Entamoeba invadens: Dynamics of DNA synthesis during differentiation from trophozoite to cyst Nishant Singh a , Sudha Bhattacharya a , Jaishree Paul b,⇑ a School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India b School of Life Sciences, Jawaharlal Nehru University, New Delhi, India article info Article history: Received 29 March 2010 Received in revised form 26 May 2010 Accepted 13 August 2010 Available online 19 August 2010 Keywords: Entamoeba invadens DNA replication Cell-cycle Thymidine incorporation Flow cytometry abstract The DNA dynamics which mediate conversion of uni-nucleate trophozoite into quadrinucleate cyst in Entamoeba histolytica is not well understood. Here, we have addressed this question in Entamoeba inva- dens (a model system for encystation) through a detailed time course study of the differentiation process. We combined flow cytometric analysis with the change in rate of thymidine incorporation and the num- ber of nuclei per cell. Our data shows that during encystment the cell population passes through three phases: (1) Early phase (0–8 h); of rapid DNA synthesis which may correspond to completion of ongoing DNA replication. Bi-nucleated cells increase with concomitant drop in uni-nucleated cells. (2) Commit- ment phase (8–24 h); in which DNA synthesis rate slows down. Possibly new rounds of replication are initiated which proceed slowly, followed by mitosis at 20 h. After this the number of bi- and uni-nucle- ated cells gradually decline and the tri- and tetra-nucleated cells begin to increase. (3) Consolidation phase (24–72 h); in which the rate of DNA synthesis shows a small increase till 32 h and then begins to decline. The G2/M peak reappears at 48 h, showing that more rounds of DNA replication may be get- ting completed, followed by nuclear division. By 72 h the encystment is virtually complete. The bi-nucle- ated stage could be an intermediate both in the conversion of trophozoite to cyst and back. Our study provides a comprehensive view of DNA dynamics during encystation and excystation of E. invadens. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Amoebiasis is the third commonest cause of death due to para- sitic infection globally, and is a major health problem in developing countries (Stanley, 2003; Troll et al., 1997). It is acquired by inges- tion of Entamoeba histolytica cysts, which get converted into the motile trophozoites in the ileo-cecal region of the intestine. Cysts are the infective stage, but so far it has not been possible to induce encystment in axenic E. histolytica trophozoites in culture. To study this differentiation process the model system used is Entamoeba invadens, the reptilian parasite which can encyst in culture (Avron et al., 1986; Sanchez et al., 1994; Vazquezdelara-Cisneros and Arroyo-Begovich, 1984). Encystation and excystation are among the simplest developmental processes in eukaryotes. Characteriza- tion of these processes may yield important information about the evolution of more elaborate developmental processes. E. invadens trophozoites typically contain one nucleus whereas mature cysts contain four nuclei (Silberman et al., 1999). During the differentiation of trophozoite to cyst, an increase in the DNA con- tent per cell is expected. Two rounds of DNA replication have been reported to occur during E. invadens encystment (Ganguly and Lohia, 2001; Kumagai et al., 1998), resulting in the tetra-nucleated cyst. However, no details of this process are known. Estimates of ploidy and the distribution of DNA in the typical cell-cycle stages in Entamoeba have been attempted previously, employing flow cytom- etry, and the results obtained have varied depending on the proto- cols used for fixing and staining the cells (Byers and Eichinger, 2005; Dvorak et al., 1995; Ganguly and Lohia, 2001; Mukherjee et al., 2008). The method employed by Dvorak’s group eliminates the high natural fluorescence of Entamoeba trophozoites. With this method it was found that exponentially growing E. invadens tropho- zoites displayed G1, S and G2 phases in proportions typical of an eukaryotic cell population (Dvorak et al., 1995; Mitchison, 1971). However, cells prepared by other methods showed more atypical patterns, with apparent ploidies ranging from 1 to 16 N (Byers and Eichinger, 2005; Ganguly and Lohia, 2001). In addition to these inconsistencies, there is a paucity of information regarding the changes that take place in the uni-nucleated trophozoite during its conversion into a tetra-nucleate cyst. Here, we have attempted to address the dynamics of DNA synthesis through a detailed time course study at different stages of the life cycle from trophozoite to cyst and back to trophozoite. We prepared the cells for flow cytometry by Dvorak’s method, and combined this analysis with the change in rate of thymidine incorporation and the number of nuclei per cell throughout the differentiation process. 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.08.013 ⇑ Corresponding author. Fax: +91 011 26742558. E-mail address: jpaul33@hotmail.com (J. Paul). Experimental Parasitology 127 (2011) 329–333 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr