Acquired Radioresistance of Hematopoietic Progenitors (Granulocyte/Monocyte Colony-Forming Units) During Chronic Radiation Leukemogenesis1

[CANCER RESEARCH 52, 1469-1476, March 15, 1992] Acquired Radioresistance of Hematopoietic Progenitors (Granulocyte/Monocyte Colony-forming Units) during Chronic Radiation Leukemogenesis1

Thomas M. Seed2 and Lillian V. Kaspar

Biological and Medical Research Division, Argonne National Laboratory, Argonne, Illinois 60439-4833

ABSTRACT modulate the leukemic response of the individual and exposed population at large (3, 8-16). Radiologically, the more impor Protracted exposure of dogs to low daily doses of whole-body ir tant modulators include dose rate, cumulative dose, and dura radiation (7.5 cGy/day for duration of life) elicits a high incidence of tion of exposure; biologically, age, sex, immunohematopoietic myeloid leukemia or related myeloproliferative disorders. Under such exposure, vital hematopoietic progenitors [granulocyte/monocyte colony- competence, and genetic/oncogenetic makeup are clearly im forming units in agar (CFU-GM)] acquire increased radioresistance along portant but are not outweighed by factor(s) that selectively control and alter hematopoietic function either during or fol with renewed proliferative capacity at an early phase of evolving myeloid lowing radiation exposure. One such factor is the "repair com leukemia. To further characterize the expression of acquired radioresist ance by CFU-GM, we evaluated the effects of various exposure rates, petence" of the hematopoietic organ against radiation damage. cumulative radiation doses, and times of exposure and postexposure in In our work with a canine model of chronic radiation-induced several groups of long-lived dogs under two conditions of irradiation: (a) myeloid leukemia, we have identified a myeloid leukemia-prone continuous, duration-of-life exposures at dose rates of 0.3-7.5 cGy/day; phenotype, of which the characteristic hallmark is a hemato and (b) discontinuous, fraction-of-life exposures at dose rates of 3.8-26.3 poietic system (specifically, hematopoietic progenitors) that has cGy/day, with cumulative doses of 450-3458 cGy and postexposure times of 14-4702 days. Results indicated that (a) under protracted continuous acquired increased but aberrant repair capacities along with renewed proliferative capacity (11, 12, 17-19). It would seem irradiation, the degree of radioresistance expressed by CFU-GM in vitro increased markedly in a biphasic pattern with rising daily rates of reasonable that the induction of these preleukemic hemato exposure; (b) under discontinuous, fraction-of-life exposure regimens, poietic changes within susceptible animals under chronic irra elevated levels of radioresistance were expressed and stably maintained diation would be dictated by the same factors that control the by CFU-GM only following large radiation doses accumulated at high expression of overt leukemic disease (e.g., dose rate, total dose, dose rates; and (c) with extended postexposure times, the magnitude of etc.). expressed radioresistance appeared to wane. These results continue to The intent of this study, therefore, is to characterize the support the hypothesis that the acquisition of radioresistance and asso expression of acquired, repair-mediated radioresistance by vital ciated repair functions by vital lineage-committed progenitors, under the hematopoietic progenitors (CFU-GM)3 within chronically ir strong selective and mutagenic pressure of chronic irradiation, is tied radiated dogs, as modulated by (a) rate of exposure, (b) cumu temporally and causally to leukemogenic transformation elicited by ra lative dose, and (c) times of exposure and postexposure. diation exposure.

