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An explanation for the ability of cytotoxic drug pretreatment to reduce bone marrow related lethality of total body irradiation (TBI)
Inr J. Radiarion Oncology Biol. Phys.. Vol. Prmted in the U.S.A. All rights reserved.
036&3016/82/03058l43$03.OQ~0 Copyright 0 1982 Pergamon Press Ltd
8. pp. S-583
0 Session III-Protectors
With Radiation
AN EXPLANATION FOR THE ABILITY OF CYTOTOXIC DRUG PRETREA’TMENT TO REDUCE BONE MARROW RELATED LETHALITY OF TOTAL BODY IRRADIATION (TBI) J.L. MILLAR,
PH.D.,
T.C.
STEPHENS,
PH.D.
Institute of Cancer Research, Sutton, Surrey SM2 5PX, England and E.A.WIST, The Norwegian
Radium
M.D.,
Hospital,
PH.D.
Montebello,
Oslo 3, Norway
Mice given 9 to ‘IOGy total body irradiation (TBI) die a hematological death 10 to 14 days after exposure. This lethality can be avoided by pretreatment with a cytotoxic drug two days before irradiation. The best example of this is seen when 200 mg/Kg cytosine arabinoside (ara-C) is given two days before TBI. Improved survival results from an earlier onset in the recovery of marrow stem cells (CFU-s) in animals given ara-C before irradiation as compared to controls. In animals given radiation alone there is a lag phase in the recovery of CFU-s; drug pretreatment before irradiation abolishes this delay. We postulate that the cells that repopulate the CFU-s compartment after irradiation are a sub-population of the CFU-s with higher self-renewal capability, lower proliferative activity and higher radiosensitivity (I),, = .8 Gy) than the overall population (D, = 1.1 Cy). Further, we suggest that drug pretreatment alters the radioselnsitivity of the first population, increasing it temporarily to that of the overall population. This may come about by ara-C triggering these CFU-s into a relatively radioresistant phase of the cell cycle. In the Lewis lung tumor ara-C pretreatment does not affect the response to radiation, even at times when the drug promotes the early recovery of the CIFU-s. It would therefore seem that a potentially useful gain in the therapeutic index may result from these findings. CFU-s, Cytotoxic drug, TBI, Protection, Lewis lung tumor, Therapeutic index.
INTRODUCTION A wide variety of compounds, some of which are themselves cytotoxic, can protect mice from an otherwise lethal dose of ionizing radiatica.5 The mechanism for this effect is not yet understood but it is known that it is not like the protection afforded by sulphydryl compounds.3,4 It has been postulateId that the bone marrow stem cell compartment is not uniform but has a heterogeneity in terms of self-renewal ca.pabi1ity.7,8 It has been postulated further that the most ‘.ancestral’ cells have the greatest self-renewal7.8 and lowest proliferative activity.’ In this paper we use this mode1 to propose a mechanism for the enhanced recovery of bone marrow stem cells after pretreatment with cytotoxil: agent and total body irradiation. An experiment using the Lewis lung carcinoma and drug/radiation modalities which spare the bone marrow is also described. METHODS,
and 26 g were used in toxicity studies and as bone marrow donors. Female mice, weighing between 20-25 g were heavily irradiated (9 Gy) and used as bone marrow recipients for the spleen colony assay. C57Bl mice were used in studies with the Lewis lung tumor. Bone marrow stem cells
The number of bone marrow stem cells (CFU-s) assessed using the method of Till and McCulloch.’
was
irradiation
All irradiations were carried out using 6oCo y radiation at the dose rate of .55 Gy/min. Drug
Cytosine arabinoside* (ara-C) and administered intraperitoneally.
AND MATERIALS
was dissolved
in saline
Animals
Tumor
Eight to 10 week old CBA/lac mice were used for normal tissue studies. Male mice, weighing between 22
The Lewis lung tumor was inoculated intramuscularly as a brei into the gastrocnemius muscle, and tumor
Accepted for publicatioln 5 November I98 I. Acknowledgements-The authors wish to thank Mr. John Peacock for his help with this study. The work was supported by a
grant from the Medical Research Council of Great Britain. E.W. was a Research Fellow of the Norwegian Cancer Society. *Cytosar (Upjohn, Ltd.). 581
measurement
Radiation
582
%
Oncology
??Biology 0 Physics
1982, Volume 8, No. 3 and No. 4
March-April
normal
+ differentlate self -renewal
low self -renewal Fig. 2. Two compartment
0
5
IO
days Fig. I. Recovery of CFU-s after 9 Gy (0) or 200 mg/Kg ara-C two days before 9 Gy (M). Data reproduced from reference 3 by kind permission of the editor of Cell and Tissue Kinet. Hypothetical decline of more radioresistant, low self-renewal population --------, hypothetical repopulation from more radiosensitive population with high self-renewal -.-.-.-.-.
