•
•
Radiation Biology
Adriamycin: A Possible Indirect Radiosensitizer of Hypoxic Tumor Cells 1 Ralph E. Durand, Ph.D. Multicellular spheroids grown in vitro provide a convenient model of nodular tumors for experimental tumor therapy studies. Adriamycin was found to inactivate cells grown as spheroids less efficiently than single cells, presumably due to enhanced cellular resistance analogous to the increased radioresistance observed when these cells are grown in close contact. Spheroid growth was retarded by a minimally toxic (0.005 J,Lg/ml) chronic level of adriamycin; irradiation and exposure to that drug concentration were not found to be synergistic. Larger adriamycin concentrations (0.5 J,Lg/ml) present during radiation exposure produced a marked "radiosensitization," presumably due to the druginhibiting cellular oxygen consumption and thus permitting reoxygenation of the previously hypoxic spheroid cells. INDEX TERMS: Adrlamycin s Chemotherapy • Radiobiology, cell and tissue studies • Radiobiology, lethality studies • Radiobiology, time-dose studies Radiology 119:217-222, April 1976
•
T
HE CYTOTOXIC EFFECTS of new anticancer agents
Table I: Summary of Regrowth Data for Radiation-Adriamycin Treatment
can easily be examined in vitro and in selected animal systems but these efforts are generally limited to studying the drug alone. Often, however, the cancer patient will receive the drug as only one aspect of a combined therapy regimen, and it thus becomes critical to determine whether any interaction can be expected between the modalities employed, and further, to develop temporal schemes which maximize the therapeutic ratio. Since radiotherapy forms an important aspect of cancer management, the effects of chemotherapeutic agents in parallel with a course of radiotherapy is of fundamental importance. The present report describes some unexpected interactions between adriamycin, a drug of current chemotherapeutic interest, and radiation, using an in vitro tumor model. Chinese hamster V79-171 cells placed in suspension culture at appropriate cell densities will aggregate into small ciumps of cells, then grow by cell division to multicellular structures or "spheroids" as large as 106 cells (1). As the spheroids enlarge, the internal cells presumably receive nutrients and oxygen only by diffusion processes, leading to the development of noncycling and eventually hypoxic cell subpopulations (2, 3). Large numbers of virtually identical spheroids can be grown within a single culture flask, and, after treatment with drugs or radiation, can be reduced to single cells using trypsin and mechanical agitation. The viability of these cells can be determined by using conventional colony-formation criteria (4). Tumor-like endpoints, including volume regression rate, regrowth rate, and cell loss can also be followed as a function of time and treatment (4). The spheroid system should be extremely useful for predicting the interactions of drugs and radiation in a tissue-like environment, since the agents investigated must act on cycling, noncycling and even hypoxic cells
Dose
o Rads GD*
1.6 Volume. 2.6 Total cells 7.0 Viable cells
1,600 Rads 2,500 Rads RV RV GO GO
RVt
800 Rads GO RV
0.92
0.6
0.92
0.4
0.85
**
1.6
0.81
0.8
0.88
0.4
0.83
**
0.95
0.73
2.2
0.76
0.4
0.85
**
1.4
* Growth delay in days, defined as the interval between control and drug-treated populations reaching a threefold increase in the given parameter. t Ratio of drug-treated to control parameters 13 days after irradiation. ** Increase in parameter insufficient to evaluate GO as defined.
in order to be effective. However, the inherent complexity of the system precludes the study of mechanisms of interaction; only the net effect can be determined, and mechanisms must be inferred. These latter considerations led to the design of the experiments reported, addressed to the following questions which may have clinical relevance: (a) Is adriamycin toxicity modified by a tlssue-llke environment (e.g., intercellular contact)? (b) Will chronic exposure to minimal drug concentrations lead to inhibition of regrowth following irradiation? (c) Will adriamycin and radiation be synergistic for any administration schemes? MATERIALS AND METHODS Chinese hamster V79-171 cells were used exclusively in this study. All growth and assay techniques have previously been described in detail (2, 3). Briefly, the cells were grown as asynchronous monolayers on 100mm Falcon plastic Petri dishes with Eagle's Basal Medium (BME) supplemented with 1% L-glutamine, 15 % fetal calf serum (FCS, purchased from GIBCO) and 100 IU/ml plus 100 J,Lg/ml penicillin and streptomycin, respectively. Spheroids were grown in magnetic stirring flasks with BME + 5% FCS.