MATERIALS AND METHODS INTRODUCTION Animals. Outbred beagles were derived from the closed Argonne Under appropriate exposure conditions, ionizing radiation National Laboratory colony; their status, origin, and general manage can act as a potent leukemogen, both in animals and in humans ment have been described (20). A total of 105 beagles were used in this (1-3). In the classic radiation leukemogenesis studies by Upton study: 34 (28 males, 6 females) served as nonirradiated controls, while et al. (4-6), the leukemogenic potential of a standard dose of the remaining 71 (60 males, 11 females) served as irradiated test ionizing radiation was shown to be markedly different when the animals. All dogs were anatomically and physiologically normal and in dose was delivered either acutely (in large single doses) or good health prior to entry into the experiment(s). These animals were part of larger groups under general toxicological evaluation of the long- chronically (in small daily doses over an extended period). At a term effects of chronic, low-dose irradiation. Various hematopathol- total exposure dose of 300 cGy, for example, acute exposures ogical aspects of the latter work, including interim survival and leuke were demonstrated to be 3-fold more effective than chronic mia patterns, have been reported (8-13,15-19,21-27). These extended exposures in inducing myeloid leukemia in RF male mice (3). The "response-sparing" (in this case, leukemia-sparing) effect animal groups comprised 1079 animals (292 nonirradiated control animals and 787 chronically irradiated animals, of which 369 received of protracting the course of exposure is commonly attributed continuous radiation exposures and the remaining 418 received frac to time-dependent repair of radiation-induced damage, regard tionated exposures). These extended animal groups were used in this less of whether the damage is prelethal, premutational, or study for the sole purpose of developing and illustrating leukemic pretransformational in nature (7). incidence rates under various regimens of chronic radiation exposure. From these and related studies, as well as from work in our Animal Irradiation. All irradiated dogs were housed in standard-size own laboratory with canines and chronic radiation exposure, it fiberglass cages just prior to (approximately 2 weeks) and throughout is clear that numerous radiological and biological variables the course of exposure. All experimental procedures, including the initiation of chronic radiation exposure (designated as day 0), were begun when the animals reached young adulthood (i.e., about 400-500 Received 9/13/91; accepted 1/8/92. The costs of publication of this article were defrayed in pan by the payment days of age). Dogs receiving a preset total dose of irradiation (i.e., of page charges. This article must therefore be hereby marked advertisement in discontinuous, fraction-of-life exposures) were maintained during the accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by the U.S. Department of Energy, Office of Health postirradiation period in wire pens. Control animals were caged simi and Environmental Research, under Contract W-31-109-ENG-38 and by U.S. larly. Irradiations were carried out in specially designed live-in exposure Department of Health and Human Services Interagency Agreement Y01-CP- 50503. 3The abbreviations used are: CFU-GM, granulocyte/monocyte colony-form *To whom all requests for reprints should be addressed, at Radiation Hema- ing unit in agar; GM, granulocyte/monocyte; ML, myeloid leukemia; MPD, tology Group, Biological and Medical Research Division (B1M/202), Argonne myeloproliferative disorders; SLD, sublethal damage; !>â€ž.doseestimate to reduce National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439-4833. cell survival by e'1; D,, dose estimate to reduce cell survival below 100%. 1469

Table 1 Test groups, number and sex of animals, number of marrows sampled, and radiobiological and temporal variables range No. marrows at start Age at testing" at testing rate time* dogsSex(M/F)No. sampledAge (days)" (days)Test(days)Age (days)Dose (cGy)Total doseÂ°(cGy)Exposuretime^days)Postexposure

