Int. 1. Radiation Oncology Bid. Phys., Vol. 21. pp. 1553-1560 Printed in the U.S.A. All rights reserved.
Copyright
0360-3016/91 s3.ca + .oo 0 1991 Pqamon Press plc
0 Biology Original Contribution
RADIATION SENSITIVITY OF HUMAN B-LINEAGE LYMPHOID PRECURSOR CELLS FATIHM. UCKIJN, M.D., PH.D. ,lT2JAMESB. MITCHELL,PH.D., 3 VEDAT OBUZ, M.D. ,l CHAE HEONJOOPARK, M.D. ,l KEVIN WADDICK, M.S., ’ NORMANFRIEDMAN,M.S. ,3 LAHCENOUBAHA, M.S., ’ Woo SUNG MIN, M.D. ’ AND CHANGW. SONG, PH.D. ’ ‘Sectionsof Cancerand LeukemiaBiology and RadiationBiology, Departmentof TherapeuticRadiology-Radiation Oncology, ‘Division of Hematology-Oncology-Bone Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455; and 3Experimental Phototherapy Section, Radiation Oncology Branch, National Cancer Institute, Bethesda, MD 20892
We studied the radiation sensitivity of eight immunophenotypicaily distinct B-lineage lymphoid precursor cell (LPC) lines of acute lymphohlastic leukemia (ALL) or fetal liver origin corresponding to discrete developmentai stages of human B-ceii ontogeny. The radiation sensitivity of B-lineage LPC showed a temporal association with the distinct stages of development. FL112 and FL114 fetal liver pro-B ceiis (Stage 0 B-Lineage LPC) with germline immunogiobuUn heavy chaht (&II) genes but rearranged T-cell receptor gamma (T,) genes (Do of FL112 = 80.3 cGy, Do of FL114 = 50.2 cGy), REH ALL pre-pre-B ceiis (Stage I B-lineage LPC) with rearranged IgH and T, genes (D, = 66.1 cGy), and NALM-6 ALL pre-pre-B/pre-B ceiis (Stage II B-lineage LPC) (Do = 50.5 cGy) corresponding to the earliest three stages of human B-lymphocyte development were the most radiation sensitive B-lineage LPC populations. By comparison, KM-3 ALL pre-B (Stage III B-lineage LPC) (D, = 194.7 cGy), IIPB-NULL ALL pre-B (Stage IV B-lineage LPC) (D, = 134.6 cGy), and s&M+ RAJIJNAMALWA early B (Stage Va/b B-lineage LPC) ceii lines (Do of RAJI = 144.0 cGy, Do of NAMALWA = 165.5 cGy) corresponding to the later stages of human B-lymphocyte development were much more radiation resistant. These resuits indicate that the radiation sensitivity of B-lineage LPC decreases during maturation within the B-lineage lymphoid precursor pathway. By comparison, the S-phase index (% of S-phase cells as determined by DNA flow cytometry) or proliferation index (% S + G,M), celluiar protein content, intracelluiar giutathione (GSH) level, glutathione-S-transferase (GST) activity, intracellular pH, or free cytoplasmic calcium concentration did not correlate with the radiation sensitivity of the B-Lineage LPC. Radiation sensitivity, Lymphoid precursor cell, B-lymphocyte ontogeny, B-lineage lymphopoiesis.
The maturation of human B-lineage lymphoid precursor cells into functional B-lymphocytes represents a developmentally programmed multistep process, which is accomparried by a cascade of somatic immunoglobulin and T-cell receptor gamma (y)/delta (6) gene rearrangements (15, 18, 29, 30), as well as a coordinated acquisition and loss of B-lineage differentiation antigens (4, 5, 11, 15, 16, 28, 32, 44, 46-48, 59). The current hypothetical models of human B-lymphocyte ontogeny are based on studies performed on leukemic B-lineage lymphoid precursors from B-lineage acute lymphoblastic leukemia (ALL) patients (3, 11, 15, 44). B-lineage ALL’s are thought to originate from developmental lesions in normal B-lineage lymphoid precursors cells (LPC) during early phases of ontogeny, which
allegedly lead to a maturational arrest at discrete stages of B-lineage lymphopoiesis (3, 15, 44). Therefore, immunophenotypically and genotypically distinct B-lineage ALL cell lines, which reflect normal B-lineage LPC phenotypes corresponding to sequential stages of early B-lymphocyte ontogeny, provide a unique opportunity for radiobiologic studies on subpopulations of B-lineage LPC in the human B-lineage lymphoid precursor cell compartment (44). In addition, EBV-transformed B-lineage LPC cell lines corresponding to the earliest stages of human B-lineage lymphopoiesis have been recently established from human fetal livers (44, 46, 47). Despite the wealth of information regarding the radiobiologic features of human and murine myeloid/erythroid precursor cell populations, very little information is available regarding the radiation sensitivity of human B-lineage
Reprint requests to: Fatih M. Uckun, M.D., Ph.D., Associate Professor, Department of Therapeutic Radiology-Radiation Oncology, Box 356 UMHC, Harvard Street at East River Road, Minneapolis, MN 55455. Supported in part by US Public Health Service Grants R29 CA 42111 and ROl CA-42633, and PO1 CA-21737.
from the National Cancer Institute, DHHS, and Special grants from the Minnesota Medical Foundation, Children’s Cancer Research Fund and Bone Marrow Transplant Research Fund, University of Minnesota. F. M. Uckun is a scholar of the Leukemia Society of America. Accepted for publication 29 March 1991.
