Augmentation of the number of nucleolar organizer regions in human megakaryocyte cell lines after induction of polyploidization by a microtubule inhibitor

Megakaryocyte polyploidization is an advantageous and regulated mechanism that leads to an increase in platelet production. In megakaryocytic cell lines, polyploidization can be obtained by using colchicine, an inhibitor of the tubulin spindle. The nucleolar organizer regions (AgNORs) are parts of nucleolar DNA transcribed into ribosomal RNA and are detected by the silver-staining technique. Their number is proportional to protein synthesis.To estimate protein synthesis in polyploid megakaryocytes, AgNORs are measured in three cell lines with megakaryocyte properties (DAMI, HEL and K562) after a 4-day culture in the presence or absence of colchicine. The mean number of AgNORs per cell was 16+/-4 (mean+/-SEM), 24+/-3 and 14+/-3 for DAMI, HEL and K-562 cell lines respectively. The addition of colchicine (10 ng mL[-1]) significantly increased the number of AgNORs per cell (DAMI 556%, HEL 338% and K-562 300% of controls, P0.05 using the t-test). Moreover, the number of nucleoles per cell after the addition of colchicine was augmented significantly (DAMI 246%; HEL 237% and K-562 148% of controls, P0.05 using the t-test). The total protein content estimated by Bradford's method increased significantly to 226%, 215% and 304% of controls in DAMI, HEL and K562 respectively (P005 using the t-test). After treatment with colchicine, the endomitotic index (EI) [mean of (log2 DNA content expressed in N)-1] measured by flow cytometry (and reflecting ploidy) increased to 234%, 255% and 301%, respectively, in DAMI, HEL and K-562 cell lines (P0.05 using the t-test). Concomitantly, the number of AgNORs per unit of DNA increased in the DAMI and HEL cell lines (P0.05 using the t-test) from 48+/-8 and 39+/-5, respectively, to 79+/-11 and 61+/-10. In contrast, the number of nucleoles and the total protein content per endomitotic index were not affected by colchicine (P0.05 using the t-test), but the number of nucleoles per endomitotic index of DAMI cells was affected.The increase in the number of the NORs induced by an agent known to stimulate polyploidization of megakaryocytic cell lines suggests that polyploidization occurs by a proportional increase in protein synthesis per DNA unit.