exposures26766624'20/67/06/05/15/117/78586911Â»19"484 Group I. Continuous duration-of-life Â±101400 Â±5134111 Â±1400 Â±3163943 +952657 Â±3173543 Â±0466 Â±2022505 Â±1523805 Â±2022024 Â±117474 Â±9531846 Â±20245096 Â±10771359 Â±46499 Â±5021143 Â±21884922 +583656 Â±1031081409000000TestÂ±390401-34703650-43993781-43371447-35381369-2694393-18400.000.300.751.883.757.5001113Â±306903710 Â± exposuresg22242222228/02/01/12/04/02/02/02/02/01/12/01822242142222403Group II. Discontinuous fraction-of-life Â±10407 Â±15584313 Â±10412 Â±974792 Â±873980 Â±1452 Â±1124502 Â±1133990 Â±17426 Â±1554841 Â±1724276 Â±12558 Â±5424941 Â±5524183 Â±90432 Â±6451039 Â±735300 Â±4475 Â±3754650 Â±3244140 +11426 Â±114749 Â±04241 Â±47400 Â±1763836 Â±1293318 Â±0400 Â±04192 Â±03775 Â±02309 Â±74864-50804244-43814712-48714392-46114032-51494485-5387406-17734642-46574624-487338364140-42440.003.753.757.507.507.507.5012.7512.7512.7526.25045015004501050150034584501050150045001204006014020046135821181703786Â±73 " Average values + SD. * One sample (1/11) excluded from final analyses due to unacceptably low progenitor plating efficiency. ' Three animals (3/24) excluded from final group due to poor marrow/progenitor yields. "*Eleven samples (11/79) excluded from final analyses due to unacceptably low progenitor plating efficiencies. facilities equipped with attenuated MCo -y-ray sources (Gamma Beam The cloning method uses a "feeder" layer [containing IO6 buffy coat Irradiatoâ„¢, Atomic Energy of Canada, Ltd., Ottawa). Daily absorbed leukocytes from control donor dogs and 10% pooled plasma from doses were estimated to be 75% of the delivered air dose. Dosimetrie acutely irradiated (400 cGy) dogs] to support CFU-GM colony forma methods and calculations have been described in detail elsewhere (28). tion in the upper agar layer (containing 0.5 x 10s to 1 x 10s "target" Irradiations were carried out in a near-continuous mode (i.e., 22 h/day, marrow cells/ml). Plates were incubated in 5% CO2 at 37"C for 8 days. 7 days/week, either for duration of life or until predetermined doses had been accumulated). During the 2-h period when the animals were Colonies were counted using an inverted light microscope. Colony type was checked by double-esterase cytochemical staining of whole- not exposed, they were fed, watered, examined, and clinically manipu lated for blood or marrow collections. Nonirradiated control animals mounted agar slabs of randomly selected cultures. Colonies were pre dominately and consistently (>90%) of the granulocyte/monocyte were handled in a similar fashion, except that they were maintained in mixed variety. Colony counts per 10* marrow cells plated were used to cages (or in pens) in adjoining, shielded rooms. Forty-nine animals were irradiated chronically for duration of life at estimate concentrations (or surviving fractions) of CFU-GM within dose rates of 0.30, 0.75, 1.88, 3.75, or 7.50 cGy/day. Twenty-six given marrow cell preparations. animals remained unirradiated and served as zero-dose rate (0 cGy/ To assess the radiosensitivity of CFU-GM in vitro, aliquots of stock day) controls for this group (Table 1). Twenty-two dogs were chronically cell suspensions were irradiated with MCo 7-rays at 25 cGy/min to irradiated for preset exposure periods at dose rates of 3.75, 7.50, 12.75, total doses of 0, 10, 25, 50, 75, 150, or 300 cGy. The surviving fraction or 26.25 cGy/day until doses of 450, 1050, 1500, or 3458 cGy had of CFU-GM within each cell sample was determined by the cloning been accumulated. Following termination of exposure, these dogs were procedure described above. The radiation-induced CFU-GM killing at removed from the irradiation facility and placed in wire pens in shielded each dose level was assessed in terms of inhibition of clonogenic activity anterooms. Eight additional animals remained unirradiated and served relative to the zero-dose control. Minimum cloning efficiencies of more as zero-dose rate/zero-dose (OcGy/day; OcGy) controls for this group. than 10 CFU-GM/10* marrow cells plated (at zero dose) were required Hematology. Hemograms were performed periodically (at about 30- for the analysis and inclusion of dose-response data analyses. Minimum day intervals or depending on clinical status) by standard methods on cloning efficiencies were not reached in approximately 5% of the total each animal (15). Bone Marrow Sampling and Progenitor Cell Enrichment. Bone mar marrow samplings; these samples were restricted to marrows obtained row samples were obtained from either the ilia or humeri of dogs under from animals under irradiation rates of 3.75 and 7.5 cGy/day delivered general anesthesia (Surital, sodium thiamylal; Parke-Davis) by "snap" in the continuous duration-of-life exposure mode. From the calculated aspiration through a pediatrie spinal needle into a 25-ml syringe con fraction of surviving CFU-GM at each of the radiation doses, dose- taining 4 ml of anticoagulant solution (1.1% EDTA, 0.7% NaCl). response curves were constructed via linear regression analyses. The Marrow samples from irradiated dogs, along with samples from non- response parameters of DO,l\, and subcellular target number (n) were irradiated controls, were collected and processed at exposure/postex- derived from these analyses. posure periods listed in Table 1. Granulocyte/monocyte progenitors Data Analyses. All marrow response data, regardless of the parameter (CFU-GM) were enriched from the marrow samples by Ficoll-sodium under assessment, were analyzed in terms of the time-constrained diatrizoate density gradient (1.077 g/liter) sedimentation (11). CFU- response of individual animals. Multiple marrow responses for individ GM-enriched mononuclear cell fractions were collected, washed, and adjusted to final working cell concentrations of 106/ml. ual animals recorded over specific time intervals were pooled, averaged, and assessed against appropriate control groups. These averaged re Progenitor Quantitation and Radiosensitivity Determinations. Stock CFU-GM-enriched marrow cell suspensions (10'/ml) were prepared sponse values, along with calculated SEs, were plotted against either and maintained at or below 10'C prior to, during, and following all in time of radiation exposure (or postexposure time) or daily rate of vitro manipulations that preceded in vitro CFU-GM cell culture assays. exposure. Statistical analyses of the comparative responses of irradiated To quantitate the number of viable CFU-GM within either original and nonirradiated animals were performed by using Student's t test, marrow cell preparations or those irradiated in vitro, a modified Pike/ with levels of significance between group responses recorded as values Robinson double agar layer cloning method was used (11, 12, 29, 30). of P. 1470

