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[CANCER RESEARCH 43, 2339-2341, 0008-5472/83/0043-0000$02.00 May 1983] Radiation Sensitivity of Leukemic Progenitor Cells in Acute Nonlymphocytic Leukemia1 Keiya Ozawa,2 Yasusada Miura, Toshio Suda, Kazuo Motoyoshi, and Fumimaro Takaku3 Division of Hemopoiesis, Institute of Hematology [K. 0., Y. M., T. S., K. M.], and Department of Internal Medicine [F. T.], Jichi Medical School, Tochigi-ken, 329-04, Japan ABSTRACT The radiation sensitivity of leukemic progenitor cells in 12 cases of acute nonlymphocytic leukemia was compared with that of normal myeloid progenitor cells (colony-forming units in culture), using in vitro cloning techniques. The D0 value for normal colony-forming units in culture was almost constant (130 ±14 rads). On the other hand, marked patient-to-patient variations were observed in the radiosensitivity of leukemic progenitor cells; namely, the D0 values in the present cases ranged from 30 to 210 rads. These variations seemed to be partly due to different cell cycle status and partly due to the intrinsic nature of leukemic progenitor cells. Moreover, in six of seven clinically drug-sensitive cases, the leukemic progenitor cells were proved to be relatively radiosensitive. Similar to in vitro drug sensitivity tests, this test may serve to predict the clinical response to chemoradiotherapy. INTRODUCTION In vitro cloning techniques have shown that the bone marrow and peripheral blood of patients with ANLL4 contain progenitor cells capable of forming colonies in culture (2, 8, 15). These colonies were considered to be leukemic in origin for a number of reasons, including the facts that cells within the colonies remained blast-like in morphology (5, 9) and that chromosomal markers characteristic of leukemic clones were identified in some colonies (5,10). In the present study, we applied this culture method to evalu ate the in vitro radiation sensitivity of leukemic progenitor cells in comparison with that of normal myeloid progenitor cells (CFUC). Radiation sensitivity of normal hemopoietic progenitor cells has been measured by several investigators (11,18), but there has been no information about leukemic progenitor cells. Inter estingly, marked patient-to-patient variations were observed in the radiosensitivity of leukemic progenitor cells, in contrast to the almost constant results observed for normal myeloid progen itor cells. In this paper, we discuss these variations in radiation sensitivity of leukemic progenitor cells. MATERIALS AND METHODS Patients. Twelve patients with ANLL were studied (Table 1). In our laboratory, about 70% of patients showed leukemic colony growth by 1Supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and from the Ministry of Education. Science, and Culture of Japan. 2 Present address: Division of Hematology, The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113, Japan. To whom requests for reprints should be addressed. 3 Present address: The Third Department of Internal Medicine, Faculty of Medi cine, University of Tokyo, Hongo, Tokyo 113, Japan. 'The abbreviations used are: ANLL, acute nonlymphocytic leukemia; CFU-C, colony-forming units in culture; E-rosette, erythrocyte rosette; PHA, phytohemagglutinin; dThd, thymidine. Received June 3, 1982; accepted February 1, 1983. the leukemic progenitor assay (13). Peripheral blood or bone marrow specimens obtained from the patients in the present study were proved to contain a sufficient number of leukemic progenitor cells for the analyses described below. The type of leukemia was classified according to the clinical data and cytochemical findings obtained by dual esterase staining (7). The present cases were treated with combination therapy consisting of Ar*-behenoyl-1-/3-D-arabinofuranosylcytosine (170 mg/sq m/day from Day 1 to Days 10 to 15), daunomycin (25 mg/sq m/day on Days 1 and 2) or aclacinomycin (14 mg/sq m/day from Day 1 to Days 10 to 15), 6mercaptopurine (100 mg/body/day from Day 1 to Days 10 to 15), and prednisone (20 mg/sq m/day from Day 1 to Days 10 to 15). If sufficient cytoreduction in bone marrow (total nuclear cell count, <15,000/cu mm) and peripheral blood (WBC, <1,200/cu mm) was obtained by the usual or smaller doses of chemotherapeutic agents, such a case was consid ered a drug-sensitive case. If sufficient cytoreduction was obtained by the prolongation of the period of chemotherapy or was not obtained by the intensive chemotherapy, such a case was considered a drug-resistant case. This judgment was used in the first course of induction chemo therapy performed after obtaining the samples. Leukemic