Photodynamic therapy induces rapid cell death by apoptosis in L5178Y mouse lymphoma cells.

The mode of cell death of two strains of mouse lymphoma L5178Y cells was studied following photodynamic therapy (PDT) sensitized by chloroaluminum phthalocyanine. Strains LY-R and LY-S differ in their relative sensitivities to UVC radiation, X-radiation, and PDT; both responded to PDT by undergoing apoptosis. The DNA was degraded into fragments with lengths which are multiples of approximately 180-190 base pairs (i.e., oligonucleosome size), a biochemical marker of apoptosis. The DNA fragmentation was dose and time dependent which indicates this response to be an enzymic process related to cell killing. Cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor, enhanced the endonucleolytic DNA fragmentation. Transmission electron microscopy revealed chromatin condensation around the periphery of the nucleus, which is also characteristic of apoptosis. The induction of apoptosis in L5178Y cells by PDT was rapid, with marked degradation of DNA occurring in as little as 30 min. The rapidity of the response to PDT suggests that cellular damage produced by PDT can directly activate endonucleolysis and chromatin condensation, thereby by-passing many of the early steps in the signal transduction program which are acted upon by other agents causing apoptosis.

Detection of chromosome aberrations by FISH as a function of cell division cycle (harlequin-FISH).

Chromosome aberrations are a sensitive indicator of genetic change, and the measurement of chromosome aberration frequency in peripheral blood lymphocytes is often used as a biological dosimeter of exposure (1,4). The length of time that cells are maintained in culture before cytogenetic analysis is probably the most important in vitro factor that influences both the frequency and types of aberrations that are seen following exposure to mutagens. Therefore, for accurate cytogenetic measurements of genetic damage, cells must be analyzed in their first mitosis following exposure. As cells progress through subsequent mitotic division cycles, cells with unstable types of aberrations, e.g., dicentrics and acentric fragments, are eliminated (1,3,4). Even the use of synchronized populations of cells does not guarantee that all cells analyzed will be in their first division following treatment. Small variations in growth rate after irradiation can lead to large variations in the proportion of cells that are in their first vs. a subsequent mitosis. For example, 48 h after G0 lymphocytes are stimulated to enter the cell cycle (the standard sampling time for cytogenetic analysis), up to 50% of the cells in mitosis can be in their second division cycle (10). While there are methods available to distinguish cells in different division cycles (see Introduction), they are not easily adapted for use with standard fluorescence in situ hybridization (FISH) procedures. The goal of this study was to develop a simple approach to detect aberrations by FISH whereby cells in different division cycles could be distinguished.

Phthalocyanine 4 photodynamic therapy-induced apoptosis of mouse L5178Y-R cells results from a delayed but extensive release of cytochrome c from mitochondria.

Photodynamic therapy (PDT) activates the mitochondrial pathway of apoptosis, for which the release of cytochrome c into the cytosol is considered critical. To further elucidate the role of cytochrome c release in PDT-induced apoptosis, we monitored cytochrome c localization immunocytochemically and related it to nuclear apoptosis of the same cells. When mouse L5178Y-R cells were treated with 300 nM phthalocyanine (Pc) 4 and 0-75 mJ/cm(2) red light, cytochrome c release had a dose response similar to that of clonogenic cell killing, with nearly identical threshold doses. Within individual cells, the release of cytochrome c appeared to be an all-or-none phenomenon. Moreover, it was tightly associated with activation of a caspase-3-like protease and changes in nuclear morphology. Thus, in response to Pc 4-PDT, the release of cytochrome c from mitochondria is a key determinant of apoptotic cell death.

Failla Memorial Lecture. The prevalence of multilocus lesions in radiation-induced mutants.