GM was noted (Fig. 2): a precipitous decline in the number of marrow CFU-GM during the initial period of exposure (~50- 'ing subgroup 125 days) was followed by a gradual recovery to nearly control 40 levels following extended periods of radiation exposure (>700 UJ days). Z LJ Change in Progenitor Radioresistance as a Function of Expo Q sure Time. Under a continuous exposure rate of 7.5 cGy/day, Ãœ the radiosensitivity of marrow CFU-GM from long-lived MPD- prone dogs markedly changed with both time of exposure and preclinical phase progression (Fig. 3). During the initial phase of exposure (<100 days), marrow CFU-GM from these dogs expressed a high degree of radiosensitivity (D0 Â«50 cGy), O 0.30 0.75 1.88 3.75 7.50 12.8 26.3 whereas following extended exposure periods (>300 days), pro RATE OF EXPOSURE genitors expressed markedly increased levels of radioresistance (cGy/doy) (Do ~ 150 cGy; P < 0.02 relative to either age-matched controls Fig. 1. Incidence of MPD versus the daily radiation dose rate under continuous, or expÃ©rimentaisless than 100 days). The time frame for the duration-of-life exposure. transition between radiosensitive and radioresistant clonotypes was 100-200 days and occurred in concert with the initial preclinical phase transition, i.e., preclinical phase 1 (suppres sion) to preclinical phase 2 (partial recovery). (See Refs. 13,17, 19, 23, and 24 for additional information on preclinical phase characteristics.) The SLD capacity of marrow CFU-GM from long-lived dogs 2 Ãœ showed similar changes dependent on time of exposure, i.e., marginal SLD capacities (Dv < 10 cGy) expressed during early phases of exposure (<100 days) and extended capacities (D9 Â«

180

160 DAYS OF EXPOSURE Fig. 2. Time-dependent changes in number of marrow-derived GM-committed hematopoietic progenitors (CFU-GM) of long-lived (>200 days), MPD-prone UJ dogs under a continuous, duration-of-life radiation exposure regimen (7.5 cGy/ T day). Average values Â±SEs plotted. A i

RESULTS \

Responses under Continuous, Duration-of-Life Exposure 20 Regimens 200 400 600 800 Incidence of Myeloproliferative Disease. The overall incidence DAYS OF EXPOSURE of MPD within groups of dogs continuously exposed to low Fig. 3. Time-dependent changes in the radiosensitivity of GM progenitors of daily doses of whole-body 7-irradiation increased from a low long-lived dogs under continuous, duration-of-life exposures (at 7.5 cGy/day). value of 1.4% at the lowest dose rate tested (0.30 cGy/day) to Average values Â±SEs plotted. a high value of 41.7% at 3.75 cGy/day (Fig. 1). At higher rates (7.5-26.25 cGy/day), the incidence of MPD declined concom- itantly with a marked reduction in average survival time result ing from hematopoietically ablative hypoplastic/aplastic mar row diseases. In contrast, eliminating the short-lived dogs (i.e., dogs surviving less than 300 days, which represent 60.3% of the group at 7.5 cGy/day and 73.3% of the group at 12.75 cGy/ day) from the analyses, the incidence of MPD continued to rise at the 7.5 and 12.75 cGy/day dose rates, reaching incidences of 45.5% and 50.0%, respectively (Fig. 1). At the highest dose rate tested (26.25 cGy/day), all of the dogs (100%) exhibited ablative marrow responses to chronic radiation exposure and associated V short-term survival patterns (<300 days of survival) without any evidence of evolving MPD. -200 400 600 Change in Progenitor Number as a Function of Exposure Time. 800 In long-lived, MPD-prone dogs continuously irradiated under DAYS OF EXPOSURE Fig. 4. Time-dependent changes in sublethal damage capacity of GM progen the potent leukemogenic exposure regimen of 7.5 cGy/day, a itors of long-lived dogs under continuous, duration-of-life exposures (at 1.5 cGy/ common time-dependent response pattern of marrow CFU- day). Average values Â±SEs plotted. 1471

200 range of higher exposure rates (7.50 to 1.88 cGy/day). At the 180 lower range of exposure rates tested (0.75 to 0 cGy/day), the 160 SLD capacity again was substantially elevated at 0.75 cGy/day

140 relative to the controls (P < 0.0001) and decreased at an estimated rate of ~24 cGy with further unit reductions in the 120

100. daily dose rate (Fig. 6).