INTRODUCTION
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I. J. Radiation Oncology 0 Biology 0 Physics
LPC. The purpose of this study was to examine the radiation sensitivity of human B-lineage LPC at multiple developmental stages. To this end, we analyzed in detail the radiation response of eight immunophenotypically distinct B-lineage LPC cell lines of ALL or fetal liver origin reflecting discrete stages of maturation within the B-lineage lymphoid precursor pathway. Our findings provide unprecedented evidence that the radiation sensitivity of B-lineage LPC decreases during maturation in a developmentally programmed fashion. Stage 0 (Pro-B), Stage I (Pre-pre-B), and Stage II (Pre-pre-B/Pre-B) B-lineage LPC were more radiosensitive than Stage III/IV (Pre-B) or Stage V (Early B) B-lineage LPC. To our knowledge, the present study represents the first report on the radiation sensitivity of human B-lineage LPC and provides the first evidence for its temporal association with discrete developmental stages in human B-lymphocyte ontogeny.
METHODS AND MATERIALS Cell lines The following LPC cell lines were used in this study: FL1 12, pro-B; FL1 14, pro-B; REH, pre-pre-B; NALM-6, pre-pre-B/pre-B; KM3, pre-B; HPB-NULL, pre-B; RAJI, early B; NAMALWA, early B; CEM, pre-T; and MOLT-3, pre-T. FL1 12 and FL1 14 cell lines were established as previously described by EBV-transformation using LPC-enriched mononuclear cells from two fetal livers of 8 week and 14 week gestational age, respectively. NALM-6, KM3, HPB-NULL, CEM, and MOLT-3 cell lines were established using leukemic blasts from ALL patients, while RAJI and NAMALWA cell lines were established using malignant lymphoma cells from Burkitt’s lymphoma patients (29, 30, 32, 34). All cell lines were maintained at 37°C in a humidified 5% CO2 atmosphere in RPM1 1640 medium* supplemented with 10% (v/v) heat-inactivated fetal bovine serum?, 50 pg/mL streptomycin, 50 IU/mL penicillin, 2 mM L-glutamine, and 10 mM Hepes buffer. Irradiation of cells Cells were irradiated using a 13’Cs itradiatorS, as previous described (38, 45, 49, 50). In brief, cell suspensions (1 X lo5 cells/ml) in plastic tissue culture flasks4 were exposed to graded doses of gamma rays ranging from 25 cGy to 400 cGy at 109 cGy/min in a single exposure at 37°C. Irradiations were performed during a log phase and under aerobic conditions. Clonogenic assays The radiation responses of LPC cell lines were evaluated using clonogenic assays, as previously described in detail (34, 5 l-54). In brief, serial dilutions of the control and irradiated test cell suspensions were made, and six replicate samples from each dilution were cultured for 10 *Gibco Laboratories, Grand Island, NY. tHyclone Laboratories, Logan, UT.
