0360-301@79/091509-04$02.00/0
Oncology Bid. Phys:, Vol. 5, pp. 150%1512 c Pergamon Press Ltd., 1979. Printed m the U.S.A.
Inr. J. Radiation
??Bleomycin
THE INTERACTION OF RADIATION AND BLEOMYCIN IN INTESTINAL CRYPT CELLS? THEODORE L. PHILLIPS, M.D., GLENDA Y. Ross, B.S., LAWRENCE S. GOLDSTEIN, Ph.D. and ADRIAN C. BEGG, Ph.D. Department of Radiation Oncology, University of California, San Francisco, CA 94143 Using the intestinal crypt cell system, the effects of bleomycin (BLM) with radiation have heen determined.
The LD,,,&,in LAF, mice used was 167 units&g and the MTD (LD,,) was 100 units/kg. Survival of crypt cells was reduced to 40% of radiation alone when drug was given 3 hr after a dose of 1100 rad, but to less than 1% when the drug was given 2 hr before. When maxhnum tolerated doses (MTD) were given from -48 to +48 hr relative to a dose of I343rad, the combination was much more effective when drug was given from -12 to -2 hr before irradiation, survival dropping from 100 to 5 cells per circumference. This increased cell kill could be due to synchrony, interference with repair of BLM damage, or other factors. A radiition survival curve determined 2 hr after drug administration had a slope similar to controls, but was shifted toward lower doses. The extrapolated D, was 0 rad. A 3 hr split dose experiment yielded survival ratios of 6 for controls and 2 for mice given the MTD 2 hr before irradiation. There appears to be a marked interaction of bleomycin and radiation, with possible inhibition of repair of the radiation damage by bleomycin and possible inhibition of repair of the bleomycin damage by radiation. Bleomycin, Intestinal crypt cells, Maximum tolerated dose. INTRODUCTION
mice, age 8-12 weeks. Animals were maintained on laboratory chow with access to water ad libitum on a 12 hr light/dark cycle. One set of split dose experiments was performed with B,AF, female mice obtained from the same source and of similar age.
Bleomycin (BLM) has proven to be a very useful antitumor agent in lymphomas and in certain squamous cell carcinomas. It has appeared to be an enhancer of radiation response in oral and other mucosal sites, as well as in the skin,6 and its method of action appears to be radiomimetic in that it causes single strand DNA breaks.5 It appears to block cell progression in the G, area, and the time of block relative to cell division appears to be dose related.3 There are conflicting reports as to whether it simply adds similar injury to that of radiation injury or may indeed impair repair of radiation injury. These studies were designed to determine whether BLM would be effective in a rapid cell renewal system, the intestinal crypt cell, and whether there would be interaction between radiation and BLM injury as determined by experiments involving varying time intervals between radiation and drug, varying radiation doses, and varying drug doses, as well as split dose radiation studies.