Progenitor Assay. Peripheral blood or bone marrow spec imens were obtained from the patients prior to treatment. Clonogenicity was determined by the method of Minden et al. (8) with minor modifica tions. Mononuclear cells were isolated by Ficoll-Metrizoate (Lymphoprep; Nyegaad, Oslo, Norway) with density centrifugation at 400 x g. In order to avoid the formation of T-lymphocyte colonies, sheep E-rosette-forming cells were subsequently removed by the technique described by Minden eÃ-al. (8). About 2% or less of E-rosette-positive cells were left after this resetting technique. Cells from the E-rosette-negative fraction were cultured for colony formation, and the rest were stored in 10% dimethyl sulfoxide (Sigma Chemical Co., St. Louis, Mo.) and 10% fetal calf serum (Flow Laboratories, Inc., Rockville, Md.) at -80° until use. Cryopreserved cells were thawed, washed, resuspended, and then used for culture. These fresh or cryopreserved cells were cultured at an appropriate concentration in 0.3% agar in «-medium (Flow) containing 20% fetal calf serum and 10% PHA-stimulated leukocyte-conditioned medium. PHAstimulated leukocyte-conditioned medium was obtained from the supernatants of cultured leukocytes (1 x 106 cells/ml) incubated for 7 days in «-medium with 10% fetal calf serum and 1% PHA (Wellcome HA-15). Culture plates were incubated for 7 to 9 days at 37°in a fully humidified atmosphere containing 5% COi in air. Colonies containing 20 or more cells were counted with an inverted microscope. Fresh cells were cultured also in 0.8% methylcellulose (Dow Chemical Co.) instead of agar under the otherwise same conditions. Cells within a part of the colonies were pooled and tested for E-rosette formation. T-lymphocyte colony forma tion was not observed in the present cases. Normal CFU-C Assay. Normal bone marrow specimens were obtained from healthy human volunteers after written informed consent. CFU-C assay was performed by the single-layer soft agar method of Robinson ef al. (17) with minor modifications. Human placenta! conditioned medium was used as the source of colony-stimulating factor (3). Morphological were obtained by by the membrane The preparations Examination. Permanent preparations of colonies the method of Kubota ef al. (6) for agar cultures and filtration technique for methylcellulose cultures (12). were stained with Wright-Giemsa, and microscopic examination was performed. Irradiation. The radiation sensitivity of leukemic progenitor cells and MAY 1983 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research. 2339 K.Ozaiva ei al. Table 1 Clinical data and the nature (radiosensitivity, cell cycle status, and plating efficiency) of leukemic progenitor cells in 12 cases of ANLL cell phase" of colonies/ count re value021018019519018514013010013512090707055503 sponse"DROSDRunDRUDRD (x 10»/liter)30529420.26.0840117.625.14.5269308.465.289.4119.613.6Blasts cells1750 1 x 10» (%)4289737594872935739486926994No. (%)1253SBND241800ND9ND41444947NDRadiosensitivityDa statusUURUURUuuRURSampleBM'BMPBPB"PBBM*PB"PBPBPBBMPBPBPBPBPBNuclear Case123456789101112DiagnosisAMML"APLAMMLAMMLAMLAMLAMLAMMLAMMLAMMLAMLAMLClinical ±78 ±52 ±340 ±437 ±652 ±1068±1373±28 ±44±23 ±4260 ±1093 ±940 ±390 ±261 ±102"882855825624665173223112745S 8 Proportion of leukemic progenitor cells in the S phase of the cycle [normal CFU-C, 29 ±6 (mean ±S.D. of 5 experiments)]. " It was judged by the degree of cytoreduction after chemotherapy. Further details are described in "Materials and Methods." c Thirty-seven % dose slope of survival curves (rads) [normal CFU-C, 130 ±14 (mean ±S.D. of 6 experiments)]. d Extrapolation number of survival curves [normal CFU-C, 1.1 ±0.1 (mean ±S.D. of 6 experiments)]. "AMML, acute myelomonocytic leukemia; U, untreated case; BM, bone marrow; DR, drug resistant; APL, acute promyelocytic peripheral ' These 9 Mean " Fresh leukemia; DS, drug sensitive; PB, blood: R, case ¡nrelapse; ND, not done; and AML, acute myeloblastic leukemia. samples obtained before treatment. ±S.D. cells were cultured for colony formation. normal CFU-C was assessed by in vitro irradiation with 137Cs at 131 too rads/min (Gammacell-40; Atomic Energy of Canada, Ltd.) prior to culture (18). Cell Cycle Status. The proportion of progenitor cells synthesizing DNA was determined using the [3H]dThd suicide technique (4). Test cells were incubated for 20 min at 37°¡nthe presence of [3H]dThd (Amersham 10- International, Ltd.) at a final concentration of 20 «¿Ci/ml. The specific activity of the radioisotope was 25 to 50 Ci/mmol. Control study without [3H]dThd was done in parallel. In early experiments, additional