Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase

Abstract

Nasopharyngeal carcinoma (NPC) is a common disease in Hong Kong and southern provinces of China. EBV infection is believed to play a critical role in the development of NPC. Previous studies on the transformation mechanism of EBV genes were mostly performed in either NPC or nonnasopharyngeal epithelial cells which may not be representative of premalignant nasopharyngeal epithelial cells. Establishment of a representative cell system would greatly facilitate the elucidation of the role of EBV infection in the development of NPC. Using telomerase alone, we were able to establish an immortalized nasopharyngeal epithelial cell line from primary nonmalignant nasopharyngeal biopsies. The telomerase‐immortalized nasopharyngeal epithelial cells are largely diploid in karyotype. Interestingly, this newly immortalized nasopharyngeal epithelial cell line, referred as NP460hTert, harbors genetic alterations previously identified in premalignant and malignant nasopharyngeal epithelial cells. These include inactivation of p16 by homozygous deletion of the p16INK4A locus and downregulation of RASSF1A expression. The deletion of the p16INK4A locus appears to be the most crucial event for the immortalization of nasopharyngeal epithelial cells by telomerase and precedes RASSF1A downregulation. In addition, detailed analysis of the cytogenetic changes by conventional cytogenetics, spectral karyotyping (SKY) and array‐based CGH revealed a gain of a 17q21‐q25 fragment on 11p15 chromosome in all NP460hTert cells which occurred before deletion of the p16INK4A locus. Gain of 17q has been previously reported in NPC. In addition, activation of NF‐κB was observed in immortalized NP460hTert cells at the later population doublings, and may play a role in the survival of immortalized NP epithelial cells. Id1 which is commonly expressed in various human cancers, including NPC, was also upregulated in the immortalized NP460hTert cells. Thus, the establishment of an immortalized nasopharyngeal epithelial cell line harboring common genetic alterations present in premalignant and cancerous nasopharyngeal epithelial cells may provide a valuable cell system to examine for early events involved in NPC carcinogenesis, particularly in elucidating the role of EBV infection in NPC development. © 2006 Wiley‐Liss, Inc.

Mechanisms of cell immortalization mediated by EB viral activation of telomerase in nasopharyngeal carcinoma

“…As the rate-limiting component of telomerase, hTERT induces immortalization of a number of cell types in culture [49] (reviewed in [50]). Recently, hTERT has also induced an immortalized nasopharyngeal epithelial cell line from non-malignant nasopharyngeal biopsies, which is accompanied by other genetic alterations seen in NPC including homozygous deletion of the p16 INK4A [12]. Consistently, hTERT is below detectable levels in most normal human somatic cells, but is expressed concomitantly with telomerase activity during cellular immortalization [40].…”

“…Nonetheless, recent studies attempting to target LMP1 have shown that silencing LMP1 by siRNA induces apoptosis in EBV-positive lymphoma cells in association with telomerase inhibition, suggesting that molecular targeting of LMP1 may be of therapeutic value in EBV-associated tumours [15]. These concepts may be reinforced by the data that expression of hTERT alone immortalizes non-malignant nasopharyngeal epithelial [12].…”

“…EBV may cause the NPC cell oncogenic clonal evolution by interfering with several intracellular mechanisms in the npg infected cells [1,[7][8][9][10][11]. Recently, evidence suggests that the enzyme telomerase required for cell immortalization is activated in NPC and the EBV proteins are involved in regulating telomerase activity [12][13][14], consistent with the findings that EBV proteins also play a key role in immortalization of infected B-lymphocyte [15][16][17]. Here we review the recent progress in our understanding of regulation of telomerase activity by EBV proteins, a critical step in cell immortalization.…”

Impact of G2 checkpoint defect on centromeric instability

“…Overstimulation of cell proliferation pathways has been shown to generate replication stress and DNA double-strand breaks at regions difficult to replicate, due to the conflict between unscheduled DNA synthesis and uncoordinated prereplicative complex assembly (Bartkova et al, 2005;Gorgoulis et al, 2005). Indeed, p16 INK4a deletion, which promotes cell proliferation, is detected in both of our telomerase-immortalized cell lines (Li et al, 2006;Cheung et al, 2010).…”

“…Cyclin B1 overexpression promotes G 2 checkpoint defect and centromeric instability To study the causative role of G 2 checkpoint defect in centromeric instability, we stably overexpressed cyclin B1 in two human telomerase-immortalized cell lines derived from normal esophageal epithelial cells (NE2-hTERT) (Cheung et al, 2010) and nasopharyngeal epithelial cells (NP460-hTERT) (Li et al, 2006), which were chosen because they had low background levels of centromeric instability. The cells were infected with retroviral plasmids, expressing cyclin B1 or empty vector, and selected with puromycin for 6 days.…”