Irradiation
Irradiation was carried out with a 13’Cs selfcontained irradiator at a dose rate of 235 rad/minute. All mice were unanesthetized for irradiation and restrained in rotating circular lucite cages containing adequate ventilation. The stack of cages was located parallel to the linear cesium source, thus the irradiation stack was perpendicular to the radiation beam. TLD measurements in mouse phantoms assured that dose was uniform throughout the mice and at each cage position during the rotation. Dosimetry was compared to standard dose measurements traceable to NBS. Drug
As advised by the manufacturer and the National Cancer Institute, all BLM dosages were determined in units and related to the standards as described on each batch of drug. No great variations in the effec-
METHODS AND MATERIAL Animals
All experiments
were performed
in LAF,
male
*This investigation was supported by National Cancer Institute Research Grants CA17227 and CA 20529. 1509
Radiation Oncology 0 Biology 0 Physics
1510
tiveness of the various drug batches were noted, although some were recently outdated clinical ampules. The BLM was reconstituted with water and given intraperitoneally at a constant volume of 0.01 ml/gm of body weight. Histology and Scoring
Four to six mice were studied in each experimental group, with the exception of the LD,, evaluations in which larger groups of 10-15 mice were employed. Whole body irradiation was used, animals were sacrificed 32/3 days after irradiation by cervical dislocation, and the jejunum was removed, pinned on waxed boards, and fixed in Tellyesniczky’s solution. Twenty-four hours later they were sliced, embedded, and processed for light microscopy. Eight to ten 5 micron-thick cross sections were prepared per mouse, so that a total of approximately 32 sections were available per experimental point. The number of regenerating crypts was scored, as described originally by Withers and Elkind,s and the number of surviving crypt cells calculated. Survival curves were determined by least squares regression calcuiations. The D, was determined by extrapolating the survival curve back to a cell number assumed to be the number of intestinal crypt cells present in the unit-radiated gut. This number has been described as approximately 20,000 by Masuda et al. .4 Because of the somewhat smaller number of cells per circumference (120) in these mice, we have used a figure of 15,000 cells. This is in good agreement with the actual extrapolation of linear survival curves for high LET carbon and neon peak irradiation, where no shoulder is seen on the survival curve. Experimental
September
1979, Volume 5, Number 9
or followed by 3 hr of a BLM dose ranging from 6100% of the MTD. Split dose studies were carried out in both LAF, and B,AF, mice by administering a single or 3 hr split dose of radiation of the same total dose, with or without a preceding dose of 25 units/kg of BLM. The doses were 1000 rad (or 500 x 2) for BLM treated mice and 1200-1260 rad (600-630 x 2) for controls.
RESULTS The drug LD,,, experiments yielded an LD,,J, of 250 units/kg and an LD,,J,,, of 167 units/kg. From the analysis of the LD,,J,,, data, a maximum tolerated dose (LD,,) of 100 unit&g was established. At this dose very few mice in large experiments will die from drug alone. Experiments in which the time interval was varied between a dose of 100 units/kg of BLM and a dose of 910 rad of radiation yielded extremely interesting results, as shown in Fig. 1. Although BLM was somewhat cytotoxic in combination with radiation at all time intervals, there was a marked enhanced degree of cell kill at time intervals from -24 to -2 hr prior to irradiation, with maximum effect at -6 and -2 hr. Small degrees of fluctuation occurred at other times, but not to any marked degree. 1000
BLEOYYCIN,
100 units/Kg
r
I
Protocols
LDSO/S and LDs0/60 values were determined by giving graded doses of drug to groups of 10-15 mice, yielding survival between lO-90% at each of the time intervals. The resultant death data were used to calculate the 50% lethal dose. The influence of time of drug administration relative to time of irradiation was determined by giving a fixed drug dose and fixed radiation dose, separated by intervals of +48 hr. The radiation dose employed in these experiments was 910 t-ad and the BLM dose 100 units/kg. Radiation survival curves were determined by administering radiation doses ranging from 400-1900 rad alone or 2 hr following a BLM dose of either 3 units/kg or 100 units/kg. The survival curves were extrapolated back, as described previously, to determine the D, after correction for cell kill by drug alone. Drug survival curves were determined by administering a radiation dose of 1100 rad, preceded by 2 hr
11”“““““““‘J -48
-36
-24 TIME
-12 OF
0 DRUG
+12 INJECTION.
+24
+3G
+40
HR.
FIG. 1. Crypt cell survival (0) as a function of time between administration of Bleomycin (100 units/kg) and *3TCsgamma rays (910 rad).
Radiation survival curves determined 2 hr after 3 and 100 units/kg of BLM are shown in Fig. 2. Because of the marked sensitivity of cells irradiated 2 hr after BLM administration, the curves are shifted to the left, but the slopes do not change. When
1511
Interaction of radiation and bleomycin 0 T. L. PHILLIPS PI cd.
BLEOMYCIN 0 INJ. 2 HR.
BLEOMYCIN 2 HR. BEFORE
IRRADIATION
0
INJ.
