Using methodology recently developed to assess gene-specific DNA repair, we have demonstrated that it is possible not only to study mitochondrial DNA repair, but also directly to compare mitochondrial and nuclear DNA repair in the same biological sample. Complex enzymatic mechanisms recognize and repair nuclear DNA damage, but it has long been thought that there was no DNA repair in mitochondria. Therefore, in an attempt to delineate more clearly which DNA repair mechanisms, if any, are functioning in mitochondria, we have investigated the repair of several specific DNA lesions in mitochondrial DNA. They include cyclobutane dimers, cisplatin intrastrand adducts, cisplatin interstrand crosslinks and alkali-labile sites. We find that pyrimidine dimers and complex alkylation damage are not repaired in mitochondrial DNA, and that there is minimal repair of cisplatin intrastrand crosslinks. In contrast, there is efficient repair of cisplatin interstrand crosslinks as evidenced by approximately 70% of the lesions being removed by 24 h. Additionally, there is efficient repair of N-methylpurines following exposure to methylnitrosourea with approximately 70% of the lesions being removed by 24 h. The results of these studies reveal that repair capacity of mitochondrial DNA damage depends upon the type of lesion produced by the damaging agent. We speculate that a process similar to the base excision mechanism for nuclear DNA exists for mitochondrial DNA but that there is no nucleotide excision repair mechanism to remove more bulky lesions in this organelle.

We have measured the capacity of highly-purified, paraffin oil-elicited neutrophils to induce DNA single-strand breaks in a newly established plasmacytoma cell line, RIMPC 2304, which was induced by a retrovirus containing the c-myc and V-Ha-ras oncogenes. This cell line effectively repairs DNA damage induced by gamma-irradiation. DNA damage induced by neutrophils was correlated with the oxidative burst of the neutrophils. The levels of superoxide anion, H2O2, and HOCl produced after stimulation of the neutrophils (6 X 10(5)/cm3) with the tumor promoter phorbol myristate acetate (100 nM) were 33.8 microM, 12.8 microM and 1.7 microM respectively in 15 min, and 98 microM, 20 microM and 8.7 microM respectively in 90 min. The results of alkaline elution experiments revealed that when the same concentration of neutrophils was co-incubated for 15 min in serum-free medium with an equal number of radioactively labeled RIMPC 2304 cells, the latter incurred a level of damage that approximated that caused by 300 rad equivalents of gamma-irradiation or by a 1-min treatment with 20 microM H2O2 at 37 degrees C. Damage from neutrophils was coincident with the oxidative burst; it was induced rapidly (within 5 min) but remained high for more than 90 min. The level of damage achieved was dependent upon the ratio of neutrophils: target cells and was clearly detectable at ratios as low as 0.25:1. Induction of single-strand breaks was completely inhibited by catalase and partially inhibited by superoxide dismutase, mannitol, and reduced glutathione but not by Na azide. Addition of the non-steroidal anti-inflammatory drug indomethacin either enhanced (at 50 microM) or had no effect (at 2 microM) on the damage detected. Finally, repair of strand breaks induced by neutrophils was significantly slower (half-time approximately 10 min) than that observed for repair of similar levels of damage induced by H2O2 or gamma-irradiation (half-times approximately 3 min, each). The results indicate that neutrophils cause prolonged DNA damage in neighboring cells. Moreover, they indicate that although H2O2 produced in the oxidative burst is an essential mediator of the damage observed, additional reactive oxygen intermediates including the superoxide anion are also implicated. The data are discussed in relation to the possible role of neutrophils in chronic inflammation and in pristane-induced plasmacytoma formation in mice.

Immunoglobulin T-cell receptor (IgTCR) molecules are potentially potent immune response modifiers because they allow T cells to bypass tolerance. Tolerance to self antigens has been one of the major barriers to the development of effective adoptive immunotherapies for treating cancer. In vitro studies in several laboratories have shown that cross-linking IgTCR molecules with the target antigen leads to cytolytic activity, cytokine release, and T-cell proliferation in model systems. However, many of these studies have used established T-cell lines rather than normal T cells or indirect assays of cytotoxicity, proliferation, and cytokine release. We have sought to establish the validity of these model systems while developing more effective adoptive immunotherapies using normal human T cells. In the present study the activation of T-cell proliferation after IgTCR cross-linking was evaluated. The results show that, in addition to IgTCR signals, CD28 costimulation is required to induce expansions of normal peripheral blood mononuclear cell-derived T cells. Signals from IgTCR alone can induce transient cell division, but they do not induce the prolonged polyclonal expansions that are characteristic of native immune responses. Very strong IgTCR signals could circumvent the CD28 requirement, but only at levels that are unlikely to be physiologically relevant. CD28 costimulation also suppressed the deletion of tumor-reactive subclones by activation-induced cell death. These studies confirm the importance of CD28 costimulation to the proliferation of IgTCR-modified human T cells, a key feature of an effective, reconstructed antitumor response.