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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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CDK2 is involved in regulating CDKN3-mediated leukemic cell survival.(A) Shown is lentiviral vectors constructed in this study that encode luciferase shRNA (sh-luc) control, CDKN3 shRNA (sh-CDKN3), sh-CDKN3 and either wild type CDK2 (CDK2-WT) or CDK2 dominant-negative mutant (CDK2-D145N). (B) RT-PCR and Western blotting were performed to examine the expression of CDKN3, CDK2, and XIAP in K562 cells expressing sh-CDKN3 alone, sh-CDKN3 and CDK2-WT, sh-CDKN3 and CDK2-D145N, or control. (C) Survival of K562 cells expressing sh-CDKN3, sh-CDKN3 and CDK2-WT, sh-CDKN3 and CDK2-D145N, or the control was analyzed by flow cytometry after treatment with or without 10 µM of imatinib for 48 h. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05.
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pone-0111611-g006: CDK2 is involved in regulating CDKN3-mediated leukemic cell survival.(A) Shown is lentiviral vectors constructed in this study that encode luciferase shRNA (sh-luc) control, CDKN3 shRNA (sh-CDKN3), sh-CDKN3 and either wild type CDK2 (CDK2-WT) or CDK2 dominant-negative mutant (CDK2-D145N). (B) RT-PCR and Western blotting were performed to examine the expression of CDKN3, CDK2, and XIAP in K562 cells expressing sh-CDKN3 alone, sh-CDKN3 and CDK2-WT, sh-CDKN3 and CDK2-D145N, or control. (C) Survival of K562 cells expressing sh-CDKN3, sh-CDKN3 and CDK2-WT, sh-CDKN3 and CDK2-D145N, or the control was analyzed by flow cytometry after treatment with or without 10 µM of imatinib for 48 h. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05.

Mentions: To confirm the above findings, we generated CDKN3-knockdown K562 cells stably expressing either CDK2-WT, CDK2-D145N (dominant-negative mutant), or the control (Figures 6A and 6B). These cells were subjected to imatinib treatment and analyzed for cell survival. As shown in Figure 6C, without imatinib treatment, depletion of CDKN3, overexpression of CDK2-WT or CDK2-D145N did not significantly affect cell viability in K562 cells as compared to the control cells. However, consistently with our aforementioned findings, depletion of CDKN3 significantly increased the survival of imatinib-treated leukemic cells (Figure 6C). Overexpression of functional CDK2 further increased the survival of CDKN3-depleted leukemic cells after treatment with imatinib, whereas ectopic expression of the catalytically inactive CDK2-D145N mutant had the opposite effect on survival of the CDKN3-deficient leukemic cells exposed to imatinib. Together, these results reveal that activity of CDK2 regulated by CDKN3 is involved in imatinib-induced apoptosis in K562 leukemic cells.


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)

CDK2 is involved in regulating CDKN3-mediated leukemic cell survival.(A) Shown is lentiviral vectors constructed in this study that encode luciferase shRNA (sh-luc) control, CDKN3 shRNA (sh-CDKN3), sh-CDKN3 and either wild type CDK2 (CDK2-WT) or CDK2 dominant-negative mutant (CDK2-D145N). (B) RT-PCR and Western blotting were performed to examine the expression of CDKN3, CDK2, and XIAP in K562 cells expressing sh-CDKN3 alone, sh-CDKN3 and CDK2-WT, sh-CDKN3 and CDK2-D145N, or control. (C) Survival of K562 cells expressing sh-CDKN3, sh-CDKN3 and CDK2-WT, sh-CDKN3 and CDK2-D145N, or the control was analyzed by flow cytometry after treatment with or without 10 µM of imatinib for 48 h. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4216094&req=5

pone-0111611-g006: CDK2 is involved in regulating CDKN3-mediated leukemic cell survival.(A) Shown is lentiviral vectors constructed in this study that encode luciferase shRNA (sh-luc) control, CDKN3 shRNA (sh-CDKN3), sh-CDKN3 and either wild type CDK2 (CDK2-WT) or CDK2 dominant-negative mutant (CDK2-D145N). (B) RT-PCR and Western blotting were performed to examine the expression of CDKN3, CDK2, and XIAP in K562 cells expressing sh-CDKN3 alone, sh-CDKN3 and CDK2-WT, sh-CDKN3 and CDK2-D145N, or control. (C) Survival of K562 cells expressing sh-CDKN3, sh-CDKN3 and CDK2-WT, sh-CDKN3 and CDK2-D145N, or the control was analyzed by flow cytometry after treatment with or without 10 µM of imatinib for 48 h. Plotted are results from three independent experiments. Error bars represent SEM, n = 3; *P<0.05.
Mentions: To confirm the above findings, we generated CDKN3-knockdown K562 cells stably expressing either CDK2-WT, CDK2-D145N (dominant-negative mutant), or the control (Figures 6A and 6B). These cells were subjected to imatinib treatment and analyzed for cell survival. As shown in Figure 6C, without imatinib treatment, depletion of CDKN3, overexpression of CDK2-WT or CDK2-D145N did not significantly affect cell viability in K562 cells as compared to the control cells. However, consistently with our aforementioned findings, depletion of CDKN3 significantly increased the survival of imatinib-treated leukemic cells (Figure 6C). Overexpression of functional CDK2 further increased the survival of CDKN3-depleted leukemic cells after treatment with imatinib, whereas ectopic expression of the catalytically inactive CDK2-D145N mutant had the opposite effect on survival of the CDKN3-deficient leukemic cells exposed to imatinib. Together, these results reveal that activity of CDK2 regulated by CDKN3 is involved in imatinib-induced apoptosis in K562 leukemic cells.

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