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Residual injury to the hemopoietic microenvironment following sequential radiation and busulfan
??Original Contcibution RESIDUAL INJURY TO THE HEMOPOIETIC MICROENVIRONMENT FOLLOWING SEQUENTIAL RADIATION AND BUSULFAN LYNNE M. WATHEN, Radiation
Research
Laboratory
PH.D., RICHARD L. DEGOWIN, M.D., B.S. AND SHIRLEY A. KNAPP and the Division of Hematology-Oncology, Medicine, Iowa City, IA 52242 USA
D. PHILLIP GIBSON,
The University
of Iowa College of
Our earlier studies in mice showed,that sequential radiation and cyclophosphamide suppressed marrow stromal cells (MSC) and prevented local hemopoietic repopulation for several months. Becauseothers haveshown that busulfan administration caused marrow aplasia, we studied its ability, combined with radiation, to produce a persistent microenvironmental defect in mice. Intraperitoneal administration of four weekly doses of 20 mg/kg busulfan, starting one week after 1500 rad leg irradiation, produced a severe microenvironmental lesion for 6 months reflected by lack of repopulation in femoral marrow to greater than 50% of normal by MSC, hemopoietic stem cells (CFU-S), and granulocyte-macrophage precursors. Differential marrow cell counts revealed that precursors of hemopoietic cells were more profoundly affected than their progeny. Hemopoietic stem cells and MSC failed to recover in busulfan-treated mice at 6 months to the same extent as those treated with cyclophosphamide. In addition, the busulfan-treated mlice had an excessive number of myeloid blast cells and a severe erythroid depletion suggesting that these animals were preleukemic. We conclude that: I) sequential radiation and husulfan administration caused long-term microenvironmental damage reflected by incomplete repopulation of the femoral marrow and suppression of MSC, and 2) multiple courses of busulfan prevented hemopoietic repopulation longer than a similar regimen of cyclophosphamide. Hemopoiesis, Microenvironment,
Busulfan.
INTRODUCTION
earlier studies in mice showed that sequential radiation and cyclophosphamide suppressed marrow stromal cells (MSC) and prevented hemopoietic repopulation in the irradiated field for several months.23 Because of the marrow asplasia experienced with busulfan (BU) administration,12 we explored its ability, combined with radiation, to produce a residual microenvironmental defect in mice. Though cyclophosphamide and busulfan are both considered alkylating agents, they are substantially different chemotherapeutic drugs. Cyclophosphamide is related to nitrogen mustard and has recently been used as an immunosuppressive pretreatment for bone marrow transBusulfan is a member of the sulfonic acid plantation.“.‘” esters and differs in structure from nitrogen mustard. It is neither immunosuppressive nor cycle-dependent and is considered ineffective against solid tumors. Busulfan is primarily used for palliative treatment of chronic myelocytic leukemia. Recent clinical use of busulfan plus cyclophosphamide’,’ instead of total body irradiation plus cyclophosphamide, as pretreatment for bone marrow
transplantation, underscored the need for studies on the long-term hemopoietic effects of busulfan. Repeated intraperitoneal injections of busulfan were found by other investigators to suppress granulocytic progenitor cells (CFU-C), pluripotent stem cells (CFUS) and erythroid progenitors (CFU-E) in mice.2~‘3 Repopulation of CFU-C and CFU-E in busulfan-treated mice preceded recovery in the CFU-S po01.~,‘.* Busulfan appeared to have a dual effect on CFU-S; it depressed their number and limited their proliferative capacity.3 The slow recovery of CFU-S after BU administration was attributed by Udupa et al.,” to a predilection of BU for noncycling cells. Castro-Malaspina et a1.,4 using the thymidine suicide technique, concluded that the marrow stromal cell was noncycling in individuals with undisturbed bone marrow function. Busulfan might, therefore, be expected to cause severe suppression of marrow stromal cells. Preliminary studies in our laboratory showed that a single intraperitoneal dose of BU (20 mg/kg) suppressed the total femoral nucleated cells to 85% and the marrow stromal cells to 64% of normal at one week post-injection.
This work was supported by a grant from the National Cancer Institute CA 11472. Reprint requests to: Richard L. DeCowin, M.D., Dept. of Internal Medicine, University of Iowa Hospitals, Iowa City, IA 52242.
Acknowledgements-The authors wish to thank Mr. Harold Plate and Mr. Robert Wickham for the care of the mice used in these studies, and Ms. Carolyn Malone for preparation of this manuscript. Accepted for publication 14 March 1982.
Our
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We concluded that BU caused a systemic marrow stromal insufficiency and wished to test the agent’s ability to produce residual damage in the hemopoietic microenvironment and alter the repopulation of a local irradiationinduced hemopoietic lesion. Accordingly, mice were treated with 1500 rad leg irradiation (1500 rad L.I.) followed by four weekly injections of 20 mg/kg BU and compared to control mice receiving either BU or 1500 rad L.I. or neither. METHODS
AND MATERIALS
Mice
Seven to ten week-old female CF-I mice, weighing 22-25 gm, were housed in plastic cages of 8 mice each with 12 hr of light/day and allowed standard mouse chow and acidified tap water (pH 2.5) ad libitum. Irradiation
On day 0, mice anesthetized with sodium pentobarbito], received 1500 rad x-irradiation to the right leg (I 500 rad L.I.) while the rest of the body was shielded with 6 mm of lead. Doses to the hind limb were measured with an ionization chamber.* The mice were irradiated at a target distance of 55 cm and 250 kVp with an x ray machine? at 30mA with filtration of 0.25 mm Cu plus I .O turntable at approximately 80 mm Al on a rotating rad/minutc. Busulfun
The dose of busulfan used in these studies was determined by the pilot investigations summarized in Table 1. Beginning one week post-irradiation, the mice received one intraperitoneal dose of busulfan (20 mg/kg) each week for four consecutive weeks. Weight and peripheral
blood
Animals were weighed, and leukocyte, hematocrit and differential blood smear values were obtained by usual methods using blood from the tail vein. Bone marrow
The mice were killed by cervical dislocation. Their femurs were removed and the femoral contents were flushed into centrifuge tubes with 5 ml of M-199 tissue culture medium* containing 10%) fetal calf serum (FCS) and L-glutamine.24 Total nucleated cell determinations and bone marrow differential counts were obtained as described previously.24 The marrow from each treatment group was pooled, an aliquot was counted on a Coulter Counter, and diluted to I x lOh nucleated cells/ml with culture medium. Culture
of‘marrow
stromal
cells (MSC)
Two miliiliters of M-199 containing I x lOh nucleated cells/ml of pooled bone marrow from each treatment *Victoreen. tMaxitron.
