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The effect of chemical radiation protectors on cell cycle progression after gamma or neutron irradiation

The effect of chemical radiation protectors on cell cycle progression after gamma or neutron... Biological & Medical Research Division, Argonne National Laboratory, Argonne, IL and 'Radiation Oncology Departmenr, J . Graham Brown Cancer Center, University of Louisville, Louisville, K Y , +Department of Biology, University of Northern Iowa, Cedar Falls. IA and $Department of Radiation and Cellular Oncology, University of Chicago, Chicago, IL, USA (Received 10 October 1990; accepted 13 November 1990) Abstract. The effects of two chemical radiation protectors, WR-1065 and WR-151326, were characterized in V79 Chinese hamster cells after either cobalt-60 (6"Co) gamma or fission spectrum neutron irradiation. Each protector was administered at a concentration of 4 mM t o exponentially growing cultures for 30 min prior t o and during irradiation with either 6oCogamma or JANUS fission spectrum neutrons. After irradiation the cells were either plated immediately for survival or returned t o the incubator and assayed for cell progression. Aliquots of cells were removed at selected times, counted, fixed and stained with 4'6-diamidino-2-phenylindole (DAPI). Analysis of D N A histograms indicate that the presence of the protector during irradiation reduced the division delay experienced at the G;?-M interface. Implications of these effects are discussed. There is considerable interest in the phosphorothioate class of radiation protective agents as an adjunct in the treatment of cancer. This was originally based on the findings of Yuhas & Storer (1969), who found that WR-2721 protected mice against radiation-induced lethality by a factor of 1.6, while tumour protection was only 1.15. The mechanism of action, although poorly understood, is widely believed to result from scavenging OH radicals in and about the presumed target molecule DNA. Exposure to high linear energy transfer (LET) radiations, such as neutrons or other heavy particles, is known to produce more biological damage than an equal dose of low LET radiation (Barendsen & Walter 1964). Chemical protectors, however, are not as useful in reducing these high LET effects as they are in reducing low LET effects (Sigdestad et al. 1986, Afzal & Ainsworth 1987). We have previously shown (Sigdestad etal. 1988) that WR-1065 and WR-151326, which are the dephosphorylated derivatives of the more familiar WR-2721 and WR-15 1327, effectively perturb cell cycle progression in the absence of radiation. During drug exposure, there was a build-up of cells in S and G2phases. Chatterjee & Jacob-Raman (1986) reported that reduced glutathione (GSH) treatment produced a cell cycle delay in lymphocytes. When these cells were irradiated 30 min later, however, the expected radiation-induced cell cycle delay was dramatically reduced. These findings prompted us to assess the role of the thiophosphoroate class of protectors in progression delay after high or low LET radiation. Correspondence: Dr D. J . Grdina, Biological & Medical Research Division, Argonne National Laboratory, Argonne, IL 60439, USA. C. P. Sigdestad, B. L. Bergquist and D. J . Grdina MATERIALS AND METHODS Cell system V79-B310H Chinese hamster cells were grown at 37°C in plastic T-flasks or in plastic 100 mm petri dishes containing alpha MEM-10 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Biologos, Naperville, IL, USA) in a humidified atmosphere of 5% C 0 2 and 95% air. A complete description of the culture condition is given elsewhere (Suzuki et al. 1981). Radiation protectors WR-1065 (2-[aminopropylamino]ethyl mercaptan dihydrochloride) and WR-151326 (3-[3-methylaminopropylamino]propyl mercaptan dihydrochloride) were used in these studies and were obtained from the Division of Experimental Therapeutics, Walter Reed Army Medical Center, Washington, DC, USA. Each was originally dissolved in phosphate-buffered saline (PBS) at a 1 M concentration and sterilized by filtration immediately before use. Cells were treated with either protector, at a concentration of 4 mM, for 30 min prior to and during irradiation. Immediately after irradiation, the cells were washed twice with PBS and either plated for survival or returned to the incubator for cell doubling time assay using flow cytometry . irradiation procedure Exponentially growing V79 cells were trypsinized and irradiated in 15 ml centrifuge tubes at