controls were used; i.e., test cells were incubated with [3H]dThd (20 /¿Ci/ml)in the presence of an excess of unlabeled dThd (100 ng/ml). Killing effect of [3H]dThd was completely abrogated by the addition of unlabeled dThd. Following incubation, the cellular uptake of the [3H]dThd was stopped by the addition of ice-cold «-medium containing unlabeled dThd at 100 Mg/ml, and the cells were washed 3 times. Then, the cells were cultured for colony formation. Unlabeled dThd (10 i»g/ml)was added to the culture mixture to exclude the possibility of inactivation of colony formation due to carry-over of radioactive nucleotides. The percentage of kill was calculated as (control - experiment)/control. 100 200 300 400 500 Radiation dose, rads Chart 1. Radiation survival curves of leukemic progenitor cells (•)and normal CFU-C (O). RESULTS Colonies were formed ¡n12 cases of ANLL. As summarized in Table 1, there were marked individual variations in plating efficiency. A large number of colonies were formed in acute myelomonocytic leukemia cases. Cells within the colonies formed in leukemic patients were more immature in morphology than those in normal myeloid colonies when examined in WrightGiemsa-stained preparations (12), although some degree of mat uration of cells was observed in most cases. Radiation survival curves for colony-forming ability of leukemic progenitor cells and normal CFU-C were shown in Chart 1. The radiosensitivity of normal CFU-C was almost constant with the DO value (the dose of irradiation required to reduce the cell survival to 37% in the exponential portion of survival curve) of 130 ±14 rads (mean ±S.D. of 6 experiments; range, 110 to 155 rads). The n value (extrapolation number of survival curve) was 1.1 ±0.1 (S.D.). 2340 In contrast, marked patient-to-patientvariations were ob served in radiation sensitivity of leukemic progenitor cells; namely,theD0valuesrangedfrom 30 to 210 rads(Table1).The DOvaluesof cryopreservedcells were similarto those of fresh cells(Cases3 and4), indicatingthat thefreezingproceduredoes not significantlyaffectthe resultsof the radiosensitivitytest. The Dovalueswerealsosimilarfor bonemarrowandperipheralblood of the samepatientstaken at the sametime (Cases2 and 4). The variationsof radiosensitivityof leukemicprogenitorcells were not relatedwith the clinicaltypes of leukemiaor clinical status (untreatedor relapse)(Table1).Therewas also no rela tionshipbetweenthe radiosensitivityandplatingefficiency. Cellcyclestatusof leukemicprogenitorcellswas determined in 9 cases,usingthe [3H]dThdsuicidetechnique,to investigate whetherradiosensitivitywas influencedby the cell cyclestatus. Proportionof normalCFU-Cin S phasewas almostconstant(29 ±6%, mean±S.D.of 5 experiments).As summarizedin Table CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research. Radiation Sensitivity of Leukemic Progenitors 1, however, there were patient-to-'patient variations in cell cycle status. In patients who had a high proportion of leukemic pro genitor cells in S phase, the D0 values of leukemic progenitor cells were low (Cases 7 to 12), except in Case 2. The leukemic progenitor cells of some patients were in a non- or slowly cycling state (Cases 1, 3, 4, and 6). In these patients, the leukemic progenitor cells were relatively radioresistant. The Do values of leukemic progenitor cells were compared between 4 drug-resistant cases (Cases 1, 3, 4, and 6) and 7 drug-sensitive cases (Cases 2 and 7 to 12). The mean D0 values were 160 ±39 (S.D.) and 79 ±48 (S.D.) rads, respectively. Particularly, in 6 of 7 drug-sensitive cases, the leukemic progen itor cells were relatively radiosensitive. Cell cycle status of leukemic progenitor cells seemed to be also related with the clinical response to chemotherapy. The leukemic progenitor cells of 4 drug-resistant cases were in a non- or slowly cycling state. In patients who had a high proportion of leukemic progenitor cells in S phase, chemotherapy was apparently effective (Cases 2 and 8 to 11). DISCUSSION Radiation sensitivity of normal hemopoietic progenitor cells has been reported to be almost constant by several investigators (11, 18), although a slight difference in the D„values was observed between pluripotent hemopoietic progenitor cells (CFUGEMM) and committed myeloid progenitor cells (CFU-C) (11). The former cells were shown to be slightly radiosensitive. The present study showed that the radiosensitivity of leukemic pro genitor cells was markedly different from patient to patient. On the other hand, the D0 value for normal CFU-C was almost constant, as reported by other investigators. The variations of