population.
model of the CFU-s
compartment is divided into two subsets differing in radiosensitivity and self-renewal capacity. After a high dose of radiation (e.g. 9 Gy), these subsets are depleted disproportionately, only 4% of the surviving CFU-s have a high self-renewal capacity and 96% have low self-renewal capability. This leads to a decline in the overall number of CFU-s because of loss through differentiation of the CFU-s subset with low self-renewal capability before the CFU-s with high self-renewal can repopulate the compartment. In Figure 1 these processes are represented by the broken lines and the sum of these gives the number of measurable CFU-s at any particular time. This would explain the post-irradiation dip in marrow stem cells in animals receiving radiation alone. If, however, ara-C is given two days before irradiation and the D, of the more radiosensitive CFU-s increased to around 1.1 Gy then both populations would be reduced in proportion and recovery of the CFU-s population would begin promptly (Figure I). The surviving fraction of the CFU-s population after dose of radiation D given by: s.f. = .55 em”‘* + .45 emDi’.‘3 in normal
mice
and by: growth was monitored by fitting the leg through standard apertures. Tumor mass, at various times during the course of the experiment, was established from a calibration curve. RESULTS
AND DISCUSSION
After a dose of radiation the bone marrow stem cells do not recover promptly, but may even show a post-irradiawith a cytotoxic agent tion dip in CFU-s.*.’ Treatment such as ara-C two days before irradiation removes this lag and CFU-s recovery begins promptly (Figure 1). This, in kinetic terms, is how pretreatment with various cytotoxic agents improves animal survival after irradiation, as the post-irradiation lag phase in the bone marrow stem cells is pronounced at high doses and become life-threatening. We now propose a mechanism to explain the postirradiation lag phase and how pretreatment with certain cytotoxic agents over-rides it. In Figure 2, the CFU-s
s.f. = .5 emD”,” in ara-C pretreated
mice
(where ara-C itself reduces the population by 50%) fits the existing data well. For instance, after 9 Gy the first equation predicts a surviving fraction of 1.6 1 x 1Om4and the second a surviving fraction of 1.74 x 10e4; this is in good agreement with the day 0 values in Figure 1. Table I. The effect of a second dose of ara-C (200 mg/kg) at the time of irradiation on four day recovery of CFU-S CFU-S % normal Treatment
Exp I
Exp II
ara-C 2d 9 Gy 9 Gy alone ara-C 2d ara-C + 9 Gy ara-C + 9 Gy
.23 .Ol5 .0.5 .Ol3
.I0 .008 .029 0
Drug pretreatment
reduces lethality after
Tumour wt Ig)
, .
’ 12
6
4’
10
11
days Fig. 3. Growth delay of Lewis lung tumor grown intramuscularly. Control (0) 25 Gy alone (0) ara-C 96 hours before 25 Gy (x), ara-C 48 hours before 25 Gy (A), ara-C I2 hours before 25 Gy (m). ara-C given at the same time as 25 Gy (A).
TBI 0 J.L.
MILLAR et al.