8Â° Responses under Discontinuous, Fraction-of-Life Exposure 60- Regimens Incidence of Myeloproliferative Disease. Low incidences of MPD occurred within the discontinuous, fraction-of-life expo sure groups, whether the groups were analyzed in their entirety -101 23456789 10 or in segregated "high-risk" subpopulations, e.g., long-term RATE OF EXPOSURE (cGy/day) survivors (Fig. 7). Relatively low peaks of MPD incidence (about 11%) occurred in groups irradiated at 7.5 cGy/day to Fig. 5. Change in radioresistance of CM progenitors of long-lived dogs (>600 days) relative to the daily rate of radiation exposure delivered continuously for total doses of 1500 and 3000 cGy. Incidences were further duration of life. Average values Â±SEs plotted. reduced (to about 5%) at lower cumulative doses (1050 and 450 cGy), as well as at both lower (3.8 cGy/day) and higher (12.8 and 26.3 cGy/day) dose rates (Fig. 7). Change in Progenitor Number as a Function of Postexposure Time. Regardless of initial daily rate of exposure or cumulative radiation dose, at extended postexposure periods, the number of marrow GM progenitors either approached or slightly ex ceeded levels found in marrow from comparably long-lived, nonirradiated control animals (Fig. 8). These minimal differ ences between irradiated and nonirradiated animals were not

10- 60 5 50 -101 23456789 10 RATE OF EXPOSURE 40 LU (cGy/day) g S 30 Fig. 6. Change in sublethal damage capacity of GM progenitors of long-lived o 2 dogs (>600 days) relative to the daily rate of radiation exposure delivered O * 20 continuously for duration of life. Average values Â±SEs plotted. Z io 25-50 cGy) following prolonged exposures (>400 days; P < 0.02) (Fig. 4). In contrast to marked changes in radiosensitivity and suble thal damage capacity exhibited by marrow CFU-GM from the O 0.30 0.75 1.88 3.75 7.50 12.8 26.3 long-lived, MPD-prone dogs, marrow CFU-GM from chroni EXPOSURE RATE cally irradiated, short-lived (<300 days) dogs with evolving (cGy/day) Fig. 7. Incidence of MPD versus the daily radiation dose rate under discontin hypoplastic/aplastic marrow disease(s) failed to exhibit such uous, fraction-of-life exposures. changes (data not shown). Change in Progenitor Radioresistance as a Function of Expo sure Rate. Decreasing the rate of exposure from 7.5 cGy/day to lower dose rates (e.g., about 1.9-0.3 cGy/day) resulted in a 140- substantial reduction (P < 0.001) in the level of radioresistance of marrow CFU-GM from long-lived, chronically irradiated 120-100- dogs (656-3773 days of chronic exposure) (Fig. 5). The net reduction in radioresistance was ~68 cGy (representing about 80- a 40% decline as estimated by the change in D0; Fig. 5). The dose rate-dependent decline in radioresistance occurred in a 60-40-20- biphasic pattern; between the dose rates of 7.5 and 1.0 cGy/ day, the decrement in radioresistance was estimated at ~6 cGy/ cGy of daily dose rate; between 1.0 and 0 cGy/day, the rate of decline was estimated at ~61 cGy/cGy of daily dose rate (Fig. 0-con-0 5). dis-7.50CONTROLdis-0 con-3.75 dis-3.75 con-7.50 The progenitor's SLD capacity declined from the high levels IRRADIATED IRRADIATED observed at 7.5 cGy/day (P < 0.0001) to control-like levels at Fig. 8. Comparison of GM progenitor number in long-lived nonirradiated dogs with dogs chronically irradiated (3.75 or 7.5 cGy/day) under either the continuous 1.88 cGy/day (Fig. 6). A relatively low decrementing rate of (con) or discontinuous (dis) exposure modes. Both control responses and dose ~2.5 cGy/cGy of the daily dose rate was estimated over this rate-specific responses represent pooled data sets. Average values Â±SEs plotted. 1472