In L5178Y mouse lymphoblasts, ionizing radiation-induced mutant frequencies were dramatically higher when the genetic marker analyzed was heterozygous (tk+/tk-) than when hemizygous (tk+/tk0 or hprt+/hprt0). In contrast, base-change mutagens induced similar mutant frequencies at heterozygous and hemizygous loci. These results indicate that the majority of radiation-induced mutants harbor multilocus lesions, and that these mutants are poorly recovered when the target gene is in a hemizygous chromosomal region. Dose-rate dependence of radiation-induced mutant frequency was demonstrated at the heterozygous tk locus but not at the hemizygous hprt locus; in a cell line deficient in the rejoining of DNA double-strand breaks (DSBs), no dose-rate dependence was observed for either locus. The majority of TK-/- mutants, whether spontaneous or induced by X, alpha-particle or UV radiation, or by photosensitization, showed loss of the entire active tk allele. The percentage of TK-/- mutants exhibiting inactivation of galactokinase, encoded by the neighboring gk gene, was high in UV repair-deficient cells exposed to UV radiation and in DNA DSB repair-deficient lines exposed to X radiation. Thus the presence of unrepaired DNA lesions, whether DSBs or pyrimidine dimers, appears to result in an increase in the percentage of mutants harboring multilocus lesions.

The repair of potentially lethal damage and sublethal damage in strains of mouse L5178Y lymphoma cells differing in radiation sensitivity.

Mouse lymphoma strains L5178Y-R (LY-R) and L5178Y-S (LY-S), which are differentially sensitive to the cytotoxic effects of ionizing radiation, were found to differ in their abilities to repair potentially lethal damage (PLD) and sublethal damage (SLD). The results showed that strain LY-R was more proficient than strain LY-S in the repair of SLD. The split dose recovery observed in strain LY-S could be accounted for by its recovery during postirradiation incubation. In contrast, SLD repair occurred in the absence of PLD repair in strain LY-R. The possibility that the repair of PLD might be completed prior to the postirradiation incubation in strain LY-R was suggested by the decreased survival observed when the cells were irradiated in a hypotonic solution. The repair of PLD and SLD in strain LY-S was temperature sensitive, occurring during postirradiation incubations between 15 and 34 degrees C, but not at 37 or 40 degrees C. This temperature sensitivity is very similar to the temperature sensitivity of the repair of pH 9.6-labile lesions in DNA in strain LY-S, as reported previously. Thus postirradiation cellular recovery processes in strain LY-S may involve the repair of pH 9.6-labile lesions in DNA. Temperature-dependent changes in the postirradiation distribution of cells throughout the cell cycle were observed which could contribute to the temperature sensitivity of the postirradiation recovery of strain LY-S.

The photobiology of photodynamic therapy: cellular targets and mechanisms.

Photodynamic therapy (PDT) is dependent on the uptake of a photosensitizing dye, often a porphyrin-related macrocycle, by the tumor or other abnormal tissue that is to be treated, the subsequent irradiation of the tumor with visible light of an appropriate wavelength matched to the absorption spectrum of the dye, and molecular oxygen to generate reactive oxygen intermediates. The initial oxidative reactions lead to damage to organelles in which the dye is bound, culminating in cell death and destruction of the tumor or abnormal tissue. Apoptosis is a common mechanism of cell death after PDT both in vitro and in vivo. PDT also triggers the activation of several signal transduction pathways in the treated cells; some of these are stress responses aimed at cell protection, while others are likely to contribute to the cell death process. The photosensitizers of greatest interest in PDT bind to various cytoplasmic membranes but are not found in the nucleus and do not bind to DNA. Nevertheless, some DNA damage is produced that can lead to mutagenesis, the extent of which is dependent on the photosensitizer, the cellular repair properties and the target gene. Thus, in spite of generating some responses common to ionizing radiation and other oxidative stresses, PDT is unique in the subcellular localization of damage, the combination of signaling pathways that are activated, and rapid kinetics of the induction of cell death processes.

Poly(ADP-ribose) and the response of cells to ionizing radiation.

The activity of poly(ADP-ribose) polymerase is stimulated by DNA damage resulting from treatment of cells with ionizing radiation, as well as with DNA-damaging chemicals. The elevated polymerase activity can be observed at doses lower than those necessary for measurable reduction in cellular NAD concentration (less than 20 Gy). Several nuclear proteins, including the polymerase itself, are poly(ADP-ribosylated) at elevated levels in irradiated Chinese hamster cells. The addition of inhibitors of poly(ADP-ribose) polymerase to irradiated cells has been found to sensitize the cells to the lethal effects of the radiation, to inhibit the repair of potentially lethal damage, and to delay DNA strand break rejoining. Because of the nonspecificity of the inhibitors, however, it is as yet unknown whether their effects are directly related to the inhibition of poly(ADP-ribose) polymerase, to interference with the poly(ADP-ribosylation) of one or more chromosomal proteins, or to effects unrelated to the poly(ADP-ribosylation) process. The data are consistent with the involvement of poly(ADP-ribose) in the repair of radiation damage, but the nature of this involvement remains to be elucidated.