ice-bath temperature, in order to prevent cell cycle progression during the irradiation procedure. Fission-spectrum neutrons were obtained from the JANUS reactor of the Biological, Environmental and Medical Research Division, Argonne National Laboratory. The characteristics of the JANUS source are described in detail elsewhere (Williamson & Frigerio 1972). The average neutron energy was 0.85 MeV, and contamination by gamma rays was 3 4 % of the total absorbed dose. All neutron irradiations were performed at a dose rate of 0.24 Gy/min. Cells were irradiated, under similar conditions, using cobalt-60 gamma rays from a Gamma Beam 650 (Atomic Energy of Canada) at a dose rate of 0.5 G y h i n . Cell doubling time Cells were recovered from stock cultures, replated in 100 mm Petri dishes at a density of 7.5 x lo4 and, 24 h later, when the cell density approximated 4 x lo5 cells per plate, the medium was replaced with medium containing either WR-1065 or WR-151326 at a concentration of 4 m for 30 min. The cells were then irradiated with either 7 Gy gamma o r 2 Gy M fission spectrum neutrons. Unirradiated cells treated with protector served as positive controls. The medium was removed and replaced with fresh medium without protector. At closely spaced time intervals, plates were trypsinized and the number of cells was determined using a Coulter counter with appropriate correction for coincidence. The cells from each group were fixed in ice-cold 70% ethanol in preparation for flow cytometric (FCM) analysis. FCM analysis The determination of the D N A content of irradiated and unirradiated V79 cells as well as the evaluation of the effects of WR-1065 and WR-151326 on cell cycle progression was made using FCM techniques. Cells were stained with 4' ,6-diamidino-2-phenylindole-DAPI (Russel, Newman & Williamson 1975) in a 0.1% citrate solution according to a method described Chemical protectors and the cell cycle elsewhere (Gohde, Schuman & Zante 1978, Gohde et al. 1979). Flow cytometry patterns were obtained using a Partec PAS-I1 (Particle Analyzing System, Partec A G , Basel, Switzerland) and were analysed using a computer program obtained from Techno System-GMBH (Darmstadt, West Germany). T h e coefficient of variation of the G , peaks obtained for unperturbed cell samples routinely ranged from 1.5 to 2.5%. RESULTS Cell survival V-79 cells grown under standard tissue culture conditions were examined for cell survival after irradiation with either JANUS fission spectrum neutrons or 6oCo gamma rays. The two radiation protectors WR-1065 and WR-151326 were studied to determine their ability to reduce radiation cell killing. The results are shown in Figures 1& 2. The curves were fitted using the linear quadratic model and the salient survival data are presented in Table 1. Because this model assumes a continuously downward bending of the survival curves, protection factor (PF) values were calculated on a basis of a method described by Chaplin et al. (1987). P F values of 1.6 were obtained by applying either WR-1065 o r WR-151326 for 30 min prior to and during gamma irradiation. Similar measurements after irradiation with JANUS fission neutrons produced P F values of about two. Cell cycle progression Progression of cells through the cell cycle was determined using flow cytometry. Preliminary studies indicates that 7 G y gamma radiation was approximately equal to 2 Gy fission neutrons, which explains the choice of radiation doses. Progression was examined at 2 , 4 , 8 , 1 2 , 1 6 , 2 0 , 2 4 and 28 h after irradiation. Figure 3 shows typical flow cytometry patterns (12 h postI 1 $. Dose (Gy) Figure 1. The effect of WR-1065 chemical protection on the survival of V79 cells in or culture after irradiation with either cobalt-60 gamma (0) JANUS fission spectrum Survival of untreated cultures after gamma ( V ) and neutron (V) neutrons (0). irradiation is also shown. C. P . Sigdestad, B . L . Bergquist and D . J . Grdina Dose (Gy) Figure 2. The effect of WR-151326chemical protection on the survival of V79 cells in culture after irradiation witheithercobalt-@gamma (0) JANUS fission spectrum or neutrons (0). Survival of untreated cultures after gamma ( V ) and neutron (V) irradiation is also shown. irradiation) in unprotected, as well as WR-1065 or WR-151326 treated V-79 cells, after either gamma or neutron irradiation. The most striking characteristic of these curves is the effect of the protectors on the G2 M compartment. It is apparent in the unprotected but irradiated