1 From the Radiobiology Research Laboratory, Departments of Human Oncology and Radiology, Wisconsin Clinical Cancer Center, Madison, Wis. Accepted for publication in October 1975. Supported in part by Cancer Center Grant CA-14520 from the National Cancer Institute, NIH. shan
217
218
RALPH
E.
DURAND
April 1976
O.OS,..g/ml
30 MIN.
z o
i= u a:::
«
z
o
t;
1.L
0.001
10.0,..1I/ ml
,
0.001
\~.--CONTROL \
:::>
\
l/l
o \ \
o
1200
2400
3600
DOSE (RAD)
Fig. 5. Cell survival as a function of radiation dose for spheroids exposed to 0.5 jLg/ml adriamycin for 30 minutes prior to and during irradiation. The broken survival curves indicate the response of untreated spheroids and totally reoxygenated spheroids, respectively.
Vol. 119
AORIAMYCIN:
A POSSIBLE INDIRECT RADIOSENSITIZER OF HYPOXIC TUMOR CELLS
diation (Figs. 4 and 5) could perhaps be exploited in the clinical situation. From the complete survival curves in Figure 5, it appears that adriamycin selectively' 'sensitizes" only the hypoxic cells. This could be achieved by one of three mechanisms: (a) selective cytolethality to the hypoxic cells; (b) "radiosensitization" of the hypoxic population by a mechanism similar to oxygen; or (c) metabolic action leading to reoxygenation of the hypoxic population. The first of these suggestions was not supported by the toxicity data-about 15 % of the cells were hypoxic, yet a decrease in plating efficiency of only 2-3 % was noted after incubation in the drug. The second possibility was refuted by control experiments in which adriamycin was added to hypoxic single cells; no oxygen-like radiosensitization was observed. Since the hypoxic population in the spheroids is a direct result of oxygen consumption by the external cells (2, 3), modification of the respiration rate can lead to dramatic changes in hypoxic fractions (8). Adriamycin is known to exercise marked depressive effects on both cellular and mitochondrial oxygen utilization rates (9); hence; the apparent sensitization is probably an indirect effect due to drug interference with normal metabolic processes. The net effect may nonetheless have therapeutic potential for combined therapy of tumors which have large numbers of radioresistant, hypoxic cells. Growth and repopulation data for irradiated spheroids subsequently exposed to adriamycin were somewhat more difficult to interpret. Despite the ambiguities in defining meaningful endpoints, the data generally suggested that adriamycin was progressively less effective in inhibiting growth as the "tumor burden" was decreased by irradiation (TABLE I). It seems likely that this may be explained by re-examination of the data relating response of spheroids to the drug alone (Fig. 3, A). The response can be described as occurring in three separate phases. Virtually no effect was observed during the first 2-3 days of treatment. This was followed by an apparent "inhibition" of growth, in the absence of cell killing. After a further 3 days' exposure to adriamycin, the subsequent growth rate (even in the presence of the drug) was not significantly different than that of the controls. This similarity of growth rate was even more markedly illustrated in the regrowth data after 800 or 1,600 rads (Figs. 3, B and C). Since the net effect can be characterized as a transient, drug-induced inhibition of growth followed by growth at "normal" rates, one might postulate that either (a) an adriamycin-resistant population of cells develops, or (b) all cells apparently "adapt" to growth in the presence of the drug. Preliminary experiments involving a second adriamycin treatment to progeny of those cells which survived the first exposure have shown no heritable resistance; the second postulate thus appears more attractive. It is also worthy of note that this "adaptive" period was less than the time required for the hypoxic cells surviving 2,500 rads to begin to repopulate the spheroid, even in the absence of the drug. Presumably, even noncycling cells
221
Radiation Biology
