Screening of Cytotoxic Drugs for Residual Bone Marrow Damage 1,2 K. J. Trainor 3 and A. A. Morley3,4,5 ABSTRACT-Several cytotoxic drugs were tested for their ability to produce permanent residual damage to the bone marrow. A short course of the drug was given to BALB/c female mice, and the numbers of various types of bone marrow cells were deter· mined at least two months later. Evidence of residual damage was found after administration of busulfan and 1,3· bis(chloroethyl)·1·nitrosourea, but not after administration of cyclophosphamide, 5-fluorouracil, 6-mercaptopurine, methotrexate, or vinblastine.-J Natl Cancer Inst 57: 1237-1239, 1976.
The most commonly accepted model of the physiologic basis of action of cytotoxic drugs is that proposed by Skipper and co-workers (1, 2) who postulated that administration of a cytotoxic drug kills a constant fraction of normal and tumor cells and that, following cessation of drug administration, the surviving cells sustain no permanent damage and recommence proliferation at their previous rate. According to this model, any differential effect of the drug on normal and neoplastic cells is attributed to a difference in cell sensitivity due either to inherent characteristics or difference in kinetic state or to a greater ability of normal cells to increase their rate of proliferation and thus restore their number following depletion. Though the Skipper model is widely accepted, several observers have suggested that cytotoxic drugs may occasionally cause permanent alteration to the proliferative rate in tumor cells (3 -8). However, we know of no comparable studies on normal cells. Recently we found that the cytotoxic drug B U, in addition to having a direct lethal effect on normal bone marrow cells, produces a form of residual injury (9). This form of injury appears to persist indefinitely and is characterized by mild or moderate marrow failure that may terminate in complete aplasia. The principal factor in the genesis of bone marrow failure is permanent impairment of proliferation and differentiation of the stem cell, which may lead to complete stem cell failure (10, 11). Since bone marrow failure has not been a clearly recognized side effect of cytotoxic drugs and since it has both theoretical and practical implications, we screened some agents other than B U for their ability to cause similar injury. MATERIALS AND METHODS
Mouse cells. - Virgin female BALB/c mice, 10-16 weeks of age, were obtained from the Institute of Medical and Veterinary Science, Adelaide, Australia. Groups of mice were inoculated ip at various intervals for 8 weeks with the drug being tested; uninoculated mice served as controls. At least two months after cessation of drug administration, groups of 4 or 5 treated and control mice were killed. Nucleated cell counts were performed on one tibia from each animal, and bone marrow cells from the other tibia were pooled with those from mice in the same group for assay of granulocytic progenitor cells (CFC) and of pi uri potential stem cells (CFU), per tibia, as previously described (10). In several VOL. 57, No.6, DECEMBER 1976 Downloaded from https://academic.oup.com/jnci/article-abstract/57/6/1237/1034216 by Insead user on 08 March 2018
1237
experiments, the ability of CFU in the spleen to form further CFU and CFC was measured by retransplantation, as described (10). Drugs. - For drugs other than B U a pilot study was done to determine the approximate LD50 of a single ip dose and the duration of bone marrow suppression as assessed by tibial cell counts. For the formal experiment to test the drug, mice were then randomized into 3 groups: 1) One group served as the control. 2) The high dose group was treated ip with approximately one-third of LD50 at regular intervals for 8-10 weeks. The dose of the drug was progressively increased or decreased so that, by the end of treatment, approximately one-third of the mice in the group had died from drug overdose. 3) The low dose group received approximately twothirds of the dose given on the same day to the high dose group. Two groups were used so that the dose of the drug in the high dose group could be increased to lethal levels while survivors remained in the low dose group. Administration of BU to 2 groups of mice was not necessary, since the appropriate dose had been determined previously. The drugs tested, the methods of administration, and the doses given were as follows. BU: Ten or 20 mg was dissolved in 1.5 ml acetone; sterile water was added to make 10 ml solution, and 0.01 mllg was injected ip. Doses of20, 20, 20, and 10 mg BU/ kg were given at intervals of 2 weeks. BCNU: Thirty mg powder was dissolved in 0.9 ml ethanol; sterile water was added to make 10 ml solution; and 0.01 mllg was injected. Doses of 30 mg BCNU/kg were given at intervals of 2 weeks. Mortality was 50 %. 