Effect of phorbol myristate acetate on c-myc, beta-actin, and FV gene expression in morphologically recognizable human megakaryocytes: a kinetic analysis employing in situ hybridization

Abstract

Phorbol esters are reported to modulate numerous megakaryocyte maturation maturation parameters, but the effects of these compounds on normal human megakaryocyte gene expression are unknown. We therefore determined if phorbol myristate acetate (PMA)-induced changes in megakaryocyte gene expression could be detected and semi-quantified in single cells using the techniques of in situ hybridization and photodensitometry. Megakaryocytes were isolated from normal human bone marrow by counterflow centrifugal elutriation. After 2, 15, and 24 h of culture in 8 nM PMA, megakaryocytes were probed by in situ hybridization with biotin-11-dUTP-labeled cDNA probes for human c-myc, beta-actin, and coagulation cofactor V (FV) mRNA. Hybridization was detected by an enzyme-catalyzed colorimetric reaction that was photometrically quantified as an inverse function of light transmission through labeled cells. In control megakaryocytes, steady state levels of c-myc and FV mRNA did not change over the 24-h observation period, whereas that of beta-actin appeared to increase slightly. In contrast, after 2 h of exposure to 8 nM PMA, a statistically significant increase (p less than 0.001) in c-myc and beta-actin mRNA expression was observed, whereas FV mRNA expression appeared to be unchanged (p = 0.207). These inductions were transient, however, because by 15 or 24 h, beta-actin and c-myc expression levels, respectively, no longer exceeded those measured in untreated controls. However, in the presence of higher PMA doses (160 nM), beta-actin mRNA levels remained elevated at 24 h. The relationship between megakaryocyte maturation and apparent level of beta-actin mRNA expression was also examined. Although cell size and stage, and size and beta-actin mRNA levels showed a modest relationship, mRNA levels and morphologic maturation stage were poorly correlated. These results demonstrate that PMA has complex effects on gene expression in morphologically recognizable human megakaryocytes. Of equal importance, they also demonstrate that in situ hybridization can be employed as a useful tool for studying human megakaryocyte cell biology at the molecular level.