In addition to the ARF/p53 pathway, the DNA damage response (DDR) has been recognized as another oncogeneprovoked anticancer barrier in early human tumorigenesis leading to apoptosis or cellular senescence. DDR mutations may promote tumor formation, but their impact on treatment outcome remains unclear. In this study, we generated ataxia telangiectasia mutated (Atm)-proficient and -deficient B-cell lymphomas in E-myc transgenic mice to examine the role of DDR defects in lymphomagenesis and treatment sensitivity.Atm inactivation accelerated development of lymphomas, and their DNA damage checkpoint defects were virtually indistinguishable from those observed in Atm ϩ/ϩ -derived lymphomas that spontaneously inactivated the proapoptotic Atm/p53 cascade in response to Myc-evoked reactive oxygen species (ROS). Importantly, acquisition of DDR defects, but not selection against the ARF pathway, could be prevented by lifelong exposure to the ROS scavenger N-acetylcysteine (NAC) in vivo. Following anticancer therapy, DDR-compromised lymphomas displayed apoptotic but, surprisingly, no senescence defects and achieved a much poorer long-term outcome when compared with DDRcompetent lymphomas treated in vivo. Hence, Atm eliminates preneoplastic lesions by converting oncogenic signaling into apoptosis, and selection against an Atm-dependent response promotes formation of lymphomas with predetermined treatment insensitivity. IntroductionCancer development is a process against which normal cells appear to be well protected. 1 As an early cellular barrier against imminent transformation, normal cells monitor latent oncogenic signals, which may result in activation of the ARF/p53 axis as an antiproliferative constraint, ultimately leading to apoptosis or cellular senescence. 2 In turn, genetic defects in these oncogeneinduced fail-safe programs are prerequisites for malignant conversion and thus are selected for in manifest malignancies. [3][4][5] In addition, oncogene-related cellular stress enacting a DNA damage response (DDR) has been proposed as an ARF-independent constraint to limit aberrant cell division in early tumorigenesis via induction of apoptosis or senescence. 6-10 Indeed, prototypic oncogenes such as Myc and Ras have been reported to cause DNA damage, possibly via the production of reactive oxygen species (ROS) 11,12 or by generating aberrant DNA replication intermediates. 13,14 Conversely, inherited DDR defects as known from ataxia telangiectasia (A-T), 15 the Nijmegen breakage syndrome (Nbs), 16 or the Li-Fraumeni syndrome (affecting Chk2 or p53) 17,18 predispose to cancer, presumably by facilitating mutagenic activation and permitting transforming action of mitogenic oncogenes. 19 Immunohistochemical studies on epithelial tumor specimens of various developmental stages driven by unspecified oncogenic moieties found hyperproliferative precursor lesions to display activated components of the Atr/Nbs1/Chk1 and the Atm/Chk2/p53 DDR cascades, whereas this activation was lost in more advanced neoplastic lesions, thereb...

The modulated expression of MHC class I on tumour tissue is well documented. Although the effect of MHC class I expression on the tumorigenicity and immunogenicity of MHC class I negative tumour cell lines has been rigorously studied, less is known about the validity of gene transfer and selection in cell lines with a mixed MHC class I phenotype. To address this issue we identified a C26 cell subline that consists of distinct populations of MHC class I (H-2D/K) positive and negative cells. Transient transfection experiments using liposome-based transfer showed a lower transgene expression in MHC class I negative cells. In addition, MHC class I negative cells were more sensitive to antibiotic selection. This led to the generation of fully MHC class I positive cell lines. In contrast to C26 cells, all transfectants were rejected in vivo and induced protection against the parental tumour cells in rechallenge experiments. Tumour cell specificity of the immune response was demonstrated in in vitro cytokine secretion and cytotoxicity assays. Transfectants expressing CD40 ligand and hygromycin phosphotransferase were not more immunogenic than cells expressing hygromycin resistance alone. We suggest that the MHC class I positive phenotype of the C26 transfectants had a bearing on their immunogenicity, because selected MHC class I positive cells were more immunogenic than parental C26 cells and could induce specific anti-tumour immune responses. These data demonstrate that the generation of tumour cell transfectants can lead to the selection of subpopulations that show an altered phenotype compared to the parental cell line and display altered immunogenicity independent of selection marker genes or other immune modulatory genes. Our results show the importance of monitoring gene transfer in the whole tumour cell population, especially for the evaluation of in vivo therapies targeted to heterogeneous tumour cell populations.