6-MP: Seventy to 120 mg powder was dissolved in 1 ml dimethyl sulfoxide, and sterile water was added to make 10 ml solution. Injection of 0.01 mg/g of the resulting suspension provided weekly doses of 70-120 mg 6-MP/ kg. Mortality was 62%. CY: The drug was appropriately diluted in sterile water and injected weekly in a dose of 60-160 mg/kg. Mortality was 24 %. 5-FU: The drug was appropriately diluted in sterile ABBREVIATIONS USED: BU = busulfan; CFC = colony-forming celI(s); CFU = colony-forming unit(s); LD50 = mean lethal dose' BCNU = 1,3-bis(chloroethyl)-I-nitrosourea; 6-MP = 6-mercaptopu:ine; CY = cyclophosphamide; 5-FU = 5-fluorouracil; MTX = methotrexate; VBL = vinblastine. Received November 11, 1975; accepted April 27, 1976. Supported by the Michell and Anti-Cancer Foundation of the University of Adelaide and by the National Health and Medical Research Council of Australia. 3 Department of Medicine, University of Adelaide, Adelaide, Australia. 4 Present address: Department of Haematology, Flinders Medical Centre, Bedford Park, South Australia 5042. 5 We thank Mrs. J. Remes and Mrs. R. Lloyd for excellent technical assistance and Dr. N. D. Harvey for help with the radiation facilities used. 1
2
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TRAINOR AND MORLEY
water and injected weekly in a dose of60 mg/kg. Mortality was 35%. MTX: The drug was appropriately diluted in sterile water and injected twice weekly in a dose of 5-10 mg/kg. Mortality was 32 %. VBL: The drug was appropriately diluted in sterile normal saline and injected twice weekly in a dose of 0.91.5 mg/kg. Mortality was 55 %. RESULTS
At the times of final analyses, the number of mice surviving from all high dose groups was sufficient to enable all assays to be done on these groups; assays were not performed on the low dose groups. All assays were done at least 2 months after cessation of drug administration, and each assay involved killing a group of 4 or 5 treated and control mice. The absolute numbers of nucleated cells were determined (text-fig. 1). The numbers of CFC (granulocytic progenitor cells) and CFU (pluripotential stem cells) were expressed as percentages of the controls, and the standard errors of the percentages were calculated (12, 13). The results for CFC are shown in text-figure 2 and for CFU in text-figure 3. The assays measuring selfrenewal and differentiation ability of CFU (text-fig. 4) were based on the principle that a single CFU gives rise to a spleen colony which contains further CFU and CFC. The number of CFU per spleen colony was therefore an index of the ability of the original CFU to renew themselves; the number of CFC spleen colony was an index of the ability of the original CFU to differentiate. The ratio of control/treated CFU/spleen colony compared the ability of control and treated CFU to renew themselves; the ratio of the control/treated CFC/spleen colony compared ability of control and treated CFU to differentiate. High ratios suggested impairment of selfrenewal and/or impairment of differentiation; ratios of approximately 1 suggested no impairment. The assays used in this study are not particularly precise; measurement of nucleated cells per tibia was probably the most accurate, and assay of splenic self-
BU
BCNU
CY
5-FU
--- -J-'-
100 ---
!
I-ir
! !i
e1:
6-MP
!
- - - -!--
BCNU
CY
5-FU
6-MP VBL
!
!
MTX
-!,
!
0
()
"'6 o-!?
!
I
10
(.) lJ.. (.)
TEXT-FIGURE 2.-CFC/tibia (mean ±SE) in mice treated with various cytotoxic drugs. Each point refers to a CFC assay on a bone marrow suspension prepared with pooled cells from tibia of 4 or 5 mice. BU
BCNU
100 --- ---
e1:
I
!
0
t!
CY
5-FU
_L!_
Jrt-
6-MP VBL
-r
f-
MTX
I-!-- ll_ f
!
!
()
"'6
~
10
::J
lJ.. (.)
TEXT-FIGURE 3.-CFU/tibia (mean ±SE) in mice treated with various cytotoxic drugs. Each point refers to a CFU assay on a marrow suspension prepared with pooled cells from tibias of 4 or 5 mice. 20
au
aCNU
CY
5-FU
6-MP VBL
MTX
~
10
•
8 •
BU
VBL
MTX CONTROL TREATED 1
•
--
0
8
• CFU o CFC
------r-•
0
•
x
«
iIi i=
~ ...J
5
f
UJ
(.)
