Telomere homolog oligonucleotides induce apoptosis in malignant but not in normal lymphoid cells: Mechanism and therapeutic potential

Telomeres are tandem repeats of a 6-nucleotide sequence (TTAGGG) that protect the ends of linear chromosomes from being recognized as double-stranded DNA breaks. In eukaryotic DNA replication, the DNA at the ends of telomeres is not completely replicated and, therefore, telomeres shorten by 50–100 bp with each mitotic cycle. In somatic cells, when the shortened telomeres have been reduced beyond a critical minimal length, the telomere structure is thought to become unstable, thereby activating DNA damage responses that lead to cell cycle exit and irreversible replicative senescence.1–3 This permanent limitation on growth likely protects organisms from the cancer risks associated with unlimited replicative potential.4 Treatment of cultured cells with DNA oligonucleotides that are homologous to the TTAGGG repeat sequence of the chromosomal telomere, termed “T-oligos,” induces differentiation, apoptosis or senescence.1,3,5 We and others have shown that T-oligo treatment induces DNA damage-like responses: phosphorylation of p53, histone H2AX (γH2AX), p95/Nbs1, Chk1 and Chk2 [downstream effectors of the ataxia telangiectasia mutated (ATM) and ATM-related protein kinases], inhibition of DNA synthesis, a senescent phenotype in fibroblasts and apoptosis or senescence in multiple transformed cells.1–3,5–9 Specifically, in normal fibroblasts, prolonged T-oligo treatment induces irreversible cell cycle arrest, flat cell morphology, induction of p16, p21, p53 and p95/Nbs1 and increased expression of senescence-associated β-galactosidase,2 indistinguishable from late-passage in vitro replicative senescence. 2,10,11 This phenotype has been suggested to arise because T-oligo treatment may mimic the exposure of single-stranded DNA during telomere crisis that follows telomere shortening; indeed, telomere loop disruption by dominant negative (DN) TRF2 produces a similar senescent phenotype in these cells.2 However, T-oligos induce these responses independently of telomerase expression and without shortening endogenous telomeres, loss of the telomere 3′ G-rich overhang or disrupting telomere structure.2,5–7,12 T-oligo treatment does not inhibit telomerase5 and its effects are specific to the telomeric DNA sequence, because control scrambled, unrelated or complementary oligonucleotides of the same length are ineffective.1–3,5 Remarkably, T-oligos cause apoptosis in many malignant cell types, instead of cell cycle exit and senescence,2,3,13 again mimicking experimental telomere loop disruption by DN TRF2.14 By an unknown mechanism, T-oligos rapidly concentrate in the nucleus,1,2,13 where such oligos appear to have a half life of at least several days.15 Within 24 hr, T-oligos induce S-phase cell cycle arrest, H2AX phosphorylation and cause apoptosis in breast, pancreatic and ovarian carcinoma and melanoma cell lines, including lines that lack p53 and/or p16 and harbor a variety of other abnormalities in key regulatory signaling pathways.1,3,13,16 For example, cultures of malignant melanoma (MM-AN) cells3 or breast cancer (MCF-7) cells13 show dramatically increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and sub-G1 DNA content upon T-oligo exposure. Furthermore, these responses occur selectively in malignant cells and not in their nontransformed, normal counterparts.3,6,13 This difference between the responses of normal and malignant cultured cells has suggested that T-oligos may have therapeutic potential as anticancer agents. T-oligos have been tested as an anticancer therapy in vivo in preclinical models in mice. T-oligo administered by intralesional, intravenous (i.v.) or intraperitoneal (i.p.) injection in severe combined immunodeficiency mice bearing human MM-AN melanoma or MCF-7 breast cancer xenografts reduced primary tumor volumes and metastases by 85–90%.3,13 These reductions in tumor burden were achieved at least in part through T-oligo-specific apoptosis as assayed by TUNEL staining.3,13 Yet, under these conditions, no toxicity to normal tissue was apparent by histologic examination at autopsy, including intestinal mucosa, hair follicles, bone marrow, liver, jejunum, brain, lung or kidney,3,13 confirming efficacy with no detectable toxicity. These data suggested that, in addition to the solitary solid tumors studied to date, T-oligos might also cause apoptosis effectively in lymphoid malignancies. Indeed, at least in vitro, human Jurkat T-leukemic cells undergo T-oligo-specific apoptosis.1 We, therefore, asked whether diverse human and murine T-lymphoid and B-lymphoid tumor cell lines might also exhibit T-oligo-specific apoptosis. We then expanded the studies to a recently developed mouse model with systemic, aggressive B-cell lymphoma/leukemia.