August 1982. Volume 8, Number 8
Table
I. Survival
and marrow stromal colonies of mice after four weekly injections of busulfan*
Doses of busulfan (mg/k)
Marrow stromal colonies* (7%of control)
Survival ratiost
69 32 9
16/16 16/16 l/l6
IO 20 40
*MSC cultured I hour after the last injection. TExpressed as number of survivors over number injected.
of mice
group was cultured in 35 mm tissue culture dishes. The cells were incubated and the media changed as described previously.24 The cells were fixed using methanol, stained with Mallory’s Azure II Methylene Blue and the adherent colonies of 50 or more cells in each dish were counted on day 7. Approximately 20 marrow stromal colonies per dish were obtained from 2 x IO6 normal murine bone marrow cells.
Spleen
colony assay
A modified spleen colony assay of Till and McCulloch,‘” using 750 rad total body irradiation for recipient mice, was used to determine the hemopoietic stem cell count of the right femur following 4 weekly doses of BU, I500 rad L.I.. or leg irradiation followed by the 4 doses of BU. Total body x-irradiation (750 rad) one day before prepared the assay mice for the intravenous injection of I x IO” pooled bone marrow cells from the treated donor mice. On day 7, the spleens from the recipient mice were removed, placed in Bouin’s fluid and the colonies were counted.
Colony forming
units-Culture
granulocyte-macrophage
CFU-C or
colonies
(CFU-GM)
Using 0.3% agar in the method reported by Metcalf,” the pooled bone marrow samples were evaluated for CFU-C content. Colony stimulating activity was obtained using postendotoxin serum from mice which had been injected with 5 pg of E. coli endotoxin.§‘4 The 35 mm culture dishes containing I x IO’ bone marrow cells were incubated for seven days and colonies of 50 or more cells were counted. The average number of CFU-C per I x I OS normal bone marrow cells was 53 -+ 1.4. Assay and “Cr labeling of red blood cells
Autologous red blood cells were labeled using a method similar to that described by Ultmann and Gordon.” Briefly, blood was drawn from the normal and treated mice via cardiac puncture and injected into a sterile vial fM-199, @&ma.
Gibco.
X-ray and busulfan stromal damage 0 L. M. WATHEN et al.
120
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BU BU BU BU
0 X r(
011
0
1
2
3 Months
BU 20 mglkg x 4 1500 rad L.I. BU + L.I.
4
5
6
Postirradiation
Fig. I. Total nucleated cells per femur. For this and subsequent figures: L.I. = right hind leg irradiation, BU = busulfan injection, arrow indicates that irradiation was given on day zero, n = number of mice per point, shaded areas = normal + standard error of the mean (SEM), error bars = *SEM.
0.15 ml of acid citrate dextrose.? The red blood cells (RBC) were labeled in vitro by the addition of 5’Cr (Na, “CrO,$ 20-30 $Zi/ml of blood) and incubated for 30 minutes at room temperature (26OC). The RBC incubation was terminated with the addition of 8 to 10 mg of ascorbic acid/ml of blood. The RBC were washed twice with sterile saline and reconstituted with saline to the original volume withdrawn from the mouse. The labeled blood (0.5 ml) was injected via the tail vein into the same mouse from which it was withdrawn. At specified times following “0-RBC injection, tissue and blood samples from the recipient animals were removed and counted in an Auto-gamma Spectrometer* using a window from 280400 keV. containing
Vascular patency following 1500 rad leg irradiation and busulfan Mice were irradiated and treated with busulfan in the same regimen as that describe above. Briefly, on day 0, mice received 1500 rad leg irradiation followed by 4 weekly doses of busulfan (20 mg/kg) beginning on day 7. On day 60, the mice were injected with autologous “Cr-RBC. Twenty-four hours after the “Cr-RBC injection, 20 ~1 blood sampleis, femurs and spleens from the recipient mice were removed and counted. Statistical treatment of data These data are the result of two replicate studies. All data, except the “Cr investigation, were analyzed using a two-way analysis, of variance” to compare drug and radiation interaction. In the “Cr study, data were analyzed using the student’s t test.
7250 mg sodium citrate, 120 mg dextrose, 80 mg citric acid dissolved in 10 ml of sterile water.
RESULTS Mouse weight and peripheral blood Most of the mice gradually increased in weight during the 6 months post-irradiation. The busulfan treated animals did not experience a steady weight increase, and animal deaths occurred at 2-3 months. Peripheral blood leukocytes were transiently depressed for 2 months to less than 60% of normal and hematocrits were depressed for one month to about 85% of normal in mice treated with busulfan, but later they returned to normal. There was no sign of inflammation in the irradiated leg, though slight ephemeral thinning of hair occurred. Total nucleated cells Fifteen hundred rad leg irradiation (1500 rad L.I.) inhibited the repopulation of nucleated cells to less than 70% of normal at 1, 2, and 4 months post-irradiation indicating induction of a residual femoral marrow lesion (Fig. 1). Busulfan suppressed femoral nucleated cells to a similar extent at one month. In the busulfan treated animals gradual recovery occurred to about 80% of normal at 4 months but declined to about 60% at 6 months post-irradiation. The combined modalities produced a consistent pattern of suppression to about 40% through 4 months and rose to about 70% of normal at 6 months postirradiation. Bone marrow differential counts Radiation alone persistently suppressed myeloid (myeloblasts, myelocytes, neutrophils) cells to about 70% of normal (Fig. 2). Busulfan alone depleted the myeloid population to 50% at one month with gradual increase to
$New England Nuclear. *Packard.
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Radiation Oncology 0 Biology 0 Physics
+
1982. Volume
8, Number
8
BU BU BIJ BU
n=8-10
0 BU 20 mglkg x 4 rad L.I. M BU + L.I.
X 1600
OO 1.
* * 11
.
.
21
.
.
.
31
.
*
*
41.
.
.
51.
.
61
*.
Months Postirradiation Fig. 2. Absolute
number of myeloid (myeloblasts,
and neutrophils)
per femur
post-irradiation. Erythroblasts experienced a transient overshoot after one month of busulfan alone, remained at 75-85s at 2 and 4 months, and plunged to less than 20% at 6 months (Fig. 4). in mice receiving X ray alone, erythroid cellularity stayed at 60-70s of normal from I to 4 months post-irradiation and returned to near normal levels at 6 months. In contrast, combined therapy suppressed erythroid cells to 30P45% of normal from I to 6 months post-irradiation.
about 80% of normal through 6 months post-irradiation. At 6 months, the bone marrow of busulfan-treated mice contained 2.5 times as many early myeloid cells as the control animals suggesting an intensive myeloid hyperplasia in these mice. The combined therapy produced a myeloid diminution greater than either modality alone which remained below 60% of normal through 4 months but recovered to a single modality level (70-8091) by 6 months. There were not excessive numbers of blasts. Radiation alone and busulfan alone produced similar depletions of lymphoid cellularity and recovered to 7080% of normal at four months post-irradiation (Fig. 3). At 6 months, the busulfan-treated mice showed a pronounced suppression of lymphoid cells to 25Y~ of normal. Combined radiation and busulfan treatment persistently suppressed lymphoid cells to less than 30% of normal through 4 months with recovery to about 55% at 6 months 120
myelocytes,
Marrow srromal crlls (MSC‘) Femoral MSC were depressed to values 28% to 42% of normal during the 4 months post-irradiation following radiation alone (Fig. 5). By 6 months, they had recovered to 80% of normal. The systemic agent, busulfan, suppressed MSC values to less than 40% of normal at I, 4, and 6 months with an abortive rise in these supporting
n = 8-18
BU BU BU BU
P
01.“‘. 0
1
.