BEFORE IRRADIATION 3 HR. AFTER IRRADIATION
‘3’cs
1
Y
ICONTROL,
II00
25
SO BLEO
BOO
1200 DOSE,
1600
2000
RAD
FIG. 2. Radiation survival curves for intestinal crypt cells exposed to 13’Cs gamma rays alone (0) or 2 hr after 3 units/kg (Cl) or 100 units/kg of Bleomycin.
correction is made for the 99% cell kill caused by BLM, the D, by extrapolation is 300 rad. If one assumes the shift to the left is not due to BLM cell kill and corrects for the 40% cell kill seen when BLM is given after irradiation, the D, is zero. Thus one must know the reason for the curve shift to properly understand the D, changes. The drug dose response curves obtained by administering radiation of 1100 rad preceded or followed by graded drug doses were also of great interest, as shown in Fig. 3. Administration of drug at 3 hr following irradiation showed little effect, with maximum cell kill at the MTD of only 40-50%. However, when the drug was given 2 hr before irradiation, maximum cell kill of over 9% was seen, with survival reduced from 150 surviving cells per circumference to 1 surviving cell per circumference. This marked increased effect with prior drug administration was seen down to doses of 25% of the MTD and indeed (not shown in Fig. 2) down to 6% of the MTD. When BLM is given 3 hr before irradiation, the survival curve is not linear for cell kill by drug, and it would appear that in the first part of the curve the D,, would be about 10 units/kg, but in latter parts would be about 50 units/kg. Because of the marked difference in cell kill when
75
RID
lot
DOSE, units/Kg
FIG. 3. Drug survival curves for intestinal crypt cells with Bleomycin in increasing doses given 2 hr before (0) or 3 hr after (0) 1100 tad of 13’Csgamma rays.
drug is given before irradiation, rather than after, it could be postulated that this is due to elimination of repair of sublethal radiation damage or elimination of the shoulder on the radiation survival curve. Twodose studies do not completely support this. A 3-hr split dose experiment in LAF, mice yielded a survival ratio of 6 for controls and 2 for mice given 25% of the MTD before split dose irradiation. (The ratio of the number of surviving crypt cells after 2 split doses to that after a single dose of the same total.) With B&F, mice the survival ratios were 6 for control mice and 4 for drug treated mice. The single dose cell survival levels were 12 and 5 for control and BLM treated LAF, mice, and were 9 and 1 for the B,AF, mice. DISCUSSION Bleomycin has proven to be an extremely interesting agent in its effect in intestinal crypt cells. It was shown by Guigon et al.* that BLM somewhat enhances the response of skin, but not to a tremendous degree. The BLM dosages employed were somewhat smaller than the maximal doses employed here; but did overlap. Our previous work with esophagus had shown some enhanced response (DEF of 1.1 at BLM doses of 3 units/kg).’ Clinical studies have revealed marked enhancement of radiation effect on the lung by BLM.’ This has not been observed in the intestine clinically.‘j Thus the marked apparent potentiation of injury when radiation and
1512
Radiation Oncology 0 Biology 0 Physics
BLM are combined at certain time intervals is rather remarkable. The timing curves suggest that the time of maximum enhanced response occurs between -12 hr and just before irradiation. This time course is much too long for repair of radiation injury, but could be involved in the repair of BLM induced injury. The increased response during this time could also be due to BLM induced G, block, although an enhanced cell kill by a factor of 2 logs is somewhat large for synchronization effects alone. In addition, when synchronization occurs, the slope of the radiation survival curve is likely to change. The lack of cyclic fluctuations in the timing curve also argues against synchronization. However, both synchronization and radiation interference with the repair of BLM in-
September 1979, Volume 5, Number 9
duced injury remain possible explanations. The choice of how much cell kill to ascribe to BLM, i.e., 4% or 9%, makes interpretation of the D, changes derived from radiation survival curves difficult. The 2-dose studies suggest that BLM is not blocking repair of radiation sublethal damage, but may slow or reduce it in magnitude. Thus most of the shift in the radiation survival curve is probably due to BLM enhanced cell kill, not a change in the radiation survival curve shoulder. The results suggest that combinations of BLM and radiation may be particularly dangerous at certain time intervals for intestinal mucosal damage, but also suggest that this may be a mechanism of enhancing BLM effect on intestinal carcinomas or other tumors, a so far unexplored possibility.
