT overexpressing cells were viable under the same imatinib treatment (Figure 1B), although overexpression of CDKN3 had no significant effect on the cell survival in the absence of imatinib (Figure S2A). To rule out the possibility of off-target responses caused by the imatinib dose at 10 µM, we also treated K562 cells with 5 µM imatinib for 36 h. Similarly, overexpression of CDKN3 significantly reduced the cell viability under this condition as compared with the control (Figure S2B). Importantly, there was no significant difference in cell survival between the control cells and cells expressing CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity, in response to imatinib treatment (Figures 1C and 1D). These data suggest that CDKN3 overexpression promotes imatinib-induced apoptosis in K562 cells, and that the phosphatase activity of CDKN3 is required for its function in regulating leukemic cell survival in the presence of imatinib.

Bottom Line: Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence.Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression.Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China.

ABSTRACT
CDKN3 (cyclin-dependent kinase inhibitor 3), a dual specificity protein phosphatase, dephosphorylates cyclin-dependent kinases (CDKs) and thus functions as a key negative regulator of cell cycle progression. Deregulation or mutations of CDNK3 have been implicated in various cancers. However, the role of CDKN3 in Bcr-Abl-mediated chronic myelogenous leukemia (CML) remains unknown. Here we found that CDKN3 acts as a tumor suppressor in Bcr-Abl-mediated leukemogenesis. Overexpression of CDKN3 sensitized the K562 leukemic cells to imanitib-induced apoptosis and dramatically inhibited K562 xenografted tumor growth in nude mouse model. Ectopic expression of CDKN3 significantly reduced the efficiency of Bcr-Abl-mediated transformation of FDCP1 cells to growth factor independence. In contrast, depletion of CDKN3 expression conferred resistance to imatinib-induced apoptosis in the leukemic cells and accelerated the growth of xenograph leukemia in mice. In addition, we found that CDKN3 mutant (CDKN3-C140S) devoid of the phosphatase activity failed to affect the K562 leukemic cell survival and xenografted tumor growth, suggesting that the phosphatase of CDKN3 was required for its tumor suppressor function. Furthermore, we observed that overexpression of CDKN3 reduced the leukemic cell survival by dephosphorylating CDK2, thereby inhibiting CDK2-dependent XIAP expression. Moreover, overexpression of CDKN3 delayed G1/S transition in K562 leukemic cells. Our results highlight the importance of CDKN3 in Bcr-Abl-mediated leukemogenesis, and provide new insights into diagnostics and therapeutics of the leukemia.

Show MeSH
Related in: MedlinePlus