groups that a large proportion of cells are in the G2 compartment, whereas in the protected groups this block appears to have been released. The percent GI, S and G2 + M were computer-derived. Figures 4 & 5 show graphically the results of flow cytometric analysis for “Co and neutron irradiation respectively. The presence of the protector appeared to modify the progression pattern after irradiation regardless of the radiation source. Generally, the G I population was reduced after irradiation, reaching a nadir between 4 and 6 h, with a subsequent slow rise to control levels 28 h after exposure. The DNA synthetic phase initially increased in size, with a subsequent depression at 12 h. At 28 h the number of S phase cells was approaching control levels. As stated above, the most significant difference was noted in the G2 M population, where cells pretreated with protector did not experience the typical G2 Table 1. Salient survival curve parameters Source Neutron Treatment Alpha 1.726 0.568 0.755 0.237 0.041 0.058 SD Beta SD PF 1.oo 1.97 2.00 1.oo 1.64 1.62 None WR- 1065 WR- 151326 None WR-1065 WR- 151326 -0 0.118 0.041 Gamma PF protection factor, calculated using linear quadratic model as described by Chaplin ef al. (1987) Chemical protectors and the cell cycle 600 Channel number Figure 3. Typical flow cytometry patterns observed 12 h following irradiation with comparable doses of a, c & e 700 rad cobalt-60 gamma or b, d & f 200 rad JANUS fission spectrum neutron irradiation: a & b control cultures; c & d treated with WR-1065; e & f treated with WR-151326. C. P . Sigdestad, B . L. Bergquist and D . J . Grdina I” -I I 30 Time after y irradiation (h) Figure 4. Quantitative analysis of flow cytometry patterns, showing the effects of chemical protectors WR-1065 (A)and WR-151326 (W) on cell progression as a function of time after 700 rad cobalt-60 irradiation; controls treated only by a irradiation are also shown (0). = G I ; b = S; c = Gz + M. Chemical protectors and the cell cycle Time after neutron irradiation (h) Figure 5. Quantitative analysis of flow cytometry patterns. showing the effects of chemical protectors WR-1065 (A)and WR-151326 (W) on cell progression as a function of time after 200 rad fission neutron irradiation; controls treated only by irradiation are also shown (0). = G I ; b = S; c = GZ + M. a C. P . Sigdestad, B. L. Bergquist and D . J . Grdina block which was observed in the radiation-only group. The same effect was observed regardless of whether the cells were irradiated with gamma or fission neutron irradiation. Cell doubling time The doubling time for V79 cells was determined after irradiation, with or without addition of radiation protector. Cells treated only with protector served as positive controls. The results are presented in Table 2. Negative controls (V79 cells without radiation or drug treatment) showed a doubling time of 10 h. Positive control cells, those treated with either protector but no radiation treatment, showed increased doubling time to 16 h. There was no difference between the results obtained with either WR-10165 or WR-151326. The results obtained in irradiated cells indicate that the presence of protector during irradiation reduced the division delay by 1-2 h in neutron-irradiated cells and 5 h in gamma-irradiated cells. The response noted was similar regardless of which protector was tested. The aminothiol radiation protectors which inhibit cell progression without irradiation act to reduce radiation-induced division delay. Table 2. Doubling time (h) of V79 cells after various treatment Radiation treatment Drug treatment None Gamma (7 Gy) 20 15 15 Neutrons (2 Gy) None WR-1065 WR-15 1326 DISCUSSION We have previously reported (Sigdestad et al. 1988) the effect of WR-1065 and WR-151326 without radiation on cell cycle progression in V79 cells in culture. Drug exposure for 3 h resulted in cell cycle perturbations characterized by a build-up of cells in the S and G2phases. After the protectors were removed, cells began to redistribute throughout the cell cycle and 12 h were required before cells exposed to WR-1065 approached levels commensurable with controls. In contrast, cells treated with WR-151327 required about 24 h to redistribute to control levels. No evidence was presented which explained the mechanism of action of this phenomenon. Billen (1983), however, demonstrated that cysteamine can inhibit DNA polymerase-directed repair synthesis, which could be possibly related to chelation of selected