Oncogene (1997) 15, 2659 ± 2665 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 In¯uence of ATM function on telomere metabolism Lubomir B Smilenov1, Susan E Morgan2, Wilfredo Mellado1, Satin G Sawant1, Michael B Kastan2 and Tej K Pandita1 1Center for Radiological Research, College of Physicians and Surgeons, Columbia University, New York, NY 10032; 2The Johns Hopkins Oncology Center, Baltimore, Maryland 21205, USA The ATM gene product, which is defective in the cancer- associations (TA) also called chromosome end associa- prone disorder ataxia telangiectasia, has been implicated tions, involve telomeres which are essential for the in mitogenic signal transduction, chromosome condensa- stability and complete replication of eukaryotic tion, meiotic recombination and cell cycle control. The chromosomes (Muller, 1938; McClintock, 1941). ATM gene has homology with the TEL1 gene of yeast, Telomeric associations have been linked to genomic mutations of which lead to shortened telomeres. To test instability and carcinogenicity (Pathak et al., 1988; the hypothesis that the ATM gene product is involved in Counter et al., 1992; Pandita et al., 1995, 1996). In telomere metabolism, we examined telomeric associa- contrast to the unstable ends generated by chromo- tions (TA), telomere length, and telomerase activity in some breakage, telomeres cap chromosomes, prevent human cells expressing either dominant-negative or degradation and end-to-end ligation. Mammalian complementing fragments of the ATM gene. The telomeres are composed of tandem arrays of phenotype of RKO colorectal tumor cells expressing (TTAGGG)n repeat sequences and are maintained by ATM fragments containing a leucine zipper (LZ) motif telomerase. Telomeres shorten as a function of age in mimics that of ataxia telangiectasia (A-T) cells. These cells derived from normal blood, skin and colonic transfected RKO cells relative to transfected controls mucosa (Hastie et al., 1990; Lindsey et al., 1991; had a higher frequency of cells with TA and shortened Allsopp et al., 1992;). As a result of this shortening, it telomeres, but no detectable change in telomerase is thought that critical genes at the ends of activity. In addition, the percentage of cells with TA chromosomes either become deleted or are activated, after gamma irradiation was higher in the transfected thus leading to cell death (Levy et al., 1992; Olovnick, RKO cells with dominant negative activity of the ATM 1992). gene, compared to control cells. SV40 transformed Recently, the gene that mediates the disease ataxia ®broblasts derived from an A-T patient and transfected telangiectasia has been cloned and has been designated with a complementing carboxyl terminal kinase region of ATM (AT, mutated) (Savitsky et al., 1995a). The the ATM gene had a reduced frequency of cells with TA, predicted product of the ATM gene open reading with no eect on the telomere length or telomerase frame is a large protein of 3056 amino acids (Savitsky activity. The present studies using isogenic cells with et al., 1995b). The protein shows similarity to several manipulated ATM function demonstrate a role for the yeast and mammalian proteins involved in meiotic ATM gene product in telomere metabolism. recombination and cell cycle progression, namely the products of MEC1 in budding yeast and rad3 in ®ssion Keywords: telomeric associations; telomere length; yeast, and the TOR proteins of yeast and mammals telomerase activity; A-T; LZ; PI-3 kinase domain (Savitsky et al., 1995b; Brush et al., 1996; Morrow et al., 1996). The ATM gene product shares the PI-3 kinase signature of a growing family of proteins involved in the control of cell cycle progression, Introduction processing of DNA damage and maintenance of genomic stability (Greenwell et al., 1995; Keith and Ataxia telangiectasia (A-T) is an autosomal disorder Schreiber, 1995; Hawley and Friend, 1996). The characterized by progressive neurological degeneration, presence of leucine zipper motif in the ATM protein premature aging, growth retardation, speci®c immuno- suggests possible dimerization of the protein or de®ciencies, telangiectasias, high sensitivity to ionizing interaction with additional proteins. Because of ATM radiation, genomic instability and cancer progression homology to TEL1 mutants of yeast (Greenwell et al., (Bridges and Harnden, 1982; Sedgwick and Border, 1995), it has been suggested that mutations in ATM 1991; Harnden, 1994). Cells derived from A-T patients gene could lead to defective telomere maintenance. To exhibit a variety of abnormalities in culture such as test the hypothesis that the ATM gene product hypersensitivity to ionizing radiation, higher require- in¯uences telomere metabolism, the cell types listed in ments for serum growth