‘I*
2
.“‘. Months
Fig. 3. Absolute
3
.I*.
4
Postirradation
number of lymphoid cells per femur.
.
“*
5
”
6
X-ray and busulfan
stromal
damage
??L.
1319
M. W&THEA et al.
n = 8-16
20 -
0
BU 20 mg/Kg x 4 L.I.
*..*
X 1600 rad
“4
w. BU + L.I. 0
0
““““,““““.“...’
1
2
3
4
5
6
Months Postirradiation Fig. 4. Absolute
number of erythroblasts
cells at 2 months post-irradiation. The combined radiation and busulfan treatment caused persistent suppression of MSC to less than 25% through 4 months and rose to about 45% of normal at 6 months. Stem cells and progenitws The hemopoietic stem cells (CFU-S) declined from 75% at one month to 52% of normal at 6 months postirradiation following X ray alone (Fig. 6). CFU-S in mice treated with busulfan alone reached a nadir (5%) at one month, plateaued at 45%) at 2 and 4 months and dropped to 10% at 6 months. The combined treatment depleted CFU-S to less than 30% of hormal through 6 months post-irradiation indicating a persistent stem cell deficit. Granulocyte-macrophage precursors (CFU-C) remained below 10% for 4 months with combined modality treatment, 25% with BU alone and 45% with X ray alone (Fig. 7). At 6 months, the CFU-C recovered to 55% with
per femur.
irradiation and 43% with combined radiation and busulfan treatment but remained at 25% with BU alone. Vascular patency The ingress of “Cr-RBC into the irradiated femurs of combined modality treated mice was limited to 75% of the activity found in normal femurs. (Fig. 8) Irradiation alone caused less severe inhibition and BU alone failed to cause inhibition of “Cr-RBC entrance into the femoral vascular space. Thus, a portion of the microenvironmental damage seen at 2 months in the 1500 rad L.I. + BU study was the result of occlusion of the microvasculature. DISCUSSION
These studies have shown that sequential therapy with irradiation followed by busulfan administration caused residual injury to the bone marrow microenvironment for at least 6 months. The failure of hemopoietic and marrow
n-8-10
Months Postirradiation Fig. 5. Absolute
number of marrow stromal cell (MSC)
colonies per femur
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0 Physics
August 1982, Volume 8, Number
8
??BU 20 m2lKg x 4 X 1600 rad L.I.
M BU + L.I.
1
1.. OO
1
.
.
.
1
1
1..
“.
2 Months
Fig. 6. Absolute
stromal
cells
to repopulate
the
number
femoral
of hemopoietic
bone
*
1..
.
4
3
‘.
.
*
1
8
5
Postirradiation
stem cells or splenic
marrow
confirmed the presence of this lesion. The hemopoietic precursors (CFU-S and CFU-C) were more severely suppressed by the combined modality treatment than their progeny (myeloid, erythroid and lymphoid cells). It is possible that these precursors were more committed to differentiation than to self-replication. However, Chervenick and Boggs’ found that, in total body irradiated mice, when stem cells were depleted below 10% of normal, self-replication occurred in place of differentiation. Above IO%, both differentiation and selfreplication were present but self-replication took priority until normal CFU-S numbers were attained.5 Investigations in our laboratory demonstrated that after partial body irradiation, the stem cell compartment had to attain a critical level of repletion before responsiveness to endogenous erythropoietin could induce splenic erythropoiesis6 In addition, others have found that CFU-S surviving treatment with busulfan, self-replicated at the expense of differentiation.’ Trainor and co-workers have offered an
colonies (CFU-S)
per femur.
alternative hypothesis for the hemopoietic precursor impairment caused by busulfan.20 They stated that stem cells from busulfan-treated mice do not differ in their probability for either self-renewal or differentiation but rather suffer from impaired proliferation in the stem cell compartment leading to decreased compartment size and to a decreased absolute number of cells differentiating out of it.20 At 6 months after day zero, the mice treated with busulfan alone appear to have a more severe hemopoietic insufficiency than the mice treated with radiation or combined radiation and busulfan. The excessive number of myeloid blast cells and the severe erythroid depletion suggested that these animals were preleukemic. The characteristics of preleukemia in humans have been described by Linman and Bagby.” There is additional evidence for the preluekemic activity of busulfan in a report involving long-term administration of busulfan to patients who had had bronchial carcinoma.16 In this study, four of the 243 patients treated with busulfan
n=e-16
0 BU 2OmgIKg x 4 )( 1500 rad L.I. H BU + L.I.
Months
Fig. 7. Absolute
number
Postirradiation
of granulocyte-macrophage
colonies
(CFU-C)
per
femur.
X-ray
._._
R L FEMUR
R L FEMUR
1'500RAD LI
NORMAL
and busulfan
stromal
R L FEMUR
R L FEMUR
Bl!
AU + LI
Fig. 8. Radioactivity of 5’Cr-labeled autologous red blood cells in normal, irradiated and busulfan treated mice two months after irradiation. R = right femur, L = left femur, 1500 rad L.I. = 1500 rad right hind leg irradiation, BU = 20 mg/kg BU injected I.P. once each week for 4 consecutive weeks, BU + LI = 1500 rad right hind leg irradiation followed by busulfan injections. (n = 14-l 5 mice per treatment group), *this value is significantly different from R normal (p 5 .OS). Error bars i standard error of the mean. developed
leukemia
com.pared
It is also
possible
to none of the 243 treated
a.nd none of the 249 on placebo.‘6
with cyclophosphamide that
the
excessive
number
of myelo-
in the busulfan-treated animals was partially a result of marrow regeneration. The lack of excessive myeloblasts in the femurs receiving both irradiation and busulfan may either be due to the absence of marrow regeneration or to an insufficient microenvironment for the leukemia cells. In general, the pattern of hemopoietic suppression following sequential radiation and busulfan was similar to that following sequential radiation and cyclophosphamide.23 However, at 4 an!d 6 months post-irradiation, the busulfan-treated mice had a more severe suppression of CFU-S, CFU-C and MSC than mice treated with cyclophosphamide alone. Assessment of these data suggested that, unlike cyclophosphamide, busulfan may: 1) have limited the proliferative capacity of the CFU-S, CFU-C and MSC, or 2) have caused a morphologic or metabolic change in these cells, impeding their migratory capacity blasts
??L. M. WATtlENet al.
