Interaction between protein kinase C and actin in megakaryocyte polyploidization

Megakaryocytes undergo a peculiar and irreversible program by which they become polyploid through repeated cycles of DNA synthesis without concomitant cell division. In order to study the possible concomitant role of protein kinase C and actin in megakaryocyte polyploidization, three cell lines (DAMI, HEL and K562), expressing some properties of the megakaryocytic lineage and known to differentiate into the megakaryocytic pathway in the presence of phorbol esters, were cultivated in the presence of phorbol myristate acetate alone (PMA, 5 x 10(-9) M, activator of protein kinase C, PKC) or concomitantly with cytochalasin B (2 micrograms/ml, inhibitor of actin polymerization). We have previously shown that DAMI, HEL and K562 cells in which actin polymerization was inhibited by cytochalasin B, acquired megakaryocytic properties in the way that they became polyploid, acquired a megakaryocytic phenotype and arrested proliferation (4). After four days of culture in the presence of PMA and cytochalasin B, the number of polyploid cells (estimated by flow cytometry) increased in comparison with control or PMA-treated cells. However, it was lower than in cytochalasin B-treated cells. Indeed, control cells predominantly diploid (2N) became polyploid with the appearance of 8N, 16N and 32N cells after addition of PMA, cytochalasin B or PMA + cytochalasin B. The endomitotic index (EI, as described in 5) which corresponds to the mean of (¿log2 DNA content expressed in N¿-1) was 0.5 +/- 0.1, 0.7 +/- 0.1 and 0.3 +/- 0.1 in control DAMI, HEL and K562 cells, respectively. The EI increased to 0.9 +/- 0.2; 1.0 +/- 0.2 and 0.4 + 0.1 in cells treated with PMA and to 1.6 +/- 0.3; 1.4 +/- 0, and 0.9 +/- 0.2 when PMA was added concomitantly to cytochalasin B. Total DNA estimated from the cell content and the percentage of cells present at each ploidy stage did not change in cytochalasin B-treated cells in comparison to control conditions. However, treatment of DAMI, HEL and K562 cells with PMA alone or concomitantly with cytochalasin B revealed that the total DNA content significantly decreased in these conditions. At last, treatment of the three cell lines with PMA alone or concomitantly with cytochalasin B for 4 days caused a complete inhibition of proliferation. In conclusion, the concomitant addition of PMA and cytochalasin B to the three cell lines lead to an augmentation of cell ploidy and to a cessation of proliferation. However, we did not observe any synergistic effect of the two compounds. The possible interaction between actin and protein kinase C is discussed in the paper.