Cis-Activation of the human telomerase gene (hTERT) by the hepatitis B virus genome.

Abstract

Telomerase catalyzes the elongation of telomeric repeat DNA to maintain telomere length and structure (1). Although telomerase is a complex composed of a catalytic subunit (hTERT) and an RNA component (hTER), it is the expression of hTERT (2,3) that is the major determinant of telomerase activity in human cells (1,4). Maintaining telomere length by telomerase is associated with human cell immortalization and oncogenesis (1); however, no mutations in the hTERT or hTER telomerase genes have been found in human cancers. Hepatocellular carcinoma is often associated with both telomerase activation and viral infection. Here, we show the cisactivation of the hTERT gene by an insertion of viral DNA into the hTERT promoter region. A 66-base-pair (bp) sequence within the promoter region of hTERT gene (positions −372 to −307) (Fig. 1, A) is identical to a cellular sequence at the integration site of the hepatitis B virus (HBV) genome in a telomerase-positive (Fig. 1, B) hepatocellular carcinoma cell line, huH-4 (GenBank X51995) (5,6). This sequence corresponds to the upstream junction of the HBV integration (Fig. 1, A). Southern blot analysis confirmed an huH-4-specific rearrangement within the hTERT promoter and suggested an intact hTERT coding region in this cell line. Genomic polymerase chain reaction (PCR) by use of the primers from HBV (near the enhancer region or at the preS1 region) and hTERT sequences (in exon 1 or intron 2) generated huH-4-specific products (Fig. 1, C) that allowed identification of the downstream integration site of HBV (Fig. 1, A and E). The HBV genome was disrupted within its preS1 region and inserted at position −257 of the hTERT promoter, with an associated 49-bp deletion (positions −306 to −258) of the hTERT promoter. The integrated HBV genome was rearranged so that a HBV enhancer sequence (7) was placed about 1.6 kilobase upstream of the junction. The presence of a full-length hTERT messenger RNA starting from the endogenous transcription initiation site in huH-4 cells has been shown by the ribonuclease protection assay [see (5)] Affiliation of authors: Laboratory of Biosystems and Cancer, Cancer and Aging Section, Division of Basic Sciences, Center for Cancer Research, National Cancer Institute, Bethesda, MD. Correspondence to: Izumi Horikawa, Ph.D., National Institutes of Health, Bldg. 40, Rm. 2609, MSC-3020, Bethesda, MD 20892–3020 (e-mail: horikawi@mail.nih.gov). See “Notes” following “References.”