metal ions (Jellum, Aaseth & Eldjarn 1973). The present study was an extension of these results to include combined treatment of radiation, a well-known cell cycle modifier, and selected protectors which we have shown to affect cell cycle progression. V79 cell survival after either fission neutron or 6oCo gamma irradiation was found to be enhanced by pre-treatment with either WR-1065 or WR-151326. PF values of 1.6 and 2.0 were obtained after gamma and neutron irradiation respectively. No difference between the two protectors examined was observed, regardless of the radiation source. Flow cytometry was used as a method of assessing the combined effect of radiation and chemical protectors on cell progression. Although there were various differences in progression of cells through the cell cycle for the various groups tested, the main effect noted was a difference in the proportion of cells blocked at the G2-M interface. The well-known G2 block was observed after irradiation without the addition of chemical protector. This increased the proportion of G2cells from 10 to more than 40% at 12 h after irradiation. However, pretreating Chemical protectors and the cell cycle cells with either WR-1065 or WR-151326 reduced significantly the percentage of cells with G2 DNA content. This is seen as a reduced percentage of cells blocked at the G2-M border, as well as an earlier release of the cells experiencing inhibited progression. The significance of these results is not clear, except when one considers that protector alone induces a G2 block, as does radiation alone. When the two modalities are combined, the block is either reduced or ameliorated. Whatever the mechanism, it appears to be more sensitive to neutron irradiation, because the division delay is greater in this group than after gamma irradiation. The same phenomenon was observed, albeit to a lesser extent, in the cell doubling time experiments. Untreated cells double approximately every 10 h, while radiation-only treated cells have doubling times of 19-20 h and protector-only treated cells double every 16 h. When gamma irradiated cells are pretreated with protector the doubling time is only 15 h, less than for either of the two individual treatments. The data presented do not provide a mechanism for the observed results. A review of the literature suggests that this is not an unique finding. Chatterjee & Jacob-Raman (1986), while investigating the effect of reduced glutathione on X-ray-induced chromosome aberrations, noted that GSH pre-treatment reduced radiation-induced cell cycle delay to normal, although such treatment in unirradiated cells caused a remarkable delay in cell progression. They further state that GSH treatment after irradiation results in additive cell cycle delay. Aminothiol radiation protectors, such as those used in the present study, have been shown to bind to DNA (Brown 1967, Sigdestad et al. 1988, Grdina et al. 1988), with a possible result of reduced strand separation and a concomitant delay in replication. This is a possible mechanism for cell cycle delay in protector-treated cells. Chatterjee & Jacob-Raman hypothesized that the lack of delay noted in combined drug-radiation treatment might be a result of the radiation-induced free radicals, which would have caused delay in the cell cycle, being scavenged by the protector, and thereby being unavailable for their own delaying effect. It would appear that the results indicate that treatment with protector, without radiation, acts by some mechanism to inhibit proliferation, but if the protectors are used prior to irradiation, they function by a second mechanism to reduce cell cycle delay. ACKNOWLEDGEMENTS The authors acknowledge the expert dosimetry performed by Gordon Holmblad. We are also indebted to Ms Jane Perrin for expert technical assistance. This research was supported by the US Department of Energy, Office of Health and Environmental Research under contract No. W-31-109-ENG-38, and DHHSINCI Grant CA-37435 (to D.J.G.). This work was carried out while C.P.S. was on sabbatical leave from the Radiation Oncology Department, University of Louisville, Louisville, KY, and while B.L.B. was a participant in the Summer Faculty Research Program, Argonne National Laboratory. C.P.S. and B.L.B. are indebted to the Division of Educational Programs, Argonne National Laboratory, for partial support. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Proliferation Wiley

The effect of chemical radiation protectors on cell cycle progression after gamma or neutron irradiation