TEXT-FIGURE 1. - Nucleated cells/tibia in mice treated with various cytotoxic drugs. The results for B U are the mean of 17 assays, but all other results show the mean ±SE of individual assays. The normal range (mean ±SE) is indicated by dotted area. Each individual assay involved killing 4 or 5 mice and counting individual tibial cells.
J
NATL CANCER INST
Downloaded from https://academic.oup.com/jnci/article-abstract/57/6/1237/1034216 by Insead user on 08 March 2018
110 ~o,~--~--~--~--~--~--J-~
TEXT-FIGURE 4. - Ratios of control to treated mice in the number of CFU/spleen colony or the number of CFC/spleen colony.
renewal was probably the least precise. Therefore, we set subnormal values as the criterion for residual injury in mice following exposure to drugs, although we recognized that this might result in our overlooking the lesser degrees of residual damage. No evidence was found for the presence of residual injury following treatment with 6-MP, CY, 5-FU, MTX, or VBL; evidence was found for residual injury followVOL. 57, NO.6, DECEMBER 1976
SCREENING CYTOTOXIC DRUGS
ing treatment with BCNU and BU. Following administration of BCNU, the number of nucleated bone marrow cells, CFC, and CFU and the ability of CFU to selfrenew and to produce CFC decreased persistently. DISCUSSION
The two drugs found to produce residual marrow injury were B U and BCNU. In contrast with the other drugs tested, both BU and BCNU seem to exert prolonged and severe toxic effects on the bone marrow stem cell compartment (14, 15). Since this is the compartment principally involved in residual bone marrow injury, drugs having this type of effect on the stem cell compartment may be those which are particularly prone to cause residual injury. That B U and BCNU are both bifunctional alkylating agents might suggest that crosslinking of DNA was the biochemical mechanism involved. Yet residual damage was not detected when CY, which may act similarly, was used. Whether CY causes residual impairment of proliferation of tumor cells is disputed (2, 4); if it does, failure to find an effect on normal marrow cells may be due to a differential effect of cyclophosphamide on normal marrow and tumor cells and/or the inherent imprecision of the assays used in the present study. From the functional point of view, residual bone marrow damage after administration of B U and BCNU can be regarded as a permanent impairment of the proliferation and differentiation abilities of marrow cells, which results in permanent decrease in their number. This effect of certain cytotoxic drugs on normal marrow cells had not been clearly defined previously, and little evidence exists as to whether cells other than those of the marrow sustain similar damage. In man, chronic hypoplastic marrow failure (aplastic anemia) probably is a manifestation of residual damage, and widespread disturbances in immunity are known to occur in the disease (16, 17). This would suggest that cells of the immune system may also sustain residual damage, and preliminary results from our laboratory support that possibility. Rational use of cytotoxic drugs requires an understanding of their toxic effects on normal and on neoplastic cells. The recognition of residual injury to either cell type could therefore be of potential importance in leading one to a better understanding of the mode of action of cytotoxic drugs and thus to their more effective use.