may be able to "adapt" to the adriamycin, thus explaining the reduced effect of adriamycin with increasing radiation dose. It is interesting that the two dissimilar situations discussed above lead to a similar prediction for the optimal clinical utilization of adriamycin in combination with radiotherapy. One would predict that optimal effects would be obtained by administration of relatively large doses of adriamycin a few minutes prior to each of the first few fractions of radiotherapy. This scheme would ostensibly lead to some degree of tumor reoxygenation ("radiosensitization") as well as direct chemotherapeutic effects and might then be expected to provide a maximal therapeutic ratio. Clinical regimens involving small numbers of relatively large doses of adriamycin have been used successfully in the absence of radiation (10, 11), and can be justified on the basis of the relatively long plasma half-life of adriamycin observed by Benjamin et al. (12). In the latter study, a mean plasma concentration of 1 nM/ml (0.0006 tLg/ml) was observed 20-30 minutes after administration of a "standard" dose of 60 mg/m 2 . Recalling that the in vitro cell line used in the present study is somewhat more resistant to adriamycin (Figs. 1 and 2) than some other cells in culture (5, 6), and that cells in contact (spheroids) are additionally resistant, it seems justifiable to predict that the qualitative results reported here may well be expected in vivo within acceptable dose ranges. In addition, tissues may concentrate the drug (13), suggesting that higher net drug concentrations may be attainable. While this in vitro model system may suggest possible improvements in cancer control, it must be emphasized that the system has no vascular or physiological responses, and as such, only clinical experience can satisfactorily evaluate the safety (14) and efficacy of any new treatment protocol. If, however, only a temporal rearrangement of existing regimens with known normal tissue responses and complications is required, then the specific therapeutic gains suggested here can more easily be evaluated. ACKNOWLEDGMENTS: The expert technical assistance of Mrs. B. Roe, and the continued interest and suggestions of Drs. K. Clifton and M. Yatvin are gratefully acknowledged. Radiobiology Research Laboratory University of Wisconsin Medical School 420 N. Charter St. Madison, Wis. 53706
REFERENCES 1. Sutherland RM, McCredie JA, Inch WR: Growth of multicell spheroids in tissue culture as a model of nodular carcinomas. J Natl Cancer Inst 46:113-120, Jan 1971 2. Durand RE, Sutherland RM: Dependence of the radiation response of an in vitro tumor model on cell cycle effects. Cancer Res 33:213-219, Feb 1973 3. Sutherland RM, Durand RE: Hypoxic cells in an in vitro tumour model. Int J Radiat Bioi 23:235-246, Mar 1973
222
RALPH
4. Durand RE: Cure, regression and cell survival: a comparison of common radiobiological endpoints using an in vitro tumour model. Br J Radiol 48:556-571, Jul 1975 5. Barranco SC, Gerner EW, Burk KH, et al: Survival and cell kinetics; effects of adriamycin on mammalian cells. Cancer Res 33:11-16, Jan 1973 6. Kim SH, Kim JH: Lethal effect of adriamycin on the division cycle of HeLa cells. Cancer Res 32:323-325, Feb 1972 7. Durand RE, Sutherland RM: Effects of intercellular contact on repair of radiation damage. Exp Cell Res 71:75-80, Mar 1972 8. Durand RE, Biaglow JE: Modification of the radiation response of an in vitro tumour model by control of cellular respiration. Int J Radiat BioI 26:597-601, Dec 1974 9. Gosalvez M, Blanco M, Hunter J, et al: Effects of anticancer agents on the respiration of isolated mitochondria and tumor cells. Europ J Cancer 10:567-574, Sep 1974 10. Gottlieb JA, Baker LH, Quagliana JM, et al: Chemotherapy
E.
DURAND
April 1976
of sarcomas with a combination of adriamycin and dimethyl triazeno imidazole carboxamide. Cancer 30: 1632-1638, Dec 1972
11. Kenis Y, Michel J: Preliminary results of a clinical trial with intermittent doses of adriamycin in lung cancer. [In J International Symposium on Adriamycin, Milan. Carter SK, Di Marco A, Chione M, et al, eds. Berlin, Georg Springer-Verlag, 1972, pp 161-164 12. Benjamin RS, Riggs CE Jr, Bachur NR: Pharmacokinetics and metabolism of adriamycin in man. Clin Pharmacol Ther 14: 592-600, Jul-Aug 1973 13. Herman E, Mhatre R, Lee IP, et al: A comparison of the cardiovascular actions of daunomycin, adriamycin and N-acetyldaunomycin in hamsters and monkeys. Pharmacology 6:230-241, Oct 1971 14.
