HETEROGENEITY OF DNA CONTENT AND EXPRESSION OF CELL-CYCLE GENES IN AXENICALLY GROWING ENTAMOEBA-HISTOLYTICA HM1-IMSS CLONE-A

The cell division cycle of Entamoeba histolytica was studied using mul ti-parametric how cytometry in asynchronous and partially synchronised cells. Dynamic changes in the DNA synthesis and DNA content of axenic ally growing trophozoites were observed by using 5-bromo-2'-deoxyuridi ne (BrdU) uptake and DNA specific fluorochromes. It was observed that DNA synthesis in these cells continues beyond the typical S-phase stop point when DNA duplication is complete. Asynchronously growing E. his tolytica cells could be synchronised by serum starvation followed by s erum re-addition. BrdU incorporation in synchronised cells showed that cell synchrony is maintained for at least one generation time, in whi ch the G1 phase lasts for 2-3 h and the S-phase lasts for 5-6 h. Analy sis of our results revealed that E. histolytica trophozoites, growing in axenic medium, are made up of a heterogenous population of euploid and polyploid cells. The number of polyploid cells increases with age of the cells in culture. Expression of putative cell cycle and signal transduction markers was studied using specific antibodies and changes in their expression levels have been correlated with changes in the D NA content. Based upon our results we could identify G1, S and G2 phas es of the cell cycle of E. histolytica and also predict the mechanism underlying the generation of polyploidy in these cells, which may have significant effects on its biology and pathogenesis. (C) 1997 Elsevie r Science B.V.