Role of serum in regulating the Entamoeba histolytica cell cycle: a flowcytometric analysis

Abstract

To identify stage-specific molecules that might be important in pathogenicity, we focused on synchronizing Entamoeba histolytica HM1:1MSS axenic cultures by serum deprivation. By growing trophozoites in medium containing 2% serum for 16 h and then replacing with complete medium, we found that most of the trophozoites were in the G0/G1 phase of the cell cycle. Subsequently, the distribution of trophozoites in G0/G1, S and G2/M phases was studied for up to 24 h and it was observed that after 8 h, the trophozoites began to come out of the quiescent G0/G1 phase and the S and G2/M populations increased, indicating that certain serum factors regulate induction of cell division. It will be important to investigate these factors to find serum substitutes for cultivation media.

Entamoeba histolytica EhDEAD1 is a conserved DEAD-box RNA helicase with ATPase and ATP-dependent RNA unwinding activities

“…EhDead1 mRNA is over expressed in S phase of cell cycle EhDEAD1 exhibits 70% homology to yeast DED1 RNA helicase (Table 1), which participates in cell cycle regulation (Warbrick and Glover, 1994;Forbes et al, 1998;Grallert et al, 2000). Although axenic cultures of E. histolytica are made of a heterogeneous population of cells with varying DNA content, various studies have described cell cycle phases in log phase cultures in distinct strains or clones (Orozco et al, 1988;Dvorak et al, 1995;Gangopadhyay et al, 1997;Vohra et al, 1998;Das and Lohia 2002;Dastidar et al, 2007). Here, we used trophozoites of clone L6, which is routinely synchronized by the mitotic blocker colchicine in our laboratory and whose cell cycle phases lasting is well characterized (Orozco et al, 1988;Marchat et al, 2003).…”

“…Next, DNA synthesis was inhibited by serum starvation in transfected and non-transfected trophozoites (Vohra et al, 1998) and then, cell cycle reactivation was induced by addition of fresh medium and DNA content was analyzed by flow cytometry ( Fig. 7D and E).…”

“…Serum starvation was carried out by incubating growing trophozoites of clone A in TYI-S-33 medium with 2% serum for 16 h at 37°C (Vohra et al, 1998). Cultures of clone L6 (strain HM1:IMSS) were synchronized using complete TYI-S-33 medium with 200 μg/ml colchicine for 24 h. Then, cells were transferred to fresh medium and harvested at 0, 3, 8 and 12 h to obtain trophozoites in M, G1, S and G2 phase, respectively (Orozco et al, 1988).…”

Human hololactoferrin: endocytosis and use as an iron source by the parasite Entamoeba histolytica

“…Cells were kept for 2 h in BI-S-33 lacking BS, ammonium ferric citrate (AFC) and cysteine in order to synchronize the culture (Vohra et al, 1998). Amoebas (10 4 ) were inoculated into 1 ml BI-S-33 or low-iron with or without holoLf incubated for 96 h, and samples were collected every 24 h to test cell viability by trypan blue exclusion, using light microscopy.…”

Genome Re-duplication and Irregular Segregation Occur During the Cell Cycle of Entamoeba histolytica

“…Estimation of genome content was based on fluorescence of DNA binding dye either by flow cytometry (whole cell genome content) or scanning cytometry (individual nuclear genome content). Increased heterogeneity was observed in late log phase culture, suggesting that depletion of nutrients maybe responsible for loss of cell cycle control (Gangopadhyay et al 1997a, Vohra et al 1998. However, later studies showed that genome reduplication occurred within 2 h after initiation of DNA synthesis in freshly cultured synchronized cells .…”