M Yagi1, G J Roth. 1. Research, Seattle Division, Veterans Affairs Puget Sound Health Care System, WA 98108, USA. myagi@u.washington.edu
Abstract
BACKGROUND: During differentiation, megakaryocytes (MK), the bone marrow precursors of circulating blood platelets, undergo polyploidization, repeated rounds of DNA replication without cell division. Mature normal MK may contain a DNA content of up to 128N, in contrast to normal diploid (2N) cells. The extent of polyploidy may influence the number of platelets produced by the MK. Therefore, understanding the molecular mechanisms regulating polyploidization could identify events involved in controlling both cell division and thrombopoiesis. OBJECTIVE: We investigated the expression of several proteins involved in mitosis in cultured mouse MK, and tested the effect of expression on polyploidization. METHODS: Western blot and immunofluorescent analyses were used to assess expression of cell cycle proteins in cultured MK. Populations of polyploidizing MK were separated on the basis of DNA content by flow cytometry. The gene encoding mouse polo-like kinase 1 (PLK-1) was introduced into MK by retroviral transduction, and its effects measured by flow cytometry. RESULTS: Polyploid mouse MK expressed lower levels of two proteins, p55CDC and PLK-1, whose activity is necessary for cell cycle progression and completion of mitosis. Comparison of sorted 2N/4N and polyploid MK indicated that PLK-1 expression was absent in polyploid MK, while expression of other cell cycle proteins was similar in both populations. Forced expression of PLK-1 during MK differentiation was associated with decreased polyploidization. CONCLUSION: These experiments suggest that PLK-1 is an important regulator of polyploidization in differentiating MK.
BACKGROUND: During differentiation, megakaryocytes (MK), the bone marrow precursors of circulating blood platelets, undergo polyploidization, repeated rounds of DNA replication without cell division. Mature normal MK may contain a DNA content of up to 128N, in contrast to normal diploid (2N) cells. The extent of polyploidy may influence the number of platelets produced by the MK. Therefore, understanding the molecular mechanisms regulating polyploidization could identify events involved in controlling both cell division and thrombopoiesis. OBJECTIVE: We investigated the expression of several proteins involved in mitosis in cultured mouse MK, and tested the effect of expression on polyploidization. METHODS: Western blot and immunofluorescent analyses were used to assess expression of cell cycle proteins in cultured MK. Populations of polyploidizing MK were separated on the basis of DNA content by flow cytometry. The gene encoding mousepolo-like kinase 1 (PLK-1) was introduced into MK by retroviral transduction, and its effects measured by flow cytometry. RESULTS: Polyploid mouse MK expressed lower levels of two proteins, p55CDC and PLK-1, whose activity is necessary for cell cycle progression and completion of mitosis. Comparison of sorted 2N/4N and polyploid MK indicated that PLK-1 expression was absent in polyploid MK, while expression of other cell cycle proteins was similar in both populations. Forced expression of PLK-1 during MK differentiation was associated with decreased polyploidization. CONCLUSION: These experiments suggest that PLK-1 is an important regulator of polyploidization in differentiating MK.
Authors: Nicholas Papadantonakis; Maria Makitalo; Donald J McCrann; Kenian Liu; Hao G Nguyen; Greg Martin; Sunita Patel-Hett; Joseph E Italiano; Katya Ravid Journal: Cell Cycle Date: 2008-05-21 Impact factor: 4.534