Differential antimutagenicity of WR-1065 added after irradiation in L5178Y cell lines.

The purpose of this study was to determine the antimutagenicity of WR-1065 added after irradiation of cells of cell lines differing in their ability to rejoin radiation-induced DNA double-strand breaks (DSBs). The postirradiation antimutagenicity of WR-1065 at the thymidine kinase locus was demonstrated for L5178Y (LY)-S1 cells that are deficient in repair of DNA DSBs. Less postirradiation antimutagenicity of WR-1065 was observed in LY-R16 and LY-SR1 cells, which are relatively efficient in DSB repair. Postirradiation treatment with WR-1065 had only a small stimulatory effect on DSB rejoining. A 3-h incubation of irradiated LY cells with WR-1065 caused slight changes in the distribution of cells in the phases of the cell cycle that differed between LY-S1 and LY-SR1 cells. Both LY-S1 and LY-SR1 cells were protected against the cytotoxic and mutagenic effects of radiation when WR-1065 was present 30 min before and during the irradiation. We conclude that the differential postirradiation effects of WR-1065 in the LY-S1 and LY-SR1 cells are not caused by differences in cellular uptake of the radioprotector or in its radical scavenging activity. Possible mechanisms for the postirradiation antimutagenicity of WR-1065 are discussed.

Antimutagenicity of WR-1065 in L5178Y cells exposed to accelerated (56)Fe ions.

The ability of the aminothiol WR-1065 [N-(2-mercaptoethyl)-1,3-diaminopropane] to protect L5178Y (LY) cells against the cytotoxic and mutagenic effects of exposure to accelerated (56)Fe ions (1.08 GeV/nucleon) was determined. It was found that while WR-1065 reduced the mutagenicity in both cell lines when it was present during the irradiation, the addition of WR-1065 after the exposure had no effect on the mutagenicity of the radiation in either cell line. No marked protection against the cytotoxic effects of exposure to (56)Fe ions was provided by WR-1065 when added either during or after irradiation in either cell line. We reported previously that WR-1065 protected the LY-S1 and LY-SR1 cell lines against both the cytotoxicity and mutagenicity of X radiation when present during exposure, but that its protection when administered after exposure was limited to the mutagenic effects in the radiation-hypersensitive cell line, LY-S1. The results indicate that the mechanisms involved differ in the protection against cytotoxic compared to mutagenic effects and in the protection against damage caused by accelerated (56)Fe ions compared to X radiation.

A radon generator/delivery system.

A radon-generating system is described in which 222Rn, emanating from 226Ra stored in an aluminum containment vessel, may be pumped into a syringe for subsequent injection into a standard spinner flask containing tissue culture medium. The radium-containment vessel is sealed by an indium gasket and three metal bellows valves, one of which was used to fracture the glass capsule that contained 2.9 GBq of radium salt. A rotating piston pump transfers radon-enriched air from the radium-containment vessel to a delivery loop that includes a transfer syringe. The flow of air and radon through the loop is manipulated by three crossover ball valves, one of which may be set to fill the syringe. A charcoal trap is provided to collect residual radon left in the delivery loop after the transfer syringe has been filled. The protocol used to expose cells to radon and its progeny is described as well as the dosimetry that is used to estimate the dose delivered to the cells. A description of safety precautions taken in fabricating the generator and in conducting radiobiological studies is also presented.

Characterization of multilocus lesions in human cells exposed to X radiation and radon.

