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These data demonstrate an interaction between C225 and RT. C225-mediated apoptosis and inhibition of EGFr phosphorylation may be critical in the interaction. Studies to define the precise influence of combined modality treatment on the EGFr signal transduction cascade need to be pursued. The combination of growth factor receptor antibodies and RT has potential application in clinical oncology.

The DNA-dependent protein kinase (DNA-PK) is a trimeric enzyme consisting of a 460-kDa catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex called Ku, which is comprised of 70 (Ku70) and 86 (Ku80) kDa subunits. Mutations that affect the expression of the catalytic or Ku80 subunits of DNA-PK disrupt both V(D)J recombination and DNA double-stranded break repair pathways. In this report, we show that two previously uncharacterized rodent cell lines that are defective in DNA double-stranded break repair express catalytically inactive DNA-PK. The DNA-PKcs from the DNA double-stranded break repair mutant cell lines IRS-20 and SX-9 assembles on double-stranded DNA but fails to function as a protein kinase. In addition to the kinase defect, the abundance of the DNA-PKcs from both of these cell lines is reduced relative to wild-type controls. These results suggest that the DNA-PKcs gene from each of these cell lines contains mutations that inactivate the enzymatic activity and the expression or stability of the gene product. These data further strengthen the hypothesis that DNA-PK-mediated protein phosphorylation is a necessary component of the DNA double-stranded break repair pathway.The rejoining of double-stranded DNA breaks induced by ionizing radiation or occurring as intermediates of V(D)J recombination is performed via a biochemical pathway that includes the DNA-dependent protein kinase holoenzyme. DNA-PK 1 is a trimeric complex consisting of a DNA-binding component made up of the 70 and 86 kd subunits of the Ku autoantigen (1, 2) and a catalytic subunit of approximately 460 kDa (3). Cells from the x-ray-sensitive complementation group (xrs)-7, which includes the severe combined immunodeficiency (scid) mouse and the Chinese hamster ovary (CHO) V3 cell line, exhibit reduced expression of the DNA-PKcs, lack measurable DNA-stimulated kinase activity, and are defective for DNA double-stranded break repair and V(D)J recombination (4 -6). Similarly, cells from the xrs-6 complementation group, which contain mutations that reduce the expression of the Ku80 subunit of DNA-PK, exhibit losses of Ku-specific DNA-ending binding activity (7-9) DNA-PK kinase activity (10) and are also defective for DNA double-stranded break repair and V(D)J recombination (11-13).Molecular analysis of these DNA-repair mutant cells indicates that DNA-PK is required for the rejoining of doublestranded DNA breaks, but the mechanism by which DNA-PK functions in this process has yet to be elucidated. DNA-PK is a serine and threonine protein kinase that is activated by doublestranded DNA containing single-stranded to double-stranded transitions, such as DNA-ends, nicks, gaps, and stem-loop structures (14). In vitro, the Ku and catalytic subunits of DNA-PK assemble in a DNA-dependent manner (15), and the DNA-bound holoenzyme preferentially phosphorylates substrates that are bound to the same DNA molecule (1, 16). DNA-PK has been shown to phosphorylate a broad range of proteins in vitro, most of which are DNA-binding proteins (17), includi...

Overexpression of proapoptotic Bax favors death in cells resistant to ionizing radiation. We hypothesized that expression of Bax via adenoviral-mediated gene delivery could sensitize radiation-refractory cells to radiotherapy. An inducible Bax recombinant adenovirus (Ad/Bax) had been generated using the Cre/loxp system. Human ovarian cancer cell lines and primary, patient-derived cancer cells from ascites were irradiated and infected with the Ad/Bax and an expression-inducing vector, Ad/Cre. Cell death was evaluated by crystal violet staining, fluorescence-activated cell sorter analysis of Annexin V, and colony formation assay (cell lines only). To further characterize the mechanism of death, cell morphology was examined by nuclear staining with Hoechst 33258. Lastly, to evaluate the capacity of the combined treatment to inhibit tumor growth, mice were injected subcutaneously with ovarian cancer cells exposed to Bax, radiation therapy (RT), or both, and tumor size was measured periodically. Infection of the cancer cell lines and primary cells with both Ad/Bax and Ad/Cre significantly enhanced sensitivity to ionizing radiation, achieving high levels of cell killing in short-term assays. In addition, the combination of Bax and radiotherapy reduced the survival fraction of cell lines 2 logs in standard colony-forming assays. Investigation into the involved mechanism suggests that Bax-mediated radiosensitization occurs through both apoptosis and necrosis pathways. Further, mice subcutaneously injected with ovarian tumor cells previously treated with radiation, or with radiation and irrelevant viruses, consistently developed tumor nodules. In addition, approximately 80% of injections were followed by tumor formation after treatment with Ad/Bax and Ad/Cre alone. In contrast, tumor formation was completely inhibited after combined treatment with Ad/Bax and Ad/Cre and radiation. Augmentation of the effect of radiotherapy on human ovarian cancer cells and primary cancer cells from patients via a recombinant adenovirus encoding Bax is feasible.

We have isolated a stable mutant of CHO cells, designated irs-20, that is hypersensitive to ionizing radiation. The selection system was designed to select for mutants unable to proliferate at the low dose rate of 0.06 Gy per hour, a dose rate that has little influence on the effect of radiation on the cell cycle of wild-type cells during 1- to 2-week exposures. The irs-20 mutant cells irradiated continuously at 0.06 Gy/h showed a cell cycle redistribution, with an increasing G2 + M-phase fraction, but underwent approximately four doublings before cell population growth was completely inhibited. Dose rates three to four times higher were required to produce similar perturbation in the cell cycle of wild-type cells. Asynchronous log-phase irs-20 cells were approximately twofold more sensitive than the parental CHO cells as measured by comparing the doses required to reduce survival to 10%. The survival response of synchronized irs-20 cells after a single radiation dose of 3.8 Gy at different times during the cell cycle was qualitatively similar to the pattern for wild-type CHO cells for an approximately isosurvival dose of 7.4 Gy. The irs-20 cells were hypersensitive to the "radiomimetic" drug bleomycin but showed the wild-type sensitivity to ethyl methane sulfonate, ultraviolet light (254 nm) and mitomycin C. The irs-20 mutant cell has maintained its phenotype for over 1 year in continuous culture, indicating that the defect is genetically stable. The karyotype of the mutant cells is not different from that of its parent. Further evidence of stability is that clonal lines derived from cells surviving high radiation doses also had the irs-20 phenotype, and treatments with 5-azacytidine sufficient to cause high reversion (approximately 2 x 10(-1)) to proline independence resulted in no measurable reversion to wild-type radiosensitivity.