about 880 days postexposure (Fig. 12). Associated with this gradual shift from the acquired radioresistant to the radiosen sitive phenotype was the apparent reestablishment of normal POSTIRRAMATION hematological parameters. This response pattern was con trasted with the second dog, which retained markedly elevated levels of radioresistance during its 56-day postexposure survival O I course and died of acute leukemia. Change in Progenitor Radioresistance as a Function of Expo sure Rate and Cumulative Radiation Dose. An extended survey of the radioresistance progenitor phenotype within the marrow of long-lived dogs showed that discontinuous, fraction-of-life exposure regimens were largely ineffective in promoting (or 300 400 500 600 700 800 maintaining) the radioresistant trait (Fig. 13). Only at the DAYS OF EXPOSURE/POSTEXPOSURE relatively high dose rate of 12.75 cGy/day and doses of 1050 Fig. 9. Sequential change in marrow CM progenitor number during the time and 1500 cGy were moderately (but significantly) radioresistant surrounding the termination of chronic radiation exposure (7.5 cGy/day for 461 CFU-GM observed (P < 0.001) (Fig. 13). Dose rates (<12.75 days). Average values Â±SEs plotted. cGy/day), as well as the lower cumulative doses, consistently failed to elicit (or maintain) levels of radioresistance comparable 225-200-175-150-125- to those observed under continuous irradiation. The marrow progenitor's SLD capacity was not extended significantly from control levels (as seen under continuous, duration-of-life exposures) at the time of testing and as a consequence of prior chronic irradiation, despite the relatively

10075 to-,

5025n.IT11I 35 30

con-0 dÂ¡s-0 con-3.75 dis-3.75 con-7.50 dis-7.50 CONTROL IRRADIATED IRRADIATED Fig. 10. Comparison of GM progenitor radiosensitivity in long-lived nonirradiated dogs with chronically irradiated (3.75 or 7.5 cGy/day) dogs exposed under either the continuous (con) or discontinuous (

con-0 dis-0 con-3.75 dis-3.75 con-7.50 dis-7.50 significant, however (P > 0.2). The recovery in progenitor number following termination of chronic irradiation occurred CONTROL IRRADIATED IRRADIATED early and rapidly (<100 days), as suggested by the sequential Fig. 11. Comparison of GM progenitor sublethal damage capacity in long- lived nonirradiated dogs with chronically irradiated (3.7S or 7.5 cGy/day) dogs marrow responses of dogs specifically analyzed during this exposed under either the continuous (con) or discontinuous (dis) exposure modes. period (Fig. 9). Both control responses and dose rate specific responses represent pooled data Change in Progenitor Radioresistance as a Function of Post- sets. Average values Â±SEs plotted. exposure Time. By comparison with the levels of radioresistance of marrow progenitors from long-lived dogs continuously irra diated at 3.75 or 7.5 cGy/day, the radioresistance of marrow progenitors from comparably long-lived, chronically irradiated dogs at extended postexposure periods (>3000 days) was mark edly reduced (P < 0.01) and not significantly different from that of an age-matched, nonirradiateci control group (P < 0.05) (Fig. 10). Comparable relationships were observed in the low levels of SLD capacity in progenitors from dogs treated under discontin uous exposure regimens (Fig. 11). The time course of the reduction in progenitor radioresist ance following termination of chronic irradiation is illustrated in Fig. 12. The high levels of radioresistance noted prior to 200 400 600 800 1000 1200 1400 terminating radiation exposure not only continued during the DAYS OF EXPOSURE/POSTEXPOSURE early postexposure period but dramatically increased in one of Fig. 12. Sequential change in radioresistance expressed by GM progenitors the two dogs under study. Following extended postexposure over a time course of chronic irradiation (at 7.5 cGy/day for 461 days) and times, the latter long-lived animal (bearing the markedly radi- following extended postexposure periods. Differential posi irradiation responses oresistant CFU-GM) exhibited a prolonged, graded reduction of nonleukemic (â€¢)versus leukemic (O) dogs are shown, relative to control in radioresistance, with "control" levels being reestablished at responses of nonirradiated dogs (A). Â»,early responses (0-350 days) of dogs under continuous irradiation (7.5 cGy/day). 1473

180- times of about 125 days were required for the progenitor event 160- to be expressed (at the 7.5 cGy/day dose rate). 140- Like the exposure time variable, the daily dose rate strongly