damage
1321
or their interaction with other cells. Evidence of such structural and functional abnormalities have been reported in lymphocytes following a regimen of busulfan similar to that used in the experiments reported here.14 The mice were allowed to recover for at least 2 months after the last dose of busulfan but still demonstrated disturbances of a variety of humoral and cellular immune function. The preliminary vascular studies using “Cr-RBC reported in this investigation increased our insight into the factors responsible for residual damage after single and combined modality therapy. As we discussed above, 1500 rad leg irradiation or busulfan alone inhibited the repopulation of nucleated cells to 65-75s or normal in the femur at 2 months post-irradiation. The “Cr-RBC investigation has shown a lack of significant vascular damage in these animals indicating that these residual femoral lesions may be primarily a result of a marrow stromal cell or matrical deficit. In addition, we reported that the combined modalities suppressed nucleated cells to 40% of normal 2 months after irradiation. The present data involving autologus “Cr-RBC demonstrated a vascular defect which was responsible for a 25% decline in cellular ingress following combined modality therapy. If stem cell circulation is similar to red cell circulation, the 35% suppression of nucleated cells, unaccounted for by vascular damage, may be the marrow stromal cell portion of the microenvironmental damage following sequential radiation and busulfan. Our investigation of combined modality therapy, using 1500 rad leg irradiation followed by 4 weekly doses of busulfan, produced residual hemopoietic injury which persisted for at least 6 months post-irradiation. Busulfan alone was more deleterious to the hemopoietic precursors and microenvironment than cyclophosphamide alone. These studies have suggested that when treatment involves sequential radiation and busulfan, it may well be the busulfan dose rather than the radiation dose which determines the extent of hemopoietic damage and ultimate repair. In contrast, previous studies23 have implied that radiation may be the limiting factor following sequential radiation and cyclophosphamide. This suggests the need for further studies on the preleukemic state cause by busulfan and stresses the possibility of late complications following busulfan therapy.
REFERENCES Beschorner, W.E., Pino, J., Boitnott, J.K., Tutschka, P.J., Santos, G.W.: Pathology of the liver with bone marrow transplantation. Effec1.s of busulfan, carmustine, acute graft-versus-host disease and cytomegalo-virus infection. Am. J. Path. 99: 369-386,198O. Boggs, D.R., Boggs, S.S., Chervenick, P.A., Patrene, K.D.: Murine recovery from busulfan-induced hematopoietic toxicity as assessed by three assays for colony-forming cells. Am. J. Hamatol. 8: 43--M, 1980. Botnick, L.F., Hannons, E.C., Hellman, S.: Nature of the hemopoietic stem cell compartment and its proliferative potential. Mood Cells !i: 195-210, 1979.
4. Castro-Malaspina, H., Gay, R.E., Resnick, G., Kapoor, N., Meyers, P., Chiarieri, D., McKenzie, S., Broxmeyer, H.E., Moore, M.S.A.: Characterization of human bone marrow fibroblast colony-forming cells (CFU-C) and their progeny. Bfood 56: 289-301, 1980. 5. Chervenick, P.A., Boggs, D.R.: Patterns of proliferation and differentiation of hematopoietic stem cells after compartment depletion. Blood 37: 568-580, 197 1. 6. DeCowin, R.L.: Erythroid differentiation during stem cell proliferation. J. Lab. Clin. Med. 70: 23-25, 1967. 7. Delmonte,
L.: Effect of myleran
on murine hemopoiesis.
I.
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8.
9.
IO. II
12.
13.
14.
15.
16.
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Granulocytic cell line specificity of action on progenitory cells. Cell Tissue Kiner. 11: 347-358, 1978. Delmonte, L.: Effect of myleran on murine hemopoiesis. II. Direct and host-mediated action on proliferative capacity and differentiation bias of spleen colony forming units 1978. (CFU-S). Cell Tissue Kinet. 11: 359-367, Korbling, M., Elfenbein, G.J., Tutschka, P., Santos, G.W.: Exaggerated blood CFU-C rebound in patients with acute leukemia following busulfan (BU) and cyclophosphamide (CY) and bone marrow transplantation. (Abstract). E.xp. Hematol7: (Suppl.) 57, 1980. Linman. J.W., Bagby, G.C.: The preleukemic syndrome (hemopoietic dysplasia). Cancer 42: 854-864, 1978. Metcalf, D.: Hemopoietic colonies: In virro cloning of normal and leukemic cells. Recent Results Cancer Res. 61: 15-17.1977. Morley, A., Blake, J.: An animal model of chronic asplastic marrow failure. I. Late marrow failure after busulfan. Blood 44: 49956, 1974. Morley, A., Trainor, K.. Blake, J.: A primary stem cell lesion in experimental chronic hypoplastic marrow failure. Blood 45: 681-688, 1975. Pugsley, C.A.J., Forbes, I.J., Morley, A.A.: Immunologic abnormalities in an animal model of chronic hypoplastic marrow failure induced by busulfan. Blood 5: 601~610, 1978. Steel, R.G., Torrie, J.H.: Analysis of variance II: Multiway classifications. In Principles and Procedures of Statistics. New York, McGraw-Hill Book Company, Inc. 1960. pp. 132-160. Stott, H., Fox, W.. Girling, D.J., Stephens, R.J., Dalton, D.A.B.: Acute leukemia after busulfan. Br. Med. J. 2: l513-1517,1977.
August 1982, Volume 8. Number 8
17. Thomas, D.E., Buchner. C.D., Banaji, M., Clift, R.A., Fefer. A., Flournoy, N., Goodell. B.W., Hickman, R.O.. Lerner. K.G., Neiman, P.E., Sale, G.E., Sanders, J.E., Singer, J.. Stevens, M.. Storb, R., Weiden. P.L.: One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 49: 5 I l-533, 1977. 18. Thomas, D.E.. Buckner. C.D.. Clift. R.A., Fefer, A., Johnson, F.L., Neiman, P.E., Sale, G.E., Sanders, J.E., Singer, J.W., Shulman, H.. Storb. R., Weiden, P.L.: Marrow Transplantation for acute nonlymphoblastic leukemia in first remission. New, Eng. J. Med. 301: 5977599. 1979. 19. Till, J.E.. McCulloch, E.A.: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radial. Res. 14: 213-222. I96 I. 20. Trainor, K.J., Morley, A.A.. Seshadri. R.S.: A proliferative defect of marrow cells in experimental chronic hypoplastic marrow failure (aplastic anemia). Eup. Hemarol. 8: 674682. 1980. 21. Udupa, K.B.. Okamura, H., Reissmann, K.R.: Granulopoiesis during myleran-induced suppression of transplantable hemopoietic stem cells. Blood 39: 3 177325, 1972. 22. Ultmann. J.E., Gordon, C.S.: Life span and sequestration of normal erythrocytes in normal and splenectomized mice and rats. Acra. Hemat. 33: I I881 26. 1965. 23. Wathen, L.M.. Knapp, S.A., DeGowin. R.L.: Suppression of marrow stromal cells and microenvironmental damage following sequential radiation and cyclophosphamide. Inr. J. Radiat. 0~01. Biol. Ph>js. 7: 935-941, 1981. 24. Werts, E.D., DeGowin. R.L., Kanpp. S.A.. Gibson, D.P.: Characterization of marrow stromal (fibroblastoid) cells and their association with erythropoiesis. E.up. Hemar. 8: 4233433, 1980.