REFERENCES 1. Einhom, L., Krause, M., Hombach, N., Fumas, B.: Enhanced pulmonary toxicity with bleomycin and radiotherapy in oat cell lung cancer. Cancer 37: 24% 2416, 1976. 2. Guigon, M., Frindel, E., Tubiana, M., Hewitt, J.: Effects of the association of chemotherapy and radiotherapy on normal mouse skin. Int. J. Radiat. Onco/. Biol. Phys. 4: 233-238, 1978. 3. Kimler, B. F.: The effect of bleomycin and radiation on G, progression. Int. J. Radiat. Oncol. Biol. Phys. 5: 1523-1526, 1979. 4. Masuda, K., Withers, H. R., Mason, K. A., Chen, K. Y.: Single-dose-response curves of murine gastrointestinal crypt stem cells. Radiat. Res. 69: 65-75, 1977.
5. Meyn, R. E., Carry, P. M., Fletcher, S. E., Demetriades, M.: Thermal enhancement of DNA strand breakage in mammalian cells treated with bleomycin. Int. J. Radiat. Oncol. Biol. Phys. 5: 1487-1489, 1979. 6. Phillips, T. L., Fu, K. K.: Quantification of combined radiation therapy and chemotherapy effects on critical normal tissues. Cancer 37: 11861200, 1976. 7. Phillips, T. L., Wharam, M. D., Margolis, L. W. Modification of radiation injury to normal tissues by chemotherapeutic agents. Cancer 35: 1678-1684, 1975. 8. Withers, H. R., Elkind, M. M.: Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int. J. Radiat. Biol. 17: 261-267, 1970.
Oncology Bid. Phys:, Vol. 5, pp. 150%1512 c Pergamon Press Ltd., 1979. Printed m the U.S.A.
Inr. J. Radiation
??Bleomycin
THE INTERACTION OF RADIATION AND BLEOMYCIN IN INTESTINAL CRYPT CELLS? THEODORE L. PHILLIPS, M.D., GLENDA Y. Ross, B.S., LAWRENCE S. GOLDSTEIN, Ph.D. and ADRIAN C. BEGG, Ph.D. Department of Radiation Oncology, University of California, San Francisco, CA 94143 Using the intestinal crypt cell system, the effects of bleomycin (BLM) with radiation have heen determined.
The LD,,,&,in LAF, mice used was 167 units&g and the MTD (LD,,) was 100 units/kg. Survival of crypt cells was reduced to 40% of radiation alone when drug was given 3 hr after a dose of 1100 rad, but to less than 1% when the drug was given 2 hr before. When maxhnum tolerated doses (MTD) were given from -48 to +48 hr relative to a dose of I343rad, the combination was much more effective when drug was given from -12 to -2 hr before irradiation, survival dropping from 100 to 5 cells per circumference. This increased cell kill could be due to synchrony, interference with repair of BLM damage, or other factors. A radiition survival curve determined 2 hr after drug administration had a slope similar to controls, but was shifted toward lower doses. The extrapolated D, was 0 rad. A 3 hr split dose experiment yielded survival ratios of 6 for controls and 2 for mice given the MTD 2 hr before irradiation. There appears to be a marked interaction of bleomycin and radiation, with possible inhibition of repair of the radiation damage by bleomycin and possible inhibition of repair of the bleomycin damage by radiation. Bleomycin, Intestinal crypt cells, Maximum tolerated dose. INTRODUCTION
mice, age 8-12 weeks. Animals were maintained on laboratory chow with access to water ad libitum on a 12 hr light/dark cycle. One set of split dose experiments was performed with B,AF, female mice obtained from the same source and of similar age.