VOL. 57, NO.6, DECEMBER 1976 Downloaded from https://academic.oup.com/jnci/article-abstract/57/6/1237/1034216 by Insead user on 08 March 2018
1239
REFERENCES (1) SKIPPER HE, SCHABEL FM jR, WILCOX WS: Experimental evaluation of potential anticam:er agents. XIII. On the criteria and kinetics associated with "curability" of experimental leukemia. Cancer Chemother Rep 35:3-111, 1964 (2) WILCOX WS, SCHABEL FM jR, SKIPPER HE: Experimental evaluation of potential anticancer agents. X V. On the relative rates of growth and host kill of "single" leukemia cells that survive in vivo Cytoxan therapy. Cancer Res 26:1009-1014,1966 (3) JOHNSON RE, ZELEN M., KEMP NH: Chemotherapeutic effects on mammalian tumor cells. I. Modification of leukemia L1210 growth kinetics and karyotype with an alkylating agent. j Nat! Cancer 1nst 34:277-285, 1965 (4) JOHNSON RE, HARDY WG: Chemotherapeutic effects on mammalian tumor cells. II. Biologic and chromosomal instability of a cyclophosphamide treated murine leukemia. Cancer Res 25:604-608, 1965 (5) JOHNSON RE, HARDY WG, ZELEN M: Chemotherapeutic effects on mammalian tumor cells. III. Modification of leukemia Ll210 growth kinetics with an antimetabolite. j Nat! Cancer Inst 36:15-20, 1966 (6) HUMPHREYS SR, GOLDIN A: Investigation of tumor variants recovered from mice with systemic leukemia (LI210) after extensive therapy with 3' ,5' -dichloroamethopterin and 3' -bromo-5'chloroamethopterin. j Nat! Cancer Inst 23:633-653, 1959 (7) DE VITA VT, BRAY DA, BOSTICK F, et al: The effect of chemotherapy on the growth of leukemia L121O. II. Persistence of a nitrosourea induced change in the growth characteristics of transplant generations. Cell Tissue Kinet 5:459-466, 1972 (8) SCHMID FA, HUTCHINSON Dj: Chemotherapeutic carcinogenic and cell-regulatory effects of triazines. Cancer Res 34:16711675, 1974 (9) MORLEY A, BLAKEj: An animal model of chronic aplastic marrow failure. I. Late marrow failure after busulfan. Blood 44:49-56, 1974 (10) MORLEY A, TRAINOR K, BLAKE,]: A primary stem cell lesion in experimental chronic hypoplastic marrow failure. Blood 45:681-688, 1975 (11) MORLEY A, BLAKE]: Haemopoietic precursor cells in experimental hypoplastic marrow failure. Austj Exp Bioi Med Sci 52:909914, 1974 (12) MORLEY A, TRAINOR K, REMES ]: Residual marrow damage: Possible explanation for idiosyncrasy to chloramphenicol. Br] Haematol. In press (13) DAVIES PL: Statistical Methods in Research and Production, 3d ed. London, Oliver and Boyd, 1961, P 41 (14) DUNN CD, ELSON LA: The comparative effect of busulfan (myleran) and aminochlorambucil on haemopoietic colony forming units in the rat. Cell Tissue Kinet 3:131-141,1970 (15) REISSMAN KR, UDUPA KB, KAWODA K: Effects of erythropoietin and androgens on erythroid stem cells after their selective suppression by BCNU. Blood 44:649-657,1974 (16) MORLEY A, FORBES I: Impairment of immunological function in aplastic anaemia. Aust N Z] Med 4:53-57, 1974 (17) MORLEY A, HOLMES K, FORBES I: Depletion of B lymphocytes in chronic hypoplastic marrow failure (aplastic anaemia). Aust N Zj Med 4:538-541,1974
J NATL CANCER INST
The most commonly accepted model of the physiologic basis of action of cytotoxic drugs is that proposed by Skipper and co-workers (1, 2) who postulated that administration of a cytotoxic drug kills a constant fraction of normal and tumor cells and that, following cessation of drug administration, the surviving cells sustain no permanent damage and recommence proliferation at their previous rate. According to this model, any differential effect of the drug on normal and neoplastic cells is attributed to a difference in cell sensitivity due either to inherent characteristics or difference in kinetic state or to a greater ability of normal cells to increase their rate of proliferation and thus restore their number following depletion. Though the Skipper model is widely accepted, several observers have suggested that cytotoxic drugs may occasionally cause permanent alteration to the proliferative rate in tumor cells (3 -8). However, we know of no comparable studies on normal cells. Recently we found that the cytotoxic drug B U, in addition to having a direct lethal effect on normal bone marrow cells, produces a form of residual injury (9). This form of injury appears to persist indefinitely and is characterized by mild or moderate marrow failure that may terminate in complete aplasia. The principal factor in the genesis of bone marrow failure is permanent impairment of proliferation and differentiation of the stem cell, which may lead to complete stem cell failure (10, 11). Since bone marrow failure has not been a clearly recognized side effect of cytotoxic drugs and since it has both theoretical and practical implications, we screened some agents other than B U for their ability to cause similar injury. MATERIALS AND METHODS
Mouse cells. - Virgin female BALB/c mice, 10-16 weeks of age, were obtained from the Institute of Medical and Veterinary Science, Adelaide, Australia. Groups of mice were inoculated ip at various intervals for 8 weeks with the drug being tested; uninoculated mice served as controls. At least two months after cessation of drug administration, groups of 4 or 5 treated and control mice were killed. Nucleated cell counts were performed on one tibia from each animal, and bone marrow cells from the other tibia were pooled with those from mice in the same group for assay of granulocytic progenitor cells (CFC) and of pi uri potential stem cells (CFU), per tibia, as previously described (10). In several VOL. 57, No.6, DECEMBER 1976 Downloaded from https://academic.oup.com/jnci/article-abstract/57/6/1237/1034216 by Insead user on 08 March 2018