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Publisher
Wiley
Copyright
1991 Blackwell Science Limited
ISSN
0960-7722
eISSN
1365-2184
DOI
10.1111/j.1365-2184.1991.tb01156.x
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Abstract

Biological & Medical Research Division, Argonne National Laboratory, Argonne, IL and 'Radiation Oncology Departmenr, J . Graham Brown Cancer Center, University of Louisville, Louisville, K Y , +Department of Biology, University of Northern Iowa, Cedar Falls. IA and $Department of Radiation and Cellular Oncology, University of Chicago, Chicago, IL, USA (Received 10 October 1990; accepted 13 November 1990) Abstract. The effects of two chemical radiation protectors, WR-1065 and WR-151326, were characterized in V79 Chinese hamster cells after either cobalt-60 (6"Co) gamma or fission spectrum neutron irradiation. Each protector was administered at a concentration of 4 mM t o exponentially growing cultures for 30 min prior t o and during irradiation with either 6oCogamma or JANUS fission spectrum neutrons. After irradiation the cells were either plated immediately for survival or returned t o the incubator and assayed for cell progression. Aliquots of cells were removed at selected times, counted, fixed and stained with 4'6-diamidino-2-phenylindole (DAPI). Analysis of D N A histograms indicate that the presence of the protector during irradiation reduced the division delay experienced at the G;?-M interface. Implications of these effects are discussed. There is considerable interest in the phosphorothioate class of radiation protective agents as an adjunct in the treatment of cancer. This was originally based on the findings of Yuhas & Storer (1969), who found that WR-2721 protected mice against radiation-induced lethality by a factor of 1.6, while tumour protection was only 1.15. The mechanism of action, although poorly understood, is widely believed to result from scavenging OH radicals in and about the presumed target molecule DNA. Exposure to high linear energy transfer (LET) radiations, such as neutrons or other heavy particles, is known to produce more biological damage than an equal dose of low LET radiation (Barendsen & Walter 1964). Chemical protectors, however, are not as useful in reducing these high LET effects as they are in reducing low LET effects (Sigdestad et al. 1986, Afzal & Ainsworth 1987). We have previously shown (Sigdestad etal. 1988) that WR-1065 and WR-151326, which are the dephosphorylated derivatives of the more familiar WR-2721 and WR-15 1327, effectively perturb cell cycle progression in the absence of radiation. During drug exposure, there was a build-up of cells in S and G2phases. Chatterjee & Jacob-Raman (1986) reported that reduced glutathione (GSH) treatment produced a cell cycle delay in lymphocytes. When these cells were irradiated 30 min later, however, the expected radiation-induced cell cycle delay was dramatically reduced. These findings prompted us to assess the role of the thiophosphoroate class of protectors in progression delay after high or low LET radiation. Correspondence: Dr D. J . Grdina, Biological & Medical Research Division, Argonne National Laboratory, Argonne, IL 60439, USA. C. P. Sigdestad, B. L. Bergquist and D. J . Grdina MATERIALS AND METHODS Cell system V79-B310H Chinese hamster cells were grown at 37°C in plastic T-flasks or in plastic 100 mm petri dishes containing alpha MEM-10 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Biologos, Naperville, IL, USA) in a humidified atmosphere of 5% C 0 2 and 95% air. A complete description of the culture condition is given elsewhere (Suzuki et al. 1981). Radiation protectors WR-1065 (2-[aminopropylamino]ethyl mercaptan dihydrochloride) and WR-151326 (3-[3-methylaminopropylamino]propyl mercaptan dihydrochloride) were used in these studies and were obtained from the Division of Experimental Therapeutics, Walter Reed Army Medical Center, Washington, DC, USA. Each was originally dissolved in phosphate-buffered saline (PBS) at a 1 M concentration and sterilized by filtration immediately before use. Cells were treated with either protector, at a concentration of 4 mM, for 30 min prior to and during irradiation. Immediately after irradiation, the cells were washed twice with PBS and either plated for survival or returned to the incubator for cell doubling time assay using flow cytometry . irradiation procedure Exponentially growing V79 cells were trypsinized and irradiated