Human TK6 lymphoblasts were exposed to X radiation or radon, and thymidine kinase negative (TK-/-) mutants were selected, isolated and harvested for analysis of structural changes in the TK gene. A large majority (82%) of the radon-induced mutants, 74% of the X-radiation-induced mutants and 45% of the spontaneous mutants lost the entire active TK allele. To analyze these mutants further we measured the loss of heterozygosity at several loci neighboring the TK locus on chromosome 17q. A greater proportion (61%) of the radon-induced mutants than X-radiation-induced or spontaneous mutants harbored the smaller lesions involving the TK allele alone or extending from the TK locus to one or both of the closest neighboring sequences tested. Further, 21% of the X-radiation-induced mutants but only 5% of the radon-induced mutants lost heterozygosity at the col1A1 locus, 31 Mb from the TK gene. These results are in agreement with a recent analysis of radon- and X-radiation-induced lesions inactivating the HPRT gene of TK6 cells, in which we reported that a lower percentage of radon- than X-radiation-induced mutants showed lesions extending to markers 800 kb or more from the HPRT gene on the X chromosome (Bao et al., Mutat. Res. 326, 1-13, 1995). In the present study, we observed that the percentage of slowly growing and very slowly growing TK-/- mutants was greater after treatment with radon than after treatment with X radiation, regardless of the type of lesion present. It is possible, therefore, that the radon-induced lesions are complex and/or less easily repaired, leading to slow growth in a large proportion of the surviving mutant cells.

Relationships between mitotic delay and the dose rate of X radiation.

Upon exposure of cells to radiation delivered at a continuous low dose rate, cell proliferation may be sustained with the cells exhibiting a constant doubling time that is independent of the total dose. The doubling time or mitotic delay under these conditions has been shown to depend on the dose rate in HeLa, V79 and P388F cells (Mitchell et al., Radiat. Res. 79, 520-536, 1979; Fox and Gilbert, Int. J. Radiat. Biol. 11, 339-347, 1966). Reanalysis of the data for these particular cell lines shows that there is a threshold dose rate for mitotic delay, and that above the threshold there is a linear relationship between the length of mitotic delay and the logarithm of the dose rate which is referred to as the dose-rate response. We have observed the same relationships for L5178Y (LY)-R and LY-S cells exposed to low-dose-rate radiation. The threshold dose rates for LY-R, LY-S and P388F cells are similar (0.01-0.02 Gy/h) and are much lower than for V79 and HeLa cells. The slope of the dose-rate response curve is the greatest for HeLa cells, followed in order by LY-S, V79 and P388F cells, and finally by LY-R cells. The slopes for HeLa and LY-R cells differ by a factor of 35.

DNA double-strand break rejoining deficiency in TK6 and other human B-lymphoblast cell lines.

TK6, WI-L2, SB and three other B-lymphoblast lines were deficient in the rejoining of DNA double-strand breaks (DSBs) induced by ionizing radiation. Cells of these cell lines rejoin less than 50% of the breaks in 60 min after exposure, as assayed by filter elution at pH 9.6. The deficiency in TK6 cells was confirmed using the comet assay. IN TK6 cells the percentage of DSB rejoining did not vary markedly with dose and was similar for G1, S, and G2 + M-phase cells. Two B-lymphocyte lines (Raji and GM0606), three T-lymphoblast lines (MOLT-4, Jurkat, and CCRF-HSB-2), HL-60 promyelocytes, and GM3440 human skin fibroblasts rejoined more than 50% of the DSBs in this period after exposure. Radiation sensitivity in terms of cell survival was measured in those cells forming colonies. Of the cell lines tested, those that were deficient in DSB rejoining were radiation-sensitive (TK6 and WI-L2: D0 = 0.64 Gy). However, not all lines that were proficient in DSB rejoining were radiation-resistant, since Jurkat and GM0606 cells were relatively radiation-sensitive (D0 = 0.63-0.73 Gy). TK6 and WI-L2 cells were more sensitive to bleomycin (D0 = 8-9 micrograms/ml) than were HL-60 and Raji cells (D0 = 40-54 micrograms/ml). No relationship of DSB rejoining to V(D)J recombinase activity was observed, since no mRNA hybridizing to the cDNA probes for RAG-1 or RAG-2 was detected in any of the cell lines tested.

The effect of dose rate on X-radiation-induced mutant frequency and the nature of DNA lesions in mouse lymphoma L5178Y cells.