1237
experiments, the ability of CFU in the spleen to form further CFU and CFC was measured by retransplantation, as described (10). Drugs. - For drugs other than B U a pilot study was done to determine the approximate LD50 of a single ip dose and the duration of bone marrow suppression as assessed by tibial cell counts. For the formal experiment to test the drug, mice were then randomized into 3 groups: 1) One group served as the control. 2) The high dose group was treated ip with approximately one-third of LD50 at regular intervals for 8-10 weeks. The dose of the drug was progressively increased or decreased so that, by the end of treatment, approximately one-third of the mice in the group had died from drug overdose. 3) The low dose group received approximately twothirds of the dose given on the same day to the high dose group. Two groups were used so that the dose of the drug in the high dose group could be increased to lethal levels while survivors remained in the low dose group. Administration of BU to 2 groups of mice was not necessary, since the appropriate dose had been determined previously. The drugs tested, the methods of administration, and the doses given were as follows. BU: Ten or 20 mg was dissolved in 1.5 ml acetone; sterile water was added to make 10 ml solution, and 0.01 mllg was injected ip. Doses of20, 20, 20, and 10 mg BU/ kg were given at intervals of 2 weeks. BCNU: Thirty mg powder was dissolved in 0.9 ml ethanol; sterile water was added to make 10 ml solution; and 0.01 mllg was injected. Doses of 30 mg BCNU/kg were given at intervals of 2 weeks. Mortality was 50 %. 6-MP: Seventy to 120 mg powder was dissolved in 1 ml dimethyl sulfoxide, and sterile water was added to make 10 ml solution. Injection of 0.01 mg/g of the resulting suspension provided weekly doses of 70-120 mg 6-MP/ kg. Mortality was 62%. CY: The drug was appropriately diluted in sterile water and injected weekly in a dose of 60-160 mg/kg. Mortality was 24 %. 5-FU: The drug was appropriately diluted in sterile ABBREVIATIONS USED: BU = busulfan; CFC = colony-forming celI(s); CFU = colony-forming unit(s); LD50 = mean lethal dose' BCNU = 1,3-bis(chloroethyl)-I-nitrosourea; 6-MP = 6-mercaptopu:ine; CY = cyclophosphamide; 5-FU = 5-fluorouracil; MTX = methotrexate; VBL = vinblastine. Received November 11, 1975; accepted April 27, 1976. Supported by the Michell and Anti-Cancer Foundation of the University of Adelaide and by the National Health and Medical Research Council of Australia. 3 Department of Medicine, University of Adelaide, Adelaide, Australia. 4 Present address: Department of Haematology, Flinders Medical Centre, Bedford Park, South Australia 5042. 5 We thank Mrs. J. Remes and Mrs. R. Lloyd for excellent technical assistance and Dr. N. D. Harvey for help with the radiation facilities used. 1
2
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TRAINOR AND MORLEY
water and injected weekly in a dose of60 mg/kg. Mortality was 35%. MTX: The drug was appropriately diluted in sterile water and injected twice weekly in a dose of 5-10 mg/kg. Mortality was 32 %. VBL: The drug was appropriately diluted in sterile normal saline and injected twice weekly in a dose of 0.91.5 mg/kg. Mortality was 55 %. RESULTS
At the times of final analyses, the number of mice surviving from all high dose groups was sufficient to enable all assays to be done on these groups; assays were not performed on the low dose groups. All assays were done at least 2 months after cessation of drug administration, and each assay involved killing a group of 4 or 5 treated and control mice. The absolute numbers of nucleated cells were determined (text-fig. 1). The numbers of CFC (granulocytic progenitor cells) and CFU (pluripotential stem cells) were expressed as percentages of the controls, and the standard errors of the percentages were calculated (12, 13). The results for CFC are shown in text-figure 2 and for CFU in text-figure 3. The assays measuring selfrenewal and differentiation ability of CFU (text-fig. 4) were based on the principle that a single CFU gives rise to a spleen colony which contains further CFU and CFC. The number of CFU per spleen colony was therefore an index of the ability of the original CFU to renew themselves; the number of CFC spleen colony was an index of the ability of the original CFU to differentiate. The ratio of control/treated CFU/spleen colony compared the ability of control and treated CFU to renew themselves; the ratio of the control/treated CFC/spleen colony compared ability of control and treated CFU to differentiate. High ratios suggested impairment of selfrenewal and/or impairment of differentiation; ratios of approximately 1 suggested no impairment. The assays used in this study are not particularly precise; measurement of nucleated cells per tibia was probably the most accurate, and assay of splenic self-
BU
BCNU
CY
5-FU
--- -J-'-
100 ---
!