in 15 ml centrifuge tubes at ice-bath temperature, in order to prevent cell cycle progression during the irradiation procedure. Fission-spectrum neutrons were obtained from the JANUS reactor of the Biological, Environmental and Medical Research Division, Argonne National Laboratory. The characteristics of the JANUS source are described in detail elsewhere (Williamson & Frigerio 1972). The average neutron energy was 0.85 MeV, and contamination by gamma rays was 3 4 % of the total absorbed dose. All neutron irradiations were performed at a dose rate of 0.24 Gy/min. Cells were irradiated, under similar conditions, using cobalt-60 gamma rays from a Gamma Beam 650 (Atomic Energy of Canada) at a dose rate of 0.5 G y h i n . Cell doubling time Cells were recovered from stock cultures, replated in 100 mm Petri dishes at a density of 7.5 x lo4 and, 24 h later, when the cell density approximated 4 x lo5 cells per plate, the medium was replaced with medium containing either WR-1065 or WR-151326 at a concentration of 4 m for 30 min. The cells were then irradiated with either 7 Gy gamma o r 2 Gy M fission spectrum neutrons. Unirradiated cells treated with protector served as positive controls. The medium was removed and replaced with fresh medium without protector. At closely spaced time intervals, plates were trypsinized and the number of cells was determined using a Coulter counter with appropriate correction for coincidence. The cells from each group were fixed in ice-cold 70% ethanol in preparation for flow cytometric (FCM) analysis. FCM analysis The determination of the D N A content of irradiated and unirradiated V79 cells as well as the evaluation of the effects of WR-1065 and WR-151326 on cell cycle progression was made using FCM techniques. Cells were stained with 4' ,6-diamidino-2-phenylindole-DAPI (Russel, Newman & Williamson 1975) in a 0.1% citrate solution according to a method described Chemical protectors and the cell cycle elsewhere (Gohde, Schuman & Zante 1978, Gohde et al. 1979). Flow cytometry patterns were obtained using a Partec PAS-I1 (Particle Analyzing System, Partec A G , Basel, Switzerland) and were analysed using a computer program obtained from Techno System-GMBH (Darmstadt, West Germany). T h e coefficient of variation of the G , peaks obtained for unperturbed cell samples routinely ranged from 1.5 to 2.5%. RESULTS Cell survival V-79 cells grown under standard tissue culture conditions were examined for cell survival after irradiation with either JANUS fission spectrum neutrons or 6oCo gamma rays. The two radiation protectors WR-1065 and WR-151326 were studied to determine their ability to reduce radiation cell killing. The results are shown in Figures 1& 2. The curves were fitted using the linear quadratic model and the salient survival data are presented in Table 1. Because this model assumes a continuously downward bending of the survival curves, protection factor (PF) values were calculated on a basis of a method described by Chaplin et al. (1987). P F values of 1.6 were obtained by applying either WR-1065 o r WR-151326 for 30 min prior to and during gamma irradiation. Similar measurements after irradiation with JANUS fission neutrons produced P F values of about two. Cell cycle progression Progression of cells through the cell cycle was determined using flow cytometry. Preliminary studies indicates that 7 G y gamma radiation was approximately equal to 2 Gy fission neutrons, which explains the choice of radiation doses. Progression was examined at 2 , 4 , 8 , 1 2 , 1 6 , 2 0 , 2 4 and 28 h after irradiation. Figure 3 shows typical flow cytometry patterns (12 h postI 1 $. Dose (Gy) Figure 1. The effect of WR-1065 chemical protection on the survival of V79 cells in or culture after irradiation with either cobalt-60 gamma (0) JANUS fission spectrum Survival of untreated cultures after gamma ( V ) and neutron (V) neutrons (0). irradiation is also shown. C. P . Sigdestad, B . L . Bergquist and D . J . Grdina Dose (Gy) Figure 2. The effect of WR-151326chemical protection on the survival of V79 cells in culture after irradiation witheithercobalt-@gamma (0) JANUS fission spectrum or neutrons (0). Survival of untreated cultures after gamma ( V ) and neutron (V) irradiation is also shown. irradiation) in unprotected, as well as WR-1065 or WR-151326 treated V-79 cells, after either gamma or neutron irradiation. The most striking characteristic of these curves is the effect of the protectors on the G2 M compartment. It is apparent