Mutation Research 755 (2013) 135–140 Contents lists available at SciVerse ScienceDirect Mutation Research/Genetic Toxicology and Environmental Mutagenesis jou rn al h om ep age: www.elsevier.com/locate/gentox C om mu n i ty add ress: www.elsevier.com/locate/mutres Multinucleation regulated by the Akt/PTEN signaling pathway is a survival strategy for HepG2 cells Ananda Mukherjee a , Sandip Misra a , Niall G. Howlett b , Parimal Karmakar a,∗ a Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S.C. Mullick Road, Kolkata 700032, West Bengal, India b Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA a r t i c l e i n f o Article history: Received 21 January 2013 Received in revised form 16 May 2013 Accepted 13 June 2013 Available online 21 June 2013 Keywords: Chang Liver Cytokinesis-block micronucleus assay HepG2 Multinucleation Akt PTEN a b s t r a c t Hepatocellular carcinoma (HCC) is non-responsive to many chemotherapeutic agents including etopo- side. The aim of this study was to examine the survival strategy of the HCC cell line HepG2 after etoposide treatment. Here we analyzed and compared spontaneous and etoposide-induced DNA damage in HepG2 (-fetoprotein (AFP)-positive) and Chang Liver (AFP-negative) cell lines. Compared to Chang Liver cells, HepG2 cells exhibited a significantly higher degree of micronucleation and a higher nuclear division index, as determined by the cytokinesis-block micronucleus assay, following exposure to etoposide. HepG2 cells were also more resistant to etoposide-induced cytotoxicity compared to Chang Liver cells. We also establish that increased etoposide-induced multinucleation in HepG2 cells is dependent on the catalytic activity of Akt, as phosphatidylinositol-3-kinase inhibitors as well as the overexpression of kinase-defective Akt reversed this phenotype. Moreover, ectopic expression of wild type PTEN reduced the frequency of etoposide-induced multinucleated HepG2 cells, and restored HepG2 etoposide sensi- tivity. Taken together, these results implicate the Akt/PTEN cellular axis as a major determinant of the etoposide resistance of HCC cells. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Hepatocellular carcinoma (HCC) is aggressive and less sen- sitive toward chemotherapeutic cytotoxic agents [1]. The HCC cell line, HepG2 has tremendous growth potential and can sur- vive with a highly unstable genome [2]. A recent study indicates that deregulation of the phosphatidylinositol-3-kinase (PI3K) path- way plays an important role in the resistance of HCC cells to chemotherapeutic agents [3]. In HCC cells Akt, an oncogenic member of the PI3K pathway, is hyperphosphorylated due to inactivation of PTEN (phosphatase and tensin homologue deleted on chromosome 10). PTEN is a dual lipid and protein phos- phatase, and specifically dephosphorylates the D3 position of phosphoinositide-3,4,5-triphosphate, thus negatively regulating the PI3K/Akt signaling pathway [4,5]. PTEN can also regulate cell motility and polarity, tumor microenvironment, cellular senes- cence and self-renewal ability of stem cells [6]. A role for nuclear PTEN in the maintenance of chromosome stability has also been established [7,8]. In a recent study, we have demonstrated that depletion of PTEN leads to genomic instability in human embryonic kidney cells after etoposide insult [9]. ∗ Corresponding author. Tel.: +91 33 2414 6710; fax: +91 33 2413 7121. E-mail address: pkarmakar 28@yahoo.co.in (P. Karmakar). Multinucleation – the process of generating more than one nucleus – is a feature of neoplastic cells. Previous studies have shown that defects in the spindle assembly check-point (SAC) [10], abnormal cytokinesis [11] or acytokinetic cell division [12], chronic activation of Akt [13], loss of p53 [14,15], reduced expression of DNA repair genes [16,17] and non-genetic aneuploidy [18] can contribute to this process. Multinucleated cell formation in Dic- tyostelium has also been demonstrated to be regulated by PTEN signaling [19,20]. Additionally, DNA damage-induced multinucle- ation as a result of defective DNA repair mechanisms has also previously been reported [21]. Having defective PI3K regulation, HepG2 cells become less resis- tant to etoposide when PI3K signaling pathway is inhibited [2,3,22]. Thus, defective cell cycle checkpoints and multinucleation may both contribute to the increased drug resistance of HepG2 cells. While the role of defective cell cycle checkpoints has been well studied, the role of cytological parameters such as multinucleation, micronucleus (MNi) formation, nuclear bud(s) (NBUDs), nucleo- plasmic bridge(s) (NPBs) and nuclear division index (NDI) have not been addressed. This prompted us to analyze the cytological damage and multinucleation of HepG2 and Chang Liver cells after etoposide treatment. These two cell lines were selected on the basis of their -fetoprotein (AFP) status, a serological marker widely used in the clinical detection of HCC [23]. We also further analyze the involvement of Akt/PTEN signaling pathway in the multinucleation process of HepG2 cells. We observed that the catalytic function of 1383-5718/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mrgentox.2013.06.009