November 1991, Volume 21, Number 6
days in 96-well flat-bottom tissue culture plates and assayed for clonogenic growth (51-54). Because this is a quanta1 assay, clones are not counted individually, but a judgment is made as to whether or not clonogenic growth has occurred in a particular well (34). The frequency of culture wells with clonogenic growth (wells containing clones with > 100 cells or diffusely growing cells covering >25% of the well) was used to estimate the number of clonogenic LPC in the original cell suspension by a modification of the Spearman Karber method (51-54). The single dose radiation survival curves were constructed by linear regression analysis, and the Do, D,, and n values were determined from the linear portion of the survival curves according to standard methods (45, 49, 50). Immunophenotyping The surface antigen profiles of the B-lineage LPC cell lines or the control cell lines were determined by immunofluorescence staining and multiparameter flow cytometry, as previously described (46-49). The following monoclonal antibodies (MoAb) were used as phycoerythrin (PE) or fluoresceinisothiocyanate (FITC) conjugates to detect the particular surface antigens on the target cell populations: G3.7 (anti-CD7), 24.1 (anti-CDlO), B43 (anti-CD19), BA-1 (anti-CD24), BU41 (anti-CD72), BU43 (anti-CD74),LN-1 (anti-CD75), CRIS-4 (anti-CD76), 2C3 (anti-IgM), gTA-4 (anti-IgD). The reactivity profiles of these MoAb with human lymphoid precursor cells at various developmental stages were detailed in a recent review article (44). Cell lines were scored as positive (+) for a given antigen if > 20% of cells reacted with the respective MoAb, positive/ negative ( 2 ) if lo-20% of cells reacted with the respective MoAb, and negative ( - ) if < 10% reacted with the respective MoAb. Southern blot hybridization analyses DNA extraction and Southern blot hybridization analyses were performed as previously described (18, 46, 47). The Cp probe is a 1.5 kb clone of the germline constant region gene (kindly provided by Dr. Brian Van Ness) hybridizing a 17 kb germline fragment in BamHl restricted DNA and a 1.3 kb germline fragment in ECOR 1 restricted DNA. The TCR B probe, JUR-B (kindly provided by Dr. Harry Orr) hybridizes to both TCR B constant region gene and TCR B J region and identifies a 10 kb germline EcoRl fragment. The TCR y probe (kindly provided by Dr. John H. Kersey) is a 0.7 kb HINDIII-EcoRI genomic fragment containing the Jy 1 gene identifying 1.5 kb, 3.2 kb germline EcoRI fragments, a 13 kb germline Bar&II fragment, and 2.1, 5.0 kb germline HINDIII fragments. Rearrangements of the IgH, TCR B, and TCR y genes were detected in EcoRI and/or Ban-HI digests of high molecular weight DNA from LPC cell lines using these different probes, as previously described in detail (18, 29, 30, 46, 47). $J.L. Shephard and Assoc., $Falcon, Becton Dickinson,
Glendale, CA, Model Mark I. Oxnard, CA.
Radiation sensitivity of B-lineage LPC 0 F. M.
1555
UCKUN et al.
characterization of human B-lineage LPC cell lines
Table 1. Multiparameter
Genotype
Immunophenotype Cell line
Maturational stage
FL112 FL114 REH NALMd
CD7
CD10
CD19
Pro-B
_
+
+
Pro-B Pre-Pre-B
_
+
CD24
CD72
CD74
CD75
CD76
C&
IgM
+ +
_
_
_
_
_
_
_
+ +
+ +
_
_ _
_
RR
RG
RR
_
+
-
+
_
_
GG
GG
RR
+ *
+ +
+ +
_
_
DD
GG
RR
+
_
GG
GG
RG
_
+
+ •t
Pre-Pre-Bi Pre-B
_
+
+
* _
+
+
+
+
+
+
+
*
IgD
_
TY
TB
GIL
RR RR
GG GG
GG GR
KM-3
Pie-B
_
HPB-NULL NAMALWA
Pre-B Early B
_ _
+
+
+
+
+
+
+
-
+
-
GG
GG
DG
RAJI Controls
Early B
_
t
+
+
+
+
+
+
-
r
+
GG
GG
DG
CEM
he-T
+
_
_
_
-
_
_
_
RR
RR
GG
Pre-T
+
+ _
_
MOLT-3
_
_
_
_
_
RR
RR
GG
Proliferative
_
Biochemical
activity
%Go,,
IS
%G,M
Proliferation index J
Protein (mg/lO’ cells)
GHS level (*/mg)
features
GST activity (nmol/min/mg)
[PHI,
[Ca’+]i (Indo- 1 Ratio)
FL112
Pro-B
49.1
28.9
22.0
50.9
1.4
2.1
126.3
7.24
FL114
Pro-B
65.0
29.7
13.3
43.0
0.5
8.5
N.D
N.D
1.97
REH NALM-6
51.5 51.8
28.5 33.0
17.0 15.2
45.5 48.2
0.4 0.3
0.2 5.3
118.7 58.7
6.93 6.91
2.33 1.91
KM-3
Pre-he-B he-he-B/ pre-B Pre-B
52. I
32.6
15.3
47.9
0.4
5.8
112.4
7.13
1.88
HPB-NULL NAMALWA
Pre-B Early B
52.0 48.8
24.0 36.2
24.0 15.0
48.0 51.2
0.1 0.5
8.3 1.6
55.3 94.8
7.17 7.05
1.83 1.98
RAJI
1.98
1.39
Early B
49.2
31.1
19.7
50.8
0.3
12.7
3.2
7.05
Controls CEM
Pre-T
65.5
31.0
3.5
34.5
0.1
5.3
180.4
7.10
N.D
MOLT-3
Pre-T
73.7
23.7
2.6
26.3
0.4
2.2
N.D
7.40
N.D
Note: The multiparameter characterization of human B-lineage LPC cell lines was performed as de&bed in Materials and Methods. The biochemical parameters, except for the GST activity are presented as the mean values of multiple (3-4) measurements. The standard erors did not exceed 10% of the mean values. GST activity data were obtained by a single measurement. ND = Not determined; RR = both alleles rearranged; GG = both alleles in germline configuration; RG = Are allele rearranged, are allele in germline configuration; DG = One allele deleted are allele in germline configuration; DD = Both alleles deleted.