factors and cytoskeletal Table 1 with dominant-negative, as well as comple- defects (Gatti et al., 1991; Meyn, 1995; Shiloh, 1995). menting activity for ATM function were utilized A-T patients have a high frequency of cells with (Morgan et al., 1997). Expression of the dominant- telomeric associations seen at metaphase and show negative ATM fragments in RKO cells leads to accelerated shortening of telomeres (Kojis et al., 1991; decreased clonogenic survival, increased chromosomal Pandita et al., 1995; Metcalf et al., 1996). Telomeric aberrations and radioresistant DNA synthesis after treatment with ionizing radiation (Morgan et al., 1997). The ATM fragment containing the kinase domain Correspondence: TK Pandita complemented radiosensitivity, the S-phase checkpoint, Received 10 June 1997; revised 23 July 1997; accepted 24 July 1997 irradiation-induced activation of c-Abl and reduced ATM gene and telomeres LB Smilenov et al 2660 Table 1 Description of cells with dominant negative or complementation activity of ATM gene Cell line Cell type Functional activity RKO Colorectal carcinoma cells RKOpBABEpuro RKO with vector only RKOFB2F3 RKO with LZ motif of ATM gene Dominant negative RKOFB2F7 RKO with LZ motif of ATM gene Dominant negative RKOFB2F12 RKO with LZ motif of ATM gene Dominant negative RKOENA/FB2F18 RKO with LZ motif with entire 5' coding region of ATM gene Dominant negative GM5849 SV40 immortalized A-T cells GM5849LXSNeo GM5849 with vector only GM5849P13K4 GM5849 with P13K domain of ATM gene Complementation GM5849P13K10 GM5849 with P13K domain of ATM gene Complementation chromosomal aberrations after treatment with gamma dominant-negative ATM fragment (Table 2). In order rays in SV40 transformed ®broblasts derived from an to determine whether loss of telomeric signals was due A-T patient (Baskaran et al., 1997; Morgan et al., to a reduction in telomere length, we next evaluated 1997). telomere length in these RKO cells at early passage as well as after several population doublings. The telomere length of the RKO cells at the time of Results and Discussion infection was 7.8 kb. The earliest passage transfected cells used for the determination of telomere length were Recently, telomeric associations have been reported as at about 31 population doublings. Telomere length was a consequence of mutations in the UbcD1 gene in determined by measuring the terminal restriction Drosophila melanogaster (Cenci et al., 1997), as well as fragment (TRF) in RsaI and HinfI digested DNA mutations in a telomerase template in Tetrahymena (Figure 3). Three out of the four RKO clones thermophila (Kirk et al., 1997). To determine whether (RKOFB2F3, RKOFB2F7 and RKOENA/FB2F18) ATM function in¯uences TA, we examined metaphase expressing the dominant-negative fragment had statis- spreads and found that RKO cells expressing tically signi®cant shorter mean TRF (Table 2). The dominant-negative fragments exhibited a 3 ± 6-fold rate of telomere shortening varied in the dierent cell higher frequency of cells with TA compared to lines from 80 ± 130 base pairs per population doubling. parental and control RKO cells (Figure 1a). This The one transfected clone (RKOFB2F12) which did abnormal telomeric association phenotype was mani- not have shorter telomeres was the same clone that had fested in both G1- and G2-phase cells as determined by an unaltered telomere signal. To determine the using the premature chromosome condensation techni- in¯uence of the pBABEpuro vector on telomere que (Figure 1a) (Pandita et al., 1995). Since gamma length, we determined the mean TRF of four clones irradiation triggers TA in A-T cells (Pandita and of RKOpBABEpuro. We found that there were no Hittelman, 1993), we also evaluated TA in these cells signi®cant dierences in mean TRF from the RKO after radiation treatment and found that the frequency control among dierent clones of RKOpBABEpuro. of cells with TA also increased in the RKO cells with This suggests that accelerated telomere shortening was the dominant-negative ATM fragments, compared to not due to the presence of the vector. Furthermore, control cells (Figure 1b). when TRF was evaluated with increased population Telomeric association frequencies were also evalu- doublings in the RKO cells expressing the dominant- ated in A-T cells expressing the complementing kinase negative fragments (RKOFB2F3), both a decrease in domain fragment. The kinase-complemented A-T cells telomere signal (data not shown) and a shortening of had a 2 ± 4-fold lower frequency of cells with TA than telomere length (loss of about 22 base pairs per the parental or control-transfected cells in metaphase, population doubling) was observed with the increase as well as in G1 and G2 (Figure 2a). Similarly, in population doubling numbers