in the unprotected but irradiated groups that a large proportion of cells are in the G2 compartment, whereas in the protected groups this block appears to have been released. The percent GI, S and G2 + M were computer-derived. Figures 4 & 5 show graphically the results of flow cytometric analysis for “Co and neutron irradiation respectively. The presence of the protector appeared to modify the progression pattern after irradiation regardless of the radiation source. Generally, the G I population was reduced after irradiation, reaching a nadir between 4 and 6 h, with a subsequent slow rise to control levels 28 h after exposure. The DNA synthetic phase initially increased in size, with a subsequent depression at 12 h. At 28 h the number of S phase cells was approaching control levels. As stated above, the most significant difference was noted in the G2 M population, where cells pretreated with protector did not experience the typical G2 Table 1. Salient survival curve parameters Source Neutron Treatment Alpha 1.726 0.568 0.755 0.237 0.041 0.058 SD Beta SD PF 1.oo 1.97 2.00 1.oo 1.64 1.62 None WR- 1065 WR- 151326 None WR-1065 WR- 151326 -0 0.118 0.041 Gamma PF protection factor, calculated using linear quadratic model as described by Chaplin ef al. (1987) Chemical protectors and the cell cycle 600 Channel number Figure 3. Typical flow cytometry patterns observed 12 h following irradiation with comparable doses of a, c & e 700 rad cobalt-60 gamma or b, d & f 200 rad JANUS fission spectrum neutron irradiation: a & b control cultures; c & d treated with WR-1065; e & f treated with WR-151326. C. P . Sigdestad, B . L. Bergquist and D . J . Grdina I” -I I 30 Time after y irradiation (h) Figure 4. Quantitative analysis of flow cytometry patterns, showing the effects of chemical protectors WR-1065 (A)and WR-151326 (W) on cell progression as a function of time after 700 rad cobalt-60 irradiation; controls treated only by a irradiation are also shown (0). = G I ; b = S; c = Gz + M. Chemical protectors and the cell cycle Time after neutron irradiation (h) Figure 5. Quantitative analysis of flow cytometry patterns. showing the effects of chemical protectors WR-1065 (A)and WR-151326 (W) on cell progression as a function of time after 200 rad fission neutron irradiation; controls treated only by irradiation are also shown (0). = G I ; b = S; c = GZ + M. a C. P . Sigdestad, B. L. Bergquist and D . J . Grdina block which was observed in the radiation-only group. The same effect was observed regardless of whether the cells were irradiated with gamma or fission neutron irradiation. Cell doubling time The doubling time for V79 cells was determined after irradiation, with or without addition of radiation protector. Cells treated only with protector served as positive controls. The results are presented in Table 2. Negative controls (V79 cells without radiation or drug treatment) showed a doubling time of 10 h. Positive control cells, those treated with either protector but no radiation treatment, showed increased doubling time to 16 h. There was no difference between the results obtained with either WR-10165 or WR-151326. The results obtained in irradiated cells indicate that the presence of protector during irradiation reduced the division delay by 1-2 h in neutron-irradiated cells and 5 h in gamma-irradiated cells. The response noted was similar regardless of which protector was tested. The aminothiol radiation protectors which inhibit cell progression without irradiation act to reduce radiation-induced division delay. Table 2. Doubling time (h) of V79 cells after various treatment Radiation treatment Drug treatment None Gamma (7 Gy) 20 15 15 Neutrons (2 Gy) None WR-1065 WR-15 1326 DISCUSSION We have previously reported (Sigdestad et al. 1988) the effect of WR-1065 and WR-151326 without radiation on cell cycle progression in V79 cells in culture. Drug exposure for 3 h resulted in cell cycle perturbations characterized by a build-up of cells in the S and G2phases. After the protectors were removed, cells began to redistribute throughout the cell cycle and 12 h were required before cells exposed to WR-1065 approached levels commensurable with controls. In contrast, cells treated with WR-151327 required about 24 h to redistribute to control levels. No evidence was presented which explained the mechanism of action of this phenomenon. Billen (1983), however, demonstrated that cysteamine can inhibit DNA polymerase-directed repair synthesis, which could be possibly