Bleomycin (BLM) has proven to be a very useful antitumor agent in lymphomas and in certain squamous cell carcinomas. It has appeared to be an enhancer of radiation response in oral and other mucosal sites, as well as in the skin,6 and its method of action appears to be radiomimetic in that it causes single strand DNA breaks.5 It appears to block cell progression in the G, area, and the time of block relative to cell division appears to be dose related.3 There are conflicting reports as to whether it simply adds similar injury to that of radiation injury or may indeed impair repair of radiation injury. These studies were designed to determine whether BLM would be effective in a rapid cell renewal system, the intestinal crypt cell, and whether there would be interaction between radiation and BLM injury as determined by experiments involving varying time intervals between radiation and drug, varying radiation doses, and varying drug doses, as well as split dose radiation studies.
Irradiation
Irradiation was carried out with a 13’Cs selfcontained irradiator at a dose rate of 235 rad/minute. All mice were unanesthetized for irradiation and restrained in rotating circular lucite cages containing adequate ventilation. The stack of cages was located parallel to the linear cesium source, thus the irradiation stack was perpendicular to the radiation beam. TLD measurements in mouse phantoms assured that dose was uniform throughout the mice and at each cage position during the rotation. Dosimetry was compared to standard dose measurements traceable to NBS. Drug
As advised by the manufacturer and the National Cancer Institute, all BLM dosages were determined in units and related to the standards as described on each batch of drug. No great variations in the effec-
METHODS AND MATERIAL Animals
All experiments
were performed
in LAF,
male
*This investigation was supported by National Cancer Institute Research Grants CA17227 and CA 20529. 1509
Radiation Oncology 0 Biology 0 Physics
1510
tiveness of the various drug batches were noted, although some were recently outdated clinical ampules. The BLM was reconstituted with water and given intraperitoneally at a constant volume of 0.01 ml/gm of body weight. Histology and Scoring
Four to six mice were studied in each experimental group, with the exception of the LD,, evaluations in which larger groups of 10-15 mice were employed. Whole body irradiation was used, animals were sacrificed 32/3 days after irradiation by cervical dislocation, and the jejunum was removed, pinned on waxed boards, and fixed in Tellyesniczky’s solution. Twenty-four hours later they were sliced, embedded, and processed for light microscopy. Eight to ten 5 micron-thick cross sections were prepared per mouse, so that a total of approximately 32 sections were available per experimental point. The number of regenerating crypts was scored, as described originally by Withers and Elkind,s and the number of surviving crypt cells calculated. Survival curves were determined by least squares regression calcuiations. The D, was determined by extrapolating the survival curve back to a cell number assumed to be the number of intestinal crypt cells present in the unit-radiated gut. This number has been described as approximately 20,000 by Masuda et al. .4 Because of the somewhat smaller number of cells per circumference (120) in these mice, we have used a figure of 15,000 cells. This is in good agreement with the actual extrapolation of linear survival curves for high LET carbon and neon peak irradiation, where no shoulder is seen on the survival curve. Experimental
September
1979, Volume 5, Number 9
or followed by 3 hr of a BLM dose ranging from 6100% of the MTD. Split dose studies were carried out in both LAF, and B,AF, mice by administering a single or 3 hr split dose of radiation of the same total dose, with or without a preceding dose of 25 units/kg of BLM. The doses were 1000 rad (or 500 x 2) for BLM treated mice and 1200-1260 rad (600-630 x 2) for controls.