Eltringham JR, Fajardo LF, Stewart JR: Adriamycin cardiomyopathy: enhanced cardiac damage in rabbits with combined drug and cardiac irradiation. Radiology 115:471-472, May 1975
•
Radiation Biology
Adriamycin: A Possible Indirect Radiosensitizer of Hypoxic Tumor Cells 1 Ralph E. Durand, Ph.D. Multicellular spheroids grown in vitro provide a convenient model of nodular tumors for experimental tumor therapy studies. Adriamycin was found to inactivate cells grown as spheroids less efficiently than single cells, presumably due to enhanced cellular resistance analogous to the increased radioresistance observed when these cells are grown in close contact. Spheroid growth was retarded by a minimally toxic (0.005 J,Lg/ml) chronic level of adriamycin; irradiation and exposure to that drug concentration were not found to be synergistic. Larger adriamycin concentrations (0.5 J,Lg/ml) present during radiation exposure produced a marked "radiosensitization," presumably due to the druginhibiting cellular oxygen consumption and thus permitting reoxygenation of the previously hypoxic spheroid cells. INDEX TERMS: Adrlamycin s Chemotherapy • Radiobiology, cell and tissue studies • Radiobiology, lethality studies • Radiobiology, time-dose studies Radiology 119:217-222, April 1976
•
T
HE CYTOTOXIC EFFECTS of new anticancer agents
Table I: Summary of Regrowth Data for Radiation-Adriamycin Treatment
can easily be examined in vitro and in selected animal systems but these efforts are generally limited to studying the drug alone. Often, however, the cancer patient will receive the drug as only one aspect of a combined therapy regimen, and it thus becomes critical to determine whether any interaction can be expected between the modalities employed, and further, to develop temporal schemes which maximize the therapeutic ratio. Since radiotherapy forms an important aspect of cancer management, the effects of chemotherapeutic agents in parallel with a course of radiotherapy is of fundamental importance. The present report describes some unexpected interactions between adriamycin, a drug of current chemotherapeutic interest, and radiation, using an in vitro tumor model. Chinese hamster V79-171 cells placed in suspension culture at appropriate cell densities will aggregate into small ciumps of cells, then grow by cell division to multicellular structures or "spheroids" as large as 106 cells (1). As the spheroids enlarge, the internal cells presumably receive nutrients and oxygen only by diffusion processes, leading to the development of noncycling and eventually hypoxic cell subpopulations (2, 3). Large numbers of virtually identical spheroids can be grown within a single culture flask, and, after treatment with drugs or radiation, can be reduced to single cells using trypsin and mechanical agitation. The viability of these cells can be determined by using conventional colony-formation criteria (4). Tumor-like endpoints, including volume regression rate, regrowth rate, and cell loss can also be followed as a function of time and treatment (4). The spheroid system should be extremely useful for predicting the interactions of drugs and radiation in a tissue-like environment, since the agents investigated must act on cycling, noncycling and even hypoxic cells
Dose
o Rads GD*
1.6 Volume. 2.6 Total cells 7.0 Viable cells
1,600 Rads 2,500 Rads RV RV GO GO
RVt
800 Rads GO RV
0.92
0.6
0.92
0.4
0.85
**
1.6
0.81
0.8
0.88
0.4
0.83
**
0.95
0.73
2.2
0.76
0.4
0.85
**
1.4
* Growth delay in days, defined as the interval between control and drug-treated populations reaching a threefold increase in the given parameter. t Ratio of drug-treated to control parameters 13 days after irradiation. ** Increase in parameter insufficient to evaluate GO as defined.
in order to be effective. However, the inherent complexity of the system precludes the study of mechanisms of interaction; only the net effect can be determined, and mechanisms must be inferred. These latter considerations led to the design of the experiments reported, addressed to the following questions which may have clinical relevance: (a) Is adriamycin toxicity modified by a tlssue-llke environment (e.g., intercellular contact)? (b) Will chronic exposure to minimal drug concentrations lead to inhibition of regrowth following irradiation? (c) Will adriamycin and radiation be synergistic for any administration schemes? MATERIALS AND METHODS Chinese hamster V79-171 cells were used exclusively in this study. All growth and assay techniques have previously been described in detail (2, 3). Briefly, the cells were grown as asynchronous monolayers on 100mm Falcon plastic Petri dishes with Eagle's Basal Medium (BME) supplemented with 1% L-glutamine, 15 % fetal calf serum (FCS, purchased from GIBCO) and 100 IU/ml plus 100 J,Lg/ml penicillin and streptomycin, respectively. Spheroids were grown in magnetic stirring flasks with BME + 5% FCS.