radiosensitivity of leukemic progenitor cells were not related with clinical types of leukemia, clinical status, or plating efficiency. Since it has been reported that CFU-C in the S phase of the cell cycle are particularly sensitive to low doses of irradiation (1), we have examined the cell cycle status of leukemic progenitor cells. Minden et al. (9) reported that the blast progenitors in leukemic patients are in a rapidly cycling state. In the present study, however, the leukemic progenitor cells of some patients were in a non- or slowly cycling state. Interestingly, the leukemic progenitor cells in these patients tended to be relatively radiore sistant. However, differences in the proliferative status of leu kemic progenitor cells could not fully explain the variations in radiosensitivity. These variations may be partly due to the intrin sic nature of leukemic progenitor cells. As a clinical application of leukemic progenitor assay, the in vitro drug sensitivity of leukemic progenitor cells has been tested, and significant correlation with the clinical response to chemo therapy was reported (14,16). The radiosensitivity and cell cycle status of leukemic progenitor cells seemed to be also related with the clinical response to chemotherapy in the present limited number of cases examined. Further accumulation of data will be necessary to determine the statistical significance and confirm this preliminary observation. Our present study will be not only valuable to understand the nature of leukemic progenitor cells but also clinically important, because radiation therapy is routinely used at the time of bone marrow transplantation, which is increasingly performed for the treatment of acute leukemia. Similar to in vitro drug sensitivity tests, radiation sensitivity tests of leukemic progenitor cells may serve to predict the clinical response to pretransplant total-body irradiation. ACKNOWLEDGMENTS The authors would like to thank Sachiko Kurokawa, Michiko YoshkJa, Yohko Odaka, and Yasuko Miyazaki for their excellent technical assistance. REFERENCES 1. Broxmeyer, H. E., Galbraith, P. R., and Baker, F. L. Relationship of colonystimulating activity to apparent kill of human colony-forming cells by irradiation and hydroxyurea. Blood, 47: 403-411,1976. 2. Buick. R. N . Till, J. E., and McCulloch, E. A. Colony assay for proliferative blast cells circulating in myeloblastic leukemia. Lancet, 1: 862-863,1977. 3. Burgess, A. W., Wilson, E. A. M., and Metcalf, D. Stimulation by human placental conditioned medium of hemopoietic colony formation by human marrow cells. Blood, 49:573-583,1977. 4. Greenberg. P. L., and Schrier, S. L. Granulopoiesis in neutropenic disorders. Blood, 41: 753-769,1973. 5. Izaguirre, C. A., and McCulloch, E. A. Cytogenetic analysis of leukemic clones (abstract). Blood, 52:287,1978. 6. Kubota, K., Mizoguchi, H., Miura, Y., Suda, T., and Takaku, F. A new technique for the cytochemical examination of human hemopoietic cells grown in agar gel. Exp. Hematol., 8: 339-344, 1980. 7. U, C. Y., Lam, K. W., and Yam, L. T. Esterases in human leukocytes. J. Histochem. Cytochem., 21:1-21,1973. 8. Minden, M. D., Buick, R. N., and McCulloch, E. A. 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Properties of blast progenitors among various types of acute nonlymphocytic leukemia (abstract). Exp. Hematol., 9 (Suppl. 9V 85,1981. 14. Park, C. H., Amare, M., Savin, M. A., Goodwin, J. W., Newcomb, M. M., and Hoogstraten, B. Prediction of chemotherapy response in human leukemia using an in vitro chemotherapy sensitivity test on the leukemic colony-forming cells. Blood, 55: 595-601,1980. 15. Park, C. H., Savin, M. A., Hoogstraten, B., Amare, M., and Hathaway, P. Improved growth of in vitro colonies in human acute leukemia with the feeding culture method. Cancer Res., 37: 4595-4601,1977. 16. Freister, H. D. Prediction of response to chemotherapy in acute myelocytic leukemia. Blood, 56: 361-367,1980. 17. Robinson, W., Metcalf, D., and Bradley, T. R. Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells. J. Cell. Physiol., 69: 83-92,1967. 18. Senn, J. S., and McCulloch, E. A. Radiation sensitivity of human bone marrow cells measured by a cell culture method. Blood, 35:56-60,1970. MAY 1983 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research. 2341 Radiation Sensitivity of Leukemic Progenitor Cells in Acute Nonlymphocytic Leukemia Keiya Ozawa, Yasusada Miura, Toshio Suda, et al. Cancer Res 1983;43:2339-2341. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/43/5/2339 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1983 American Association for Cancer Research.