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Purpose: Enhanced DNA repair activity is often associated with tumor resistance to radiotherapy. We hypothesized that inhibiting DNA damage repair would sensitize tumors to radiation-induced DNA damage. Experimental Design: A novel strategy for inhibiting DNA repair was tested.We designed small DNA molecules that mimic DNA double-strand breaks (called Dbait) and act by disorganizing damage signaling and DNA repair. We analyzed the effects of Dbait in cultured cells and on xenografted tumors growth and performed preliminary studies of their mechanism(s) of action. Results: The selected Dbait molecules activate H2AX phosphorylation in cell culture and in xenografted tumors. In vitro, this activation correlates with the reduction of Nijmegen breakage syndrome 1and p53-binding protein 1repair foci formation after irradiation. Cells are sensitized to irradiation and do not efficiently repair DNA damage. In vivo, Dbait induces regression of radioresistant head and neck squamous cell carcinoma (Hep2) and melanoma (SK28 and LU1205) tumors. The combination of Dbait32Hc treatment and fractionated radiotherapy significantly enhanced the therapeutic effect. Tumor growth control by Dbait molecules depended directly on the dose and was observed with various irradiation protocols. The induction of H2AX phosphorylation in tumors treated with Dbait suggests that it acts in vivo through the induction of ''false'' DNA damage signaling and repair inhibition. Conclusions:These data validate the concept of introducing small DNA molecules, which mimic DNA damage, to trigger ''false'' signaling of DNA damage and impair DNA repair of damaged chromosomes. This new strategy could provide a new method for enhancing radiotherapy efficiency in radioresistant tumors.

Summary Apoptosis can be triggered by cytotoxic agents and radiation currently used in cancer treatment. However, the apoptotic response appears to vary between cell types (normal or transformed) and between types of malignancy. Thus, irradiation induces apoptosis in normal human lymphocytes but not in lymphocytes derived from a subset of chronic lymphocytic leukaemia (CLL). Moreover, in this subset, spontaneous apoptosis is inhibited by irradiation. Why irradiation does not allow the initiation of the apoptotic death pathway could be explained, at least in part, and in agreement with recent findings on experimental models, by the activation of the transcriptional factor NF-KB, which is able to inhibit apoptotic cell response. Low doses (at which no effect is observed with normal human lymphocytes) of the highly specific proteasome inhibitor lactacystin are sufficient to trigger apoptosis in these malignant cells. Proteasome inhibition by lactacystin prevents the nuclear translocation of both p50 and p65 NF-KB subunits and sensitizes these cells to apoptosis by tumour necrosis factor (TNF)-a treatment. As this subset of CLL is totally resistant to any treatment, proteasome inhibition by lactacystin provides a new therapeutic approach to be explored, considering the sensitivity of malignant CLL-derived lymphocytes to be quite different from that of normal human lymphocytes.

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Moderate anemia appeared to be an independent prognostic factor in squamous cell carcinoma of the head and neck treated with radiation therapy alone.

Summary. Ubiquitin-proteasome-dependent protein processing appears to be an essential component in the control of radiation-induced apoptosis in human lymphocytes. This control is altered in chronic lymphocytic leukaemia (CLL), compared to that of normal human lymphocytes which mainly showed high apoptotic values after irradiation, but in some cases no sensitivity was observed. Interestingly, lactacystin activated the apoptotic pathway in both radioresistant and sensitive CLL cells, at doses which had no effect in normal cells where significantly higher concentrations were required. Therefore the resistance of some CLL cells to apoptosis initiation by radiation does not correlate to observed increased sensitivity to lactacystin. The nuclear level of the transcription factor NF-kB or the cytoplasmic level of IkBa remained unaltered upon irradiation or lactacystin CLL cells treatment, suggesting that the activity of the other factors involved in apoptotic death control were altered through proteasomal inhibition. These results strongly suggest an essential role of the ubiquitin system in apoptotic cell death control in CLL lymphocytes. The inhibition of proteasome-ubiquitin-dependent processing could be a discriminatory apoptotic stimulus between normal versus malignant lymphocytes and therefore might potentially be of use in this specific human pathology.