The induction of mutants at the heterozygous tk locus by X radiation was found to be dose-rate dependent in L5178Y-R16 (LY-R16) cells, but very little dose-rate dependence was observed in the case of strain L5178Y-S1 (LY-S1), which is deficient in the repair of DNA double-strand breaks. Induction of mutants by X radiation at the hemizygous hprt locus was dose-rate independent for both strains. These results are in agreement with the hypothesis that the majority of X-radiation-induced TK-/- mutants harbor multilocus deletions caused by the interaction of damaged DNA sites. Repair of DNA lesions during low-dose-rate X irradiation would be expected to reduce the probability of lesion interaction. The results suggest that in contrast to the TK-/- mutants, the majority of mutations at the hprt locus in these strains of L5178Y cells are caused by single lesions subject to dose-rate-independent repair. The vast majority of the TK-/- mutants of strain LY-R16 showed loss of the entire active tk allele, whether the mutants arose spontaneously or were induced by high-dose-rate or low-dose-rate X irradiation. The proportion of TK-/- mutants with multilocus deletions (in which the products of both the tk gene and the closely linked gk gene were inactivated) was higher in the repair-deficient strain LY-S1 than in strain LY-R16. However, even though the mutant frequency decreased with dose rate, the proportion of mutants showing inactivation of both the tk and gk genes increased with a decrease in dose rate. The reason for these apparently conflicting results concerning the effect of DNA repair on the induction of extended lesions is under investigation.

Diverse delayed effects in human lymphoblastoid cells surviving exposure to high-LET (56)Fe particles or low-LET (137)Cs gamma radiation.

To obtain information on the origin of radiation-induced genomic instability, we characterized a total of 166 clones that survived exposure to (56)Fe particles or (137)Cs gamma radiation, isolated approximately 36 generations after exposure, along with their respective control clones. Cytogenetic aberrations, growth alterations, responses to a second irradiation, and mutant frequencies at the Na(+)/K(+) ATPase and thymidine kinase loci were determined. A greater percentage of clones that survived exposure to (56)Fe particles exhibited instability (defined as clones showing one or more outlying characteristics) than in the case of those that survived gamma irradiation. The phenotypes of the unstable clones that survived exposure to (56)Fe particles were also qualitatively different from those of the clones that survived gamma irradiation. A greater percentage (20%) of the unstable clones that survived gamma irradiation than those that survived exposure to (56)Fe particles (4%) showed an altered response to the second irradiation, while an increase in the percentage of clones that had an outlying frequency of ouabain-resistant and thymidine kinase mutants was more evident in the clones exposed to (56)Fe particles than in those exposed to gamma rays. Growth alterations and increases in dicentric chromosomes were found only in clones with more than one alteration. These results underscore the complex nature of genomic instability and the likelihood that radiation-induced genomic instability arises from different original events.

Genotoxic effects of high-energy iron particles in human lymphoblasts differing in radiation sensitivity.

The effects of (56)Fe particles and (137)Cs gamma radiation were compared in TK6 and WTK1 human lymphoblasts, two related cell lines which differ in TP53 status and in the ability to rejoin DNA double-strand breaks. Both cell lines were more sensitive to the cytotoxic and clastogenic effects of (56)Fe particles than to those of gamma rays. However, the mutagenicity of (56)Fe particles and gamma rays at the TK locus was the same per unit dose and was higher for gamma rays than for (56)Fe particles at isotoxic doses. The respective RBEs for TK6 and WTK1 cells were 1.5 and 1.9 for cytotoxicity and 2.5 and 1.9 for clastogenicity, but only 1 for mutagenicity. The results indicate that complex lesions induced by (56)Fe particles are repaired less efficiently than gamma-ray-induced lesions, leading to fewer colony-forming cells, a slightly higher proportion of aberrant cells at the first division, and a lower frequency of viable mutants at isotoxic doses. WTK1 cells (mutant TP53) were more resistant to the cytotoxic effects of both gamma rays and (56)Fe particles, but showed greater cytogenetic and mutagenic damage than TK6 cells (TP53(+)). A deficiency in the number of damaged TK6 cells (a) reaching the first mitosis after exposure and (b) forming viable mutants can explain these results.

Isolation and characterization of BHK cells sensitive to ionizing radiation and alkylating agents.

A host-cell viral suicide enrichment procedure was used to isolate BHK strains sensitive to ionizing radiation. Of six strains surviving infection with irradiated herpes simplex virus (HSV), three were found to be more sensitive to ionizing radiation than the parental BHK cells. Thus the D0's of strains V1, V2, and V5 were 1.59, 1.41, and 1.49 Gy, respectively, while the D0 for the parental BHK strain was 1.79. Strains V1 and V2 were studied in more detail and found to exhibit hypersensitivity to ethyl methanesulfonate (EMS), methyl methanesulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine, but not to uv radiation. Susceptibility to mutation in response to EMS was also compared in BHK and strains V1 and V2. The frequency of induction of ouabain-resistant cells was 140% of the parental strain in the case of strain V1 and 58% of the parental strain in the case of strain V2.