RESULTS The drug LD,,, experiments yielded an LD,,J, of 250 units/kg and an LD,,J,,, of 167 units/kg. From the analysis of the LD,,J,,, data, a maximum tolerated dose (LD,,) of 100 unit&g was established. At this dose very few mice in large experiments will die from drug alone. Experiments in which the time interval was varied between a dose of 100 units/kg of BLM and a dose of 910 rad of radiation yielded extremely interesting results, as shown in Fig. 1. Although BLM was somewhat cytotoxic in combination with radiation at all time intervals, there was a marked enhanced degree of cell kill at time intervals from -24 to -2 hr prior to irradiation, with maximum effect at -6 and -2 hr. Small degrees of fluctuation occurred at other times, but not to any marked degree. 1000
BLEOYYCIN,
100 units/Kg
r
I
Protocols
LDSO/S and LDs0/60 values were determined by giving graded doses of drug to groups of 10-15 mice, yielding survival between lO-90% at each of the time intervals. The resultant death data were used to calculate the 50% lethal dose. The influence of time of drug administration relative to time of irradiation was determined by giving a fixed drug dose and fixed radiation dose, separated by intervals of +48 hr. The radiation dose employed in these experiments was 910 t-ad and the BLM dose 100 units/kg. Radiation survival curves were determined by administering radiation doses ranging from 400-1900 rad alone or 2 hr following a BLM dose of either 3 units/kg or 100 units/kg. The survival curves were extrapolated back, as described previously, to determine the D, after correction for cell kill by drug alone. Drug survival curves were determined by administering a radiation dose of 1100 rad, preceded by 2 hr
11”“““““““‘J -48
-36
-24 TIME
-12 OF
0 DRUG
+12 INJECTION.
+24
+3G
+40
HR.
FIG. 1. Crypt cell survival (0) as a function of time between administration of Bleomycin (100 units/kg) and *3TCsgamma rays (910 rad).
Radiation survival curves determined 2 hr after 3 and 100 units/kg of BLM are shown in Fig. 2. Because of the marked sensitivity of cells irradiated 2 hr after BLM administration, the curves are shifted to the left, but the slopes do not change. When
1511
Interaction of radiation and bleomycin 0 T. L. PHILLIPS PI cd.
BLEOMYCIN 0 INJ. 2 HR.
BLEOMYCIN 2 HR. BEFORE
IRRADIATION
0
INJ.
BEFORE IRRADIATION 3 HR. AFTER IRRADIATION
‘3’cs
1
Y
ICONTROL,
II00
25
SO BLEO
BOO
1200 DOSE,
1600
2000
RAD
FIG. 2. Radiation survival curves for intestinal crypt cells exposed to 13’Cs gamma rays alone (0) or 2 hr after 3 units/kg (Cl) or 100 units/kg of Bleomycin.
correction is made for the 99% cell kill caused by BLM, the D, by extrapolation is 300 rad. If one assumes the shift to the left is not due to BLM cell kill and corrects for the 40% cell kill seen when BLM is given after irradiation, the D, is zero. Thus one must know the reason for the curve shift to properly understand the D, changes. The drug dose response curves obtained by administering radiation of 1100 rad preceded or followed by graded drug doses were also of great interest, as shown in Fig. 3. Administration of drug at 3 hr following irradiation showed little effect, with maximum cell kill at the MTD of only 40-50%. However, when the drug was given 2 hr before irradiation, maximum cell kill of over 9% was seen, with survival reduced from 150 surviving cells per circumference to 1 surviving cell per circumference. This marked increased effect with prior drug administration was seen down to doses of 25% of the MTD and indeed (not shown in Fig. 2) down to 6% of the MTD. When BLM is given 3 hr before irradiation, the survival curve is not linear for cell kill by drug, and it would appear that in the first part of the curve the D,, would be about 10 units/kg, but in latter parts would be about 50 units/kg. Because of the marked difference in cell kill when
75
RID
lot
DOSE, units/Kg
FIG. 3. Drug survival curves for intestinal crypt cells with Bleomycin in increasing doses given 2 hr before (0) or 3 hr after (0) 1100 tad of 13’Csgamma rays.
