[CANCER RESEARCH 63, 7047–7050, November 1, 2003] Advances in Brief Telomere Dysfunction Results in Enhanced Organismal Sensitivity to the Alkylating Agent N-Methyl-N-Nitrosourea 1 Eva Gonza ´lez-Sua ´rez, Fermı ´n A. Goytisolo, Juana M. Flores, and Marı ´a A. Blasco 2 Department of Immunology and Oncology, National Center of Biotechnology [E. G. S., F. A. G., M. A. B.], and Animal Pathology II, Facultad de Veterinaria, Universidad Complutense de Madrid [J. M. F.], Madrid, Spain Abstract Here, we use telomerase-deficient mice, Terc / , to study the impact of telomerase abrogation in response to treatment with the alkylating agent N-Methyl-N-Nitrosourea (MNU), a potent carcinogen in the mouse. Wild- type mice treated with MNU developed lymphomas and carcinomas. In contrast, similarly treated G5 Terc / mice with critically short telomeres did not develop tumors and died of acute toxicity to the small intestine. G2 Terc / mice, which have long telomeres, were less susceptible to MNU- induced tumors than wild-type mice, as well as less sensitive to MNU toxicity than G5 Terc / mice. The results indicate that short telomeres suppress tumor growth and that lack of telomerase retards tumor pro- gression, even in the presence of long telomeres. Finally, G5 Terc / hypersensitivity to MNU supports the notion that short telomeres inter- fere with proper DNA damage repair. Introduction Telomeres are specialized protective structures at the ends of eu- karyotic chromosomes (1) synthesized by telomerase (2). Dysfunc- tional telomeres trigger end-to-end chromosome fusions and loss of cell viability (3). The fact that most cancer cells have high telomerase levels, not observed in somatic cells, and that telomere maintenance above a threshold length is necessary for cell viability make of telomerase an attractive target for cancer therapy. We studied here the tumor susceptibility and cytotoxic sensitivity of mice lacking the RNA component of telomerase (Terc; Ref. 4) in response to treatment with the alkylating agent MNU. 3 Terc -/- mice lack telomerase ac- tivity and show telomere shortening at a rate of 4 –5 kb/mouse generation (successive mouse generations are indicated as G1 through G6; Ref. 4). Early generation (G1 and G2) Terc -/- mice lack telom- erase activity but still show very long telomeres (4). Fifth generation (G5) Terc -/- mice lack telomerase activity and have a significant loss of telomeric sequences (4). Telomere shortening in these mice is accompanied by an increase in the number of chromosome ends with no detectable telomeres and in the frequency of end-to-end chromo- some fusions (4). G5 Terc -/- mice have been shown to be hypersen- sitive to -irradiation (5, 6), and short telomeres sensitize cells to the chemotherapeutic agent doxorubicin (7). In addition, telomerase-de- ficient mice with critically short telomeres are resistant to papilloma formation after chemical carcinogenesis of the skin (8). To determine whether this resistance to tumor formation and the hypersensitivity to DNA-damaging agents can be extrapolated to other carcinogens and other target tissues, we treated wt, G2 Terc -/- , and G5 Terc -/- mice with the alkylating agent MNU. O 6 -methylguanine is the preponder- ant toxic lesion produced by MNU, as it initiates G:C to A:T transition mutations in K-ras and other oncogenes, giving rise to a variety of tumors (9). O 6 -methylguanine lesions can be corrected by MMR, or specifically by O 6 -methylguanine methyltransferase. Alkylant lesions could be repaired by base excision repair, the major DNA repair pathway that protects mammalian cells against single-base DNA damage (10). Methylation-induced apoptosis appears to be MMR- dependent and to be triggered by secondary lesions, possibly DSBs formed during the MMR process (9). Alkylating agents, including MNU, are currently used in antitumor therapies (11); thus, treatment with MNU allows evaluation of the impact of short telomeres and the absence of telomerase on the murine response to DNA damage induced by this agent. Materials and Methods Mice. wt, G2 Terc -/- , and G5 Terc -/- mice were generated on a 60% C57BL/6, 37.5% Sv, and 2.5% SJL genetic background (4). MNU Treatment. MNU (Sigma, St. Louis, MO) was dissolved in PBS. Mice received weekly i.p. injections of MNU (30 mg/kg body weight) for 5 consecutive weeks. In experiment I, the first dose was administered to 8 females and 4 males of each genotype at 5 weeks of age. In experiment II, the first dose was administered to 10 males of each genotype (5–7 months of age). Control mice were treated in parallel with PBS. Histopathological Analysis. Mice were monitored daily and sacrificed when they showed signs of illness. Survivors were sacrificed at week 92 after the start of treatment. Tissues were fixed in 10% PBS-buffered formalin, embedded in paraffin, 4-m sections prepared, H&E stained, and examined by light microscopy. Intestinal lesions were classified according to intensity and extension of atrophy and severity of associated lesions (compensatory glan- dular hyperplasia, inflammation, ulceration, and lymphangiectasias). They were scored as mild (moderate shortening of villi in small intestine and of crypts in the large intestine, with no associated lesions), intermediate (marked reduction in villous/crypt size, with associated lesions of moderate intensity), and severe (disappearance of villi/crypts, with severe associated lesions). Statistical Analysis. To calculate the statistical significance of the differ- ences in MNU-induced tumor formation between wt and G2 Terc -/- mice, values on an arbitrary scale were assigned to each mouse according the pathologies present at death [0 = no tumor; 1 = benign tumor (adenoma or hemangioma); and 2 = malignant tumor (carcinoma, sarcoma, or lymphoma)]. Student’s t test was calculated using these values. Results G5 Terc / and G2 Terc / Mice Are Hypersensitive to MNU Genotoxic Damage. To analyze the impact of short telomeres and lack of telomerase in response to MNU treatment, 5-week-old wt, G2 Terc -/- , and G5 Terc -/- mice were treated with this alkylating agent for 5 weeks (experiment I, see “Materials and Methods”). G5 Terc -/- mice showed a drastic loss of viability after treatment (Fig. 1A). G5 Terc -/- mice began to die 26 weeks after initiation of treatment; by 54 weeks after the first MNU injection, 75% had died compared with Received 6/27/03; revised 9/11/03; accepted 9/12/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 E. G-S. received a predoctoral fellowship from the Spanish Fondo de Investigaciones Sanitarias. Research at the laboratory of M. A. B. is funded by grants from the Ministry of Science and Technology, the Regional Government of Madrid (Comunidad Autonoma de Madrid), the European Union, and by The Department of Immunology and Oncology (DIO). The DIO was founded and is supported by the Spanish Council for Scientific Research (Consejo Superior de Investigaciones Cientificas) and by Pfizer. 2 To whom requests for reprints should be addressed, at Molecular Oncology Program, Centro Nacional de Investigaciones Oncolo ´gicas (CNIO), Spanish National Cancer Cen- tre, Madrid, E28029, Spain. Phone: 917328031; E-mail: mblasco@cnio.es. 3 The abbreviations used are: MNU, N-methyl-N-nitrosourea; BER, base excision repair; DSB, double strand break; MMR, mismatch repair; Terc, telomerase RNA; wt, wild type. 7047 Research. on November 18, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from