1 From the Radiobiology Research Laboratory, Departments of Human Oncology and Radiology, Wisconsin Clinical Cancer Center, Madison, Wis. Accepted for publication in October 1975. Supported in part by Cancer Center Grant CA-14520 from the National Cancer Institute, NIH. shan
217
218
RALPH
E.
DURAND
April 1976
O.OS,..g/ml
30 MIN.
z o
i= u a:::
«
z
o
t;
1.L
0.001
10.0,..1I/ ml
,
0.001
\~.--CONTROL \
:::>
\
l/l
o \ \
o
1200
2400
3600
DOSE (RAD)
Fig. 5. Cell survival as a function of radiation dose for spheroids exposed to 0.5 jLg/ml adriamycin for 30 minutes prior to and during irradiation. The broken survival curves indicate the response of untreated spheroids and totally reoxygenated spheroids, respectively.
Vol. 119
AORIAMYCIN:
A POSSIBLE INDIRECT RADIOSENSITIZER OF HYPOXIC TUMOR CELLS
diation (Figs. 4 and 5) could perhaps be exploited in the clinical situation. From the complete survival curves in Figure 5, it appears that adriamycin selectively' 'sensitizes" only the hypoxic cells. This could be achieved by one of three mechanisms: (a) selective cytolethality to the hypoxic cells; (b) "radiosensitization" of the hypoxic population by a mechanism similar to oxygen; or (c) metabolic action leading to reoxygenation of the hypoxic population. The first of these suggestions was not supported by the toxicity data-about 15 % of the cells were hypoxic, yet a decrease in plating efficiency of only 2-3 % was noted after incubation in the drug. The second possibility was refuted by control experiments in which adriamycin was added to hypoxic single cells; no oxygen-like radiosensitization was observed. Since the hypoxic population in the spheroids is a direct result of oxygen consumption by the external cells (2, 3), modification of the respiration rate can lead to dramatic changes in hypoxic fractions (8). Adriamycin is known to exercise marked depressive effects on both cellular and mitochondrial oxygen utilization rates (9); hence; the apparent sensitization is probably an indirect effect due to drug interference with normal metabolic processes. The net effect may nonetheless have therapeutic potential for combined therapy of tumors which have large numbers of radioresistant, hypoxic cells. Growth and repopulation data for irradiated spheroids subsequently exposed to adriamycin were somewhat more difficult to interpret. Despite the ambiguities in defining meaningful endpoints, the data generally suggested that adriamycin was progressively less effective in inhibiting growth as the "tumor burden" was decreased by irradiation (TABLE I). It seems likely that this may be explained by re-examination of the data relating response of spheroids to the drug alone (Fig. 3, A). The response can be described as occurring in three separate phases. Virtually no effect was observed during the first 2-3 days of treatment. This was followed by an apparent "inhibition" of growth, in the absence of cell killing. After a further 3 days' exposure to adriamycin, the subsequent growth rate (even in the presence of the drug) was not significantly different than that of the controls. This similarity of growth rate was even more markedly illustrated in the regrowth data after 800 or 1,600 rads (Figs. 3, B and C). Since the net effect can be characterized as a transient, drug-induced inhibition of growth followed by growth at "normal" rates, one might postulate that either (a) an adriamycin-resistant population of cells develops, or (b) all cells apparently "adapt" to growth in the presence of the drug. Preliminary experiments involving a second adriamycin treatment to progeny of those cells which survived the first exposure have shown no heritable resistance; the second postulate thus appears more attractive. It is also worthy of note that this "adaptive" period was less than the time required for the hypoxic cells surviving 2,500 rads to begin to repopulate the spheroid, even in the absence of the drug. Presumably, even noncycling cells
221
Radiation Biology