583
If this is indeed the explanation, we do not know how this shift in D, comes about. The reduced lethality in mice is transient in nature, for if the ara-C is given more than 72 hours before TBI the lethality returns to control levels.’ It is possible that ara-C induces a time dependent change in the sensitive CFU-s population, perhaps by triggering them into S-phase (the period of DNA synthesis). If CFU-s are more radioresistant in S-phase, then a transient shift in D, from .8 Gy to 1 .l Gy is possible. This in turn should mean that they are susceptible to an S-phase active agent at the time of irradiation. Using ara-C itself, given at the time of irradiation, a four-fold reduction in the recovery of CFU-s was achieved (Table l), which is compatible with this hypothesis. In an experiment involving the Lewis lung carcinoma, 200 mg/Kg ara-C was given at time intervals varying from 96 to 0 hours before 25 Gy with tumor growth delay as the end-point (Figure 3). At no time interval did ara-C appear to protect the tumor, even though one of the time intervals was 48 hours, a time interval between drug and TBI maximally sparing to bone marrow. A gain in the therapeutic index may therefore be achieved under these circumstances. Further studies with other tumors are necessary to establish the proportion of tumors which respond in this way, because the pretreatment phenomenon in normal tissues appears to be widespread and may apply to man.6
REFERENCES 1. Botnick, L.E., Hannon, E.C., Hellman, S.: Nature of the hemopoietic stem cell compartment and its proliferative potential. Blood Cells S: 195-210, 1979. 2. Guzman, E., Lajtha, L..G.: Some comparisons of the kinetic properties of femoral and splenic haemopoietic stem cells. Cell Tissue Kinet. 3: 9 l-98, 1970. 3. Millar, J.L.: Mechanisms of radioprotection by L-cysteine or cytosine arabinoside in mouse marrow. (Abstr.) Br.J. Radiol. 52: 426, 1979. 4. Millar, J.L.: Recovery of irradiated mouse bone marrow can be enhanced by drug pretreatment. Applied Radiol. 10: 97-101,198l. 5. Millar, J.L., Blackett, N.M., Hudspith, B.N.: Enhanced post-irradiation recovery of the haemopoietic system in ani-
mals pretreated
with a variety of cytotoxic agents. Cell
Tissue Kinet. 11: 543-553,
1978.
6. Millar, J.L., Smith, I.E., McElwain, T.J.: Pretreatment with certain cytotoxic drugs reduces the normal tissue toxicity of anti-cancer agents in mice and man. Br.J. Cancer 43: 705 706,1981.
7. Rosendaal, M., Hodgson, G.S., Bradley, T.R.: Organization of the haemopoietic stem cells: The generation-age hypothesis. Cell Tissue Kinet. 12: 17-29, 1919. 8. Schofield, R., Lajtha, L.G.: Effect of isopropyl methane sulphonate (IMS) on haemopoietic colony-forming cells. Br.J. Haemat. 25: 195-202, 1973.
9. Till, J.E., McCulloch, E.A.: A direct measurement of the radiation sensitivity of normal mouse bone marrow. Radiat. Res. 14: 213-222,
1961.
036&3016/82/03058l43$03.OQ~0 Copyright 0 1982 Pergamon Press Ltd
8. pp. S-583
0 Session III-Protectors
With Radiation
AN EXPLANATION FOR THE ABILITY OF CYTOTOXIC DRUG PRETREA’TMENT TO REDUCE BONE MARROW RELATED LETHALITY OF TOTAL BODY IRRADIATION (TBI) J.L. MILLAR,
PH.D.,
T.C.
STEPHENS,
PH.D.
Institute of Cancer Research, Sutton, Surrey SM2 5PX, England and E.A.WIST, The Norwegian
Radium
M.D.,
Hospital,
PH.D.
Montebello,
Oslo 3, Norway
Mice given 9 to ‘IOGy total body irradiation (TBI) die a hematological death 10 to 14 days after exposure. This lethality can be avoided by pretreatment with a cytotoxic drug two days before irradiation. The best example of this is seen when 200 mg/Kg cytosine arabinoside (ara-C) is given two days before TBI. Improved survival results from an earlier onset in the recovery of marrow stem cells (CFU-s) in animals given ara-C before irradiation as compared to controls. In animals given radiation alone there is a lag phase in the recovery of CFU-s; drug pretreatment before irradiation abolishes this delay. We postulate that the cells that repopulate the CFU-s compartment after irradiation are a sub-population of the CFU-s with higher self-renewal capability, lower proliferative activity and higher radiosensitivity (I),, = .8 Gy) than the overall population (D, = 1.1 Cy). Further, we suggest that drug pretreatment alters the radioselnsitivity of the first population, increasing it temporarily to that of the overall population. This may come about by ara-C triggering these CFU-s into a relatively radioresistant phase of the cell cycle. In the Lewis lung tumor ara-C pretreatment does not affect the response to radiation, even at times when the drug promotes the early recovery of the CIFU-s. It would therefore seem that a potentially useful gain in the therapeutic index may result from these findings. CFU-s, Cytotoxic drug, TBI, Protection, Lewis lung tumor, Therapeutic index.