I-ir
! !i
e1:
6-MP
!
- - - -!--
BCNU
CY
5-FU
6-MP VBL
!
!
MTX
-!,
!
0
()
"'6 o-!?
!
I
10
(.) lJ.. (.)
TEXT-FIGURE 2.-CFC/tibia (mean ±SE) in mice treated with various cytotoxic drugs. Each point refers to a CFC assay on a bone marrow suspension prepared with pooled cells from tibia of 4 or 5 mice. BU
BCNU
100 --- ---
e1:
I
!
0
t!
CY
5-FU
_L!_
Jrt-
6-MP VBL
-r
f-
MTX
I-!-- ll_ f
!
!
()
"'6
~
10
::J
lJ.. (.)
TEXT-FIGURE 3.-CFU/tibia (mean ±SE) in mice treated with various cytotoxic drugs. Each point refers to a CFU assay on a marrow suspension prepared with pooled cells from tibias of 4 or 5 mice. 20
au
aCNU
CY
5-FU
6-MP VBL
MTX
~
10
•
8 •
BU
VBL
MTX CONTROL TREATED 1
•
--
0
8
• CFU o CFC
------r-•
0
•
x
«
iIi i=
~ ...J
5
f
UJ
(.)
TEXT-FIGURE 1. - Nucleated cells/tibia in mice treated with various cytotoxic drugs. The results for B U are the mean of 17 assays, but all other results show the mean ±SE of individual assays. The normal range (mean ±SE) is indicated by dotted area. Each individual assay involved killing 4 or 5 mice and counting individual tibial cells.
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Downloaded from https://academic.oup.com/jnci/article-abstract/57/6/1237/1034216 by Insead user on 08 March 2018
110 ~o,~--~--~--~--~--~--J-~
TEXT-FIGURE 4. - Ratios of control to treated mice in the number of CFU/spleen colony or the number of CFC/spleen colony.
renewal was probably the least precise. Therefore, we set subnormal values as the criterion for residual injury in mice following exposure to drugs, although we recognized that this might result in our overlooking the lesser degrees of residual damage. No evidence was found for the presence of residual injury following treatment with 6-MP, CY, 5-FU, MTX, or VBL; evidence was found for residual injury followVOL. 57, NO.6, DECEMBER 1976
SCREENING CYTOTOXIC DRUGS
ing treatment with BCNU and BU. Following administration of BCNU, the number of nucleated bone marrow cells, CFC, and CFU and the ability of CFU to selfrenew and to produce CFC decreased persistently. DISCUSSION
The two drugs found to produce residual marrow injury were B U and BCNU. In contrast with the other drugs tested, both BU and BCNU seem to exert prolonged and severe toxic effects on the bone marrow stem cell compartment (14, 15). Since this is the compartment principally involved in residual bone marrow injury, drugs having this type of effect on the stem cell compartment may be those which are particularly prone to cause residual injury. That B U and BCNU are both bifunctional alkylating agents might suggest that crosslinking of DNA was the biochemical mechanism involved. Yet residual damage was not detected when CY, which may act similarly, was used. Whether CY causes residual impairment of proliferation of tumor cells is disputed (2, 4); if it does, failure to find an effect on normal marrow cells may be due to a differential effect of cyclophosphamide on normal marrow and tumor cells and/or the inherent imprecision of the assays used in the present study. From the functional point of view, residual bone marrow damage after administration of B U and BCNU can be regarded as a permanent impairment of the proliferation and differentiation abilities of marrow cells, which results in permanent decrease in their number. This effect of certain cytotoxic drugs on normal marrow cells had not been clearly defined previously, and little evidence exists as to whether cells other than those of the marrow sustain similar damage. In man, chronic hypoplastic marrow failure (aplastic anemia) probably is a manifestation of residual damage, and widespread disturbances in immunity are known to occur in the disease (16, 17). This would suggest that cells of the immune system may also sustain residual damage, and preliminary results from our laboratory support that possibility. Rational use of cytotoxic drugs requires an understanding of their toxic effects on normal and on neoplastic cells. The recognition of residual injury to either cell type could therefore be of potential importance in leading one to a better understanding of the mode of action of cytotoxic drugs and thus to their more effective use.
