Bmi-1 is a polycomb group protein that was identified as c-myc cooperating oncogene in murine lymphomagenesis. The current study was undertaken to determine the role of Bmi-1 in human oral carcinogenesis. Bmi-1 protein and RNA expression levels were markedly enhanced in the cells of oral squamous cell carcinomas (OSCC) compared with that of normal human oral keratinocytes (NHOK). Enhanced-Bmi-1 expression was also detected in situ in the archived oral mucosal tissues with cancerous and precancerous histopathology, including that of mild epithelial dysplasia. Thus, Bmi-1 expression occurs at a very early stage in oral carcinogenesis. To determine the biological role of Bmi-1 in cell proliferation, endogenous Bmi-1 was knocked down in actively proliferating SCC4 cells and NHOK by RNA interference. After Bmi-1 knockdown, cell replication was severely retarded. However, the expression of p16 INK4A , a known cellular target of Bmi-1, was not changed in cells with or without Bmi-1 knockdown. Furthermore, Bmi-1 knockdown in HOK-16B-BaP-T cells, in which the p16 INK4A /pRb pathway was abrogated, led to immediate arrest of replication and loss of viable cells. Thus, our data suggest that Bmi-1 may act through p16 INK4A -independent pathways to regulate cellular proliferation during oral cancer progression.

Telomerase activity in human cells closely correlates with the expression of its catalytic subunit, telomerase reverse transcriptase (hTERT). Previously, we reported the lack of telomerase activity in normal human oral fibroblasts (NHOF) and the diminution of telomerase activity during senescence in normal human oral keratinocytes (NHOK). To investigate the underlying mechanisms of telomerase regulation in both cell types, we analysed the expression, promoter activity, and methylation status of the hTERT gene. The expression of hTERT mRNA diminished in senescent NHOK, but was not detected in NHOF at any stage of replication. An exogenous hTERT promoter was active in NHOF and in senescing NHOK, indicating that the lack of hTERT gene expression resulted from alteration of the endogenous hTERT promoter. Since methylation is involved in the silencing of numerous genes, we carried out DNA methylation assays. The assay revealed that the hTERT promoter was hypermethylated in NHOF and was gradually methylated during senescence in NHOK. Treatment of NHOF and senescent NHOK with the demethylating agent 5-aza-2 0 -deoxycytidine restored the expression of endogenous hTERT mRNA. Our results suggest that hypermethylation of the hTERT promoter plays a critical role in the negative regulation of telomerase activity in normal human oral cells. (Feng et al, 1995;Harrington et al, 1997;Meyerson et al, 1997;Nakamura et al, 1997). While hTERT is not expressed in most normal human somatic tissues, hTR is expressed constitutively in most human tissues (Kim et al, 1994;Shay and Bacchetti, 1997). In normal human cells and in tumour cells, telomerase activity is positively correlated with the expression of hTERT (Kim et al, 1994;Shay and Bacchetti, 1997). Therefore, regulation of hTERT expression appears to control telomerase activity. The hTERT promoter region has been cloned and characterised Takakura et al, 1999), permitting analysis of the molecular mechanisms involved in its regulation (Cong et al, 1999;Horikawa et al, 1999;Takakura et al, 1999;Wick et al, 1999). Transcription factors c-myc, Sp1, AP2, AP4, and c-myb act as activators and several repressors, such as WT1, p53, MZF-2, and Mad1, have been identified (reviewed by Poole et al, 2001). Analysis of the hTERT promoter region also revealed the presence of CpG islands containing CG-rich methylation sites (Cong et al, 1999;Horikawa et al, 1999;Takakura et al, 1999;Wick et al, 1999). Promoter region hypermethylation associated with transcriptional loss is an alternative mode of gene silencing for several tumour-suppressor genes (Zingg and Jones, 1997;Baylin et al, 1998;Herman, 1999). The hypermethylation status of the hTERT promoter has been examined in a variety of human normal and cancer cells (Devereux et al, 1999;Dessain et al, 2000;Guilleret et al, 2002). In these studies, hTERT promoter methylation was observed in telomerase-negative, as well as in telomerase-positive cells, suggesting different modes of regulation of hTERT expression in methylation-dependent and -independ...