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.

Summary In contrast to reports of extensive hypoxia in human gliomas in situ measured by P02 histography, non-invasive methods of assessing glioma oxygenation, including nitroimidazole binding, have yielded surprisingly contradictory results. In order to investigate the relationship of necrosis, hypoxia, nitroreductase activity and cellular respiration in human gliomas, subcutaneous models using the human glioma cell lines M059K, M006 and M01 Ob were developed in the murine SCID host. Intracranial growth of the M006 line was achieved in nude rats. The nitroreductive capacity of glioma cell lines was assessed and found to be similar to transplanted tumours previously reported in the literature. This suggests that if substantial numbers of viable hypoxic cells were present in situ in gliomas, then nitroimidazole-binding techniques should be capable of identifying them. Inter-tumour variability in the amount of necrosis was seen. M006 xenografts growing in subcutaneous and intracranial sites revealed extensive necrotic regions histologically, some of which were surrounded by cells labelled heavily for [3H]misonidazole, while other areas were lightly labelled. Similar binding patterns were seen for subcutaneous M059K tumours, while subcutaneous M01 Ob tumours display necroses of which almost all were surrounded by heavily labelled cells. The oxygen consumption rates of tumour cell lines grown in vivo, in which venous P02 concentrations are of the order of 2-5%, were two to sevenfold less than those of the same lines grown as monolayers in vitro under oxygen concentrations of 18%. We postulate that glioma cell lines behave as 'oxygen conformers', in that their rate of oxygen consumption appears to vary with the availability of oxygen. Together with the misonidazole-binding data, the results in this glioma tumour model are consistent with coordinate inhibition or down-regulation of respiration under moderate hypoxia.

Summary We have investigated in detail the effects of buthionine sulfoximine (BSO), a selective glutathione (GSH) depleting agent, on the GSH contents of a number of normal tissues and three experimental tumours in mice. Significant variations in the rate and degree of GSH depletion and recovery were observed among the normal tissues. Following a dose of 2.5 mmol kg-1 BSO, GSH nadirs were reached by approximately 5 h for the liver and kidney, 8 h for the lung and bone marrow, 12 h for red blood cells (RBCs) and by 24 h for the heart. The degree of depletion was greatest for the kidney (80%), liver (74%) and bone marrow (83%), intermediate for the heart (54%) and lung (40%), and least for RBCs (13%). Recovery of GSH content was fastest for the liver followed in descending order by the kidney, the lung, the bone marrow, RBCs and the heart. In contrast, the rate and extent of GSH depletion and recovery showed considerably less variation among the 3 murine tumours. In the tumours GSH nadirs were reached by 10-12h. The extent of depletion was about 55-65%. Recovery of GSH levels in the tumours required 48 h or more, a longer period than required by the liver, kidney and lung but shorter than that needed for the bone marrow, heart and RBCs. Attempts to preferentially deplete tumour GSH by exploiting the differences in recovery rates between normal tissues and tumours were only partially successful. Multiple BSO dosing at 16 h intervals allowed the liver to recover between doses, but the recovery in the kidney, lung and bone marrow was only partial and no recovery was seen in the heart. Finally, dose-depletion relationship investigations showed that, with the exception of the lungs, GSH depletion could be achieved in tumours with doses of BSO lower than those required for normal tissues.

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.

Hypocrellins are perylenequinone pigments with substantial absorption in the red spectral region and high singlet oxygen yield. They are available in pure monomeric form and may be derivatized to optimize properties of red light absorption, tissue biodistribution and toxicity. In vitro screening of synthetic derivatives of the naturally occurring compound, hypocrellin B (HB), for optimal properties of cyto-(dark) toxicity and phototoxicity resulted in selection of three compounds for preclinical evaluation: HBEA-R1 (ethanolaminated HB), HBBA-R2 (butylaminated HB) and HBDP-R1 [2-(N,N-dimethylamino)-propylamine-HB]. Extinction coefficients at 630 nm (epsilon 630) are 6230, 6190 and 4800, respectively; and 1O2 quantum yields, phi, 0.60, 0.32 and 0.42. Intracellular uptake is essentially complete within 2 h (HBEA-R1, HBBA-R2) and 20 h (HBDP-R1). Greatest uptake is associated with lysosomes and Golgi. The HBEA-R1 and HBBA-R2 elicit phototoxicity in vitro primarily via the type II mechanism, with some type I activity under stringently hypoxic conditions. Transcutaneous phototherapy with HBEA-R1 permanently ablates EMT6/Ed tumors growing in the flanks of Balb/c mice, with minimal cutaneous effects. The HBBA-R2 does not elicit mutagenic activity in strains TA98 and TA100 of Salmonella typhimurium. Further development of selected hypocrellin derivatives as photosensitizers for photodynamic therapy is warranted.