M. Joan Allalunis-Turner

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.

Preclinical assessment of hypocrellin B and hypocrellin B derivatives as sensitizers for photodynamic therapy of cancer: progress update.

Abstract— Hypocrellins are perylenequinone pigments with substantial absorption in the red spectral region and high singlet oxygen yield. They are available in pure monomelic 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‐dimethylami‐no)‐propylamine‐HB]. Extinction coefficients at 630 nm (φ630) are 6230, 6190 and 4800, respectively; and 1O2 quantum yields, φ, 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 EMToVEd 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 typhi‐murium. Further development of selected hypocrellin derivatives as photosensitizers for photodynamic therapy is warranted.

Frameshift Mutation in PRKDC, the Gene for DNA-PKcs, in the DNA Repair-Defective, Human, Glioma-Derived Cell Line M059J

Abstract Anderson, C. W., Dunn, J. J., Freimuth, P. I., Galloway, A. M. and Allalunis-Turner, M. J. Frameshift Mutation in PRKDC, the Gene for DNA-PKcs, in the DNA Repair-Defective, Human, Glioma-Derived Cell Line M059J. Radiat. Res. 156, 2–9 (2001). The glioma-derived cell line M059J is hypersensitive to ionizing radiation, lacks DNA-PK activity, and fails to express protein for the catalytic subunit, DNA-PKcs, while a sister cell line, M059K, derived from the same tumor, has normal DNA-PK activity. Both cell lines are near pentaploid and have multiple copies of chromosome 8, the chromosome on which the DNA-PKcs gene, PRKDC, is located. Sequence analysis of PCR-amplified exons revealed the loss in M059J cells of a single “A” nucleotide in exon 32, corresponding to the first nucleotide of codon 1351 (ACC, Thr) of PRKDC. Loss of the “A” nucleotide would terminate the DNA-PKcs reading frame early in exon 33. DNA from M059K cells had only the wild-type sequence. An analysis of sequences surrounding PRKDC exon 32 from 87 unrelated individuals revealed no polymorphic nucleotides except for a triplet repeat near the 3′ end of this exon; no individual had a frameshift mutation in exon 32. No other sequence differences in PRKDC between M059J and M059K cells were observed in ∼15,000 bp of genomic sequence including the sequences of exons 5 through 38 and surrounding intron sequence, suggesting a possible reduction to homozygosity at this locus prior to acquisition of the mutation leading to the M059J cell line.

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.

Human TP53 from the Malignant Glioma-Derived Cell Lines M059J and M059K Has a Cancer-Associated Mutation in Exon 8

Abstract Anderson, C. W. and Allalunis-Turner, M. J. Human TP53 from the Malignant Glioma-Derived Cell Lines M059J and M059K Has a Cancer-Associated Mutation in Exon 8. The protein coding segment of the TP53 genes from the glioma-derived cell lines M059J and M059K was sequenced. The sequences from both cell lines were identical over 5039 bp, including the gene segment containing exons 2 through 9, exon 10, and the proximal segment of exon 11. In both cells, the first nucleotide of codon 286 (GAA, Glu) is changed to an A (AAA, Lys). Comparison with the same TP53 segment from the A549 human lung carcinoma cell line revealed several differences in intron sequence.

Protection of Human Tumor Cells of Differing Radiosensitivity by WR-1065

Abstract Murray, D., Rosenberg, E. and Allalunis-Turner, M. J. Protection of Human Tumor Cells of Differing Radiosensitivity by WR-1065. We examined the ability of WR-1065, the biologically active aminothiol form of the clinically used drug amifostine (WR-2721, Ethyol®), to protect cultures of two human glioblastoma cell lines of greatly differing radiosensitivity from the cytotoxic effects of γ radiation. M059J cells are extremely radiosensitive compared to M059K cells (which were derived from the same tumor) and are defective in the DNA-dependent protein kinase (DNAPK)-mediated pathway for the repair of DSBs. In spite of their marked phenotypic differences, the two glioblastoma lines were protected equivalently (∼1.8-fold) after a 30-min preirradiation treatment with 4 mM WR-1065. These findings are in agreement with earlier studies that showed no relationship between the ability of another aminothiol, cysteamine, to protect human tumor cells with differing abilities to repair DSBs and/or radiosensitivity. Thus it appears that differences in intrinsic radiosensitivity and ability to repair DSBs are not important general factors in the modulation of the radiosensitivity of human cells by aminothiols. Because of a previous report that the radiosensitive mutant rodent xrs5 cell line (which, like M059J, is defective in the DNAPK-mediated pathway for repairing DSBs) is unusually refractory to the radioprotective effects of WR-1065, we re-examined the ability of WR-1065 to protect these cells. In contrast to the earlier studies, both the wild-type and mutant rodent lines were protected extensively by WR-1065. This discrepancy might be related to some unknown factor, such as differences in chromatin organization among xrs5 subclones that arise during their karyotypic evolution, possibly leading to altered DNA–drug associations.

Glutathione levels and chemosensitizing effects of buthionine sulfoximine in human malignant glioma cells

SummaryBiopsy samples and cultured cells derived from them were obtained from 39 patients with malignant glioma and were analyzed for 1) glutathione (GSH) content; 2) sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and/or nitrogen mustard (HN2) treatment and 3) the effect of buthionine sulfoximine (BSO) treatment on BCNU and/or HN2 cytotoxicity. The average GSH concentration of biopsy specimens was lower than those of cultured cells (2.36±0.44 vs. 11.42±2.32 nmol/106 cells). While some of the tumor specimens were sensitive to either BCNU or HN2, the majority were resistant to both. However, 8 of 23 tumors tested showed enhanced sensitivity to BCNU following treatment with BSO. Five of 17 tumors were similarly sensitized to HN2 by BSO. These results suggest that BSO chemosensitization may be of value for certain patients and that screening assays may help identify treatment-sensitive individuals.

Evaluation of diethyldithiocarbamate as a radioprotector of bone marrow

The radioprotective action of DDC on normal hematopoietic tissue in mice was evaluated. An increase in CFU-S and CFU-GM in DDC treated unirradiated control mice was consistently observed. When post-irradiation survival of CFU-S and CFU-GM from DDC treated animals was normalized to account for this observed increase, protection factors ranging from 0.9 to 1.6 were observed. These protection factors are significantly lower than those reported by others. Maximum radioprotection was observed when irradiations were performed 15 to 30 min after DDC treatment; maximum increase in GFU-GM occurred within one to two hr following treatment. The increase in CFU-GM is analogous to that observed in animals treated with endotoxin, a non-thiol radioprotector. When C3H/HeJ mice, which are genetically incapable of responding to endotoxin, were challenged with DDC, an average CFU-GM increase of 1.7 times was observed, suggesting that the stimulatory effects of DDC were not due to endotoxin contamination. DDC was administered daily for three days before irradiation and little or no increase in CFU-GM and no radioprotection was observed, suggesting that the marrow can become refractory to DDC. When WR-2721 was tested in similar studies, a dose-modifying radioprotection was observed, with no significant non-specific stimulation of hematopoietic cells.

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.