drug is given before irradiation, rather than after, it could be postulated that this is due to elimination of repair of sublethal radiation damage or elimination of the shoulder on the radiation survival curve. Twodose studies do not completely support this. A 3-hr split dose experiment in LAF, mice yielded a survival ratio of 6 for controls and 2 for mice given 25% of the MTD before split dose irradiation. (The ratio of the number of surviving crypt cells after 2 split doses to that after a single dose of the same total.) With B&F, mice the survival ratios were 6 for control mice and 4 for drug treated mice. The single dose cell survival levels were 12 and 5 for control and BLM treated LAF, mice, and were 9 and 1 for the B,AF, mice. DISCUSSION Bleomycin has proven to be an extremely interesting agent in its effect in intestinal crypt cells. It was shown by Guigon et al.* that BLM somewhat enhances the response of skin, but not to a tremendous degree. The BLM dosages employed were somewhat smaller than the maximal doses employed here; but did overlap. Our previous work with esophagus had shown some enhanced response (DEF of 1.1 at BLM doses of 3 units/kg).’ Clinical studies have revealed marked enhancement of radiation effect on the lung by BLM.’ This has not been observed in the intestine clinically.‘j Thus the marked apparent potentiation of injury when radiation and
1512
Radiation Oncology 0 Biology 0 Physics
BLM are combined at certain time intervals is rather remarkable. The timing curves suggest that the time of maximum enhanced response occurs between -12 hr and just before irradiation. This time course is much too long for repair of radiation injury, but could be involved in the repair of BLM induced injury. The increased response during this time could also be due to BLM induced G, block, although an enhanced cell kill by a factor of 2 logs is somewhat large for synchronization effects alone. In addition, when synchronization occurs, the slope of the radiation survival curve is likely to change. The lack of cyclic fluctuations in the timing curve also argues against synchronization. However, both synchronization and radiation interference with the repair of BLM in-
September 1979, Volume 5, Number 9
duced injury remain possible explanations. The choice of how much cell kill to ascribe to BLM, i.e., 4% or 9%, makes interpretation of the D, changes derived from radiation survival curves difficult. The 2-dose studies suggest that BLM is not blocking repair of radiation sublethal damage, but may slow or reduce it in magnitude. Thus most of the shift in the radiation survival curve is probably due to BLM enhanced cell kill, not a change in the radiation survival curve shoulder. The results suggest that combinations of BLM and radiation may be particularly dangerous at certain time intervals for intestinal mucosal damage, but also suggest that this may be a mechanism of enhancing BLM effect on intestinal carcinomas or other tumors, a so far unexplored possibility.
REFERENCES 1. Einhom, L., Krause, M., Hombach, N., Fumas, B.: Enhanced pulmonary toxicity with bleomycin and radiotherapy in oat cell lung cancer. Cancer 37: 24% 2416, 1976. 2. Guigon, M., Frindel, E., Tubiana, M., Hewitt, J.: Effects of the association of chemotherapy and radiotherapy on normal mouse skin. Int. J. Radiat. Onco/. Biol. Phys. 4: 233-238, 1978. 3. Kimler, B. F.: The effect of bleomycin and radiation on G, progression. Int. J. Radiat. Oncol. Biol. Phys. 5: 1523-1526, 1979. 4. Masuda, K., Withers, H. R., Mason, K. A., Chen, K. Y.: Single-dose-response curves of murine gastrointestinal crypt stem cells. Radiat. Res. 69: 65-75, 1977.
5. Meyn, R. E., Carry, P. M., Fletcher, S. E., Demetriades, M.: Thermal enhancement of DNA strand breakage in mammalian cells treated with bleomycin. Int. J. Radiat. Oncol. Biol. Phys. 5: 1487-1489, 1979. 6. Phillips, T. L., Fu, K. K.: Quantification of combined radiation therapy and chemotherapy effects on critical normal tissues. Cancer 37: 11861200, 1976. 7. Phillips, T. L., Wharam, M. D., Margolis, L. W. Modification of radiation injury to normal tissues by chemotherapeutic agents. Cancer 35: 1678-1684, 1975. 8. Withers, H. R., Elkind, M. M.: Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation. Int. J. Radiat. Biol. 17: 261-267, 1970.