may be able to "adapt" to the adriamycin, thus explaining the reduced effect of adriamycin with increasing radiation dose. It is interesting that the two dissimilar situations discussed above lead to a similar prediction for the optimal clinical utilization of adriamycin in combination with radiotherapy. One would predict that optimal effects would be obtained by administration of relatively large doses of adriamycin a few minutes prior to each of the first few fractions of radiotherapy. This scheme would ostensibly lead to some degree of tumor reoxygenation ("radiosensitization") as well as direct chemotherapeutic effects and might then be expected to provide a maximal therapeutic ratio. Clinical regimens involving small numbers of relatively large doses of adriamycin have been used successfully in the absence of radiation (10, 11), and can be justified on the basis of the relatively long plasma half-life of adriamycin observed by Benjamin et al. (12). In the latter study, a mean plasma concentration of 1 nM/ml (0.0006 tLg/ml) was observed 20-30 minutes after administration of a "standard" dose of 60 mg/m 2 . Recalling that the in vitro cell line used in the present study is somewhat more resistant to adriamycin (Figs. 1 and 2) than some other cells in culture (5, 6), and that cells in contact (spheroids) are additionally resistant, it seems justifiable to predict that the qualitative results reported here may well be expected in vivo within acceptable dose ranges. In addition, tissues may concentrate the drug (13), suggesting that higher net drug concentrations may be attainable. While this in vitro model system may suggest possible improvements in cancer control, it must be emphasized that the system has no vascular or physiological responses, and as such, only clinical experience can satisfactorily evaluate the safety (14) and efficacy of any new treatment protocol. If, however, only a temporal rearrangement of existing regimens with known normal tissue responses and complications is required, then the specific therapeutic gains suggested here can more easily be evaluated. ACKNOWLEDGMENTS: The expert technical assistance of Mrs. B. Roe, and the continued interest and suggestions of Drs. K. Clifton and M. Yatvin are gratefully acknowledged. Radiobiology Research Laboratory University of Wisconsin Medical School 420 N. Charter St. Madison, Wis. 53706
REFERENCES 1. Sutherland RM, McCredie JA, Inch WR: Growth of multicell spheroids in tissue culture as a model of nodular carcinomas. J Natl Cancer Inst 46:113-120, Jan 1971 2. Durand RE, Sutherland RM: Dependence of the radiation response of an in vitro tumor model on cell cycle effects. Cancer Res 33:213-219, Feb 1973 3. Sutherland RM, Durand RE: Hypoxic cells in an in vitro tumour model. Int J Radiat Bioi 23:235-246, Mar 1973
222
RALPH
4. Durand RE: Cure, regression and cell survival: a comparison of common radiobiological endpoints using an in vitro tumour model. Br J Radiol 48:556-571, Jul 1975 5. Barranco SC, Gerner EW, Burk KH, et al: Survival and cell kinetics; effects of adriamycin on mammalian cells. Cancer Res 33:11-16, Jan 1973 6. Kim SH, Kim JH: Lethal effect of adriamycin on the division cycle of HeLa cells. Cancer Res 32:323-325, Feb 1972 7. Durand RE, Sutherland RM: Effects of intercellular contact on repair of radiation damage. Exp Cell Res 71:75-80, Mar 1972 8. Durand RE, Biaglow JE: Modification of the radiation response of an in vitro tumour model by control of cellular respiration. Int J Radiat BioI 26:597-601, Dec 1974 9. Gosalvez M, Blanco M, Hunter J, et al: Effects of anticancer agents on the respiration of isolated mitochondria and tumor cells. Europ J Cancer 10:567-574, Sep 1974 10. Gottlieb JA, Baker LH, Quagliana JM, et al: Chemotherapy
E.
DURAND
April 1976
of sarcomas with a combination of adriamycin and dimethyl triazeno imidazole carboxamide. Cancer 30: 1632-1638, Dec 1972
11. Kenis Y, Michel J: Preliminary results of a clinical trial with intermittent doses of adriamycin in lung cancer. [In J International Symposium on Adriamycin, Milan. Carter SK, Di Marco A, Chione M, et al, eds. Berlin, Georg Springer-Verlag, 1972, pp 161-164 12. Benjamin RS, Riggs CE Jr, Bachur NR: Pharmacokinetics and metabolism of adriamycin in man. Clin Pharmacol Ther 14: 592-600, Jul-Aug 1973 13. Herman E, Mhatre R, Lee IP, et al: A comparison of the cardiovascular actions of daunomycin, adriamycin and N-acetyldaunomycin in hamsters and monkeys. Pharmacology 6:230-241, Oct 1971 14.
Eltringham JR, Fajardo LF, Stewart JR: Adriamycin cardiomyopathy: enhanced cardiac damage in rabbits with combined drug and cardiac irradiation. Radiology 115:471-472, May 1975