120- influenced the expression of the acquired progenitor response as well; i.e., the higher the daily rate (within the range of 0.3- 100 7.5 cGy/day), the higher the rate of expression of the aberrant 80- progenitor phenotype. However, at the higher dose rates (12.8 450 cGy cGy/day or higher), long-term survival was often compromised 60 to the extent that minimum exposure times were not reached, 40 thus curtailing progenitorial expression. 20- The total cumulative dose is inexorably linked to both the 0 time of exposure and the daily dose rate. As such, the cumula -40 4 8 12 16 20 24 28 32 tive dose-dependent threshold for the expression of the progen RATE OF EXPOSURE itorial event appeared to be about 1000 cGy, as indicated by (cGy/doy) analyses of progenitor responses from dogs under both contin Fig. 13. Relationship between the levels of radioresistance expressed (in terms uous and discontinuous radiation regimens. It is important to of Do) by CM progenitors and the initial rate of chronic radiation exposure and total accumulative radiation dose to which the animals were originally exposed. note that these time and radiation dose thresholds closely Average values Â±SEs plotted. correspond to the noted thresholds for leukemia induction. In this regard, it is remarkable that despite the range of dose rates high exposure rates (3.8-26.3 cGy/day) and sizable total cu and exposure times, cumulative doses as large as 450 cGy mulative doses of 450-1500 cGy (P < 0.2) (Fig. 14). consistently failed to elicit either the aberrant progenitor re sponse or evidence of evolving leukemic disease in these dogs. In addition to examining the induction and expression of the DISCUSSION aberrant progenitorial event, the stability of the response was examined. Stability is considered an important variable, since Previously we had demonstrated that chronic exposure of dogs to low daily doses of whole-body 7-radiation induced a the acquired progenitor response is seemingly tied to an early phase of aberrant hematopoietic repair during evolving myeloid high incidence of myelodysplasia that often progressed to overt leukemia (17, 18). From the results presented, once the rad ML (10, 13). Under optimal radiological conditions (e.g., 7.5 cGy/day for duration of life), the frequency of these late-arising ioresistance phenotype was acquired under continuous irradia tion and the daily exposure continued, the phenotype appeared myelodysplasic syndromes rose to well over 50% in selected to be extremely stable. This was borne out by the sequential subgroups of dogs (11). We further demonstrated through response of selected animals in which multiple marrow samples sequential analyses of hematopoietic function that a critical were collected and tested and the aberrant progenitor phenotype preclinical event in the evolution of the myelodysplasic syn was expressed for prolonged periods (up to hundreds of days). drome involved an early phase of aberrant hematopoietic repair In contrast, by discontinuing the daily radiation exposure after that mediated a broadly based hematological recovery following critical thresholds (time and total cumulative dose) had been an initial phase of acute hematopoietic suppression (17). Fur reached, the acquired phenotype appeared to become unstable thermore, the induction of the hematopoietic repair/recovery and was eventually lost in the majority of animals during process(es) under chronic irradiation seemingly served to seg regate the entire exposed population into distinct subpopula- prolonged postirradiation periods. Nevertheless, in a number of animals that had been previously irradiated under the more tions with characteristic hematopathological tendencies (e.g., potent leukemogenic exposure regimens (12.8 cGy/day to cu one subpopulation was prone to aplastic anemia, whereas an mulative doses of 1050-1500 cGy), the acquired radioresistance other was prone to myeloid leukemia). Subsequent analyses phenotype continued to be expressed for extremely long postir- demonstrated that the hematopoietic progenitors of the ML- radiation periods (e.g., 3300-4300 days; Table 1). prone dogs taken either during or following recovery exhibited The destabilizing action (in terms of loss of expression of the marked change not only in clonal activity but also in radiation sensitivity. 40-, The original intent of this study was to evaluate the effect of altering selected radiological and temporal variables (i.e., dose 35 rate, total dose, etc.) of chronic irradiation on the expression 30 of radioresistance by hematopoietic progenitors in ML-prone 25- dogs. The results presented here clearly indicate that select 20- changes in exposure variables affected not only the acquisition but also the stability of the rare progenitor phenotype (i.e., 15- radioresistant progenitors), along with associated changes in ID induction frequencies of myelodysplasia and preleukemic and S' leukemic syndromes. First, the extent of exposure strongly dictated the induction frequency; i.e., the more prolonged the O -5 exposure, the higher (and apparently the more stable) the -4 0 4 8 12 16 20 24 28 32 acquired radioresistance by GM progenitors. At the extremes, RATE OF EXPOSURE for example, the induction frequencies for the aberrant progen (cGy/day) itor clonotype and myelodysplasic syndrome ranged from 0% Fig. 14. Relationship between the levels of sublethal damage capacity (in terms to 100% as the duration of exposure (e.g., at 7.5 cGy/day) was of /'â€ž)expressed by GM progenitors and the rate and total dose of chronic extended from 60 to 400 days. Minimum (threshold) exposure exposure. Average values Â±SEs plotted. 1474