Research
Laboratory
PH.D., RICHARD L. DEGOWIN, M.D., B.S. AND SHIRLEY A. KNAPP and the Division of Hematology-Oncology, Medicine, Iowa City, IA 52242 USA
D. PHILLIP GIBSON,
The University
of Iowa College of
Our earlier studies in mice showed,that sequential radiation and cyclophosphamide suppressed marrow stromal cells (MSC) and prevented local hemopoietic repopulation for several months. Becauseothers haveshown that busulfan administration caused marrow aplasia, we studied its ability, combined with radiation, to produce a persistent microenvironmental defect in mice. Intraperitoneal administration of four weekly doses of 20 mg/kg busulfan, starting one week after 1500 rad leg irradiation, produced a severe microenvironmental lesion for 6 months reflected by lack of repopulation in femoral marrow to greater than 50% of normal by MSC, hemopoietic stem cells (CFU-S), and granulocyte-macrophage precursors. Differential marrow cell counts revealed that precursors of hemopoietic cells were more profoundly affected than their progeny. Hemopoietic stem cells and MSC failed to recover in busulfan-treated mice at 6 months to the same extent as those treated with cyclophosphamide. In addition, the busulfan-treated mlice had an excessive number of myeloid blast cells and a severe erythroid depletion suggesting that these animals were preleukemic. We conclude that: I) sequential radiation and husulfan administration caused long-term microenvironmental damage reflected by incomplete repopulation of the femoral marrow and suppression of MSC, and 2) multiple courses of busulfan prevented hemopoietic repopulation longer than a similar regimen of cyclophosphamide. Hemopoiesis, Microenvironment,
Busulfan.
INTRODUCTION
earlier studies in mice showed that sequential radiation and cyclophosphamide suppressed marrow stromal cells (MSC) and prevented hemopoietic repopulation in the irradiated field for several months.23 Because of the marrow asplasia experienced with busulfan (BU) administration,12 we explored its ability, combined with radiation, to produce a residual microenvironmental defect in mice. Though cyclophosphamide and busulfan are both considered alkylating agents, they are substantially different chemotherapeutic drugs. Cyclophosphamide is related to nitrogen mustard and has recently been used as an immunosuppressive pretreatment for bone marrow transBusulfan is a member of the sulfonic acid plantation.“.‘” esters and differs in structure from nitrogen mustard. It is neither immunosuppressive nor cycle-dependent and is considered ineffective against solid tumors. Busulfan is primarily used for palliative treatment of chronic myelocytic leukemia. Recent clinical use of busulfan plus cyclophosphamide’,’ instead of total body irradiation plus cyclophosphamide, as pretreatment for bone marrow
transplantation, underscored the need for studies on the long-term hemopoietic effects of busulfan. Repeated intraperitoneal injections of busulfan were found by other investigators to suppress granulocytic progenitor cells (CFU-C), pluripotent stem cells (CFUS) and erythroid progenitors (CFU-E) in mice.2~‘3 Repopulation of CFU-C and CFU-E in busulfan-treated mice preceded recovery in the CFU-S po01.~,‘.* Busulfan appeared to have a dual effect on CFU-S; it depressed their number and limited their proliferative capacity.3 The slow recovery of CFU-S after BU administration was attributed by Udupa et al.,” to a predilection of BU for noncycling cells. Castro-Malaspina et a1.,4 using the thymidine suicide technique, concluded that the marrow stromal cell was noncycling in individuals with undisturbed bone marrow function. Busulfan might, therefore, be expected to cause severe suppression of marrow stromal cells. Preliminary studies in our laboratory showed that a single intraperitoneal dose of BU (20 mg/kg) suppressed the total femoral nucleated cells to 85% and the marrow stromal cells to 64% of normal at one week post-injection.
This work was supported by a grant from the National Cancer Institute CA 11472. Reprint requests to: Richard L. DeCowin, M.D., Dept. of Internal Medicine, University of Iowa Hospitals, Iowa City, IA 52242.
Acknowledgements-The authors wish to thank Mr. Harold Plate and Mr. Robert Wickham for the care of the mice used in these studies, and Ms. Carolyn Malone for preparation of this manuscript. Accepted for publication 14 March 1982.
Our
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We concluded that BU caused a systemic marrow stromal insufficiency and wished to test the agent’s ability to produce residual damage in the hemopoietic microenvironment and alter the repopulation of a local irradiationinduced hemopoietic lesion. Accordingly, mice were treated with 1500 rad leg irradiation (1500 rad L.I.) followed by four weekly injections of 20 mg/kg BU and compared to control mice receiving either BU or 1500 rad L.I. or neither. METHODS
AND MATERIALS
Mice
Seven to ten week-old female CF-I mice, weighing 22-25 gm, were housed in plastic cages of 8 mice each with 12 hr of light/day and allowed standard mouse chow and acidified tap water (pH 2.5) ad libitum. Irradiation
On day 0, mice anesthetized with sodium pentobarbito], received 1500 rad x-irradiation to the right leg (I 500 rad L.I.) while the rest of the body was shielded with 6 mm of lead. Doses to the hind limb were measured with an ionization chamber.* The mice were irradiated at a target distance of 55 cm and 250 kVp with an x ray machine? at 30mA with filtration of 0.25 mm Cu plus I .O turntable at approximately 80 mm Al on a rotating rad/minutc. Busulfun
The dose of busulfan used in these studies was determined by the pilot investigations summarized in Table 1. Beginning one week post-irradiation, the mice received one intraperitoneal dose of busulfan (20 mg/kg) each week for four consecutive weeks. Weight and peripheral
blood
Animals were weighed, and leukocyte, hematocrit and differential blood smear values were obtained by usual methods using blood from the tail vein. Bone marrow
The mice were killed by cervical dislocation. Their femurs were removed and the femoral contents were flushed into centrifuge tubes with 5 ml of M-199 tissue culture medium* containing 10%) fetal calf serum (FCS) and L-glutamine.24 Total nucleated cell determinations and bone marrow differential counts were obtained as described previously.24 The marrow from each treatment group was pooled, an aliquot was counted on a Coulter Counter, and diluted to I x lOh nucleated cells/ml with culture medium. Culture
of‘marrow
stromal
cells (MSC)
Two miliiliters of M-199 containing I x lOh nucleated cells/ml of pooled bone marrow from each treatment *Victoreen. tMaxitron.
August 1982. Volume 8, Number 8
Table
I. Survival
and marrow stromal colonies of mice after four weekly injections of busulfan*
Doses of busulfan (mg/k)
Marrow stromal colonies* (7%of control)
Survival ratiost
69 32 9
16/16 16/16 l/l6
IO 20 40
*MSC cultured I hour after the last injection. TExpressed as number of survivors over number injected.
of mice
group was cultured in 35 mm tissue culture dishes. The cells were incubated and the media changed as described previously.24 The cells were fixed using methanol, stained with Mallory’s Azure II Methylene Blue and the adherent colonies of 50 or more cells in each dish were counted on day 7. Approximately 20 marrow stromal colonies per dish were obtained from 2 x IO6 normal murine bone marrow cells.