VOL. 57, NO.6, DECEMBER 1976 Downloaded from https://academic.oup.com/jnci/article-abstract/57/6/1237/1034216 by Insead user on 08 March 2018
1239
REFERENCES (1) SKIPPER HE, SCHABEL FM jR, WILCOX WS: Experimental evaluation of potential anticam:er agents. XIII. On the criteria and kinetics associated with "curability" of experimental leukemia. Cancer Chemother Rep 35:3-111, 1964 (2) WILCOX WS, SCHABEL FM jR, SKIPPER HE: Experimental evaluation of potential anticancer agents. X V. On the relative rates of growth and host kill of "single" leukemia cells that survive in vivo Cytoxan therapy. Cancer Res 26:1009-1014,1966 (3) JOHNSON RE, ZELEN M., KEMP NH: Chemotherapeutic effects on mammalian tumor cells. I. Modification of leukemia L1210 growth kinetics and karyotype with an alkylating agent. j Nat! Cancer 1nst 34:277-285, 1965 (4) JOHNSON RE, HARDY WG: Chemotherapeutic effects on mammalian tumor cells. II. Biologic and chromosomal instability of a cyclophosphamide treated murine leukemia. Cancer Res 25:604-608, 1965 (5) JOHNSON RE, HARDY WG, ZELEN M: Chemotherapeutic effects on mammalian tumor cells. III. Modification of leukemia Ll210 growth kinetics with an antimetabolite. j Nat! Cancer Inst 36:15-20, 1966 (6) HUMPHREYS SR, GOLDIN A: Investigation of tumor variants recovered from mice with systemic leukemia (LI210) after extensive therapy with 3' ,5' -dichloroamethopterin and 3' -bromo-5'chloroamethopterin. j Nat! Cancer Inst 23:633-653, 1959 (7) DE VITA VT, BRAY DA, BOSTICK F, et al: The effect of chemotherapy on the growth of leukemia L121O. II. Persistence of a nitrosourea induced change in the growth characteristics of transplant generations. Cell Tissue Kinet 5:459-466, 1972 (8) SCHMID FA, HUTCHINSON Dj: Chemotherapeutic carcinogenic and cell-regulatory effects of triazines. Cancer Res 34:16711675, 1974 (9) MORLEY A, BLAKEj: An animal model of chronic aplastic marrow failure. I. Late marrow failure after busulfan. Blood 44:49-56, 1974 (10) MORLEY A, TRAINOR K, BLAKE,]: A primary stem cell lesion in experimental chronic hypoplastic marrow failure. Blood 45:681-688, 1975 (11) MORLEY A, BLAKE]: Haemopoietic precursor cells in experimental hypoplastic marrow failure. Austj Exp Bioi Med Sci 52:909914, 1974 (12) MORLEY A, TRAINOR K, REMES ]: Residual marrow damage: Possible explanation for idiosyncrasy to chloramphenicol. Br] Haematol. In press (13) DAVIES PL: Statistical Methods in Research and Production, 3d ed. London, Oliver and Boyd, 1961, P 41 (14) DUNN CD, ELSON LA: The comparative effect of busulfan (myleran) and aminochlorambucil on haemopoietic colony forming units in the rat. Cell Tissue Kinet 3:131-141,1970 (15) REISSMAN KR, UDUPA KB, KAWODA K: Effects of erythropoietin and androgens on erythroid stem cells after their selective suppression by BCNU. Blood 44:649-657,1974 (16) MORLEY A, FORBES I: Impairment of immunological function in aplastic anaemia. Aust N Z] Med 4:53-57, 1974 (17) MORLEY A, HOLMES K, FORBES I: Depletion of B lymphocytes in chronic hypoplastic marrow failure (aplastic anaemia). Aust N Zj Med 4:538-541,1974
J NATL CANCER INST