INTRODUCTION A wide variety of compounds, some of which are themselves cytotoxic, can protect mice from an otherwise lethal dose of ionizing radiatica.5 The mechanism for this effect is not yet understood but it is known that it is not like the protection afforded by sulphydryl compounds.3,4 It has been postulateId that the bone marrow stem cell compartment is not uniform but has a heterogeneity in terms of self-renewal ca.pabi1ity.7,8 It has been postulated further that the most ‘.ancestral’ cells have the greatest self-renewal7.8 and lowest proliferative activity.’ In this paper we use this mode1 to propose a mechanism for the enhanced recovery of bone marrow stem cells after pretreatment with cytotoxil: agent and total body irradiation. An experiment using the Lewis lung carcinoma and drug/radiation modalities which spare the bone marrow is also described. METHODS,
and 26 g were used in toxicity studies and as bone marrow donors. Female mice, weighing between 20-25 g were heavily irradiated (9 Gy) and used as bone marrow recipients for the spleen colony assay. C57Bl mice were used in studies with the Lewis lung tumor. Bone marrow stem cells
The number of bone marrow stem cells (CFU-s) assessed using the method of Till and McCulloch.’
was
irradiation
All irradiations were carried out using 6oCo y radiation at the dose rate of .55 Gy/min. Drug
Cytosine arabinoside* (ara-C) and administered intraperitoneally.
AND MATERIALS
was dissolved
in saline
Animals
Tumor
Eight to 10 week old CBA/lac mice were used for normal tissue studies. Male mice, weighing between 22
The Lewis lung tumor was inoculated intramuscularly as a brei into the gastrocnemius muscle, and tumor
Accepted for publicatioln 5 November I98 I. Acknowledgements-The authors wish to thank Mr. John Peacock for his help with this study. The work was supported by a
grant from the Medical Research Council of Great Britain. E.W. was a Research Fellow of the Norwegian Cancer Society. *Cytosar (Upjohn, Ltd.). 581
measurement
Radiation
582
%
Oncology
??Biology 0 Physics
1982, Volume 8, No. 3 and No. 4
March-April
normal
+ differentlate self -renewal
low self -renewal Fig. 2. Two compartment
0
5
IO
days Fig. I. Recovery of CFU-s after 9 Gy (0) or 200 mg/Kg ara-C two days before 9 Gy (M). Data reproduced from reference 3 by kind permission of the editor of Cell and Tissue Kinet. Hypothetical decline of more radioresistant, low self-renewal population --------, hypothetical repopulation from more radiosensitive population with high self-renewal -.-.-.-.-.
population.
model of the CFU-s
compartment is divided into two subsets differing in radiosensitivity and self-renewal capacity. After a high dose of radiation (e.g. 9 Gy), these subsets are depleted disproportionately, only 4% of the surviving CFU-s have a high self-renewal capacity and 96% have low self-renewal capability. This leads to a decline in the overall number of CFU-s because of loss through differentiation of the CFU-s subset with low self-renewal capability before the CFU-s with high self-renewal can repopulate the compartment. In Figure 1 these processes are represented by the broken lines and the sum of these gives the number of measurable CFU-s at any particular time. This would explain the post-irradiation dip in marrow stem cells in animals receiving radiation alone. If, however, ara-C is given two days before irradiation and the D, of the more radiosensitive CFU-s increased to around 1.1 Gy then both populations would be reduced in proportion and recovery of the CFU-s population would begin promptly (Figure I). The surviving fraction of the CFU-s population after dose of radiation D given by: s.f. = .55 em”‘* + .45 emDi’.‘3 in normal
mice
and by: growth was monitored by fitting the leg through standard apertures. Tumor mass, at various times during the course of the experiment, was established from a calibration curve. RESULTS
AND DISCUSSION
After a dose of radiation the bone marrow stem cells do not recover promptly, but may even show a post-irradiawith a cytotoxic agent tion dip in CFU-s.*.’ Treatment such as ara-C two days before irradiation removes this lag and CFU-s recovery begins promptly (Figure 1). This, in kinetic terms, is how pretreatment with various cytotoxic agents improves animal survival after irradiation, as the post-irradiation lag phase in the bone marrow stem cells is pronounced at high doses and become life-threatening. We now propose a mechanism to explain the postirradiation lag phase and how pretreatment with certain cytotoxic agents over-rides it. In Figure 2, the CFU-s
s.f. = .5 emD”,” in ara-C pretreated
mice
(where ara-C itself reduces the population by 50%) fits the existing data well. For instance, after 9 Gy the first equation predicts a surviving fraction of 1.6 1 x 1Om4and the second a surviving fraction of 1.74 x 10e4; this is in good agreement with the day 0 values in Figure 1. Table I. The effect of a second dose of ara-C (200 mg/kg) at the time of irradiation on four day recovery of CFU-S CFU-S % normal Treatment
Exp I
Exp II
ara-C 2d 9 Gy 9 Gy alone ara-C 2d ara-C + 9 Gy ara-C + 9 Gy
.23 .Ol5 .0.5 .Ol3
.I0 .008 .029 0
Drug pretreatment
reduces lethality after
Tumour wt Ig)
, .