Spleen
colony assay
A modified spleen colony assay of Till and McCulloch,‘” using 750 rad total body irradiation for recipient mice, was used to determine the hemopoietic stem cell count of the right femur following 4 weekly doses of BU, I500 rad L.I.. or leg irradiation followed by the 4 doses of BU. Total body x-irradiation (750 rad) one day before prepared the assay mice for the intravenous injection of I x IO” pooled bone marrow cells from the treated donor mice. On day 7, the spleens from the recipient mice were removed, placed in Bouin’s fluid and the colonies were counted.
Colony forming
units-Culture
granulocyte-macrophage
CFU-C or
colonies
(CFU-GM)
Using 0.3% agar in the method reported by Metcalf,” the pooled bone marrow samples were evaluated for CFU-C content. Colony stimulating activity was obtained using postendotoxin serum from mice which had been injected with 5 pg of E. coli endotoxin.§‘4 The 35 mm culture dishes containing I x IO’ bone marrow cells were incubated for seven days and colonies of 50 or more cells were counted. The average number of CFU-C per I x I OS normal bone marrow cells was 53 -+ 1.4. Assay and “Cr labeling of red blood cells
Autologous red blood cells were labeled using a method similar to that described by Ultmann and Gordon.” Briefly, blood was drawn from the normal and treated mice via cardiac puncture and injected into a sterile vial fM-199, @&ma.
Gibco.
X-ray and busulfan stromal damage 0 L. M. WATHEN et al.
120
1317
BU BU BU BU
0 X r(
011
0
1
2
3 Months
BU 20 mglkg x 4 1500 rad L.I. BU + L.I.
4
5
6
Postirradiation
Fig. I. Total nucleated cells per femur. For this and subsequent figures: L.I. = right hind leg irradiation, BU = busulfan injection, arrow indicates that irradiation was given on day zero, n = number of mice per point, shaded areas = normal + standard error of the mean (SEM), error bars = *SEM.
0.15 ml of acid citrate dextrose.? The red blood cells (RBC) were labeled in vitro by the addition of 5’Cr (Na, “CrO,$ 20-30 $Zi/ml of blood) and incubated for 30 minutes at room temperature (26OC). The RBC incubation was terminated with the addition of 8 to 10 mg of ascorbic acid/ml of blood. The RBC were washed twice with sterile saline and reconstituted with saline to the original volume withdrawn from the mouse. The labeled blood (0.5 ml) was injected via the tail vein into the same mouse from which it was withdrawn. At specified times following “0-RBC injection, tissue and blood samples from the recipient animals were removed and counted in an Auto-gamma Spectrometer* using a window from 280400 keV. containing
Vascular patency following 1500 rad leg irradiation and busulfan Mice were irradiated and treated with busulfan in the same regimen as that describe above. Briefly, on day 0, mice received 1500 rad leg irradiation followed by 4 weekly doses of busulfan (20 mg/kg) beginning on day 7. On day 60, the mice were injected with autologous “Cr-RBC. Twenty-four hours after the “Cr-RBC injection, 20 ~1 blood sampleis, femurs and spleens from the recipient mice were removed and counted. Statistical treatment of data These data are the result of two replicate studies. All data, except the “Cr investigation, were analyzed using a two-way analysis, of variance” to compare drug and radiation interaction. In the “Cr study, data were analyzed using the student’s t test.
7250 mg sodium citrate, 120 mg dextrose, 80 mg citric acid dissolved in 10 ml of sterile water.
RESULTS Mouse weight and peripheral blood Most of the mice gradually increased in weight during the 6 months post-irradiation. The busulfan treated animals did not experience a steady weight increase, and animal deaths occurred at 2-3 months. Peripheral blood leukocytes were transiently depressed for 2 months to less than 60% of normal and hematocrits were depressed for one month to about 85% of normal in mice treated with busulfan, but later they returned to normal. There was no sign of inflammation in the irradiated leg, though slight ephemeral thinning of hair occurred. Total nucleated cells Fifteen hundred rad leg irradiation (1500 rad L.I.) inhibited the repopulation of nucleated cells to less than 70% of normal at 1, 2, and 4 months post-irradiation indicating induction of a residual femoral marrow lesion (Fig. 1). Busulfan suppressed femoral nucleated cells to a similar extent at one month. In the busulfan treated animals gradual recovery occurred to about 80% of normal at 4 months but declined to about 60% at 6 months post-irradiation. The combined modalities produced a consistent pattern of suppression to about 40% through 4 months and rose to about 70% of normal at 6 months postirradiation. Bone marrow differential counts Radiation alone persistently suppressed myeloid (myeloblasts, myelocytes, neutrophils) cells to about 70% of normal (Fig. 2). Busulfan alone depleted the myeloid population to 50% at one month with gradual increase to
$New England Nuclear. *Packard.
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Radiation Oncology 0 Biology 0 Physics
+
1982. Volume
8, Number
8
BU BU BIJ BU
n=8-10
0 BU 20 mglkg x 4 rad L.I. M BU + L.I.
X 1600
OO 1.
* * 11
.
.
21
.
.
.
31
.
*
*
41.
.
.
51.
.
61
*.
Months Postirradiation Fig. 2. Absolute
number of myeloid (myeloblasts,
and neutrophils)
per femur
post-irradiation. Erythroblasts experienced a transient overshoot after one month of busulfan alone, remained at 75-85s at 2 and 4 months, and plunged to less than 20% at 6 months (Fig. 4). in mice receiving X ray alone, erythroid cellularity stayed at 60-70s of normal from I to 4 months post-irradiation and returned to near normal levels at 6 months. In contrast, combined therapy suppressed erythroid cells to 30P45% of normal from I to 6 months post-irradiation.
about 80% of normal through 6 months post-irradiation. At 6 months, the bone marrow of busulfan-treated mice contained 2.5 times as many early myeloid cells as the control animals suggesting an intensive myeloid hyperplasia in these mice. The combined therapy produced a myeloid diminution greater than either modality alone which remained below 60% of normal through 4 months but recovered to a single modality level (70-8091) by 6 months. There were not excessive numbers of blasts. Radiation alone and busulfan alone produced similar depletions of lymphoid cellularity and recovered to 7080% of normal at four months post-irradiation (Fig. 3). At 6 months, the busulfan-treated mice showed a pronounced suppression of lymphoid cells to 25Y~ of normal. Combined radiation and busulfan treatment persistently suppressed lymphoid cells to less than 30% of normal through 4 months with recovery to about 55% at 6 months 120
myelocytes,
Marrow srromal crlls (MSC‘) Femoral MSC were depressed to values 28% to 42% of normal during the 4 months post-irradiation following radiation alone (Fig. 5). By 6 months, they had recovered to 80% of normal. The systemic agent, busulfan, suppressed MSC values to less than 40% of normal at I, 4, and 6 months with an abortive rise in these supporting
n = 8-18
BU BU BU BU
P
01.“‘. 0
1
.