related to chelation of selected metal ions (Jellum, Aaseth & Eldjarn 1973). The present study was an extension of these results to include combined treatment of radiation, a well-known cell cycle modifier, and selected protectors which we have shown to affect cell cycle progression. V79 cell survival after either fission neutron or 6oCo gamma irradiation was found to be enhanced by pre-treatment with either WR-1065 or WR-151326. PF values of 1.6 and 2.0 were obtained after gamma and neutron irradiation respectively. No difference between the two protectors examined was observed, regardless of the radiation source. Flow cytometry was used as a method of assessing the combined effect of radiation and chemical protectors on cell progression. Although there were various differences in progression of cells through the cell cycle for the various groups tested, the main effect noted was a difference in the proportion of cells blocked at the G2-M interface. The well-known G2 block was observed after irradiation without the addition of chemical protector. This increased the proportion of G2cells from 10 to more than 40% at 12 h after irradiation. However, pretreating Chemical protectors and the cell cycle cells with either WR-1065 or WR-151326 reduced significantly the percentage of cells with G2 DNA content. This is seen as a reduced percentage of cells blocked at the G2-M border, as well as an earlier release of the cells experiencing inhibited progression. The significance of these results is not clear, except when one considers that protector alone induces a G2 block, as does radiation alone. When the two modalities are combined, the block is either reduced or ameliorated. Whatever the mechanism, it appears to be more sensitive to neutron irradiation, because the division delay is greater in this group than after gamma irradiation. The same phenomenon was observed, albeit to a lesser extent, in the cell doubling time experiments. Untreated cells double approximately every 10 h, while radiation-only treated cells have doubling times of 19-20 h and protector-only treated cells double every 16 h. When gamma irradiated cells are pretreated with protector the doubling time is only 15 h, less than for either of the two individual treatments. The data presented do not provide a mechanism for the observed results. A review of the literature suggests that this is not an unique finding. Chatterjee & Jacob-Raman (1986), while investigating the effect of reduced glutathione on X-ray-induced chromosome aberrations, noted that GSH pre-treatment reduced radiation-induced cell cycle delay to normal, although such treatment in unirradiated cells caused a remarkable delay in cell progression. They further state that GSH treatment after irradiation results in additive cell cycle delay. Aminothiol radiation protectors, such as those used in the present study, have been shown to bind to DNA (Brown 1967, Sigdestad et al. 1988, Grdina et al. 1988), with a possible result of reduced strand separation and a concomitant delay in replication. This is a possible mechanism for cell cycle delay in protector-treated cells. Chatterjee & Jacob-Raman hypothesized that the lack of delay noted in combined drug-radiation treatment might be a result of the radiation-induced free radicals, which would have caused delay in the cell cycle, being scavenged by the protector, and thereby being unavailable for their own delaying effect. It would appear that the results indicate that treatment with protector, without radiation, acts by some mechanism to inhibit proliferation, but if the protectors are used prior to irradiation, they function by a second mechanism to reduce cell cycle delay. ACKNOWLEDGEMENTS The authors acknowledge the expert dosimetry performed by Gordon Holmblad. We are also indebted to Ms Jane Perrin for expert technical assistance. This research was supported by the US Department of Energy, Office of Health and Environmental Research under contract No. W-31-109-ENG-38, and DHHSINCI Grant CA-37435 (to D.J.G.). This work was carried out while C.P.S. was on sabbatical leave from the Radiation Oncology Department, University of Louisville, Louisville, KY, and while B.L.B. was a participant in the Summer Faculty Research Program, Argonne National Laboratory. C.P.S. and B.L.B. are indebted to the Division of Educational Programs, Argonne National Laboratory, for partial support.

Journal

Cell Proliferation – Wiley

Published: May 1, 1991

There are no references for this article.