Intact G2-phase checkpoint in cells of a human cell line lacking DNA-dependent protein kinase activity.

Cells respond to radiation-induced DNA damage in a cell cycle phase-specific manner as shown by (1) variation in radiosensitivity across the cell cycle and (2) checkpoints in G1 and G2 phase at which arrest of progression of cells through the phases of the cell cycle occurs. We studied these processes in cells of human glioma cell lines which lack (M059J(PK-)) or express (M059K(PK+)) DNA-dependent protein kinase (DNA-PK) activity. Cell populations enriched with cells of a specific cell cycle phase were y-irradiated and analyzed for cell survival. Although both cell lines were relatively sensitive in G1 phase and resistant in S phase, the differential sensitivity was greater in M059J(PK-) cells. In the studies on checkpoints, unsynchronized cells were irradiated and examined for evidence of cell cycle arrest. Neither cell line showed a postirradiation G1-phase arrest, presumably because of mutant p53 status. For M059J(PK-) cells, all doses tested (2.5-10 Gy) resulted in a significant increase in the proportion of G2/M-phase cells; however, for M059K(PK+) cells, a significant increase in G2/M phase was observed only after 10 Gy. These results suggest that the ability to activate the G2-phase checkpoint remains intact in cells which lack DNA-PK activity.

Radiation-induced DNA damage and repair in cells of a radiosensitive human malignant glioma cell line.

The induction and repair of DNA double-strand breaks were studied in cells of two isogenic human malignant glioma cell lines which vary in their SF2 values by a factor of approximately 30. M059J cells are radiosensitive (SF2 = 0.02) and lack the p350 component of DNA-dependent protein kinase (DNA-PK); M059K cells are radioresistant (SF2 = 0.64) and express normal levels of DNA-PK. Zero integrated field gel electrophoresis and alkaline sucrose gradient experiments indicated that equivalent numbers of DNA lesions were produced by ionizing radiation in M059J and M059K cells. To compare the capacity of both lines to repair sublethal damage, the split-dose recovery experiment after exposure to equitoxic doses of radiation was carried out. Significant sublethal damage repair was shown for M059K cells, with a 5.8-fold increase in relative survival peaking at 4 h, whereas M059J cells showed little repair activity. Electrophoresis studies indicated that more double-strand breaks were repaired by 30 min in M059K cells than in M059J cells. These results suggest that deficient DNA repair processes may be a major determinant of radiosensitivity in M059J cells.

Lack of correlation between DNA-dependent protein kinase activity and tumor cell radiosensitivity.

Lack of DNA-dependent protein kinase (DNA-PK) activity confers radiosensitivity and defective DNA double-strand break repair. Nine human malignant glioma cell lines were studied to determine whether differences in DNA-PK activity reflect differences in inherent radiosensitivity or are predictive of tumor treatment response. DNA-PK activity was present in all cell extracts, as were the DNA-PK proteins, DNA-PK catalytic subunit, Ku p70, and Ku p80. No correlation was found between the levels of DNA-PK activity and inherent radiosensitivity or in the tumor treatment response. These preliminary results suggest that variation in DNA-PK activity may not be a determinant of clinical response in malignant glioma.

Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line.

The radiosensitive rodent mutant cell line xrs-5 is defective in DNA double-strand break repair and lacks the Ku component of the DNA-activated protein kinase, DNA-PK. Here radiosensitive human cell lines were analyzed for DNA-PK activity and for the presence of related proteins. The radiosensitive human malignant glioma M059J cell line was found to be defective in DNA double-strand break repair, but fails to express the p350 subunit of DNA-PK. These results suggest that DNA-PK kinase activity is involved in DNA double-strand break repair.

Isolation of two cell lines from a human malignant glioma specimen differing in sensitivity to radiation and chemotherapeutic drugs.

Two aneuploid cell lines which differ in their inherent sensitivity to ionizing radiation and chemotherapeutic agents were established concurrently from a single tumor specimen obtained from a patient with glioblastoma. M059J cells are approximately 30-fold more sensitive to radiation than are M059K cells (surviving fractions at 2 Gy were 0.02 and 0.64, respectively). This relative difference in radiation sensitivity has remained a stable feature of the cell lines during 2 years in continuous culture. In addition, cells of the M059J line are more sensitive than those of the M059K line to the cytotoxic effects of bleomycin, N,N-bis(2-chloroethyl)-N-nitrosourea, and nitrogen mustard. These cell lines may prove to provide a useful model system for evaluating the cellular and molecular processes which confer resistance or sensitivity in cancer treatment.

Heterogeneity in response to treatment with buthionine sulfoximine or interferon in human malignant glioma cells.

Two tumor cell lines were established from each of three human malignant glioma biopsy specimens (M059, M067, M071) and sensitivity to treatment with radiation or chemotherapeutic agents (BCNU, nitrogen mustard) was determined. The effects of recombinant human interferon-alpha (rIFN) on the radiation response and of buthionine sulfoximine (BSO) on the drug response were investigated as well. For tumor M059, two cell lines that differed significantly in radiosensitivity were isolated (surviving fractions at 2 Gy = 0.02 and 0.64). The chemosensitivity and response to chemical modification differed as well. Cell lines established from tumor M071 differed in their response to rIFN only and were not sensitized by BSO. M067 cell lines showed little difference and were not sensitized by either agent. These results suggest that differences may exist both within and among human malignant gliomas with regard to their sensitivity to drugs, radiation, and the ability of chemical agents to modify treatment responses.

Radiosensitivity testing of human primary brain tumor specimens.

The inherent radiosensitivity of early passage cells derived from 22 patients with tumors of glial origin has been determined using a clonogenic assay system. The mean (+/- SD) surviving fraction at 2 Gy was 0.37 +/- 0.22 (range = 0.02-0.87). No correlation between inherent radiosensitivity and tumor cell plating efficiency or intracellular glutathione was observed. Tumor cells that were both resistant to nitrosoureas and expressed the Mer+ phenotype did not differ significantly in their radiosensitivity as compared to cells that were repair deficient (Mer-) and sensitive to nitrosoureas. Initial clinical follow-up suggests that factors in addition to inherent tumor cell radiosensitivity, such as performance status and age, continue to be the most important determinants of the response of patients with primary brain tumors to radiotherapy.

Inherent radiosensitivity testing of tumor biopsies obtained from patients with carcinoma of the cervix or endometrium.

The inherent radiosensitivity of tumor biopsies obtained from a series of patients with carcinoma of the uterine cervix or endometrium has been characterized. Early passage cell lines were irradiated and assayed for cell survival using a clonogenic assay system. Survival curves were generated using the alpha/beta model and the surviving fraction at 2 Gy (SF2) was estimated. A wide range of SF2 values was observed among histologically similar tumors. The mean (+/- SD) SF2 value was 0.29 +/- 0.12 (range = 0.11-0.59) for the cervical biopsies and 0.30 +/- 0.13 (range = 0.11-0.67) for the endometrial biopsies. No correlation between inherent radiosensitivity and tumor DNA index or histopathology was observed. Patient accrual continues with the expectation that these results may help to determine whether SF2 values are of clinical value in predicting the response of individual patients to treatment with radiotherapy.