‘I*
2
.“‘. Months
Fig. 3. Absolute
3
.I*.
4
Postirradation
number of lymphoid cells per femur.
.
“*
5
”
6
X-ray and busulfan
stromal
damage
??L.
1319
M. W&THEA et al.
n = 8-16
20 -
0
BU 20 mg/Kg x 4 L.I.
*..*
X 1600 rad
“4
w. BU + L.I. 0
0
““““,““““.“...’
1
2
3
4
5
6
Months Postirradiation Fig. 4. Absolute
number of erythroblasts
cells at 2 months post-irradiation. The combined radiation and busulfan treatment caused persistent suppression of MSC to less than 25% through 4 months and rose to about 45% of normal at 6 months. Stem cells and progenitws The hemopoietic stem cells (CFU-S) declined from 75% at one month to 52% of normal at 6 months postirradiation following X ray alone (Fig. 6). CFU-S in mice treated with busulfan alone reached a nadir (5%) at one month, plateaued at 45%) at 2 and 4 months and dropped to 10% at 6 months. The combined treatment depleted CFU-S to less than 30% of hormal through 6 months post-irradiation indicating a persistent stem cell deficit. Granulocyte-macrophage precursors (CFU-C) remained below 10% for 4 months with combined modality treatment, 25% with BU alone and 45% with X ray alone (Fig. 7). At 6 months, the CFU-C recovered to 55% with
per femur.
irradiation and 43% with combined radiation and busulfan treatment but remained at 25% with BU alone. Vascular patency The ingress of “Cr-RBC into the irradiated femurs of combined modality treated mice was limited to 75% of the activity found in normal femurs. (Fig. 8) Irradiation alone caused less severe inhibition and BU alone failed to cause inhibition of “Cr-RBC entrance into the femoral vascular space. Thus, a portion of the microenvironmental damage seen at 2 months in the 1500 rad L.I. + BU study was the result of occlusion of the microvasculature. DISCUSSION
These studies have shown that sequential therapy with irradiation followed by busulfan administration caused residual injury to the bone marrow microenvironment for at least 6 months. The failure of hemopoietic and marrow
n-8-10
Months Postirradiation Fig. 5. Absolute
number of marrow stromal cell (MSC)
colonies per femur
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0 Physics
August 1982, Volume 8, Number
8
??BU 20 m2lKg x 4 X 1600 rad L.I.
M BU + L.I.
1
1.. OO
1
.
.
.
1
1
1..
“.
2 Months
Fig. 6. Absolute
stromal
cells
to repopulate
the
number
femoral
of hemopoietic
bone
*
1..
.
4
3
‘.
.
*
1
8
5
Postirradiation
stem cells or splenic
marrow
confirmed the presence of this lesion. The hemopoietic precursors (CFU-S and CFU-C) were more severely suppressed by the combined modality treatment than their progeny (myeloid, erythroid and lymphoid cells). It is possible that these precursors were more committed to differentiation than to self-replication. However, Chervenick and Boggs’ found that, in total body irradiated mice, when stem cells were depleted below 10% of normal, self-replication occurred in place of differentiation. Above IO%, both differentiation and selfreplication were present but self-replication took priority until normal CFU-S numbers were attained.5 Investigations in our laboratory demonstrated that after partial body irradiation, the stem cell compartment had to attain a critical level of repletion before responsiveness to endogenous erythropoietin could induce splenic erythropoiesis6 In addition, others have found that CFU-S surviving treatment with busulfan, self-replicated at the expense of differentiation.’ Trainor and co-workers have offered an
colonies (CFU-S)
per femur.
alternative hypothesis for the hemopoietic precursor impairment caused by busulfan.20 They stated that stem cells from busulfan-treated mice do not differ in their probability for either self-renewal or differentiation but rather suffer from impaired proliferation in the stem cell compartment leading to decreased compartment size and to a decreased absolute number of cells differentiating out of it.20 At 6 months after day zero, the mice treated with busulfan alone appear to have a more severe hemopoietic insufficiency than the mice treated with radiation or combined radiation and busulfan. The excessive number of myeloid blast cells and the severe erythroid depletion suggested that these animals were preleukemic. The characteristics of preleukemia in humans have been described by Linman and Bagby.” There is additional evidence for the preluekemic activity of busulfan in a report involving long-term administration of busulfan to patients who had had bronchial carcinoma.16 In this study, four of the 243 patients treated with busulfan
n=e-16
0 BU 2OmgIKg x 4 )( 1500 rad L.I. H BU + L.I.
Months
Fig. 7. Absolute
number
Postirradiation
of granulocyte-macrophage
colonies
(CFU-C)
per
femur.
X-ray
._._
R L FEMUR
R L FEMUR
1'500RAD LI
NORMAL
and busulfan
stromal
R L FEMUR
R L FEMUR
Bl!
AU + LI
Fig. 8. Radioactivity of 5’Cr-labeled autologous red blood cells in normal, irradiated and busulfan treated mice two months after irradiation. R = right femur, L = left femur, 1500 rad L.I. = 1500 rad right hind leg irradiation, BU = 20 mg/kg BU injected I.P. once each week for 4 consecutive weeks, BU + LI = 1500 rad right hind leg irradiation followed by busulfan injections. (n = 14-l 5 mice per treatment group), *this value is significantly different from R normal (p 5 .OS). Error bars i standard error of the mean. developed
leukemia
com.pared
It is also
possible
to none of the 243 treated
a.nd none of the 249 on placebo.‘6
with cyclophosphamide that
the
excessive
number
of myelo-
in the busulfan-treated animals was partially a result of marrow regeneration. The lack of excessive myeloblasts in the femurs receiving both irradiation and busulfan may either be due to the absence of marrow regeneration or to an insufficient microenvironment for the leukemia cells. In general, the pattern of hemopoietic suppression following sequential radiation and busulfan was similar to that following sequential radiation and cyclophosphamide.23 However, at 4 an!d 6 months post-irradiation, the busulfan-treated mice had a more severe suppression of CFU-S, CFU-C and MSC than mice treated with cyclophosphamide alone. Assessment of these data suggested that, unlike cyclophosphamide, busulfan may: 1) have limited the proliferative capacity of the CFU-S, CFU-C and MSC, or 2) have caused a morphologic or metabolic change in these cells, impeding their migratory capacity blasts
??L. M. WATtlENet al.