’ 12
6
4’
10
11
days Fig. 3. Growth delay of Lewis lung tumor grown intramuscularly. Control (0) 25 Gy alone (0) ara-C 96 hours before 25 Gy (x), ara-C 48 hours before 25 Gy (A), ara-C I2 hours before 25 Gy (m). ara-C given at the same time as 25 Gy (A).
TBI 0 J.L.
MILLAR et al.
583
If this is indeed the explanation, we do not know how this shift in D, comes about. The reduced lethality in mice is transient in nature, for if the ara-C is given more than 72 hours before TBI the lethality returns to control levels.’ It is possible that ara-C induces a time dependent change in the sensitive CFU-s population, perhaps by triggering them into S-phase (the period of DNA synthesis). If CFU-s are more radioresistant in S-phase, then a transient shift in D, from .8 Gy to 1 .l Gy is possible. This in turn should mean that they are susceptible to an S-phase active agent at the time of irradiation. Using ara-C itself, given at the time of irradiation, a four-fold reduction in the recovery of CFU-s was achieved (Table l), which is compatible with this hypothesis. In an experiment involving the Lewis lung carcinoma, 200 mg/Kg ara-C was given at time intervals varying from 96 to 0 hours before 25 Gy with tumor growth delay as the end-point (Figure 3). At no time interval did ara-C appear to protect the tumor, even though one of the time intervals was 48 hours, a time interval between drug and TBI maximally sparing to bone marrow. A gain in the therapeutic index may therefore be achieved under these circumstances. Further studies with other tumors are necessary to establish the proportion of tumors which respond in this way, because the pretreatment phenomenon in normal tissues appears to be widespread and may apply to man.6
REFERENCES 1. Botnick, L.E., Hannon, E.C., Hellman, S.: Nature of the hemopoietic stem cell compartment and its proliferative potential. Blood Cells S: 195-210, 1979. 2. Guzman, E., Lajtha, L..G.: Some comparisons of the kinetic properties of femoral and splenic haemopoietic stem cells. Cell Tissue Kinet. 3: 9 l-98, 1970. 3. Millar, J.L.: Mechanisms of radioprotection by L-cysteine or cytosine arabinoside in mouse marrow. (Abstr.) Br.J. Radiol. 52: 426, 1979. 4. Millar, J.L.: Recovery of irradiated mouse bone marrow can be enhanced by drug pretreatment. Applied Radiol. 10: 97-101,198l. 5. Millar, J.L., Blackett, N.M., Hudspith, B.N.: Enhanced post-irradiation recovery of the haemopoietic system in ani-
mals pretreated
with a variety of cytotoxic agents. Cell
Tissue Kinet. 11: 543-553,
1978.
6. Millar, J.L., Smith, I.E., McElwain, T.J.: Pretreatment with certain cytotoxic drugs reduces the normal tissue toxicity of anti-cancer agents in mice and man. Br.J. Cancer 43: 705 706,1981.
7. Rosendaal, M., Hodgson, G.S., Bradley, T.R.: Organization of the haemopoietic stem cells: The generation-age hypothesis. Cell Tissue Kinet. 12: 17-29, 1919. 8. Schofield, R., Lajtha, L.G.: Effect of isopropyl methane sulphonate (IMS) on haemopoietic colony-forming cells. Br.J. Haemat. 25: 195-202, 1973.
9. Till, J.E., McCulloch, E.A.: A direct measurement of the radiation sensitivity of normal mouse bone marrow. Radiat. Res. 14: 213-222,
1961.