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. ACQUIRED RADIORESISTANCE OF CFU-GM radioresistant phenotype) of discontinuing the chronic exposure serve to block differentiative flow, enhance self-renewal, and, was only inferred initially by noting the often marked differ in turn, promote a gain in number of viable progenitors. At the ences in levels of expression within groups exposed to discon extremes, this process would provide a caricature of evolving tinuous versus continuous chronic irradiation. This clonal de- MPD. stabilization was subsequently verified within a small number In summary, the results presented support the concept that of sequentially assessed animals. This assessment served to acquisition by marrow progenitors of the aberrant radioresis highlight the apparent association between the acquisition and tant phenotype is governed by a select set of temporal and maintenance of progenitorial radioresistance and subsequent radiological parameters. Furthermore, acquisition of the pro preleukemic/leukemic transformations and, conversely, the loss genitor trait is associated with evolving myelodysplasic disease of progenitorial radioresistance and the subsequent reversal of and, therefore, many be a useful "high-risk" indicator of im any preleukemic changes that might have occurred. pending disease. Although the results presented indicate that strong temporal relationships exist between the acquisition of progenitorial rad ACKNOWLEDGMENTS ioresistance and the induction and progression of MPD, the question of causal relationships still remains. There are a num The authors acknowledge the excellent assistance of David Tolle, ber of possible causal mechanisms. For example, the acquired Susan Meyers, William Keenan, and Craig Wardrip for their hemato- radioresistance seen in targeted progenitors under chronic ir logical and clinical services; Donald Doyle for computer services and radiation might be due largely to an enhanced, error-prone data management; Gordon Holmblad for assisting in irradiating both animals and cells; June Lear for her secretarial help; and David Nad- repair capacity. As a result of the simultaneous induction and repair/misrepair of genomic lesions, critical genie errors might ziejka for his editorial assistance. We also gratefully acknowledge the general support and guidance of Dr. Tom Fritz. Finally, we thank the accumulate with time and elicit increasingly aberrant but vital progenitorial functions (e.g., self-renewal and differentiation). Argonne animal care specialists for their daily care of the animals and maintenance of the Argonne animal facilities, which are accredited by Although the latter causal mechanism is largely speculative at the American Association of Laboratory Animal Care. this point, we have obtained solid evidence that the acquired radioresistance in marrow progenitors is largely mediated by enhanced and aberrant molecular and cellular repair capacities REFERENCES (17, 18, 31). The absolute fidelity of the repair, however, re 1. Committee on the Biological Effects of Ionizing Radiations, National Re mains to be established. Nevertheless, we as well as a number search Council. BEIR V Report. Radiogenic cancer at specific sites. Leuke of other investigators consider misrepair to be a critical pre- mia. In: Health Effects of Exposure to Low Levels of Ionizing Radiation, pp. 242-253. Washington, D.C.: National Academy Press, 1990. transformational process associated with radiation carcinogen- 2. 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IK chronic radiation-induced myeloproliferative disease: exploitation of a unique Annual Report, Division of Biological and Medical Research, pp. 153-156. canine model. In: R. A. Pelroy and J. F. Park (eds.), Multilevel Health Effects Argonne, IL: Argonne National Laboratory, 1968. Research: From Molecules to Man, pp. 245-255. Columbus/Richland, OH: 29. Pike, B. L., and Robinson, W. A. Human bone marrow colony growth in Battei le Press, 1988. agar-gel. J. Cell. Physiol., 76:77-84, 1970. 18. Seed, T. M., Kaspar, L. V., and Grdina, D. Hematopoietic repair modifica 30. Kaspar, L. V., and Seed, T. M. CFU-GM colony-enhancing activity in sera tions during preclinical phases of chronic radiation-induced myeloprolifera of dogs under acute and chronic -y-irradiation regimens. Acta Hematol., 71: tive disease. Exp. Hematol. (NY), 14:443, 1986. 189-197, 1984. 19. Fritz, T. E., Tolle, D. V., and Seed, T. M. The preleukemic syndrome in 31. Seed, T. M. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. Acquired Radioresistance of Hematopoietic Progenitors (Granulocyte/Monocyte Colony-forming Units) during Chronic Radiation Leukemogenesis

Thomas M. Seed and Lillian V. Kaspar

Cancer Res 1992;52:1469-1476.

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