Download Radiation Sensitivity of Leukemic Progenitor

[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
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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
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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.
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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.
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