Interlaboratory comparison of the effects of radon on L5178Y cells: dose contribution of radon daughter association with cells.

The cytotoxic and mutagenic effects of radon and its progeny were compared in murine lymphoblast L5178Y-R16 cells after exposure at three institutions. The cells were exposed to 222Rn at Case Western Reserve University (CWRU) and Pacific Northwest Laboratories (PNL) and to 212Bi, a decay product of 220Rn, at the University of Chicago (UC). The dose to the cell nucleus was calculated using a dosimetric model which addressed both the contribution of the dose from the radioactivity in the medium and that associated with the cells. The dose-response curves for cell survival showed D0's of 0.30 Gy at CWRU, 0.20 Gy at PNL, 0.37 Gy for chelated 212Bi, and 0.13 Gy for unchelated 212Bi. Induced mutant frequencies at the thymidine kinase locus at the 37% survival level were 1470 x 10(-6) at CWRU, 1518 at PNL, and 2414 x 10(-6) at UC using combined results for chelated and unchelated 212Bi. The variation between institutions was greater than obtained in a previous interlaboratory comparison of the effects of radon on CHO cells. Since less radioactivity was associated with CHO cells than L5178Y cells, we have concluded that the variation between institutions in the case of L5178Y cells is caused by the differences in cell-associated radioactivity and errors related to the measurement of this parameter.

Cytotoxic and mutagenic effects of radon and radon daughters on murine L5178Y lines differing in DNA repair.

The effects of 222Rn were measured in mouse L5178Y (LY) lymphoblasts that differ in repair capabilities. Line LY-S1 is deficient in the repair of X-radiation-induced DNA doublestrand breaks, while lines LY-R16 and LY-R83 are presumed to be deficient in the excision of UV-radiation-induced pyrimidine dimers. Line LY-R83 is hemizygous while the other two lines are heterozygous at the thymidine kinase (tk) locus. After exposure to radon the D0's were found to be very similar for the three lines (0.30-0.31 Gy), whereas for X radiation the D0 for line LY-S1 is lower (0.7 Gy) than that for the two LY-R lines (1.3 Gy). Mutant frequencies at the tk locus were higher per gray after treatment with radon than X radiation, but at equitoxic doses the mutant frequencies were similar for X and alpha-particle radiation. A low radon-induced mutant frequency was observed for the hemizygous line, in agreement with the hypothesis that multilocus lesions were induced by the alpha-particle radiation and that mutants bearing intergenic lesions were not recovered in the TK+/- line. The entire active tk allele was lost by 81% of the TK-/- mutants of line LY-R16. In lines LY-S1 and LY-R16, 39-43% of the TK-/- mutants exhibited loss of galactokinase activity, indicating that the mutational lesion inactivating the tk gene frequently extended to the neighboring galactokinase gene.

Induction of multilocus mutations at the Tk1 locus after X irradiation of L5178Y cells at different times in the mitotic cycle.

TK1+/- L5178Y-R16 cells were separated into G1, S and G2/M-phase populations by centrifugal elutriation and were treated with 1.5 Gy X radiation. Cells irradiated in the G1 and G2/M phases were most sensitive to the cytotoxic effects of radiation, while cells irradiated in the G2/M phase showed the highest mutant frequency at the thymidine kinase (Tk1) locus. DNA isolated from independent TK1-/- mutants was analyzed for loss of heterozygosity (LOH) at the Tk1 locus and two microsatellites, D11Mit48 and D11Nds7. Homogenates of each mutant were assayed for activity of galactokinase (GLK), the product of the galactokinase (Glk) gene neighboring the Tk1 gene on chromosome 11. Irradiated G1-phase cells had the highest percentage of mutants showing no LOH. The frequency of mutants with LOH at both Tk1 and D11Nds7 with no loss of GLK activity was high in all cell populations: There was no significant difference in the observed frequency of these mutants between the populations. The frequency of mutants losing GLK activity was low, particularly in cells irradiated in the S or G2/M phases. The possibility that the loss of GLK activity is not indicative of LOH at the Glk gene under the conditions of the present experiments is discussed.