damage
1321
or their interaction with other cells. Evidence of such structural and functional abnormalities have been reported in lymphocytes following a regimen of busulfan similar to that used in the experiments reported here.14 The mice were allowed to recover for at least 2 months after the last dose of busulfan but still demonstrated disturbances of a variety of humoral and cellular immune function. The preliminary vascular studies using “Cr-RBC reported in this investigation increased our insight into the factors responsible for residual damage after single and combined modality therapy. As we discussed above, 1500 rad leg irradiation or busulfan alone inhibited the repopulation of nucleated cells to 65-75s or normal in the femur at 2 months post-irradiation. The “Cr-RBC investigation has shown a lack of significant vascular damage in these animals indicating that these residual femoral lesions may be primarily a result of a marrow stromal cell or matrical deficit. In addition, we reported that the combined modalities suppressed nucleated cells to 40% of normal 2 months after irradiation. The present data involving autologus “Cr-RBC demonstrated a vascular defect which was responsible for a 25% decline in cellular ingress following combined modality therapy. If stem cell circulation is similar to red cell circulation, the 35% suppression of nucleated cells, unaccounted for by vascular damage, may be the marrow stromal cell portion of the microenvironmental damage following sequential radiation and busulfan. Our investigation of combined modality therapy, using 1500 rad leg irradiation followed by 4 weekly doses of busulfan, produced residual hemopoietic injury which persisted for at least 6 months post-irradiation. Busulfan alone was more deleterious to the hemopoietic precursors and microenvironment than cyclophosphamide alone. These studies have suggested that when treatment involves sequential radiation and busulfan, it may well be the busulfan dose rather than the radiation dose which determines the extent of hemopoietic damage and ultimate repair. In contrast, previous studies23 have implied that radiation may be the limiting factor following sequential radiation and cyclophosphamide. This suggests the need for further studies on the preleukemic state cause by busulfan and stresses the possibility of late complications following busulfan therapy.
REFERENCES Beschorner, W.E., Pino, J., Boitnott, J.K., Tutschka, P.J., Santos, G.W.: Pathology of the liver with bone marrow transplantation. Effec1.s of busulfan, carmustine, acute graft-versus-host disease and cytomegalo-virus infection. Am. J. Path. 99: 369-386,198O. Boggs, D.R., Boggs, S.S., Chervenick, P.A., Patrene, K.D.: Murine recovery from busulfan-induced hematopoietic toxicity as assessed by three assays for colony-forming cells. Am. J. Hamatol. 8: 43--M, 1980. Botnick, L.F., Hannons, E.C., Hellman, S.: Nature of the hemopoietic stem cell compartment and its proliferative potential. Mood Cells !i: 195-210, 1979.
4. Castro-Malaspina, H., Gay, R.E., Resnick, G., Kapoor, N., Meyers, P., Chiarieri, D., McKenzie, S., Broxmeyer, H.E., Moore, M.S.A.: Characterization of human bone marrow fibroblast colony-forming cells (CFU-C) and their progeny. Bfood 56: 289-301, 1980. 5. Chervenick, P.A., Boggs, D.R.: Patterns of proliferation and differentiation of hematopoietic stem cells after compartment depletion. Blood 37: 568-580, 197 1. 6. DeCowin, R.L.: Erythroid differentiation during stem cell proliferation. J. Lab. Clin. Med. 70: 23-25, 1967. 7. Delmonte,
L.: Effect of myleran
on murine hemopoiesis.
I.
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8.
9.
IO. II
12.
13.
14.
15.
16.
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Granulocytic cell line specificity of action on progenitory cells. Cell Tissue Kiner. 11: 347-358, 1978. Delmonte, L.: Effect of myleran on murine hemopoiesis. II. Direct and host-mediated action on proliferative capacity and differentiation bias of spleen colony forming units 1978. (CFU-S). Cell Tissue Kinet. 11: 359-367, Korbling, M., Elfenbein, G.J., Tutschka, P., Santos, G.W.: Exaggerated blood CFU-C rebound in patients with acute leukemia following busulfan (BU) and cyclophosphamide (CY) and bone marrow transplantation. (Abstract). E.xp. Hematol7: (Suppl.) 57, 1980. Linman. J.W., Bagby, G.C.: The preleukemic syndrome (hemopoietic dysplasia). Cancer 42: 854-864, 1978. Metcalf, D.: Hemopoietic colonies: In virro cloning of normal and leukemic cells. Recent Results Cancer Res. 61: 15-17.1977. Morley, A., Blake, J.: An animal model of chronic asplastic marrow failure. I. Late marrow failure after busulfan. Blood 44: 49956, 1974. Morley, A., Trainor, K.. Blake, J.: A primary stem cell lesion in experimental chronic hypoplastic marrow failure. Blood 45: 681-688, 1975. Pugsley, C.A.J., Forbes, I.J., Morley, A.A.: Immunologic abnormalities in an animal model of chronic hypoplastic marrow failure induced by busulfan. Blood 5: 601~610, 1978. Steel, R.G., Torrie, J.H.: Analysis of variance II: Multiway classifications. In Principles and Procedures of Statistics. New York, McGraw-Hill Book Company, Inc. 1960. pp. 132-160. Stott, H., Fox, W.. Girling, D.J., Stephens, R.J., Dalton, D.A.B.: Acute leukemia after busulfan. Br. Med. J. 2: l513-1517,1977.
August 1982, Volume 8. Number 8
17. Thomas, D.E., Buchner. C.D., Banaji, M., Clift, R.A., Fefer. A., Flournoy, N., Goodell. B.W., Hickman, R.O.. Lerner. K.G., Neiman, P.E., Sale, G.E., Sanders, J.E., Singer, J.. Stevens, M.. Storb, R., Weiden. P.L.: One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 49: 5 I l-533, 1977. 18. Thomas, D.E.. Buckner. C.D.. Clift. R.A., Fefer, A., Johnson, F.L., Neiman, P.E., Sale, G.E., Sanders, J.E., Singer, J.W., Shulman, H.. Storb. R., Weiden, P.L.: Marrow Transplantation for acute nonlymphoblastic leukemia in first remission. New, Eng. J. Med. 301: 5977599. 1979. 19. Till, J.E.. McCulloch, E.A.: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radial. Res. 14: 213-222. I96 I. 20. Trainor, K.J., Morley, A.A.. Seshadri. R.S.: A proliferative defect of marrow cells in experimental chronic hypoplastic marrow failure (aplastic anemia). Eup. Hemarol. 8: 674682. 1980. 21. Udupa, K.B.. Okamura, H., Reissmann, K.R.: Granulopoiesis during myleran-induced suppression of transplantable hemopoietic stem cells. Blood 39: 3 177325, 1972. 22. Ultmann. J.E., Gordon, C.S.: Life span and sequestration of normal erythrocytes in normal and splenectomized mice and rats. Acra. Hemat. 33: I I881 26. 1965. 23. Wathen, L.M.. Knapp, S.A., DeGowin. R.L.: Suppression of marrow stromal cells and microenvironmental damage following sequential radiation and cyclophosphamide. Inr. J. Radiat. 0~01. Biol. Ph>js. 7: 935-941, 1981. 24. Werts, E.D., DeGowin. R.L., Kanpp. S.A.. Gibson, D.P.: Characterization of marrow stromal (fibroblastoid) cells and their association with erythropoiesis. E.up. Hemar. 8: 4233433, 1980.