INHIBITION OF ACTIN POLYMERIZATION BY CYTOCHALASIN B INDUCES POLYPLOIDIZATION AND INCREASES THE NUMBER OF NUCLEOLAR ORGANIZER REGIONS IN HUMAN MEGAKARYOCYTE CELL-LINES

Megakaryocyte polyploidization is an advantageous and regulated mechan ism which leads to an increase in platelet production. In megakaryocyt e cell lines, polyploidization can be obtained by using cytochalasin B , an inhibitor of the actin polymerization. The Nucleolar Organizer Re gions (AgNORs) are parts of nucleolar DNA transcribed into ribosomal R NA. They are detected by silver staining technique and their number is proportional to protein synthesis. In order to estimate protein synth esis in polyploidizing megakaryocytes, AgNORs were measured in three c ell lines with megakaryocyte properties (DAMI, HEL and K562) after a 4 -day culture in the presence or absence of cytochalasin B, an inhibito r of the actin polymerization. The mean number of AgNORs pet cell was 16.4+/-4.3 (m+/-SEM), 24.4+/-2.5 and 23.6+/-3.1 for DAMI, HEL and K-56 2 cell lines, respectively. The addition of cytochalasin B (2 mu g/ml) increased significantly the number of AgNORs per cell (DAMI: 437 %, H EL: 384 % and K-562: 345 % of controls, p < 0.05 by t-test). Moreover, the numbers of nucleoles per cell after addition of cytochalasin B we re augmented significantly (DAMI: 258 %, HEL: 271 % and K-562: 264 % o f controls, p < 0.05 by t-test). The total protein content estimated b y Bradford's method increased significantly to 938 %, 326 % and 388 % of controls in DAMI, HEL and K562, respectively (p < 0.05 by t-test) i n cells where actin was inhibited by cytochalasin B. In the presence o f cytochalasin B, the endomitotic index (EI) [mean of (log(2) DNA cont ent expressed in N) -1] measured by flow cytometry increased to 368 %, 207 % and 538 %, for DAMI, HEL and K-562 cell lines, respectively (p < 0.05 by t-test) after treatment with cytochalasin B. In contrast, th e number of AgNORs per unit of DNA (EI) and the total protein content pel unit of DNA did not change for DAMI, HEL and K-562 cell lines (p < 0.05 by t-test) after treatment with cytochalasin B. In conclusion, t he increase in the number of the Nucleolar Organizer Regions by an age nt known to stimulate polyploidization of megakaryocytic cell lines su ggests that polyploidization occurs by enhanced protein production pro portionally to DNA synthesis.