Analysis of cell cycle kinetics by quantitative DNA j7ow cytometry The UV excited dye, Hoechst 33342, was used to quan-
tify the DNA content of the B-lineage LPC populations, as previously reported (23). Quantitative DNA analysis was performed on a FACStar Plus flow cytometer equipped with Consort 40 computer using the COTFIT Program, which includes CELLCY, a cell cycle distribution function that fits DNA content histograms and calculates the percentages of cells in the Go,, , S, and GzM phases of the cell cycle, as originally described by Fox (12). Measurement of intracellular protein, GSH, GST activity, pH, and free calcium concentration The intracellular protein concentrations were determined
using the Bicinchoninic Acid Protein Assay Kit?. Bicinchoninic acid is a chromogenic reagent highly specific for Cu (I), which forms a purple complex with an absorbance at 562 nm that is directly proportional to the protein con*Becton Dickinson Immunocytometry
Systems,
tSigma Chemical Co., St. Louis, MO. SMolecular Probes, Eugene, OR.
San Jose, CA.
centration. GSH (8, 13, 22, 33, 35, 39, 55) determinations were performed using the GSH reductase procedure (43). The GST activity (2, 24, 56) was quantified by monitoring thioether formation between 1-chloro-2,4-dinitrobenzene and GSH at 340 nm and 25°C with the aid of a recording spectrophotometer, as previously described (20, 22). The intracellular pH in B-lineage LPC populations was determined using the fluorescent indicator 2’-7’-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and a fluorometer, as previously described (14, 31). Cytoplasmic free calcium concentration was measured with the dye Indo-ls and a flow cytometer,** as previously described (46-48). All of these analyses were performed on cells obtained during log phase. RESULTS AND DISCUSSION We studied the radiation responses of eight B-lineage LPC cell lines corresponding to five discrete stages of hu§Perkin-Elmer LS-5, Norwalk, CT. **FACStar Plus, Becton Dickinson, San Jose, CA.
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I. J. Radiation Oncology 0 Biology 0 Physics Table 2. Radiobiological
November 1991, Volume 21, Number 6
features of human B-lineage lymphoic cell lines Radiobiologic
Cell line
Experiment
FL1 12, Pro-B
FL114, Pro-B
REH, Pre-pre-B
no.
Pre-Pm-B/Pre-B
D, (ICY)
2 Mean csc 1 2 3 Mean f SE csc 1 2 3 4 Mean ? SE csc 1 2 Mean csc
92.6 67.9 80.3 67.7 44.7 55.6 50.2 52.0 77.4 62.0 59.0 66.1 e4.7 65.0 45.8 63.8 45.5 47.2 50.5k3.8 58.6 167.8 221.7 194.7 182.9 169.3 120.6 113.8 134.6? 14.5 154.4 204.5 201.3 132.7 123.3 165.5 29.4 169.1 168.3 119.7 144.0 144.4
0.0 -21.0 - 11.0 - 13.3 - 17.8 -31.3 - 24.6 - 27.2 3.1 15.7 - 16.2 0.927.7 7.6 37.2 33.7 - 1.9 49.2 29.5k9.5 23.1 28.1 -51.5 - 11.7 13.4 50.5 78.2 22.9 50.5 * 13.3 27.8 -39.3 -20.3 -24.1 50.1 - 8.4+ 17.3 -6.7 30.9 32.9 31.9 32.3
1.0 0.7 0.9 0.8 0.7 0.6 0.7 0.6 1.0 1.3 0.8 l.O?O.l 1.1 2.2 1.7 1.0 2.8 1.920.3 1.5 1.2 0.8 1.0 1.1 1.3 1.9 1.2 1.520.2 1.2 0.8 0.9 0.8 1.5 1.0+0.1 1.0 1.2 1.3 1.3 1.3
1 2 3 Mean t SE 1 2 3 Mean f SE
65.5 62.0 63.2 63.6r 1.0 51.9 43.2 47.9 47.7k2.5
-5.0 - 24.4 24.9 - 1.5 +- 14.3 - 15.7 1.5 16.3 0.7k9.2
0.9 0.7 1.5 l.OkO.2 0.7 1.0 1.4 l.OkO.2
1 2 Mean csc 1 2 Mean csc 1 3 Mean ? SE csc 1
; 4 Mean ? SE csc
1
KM-3, Pre-B
HPB-NULL,
Pre-B
NAMALWA,
Early B
RAJI, Early B
Controls CEM, Pre-T
MOLT-3, Pm-T
n
Do (cCy1
L
NALM-6,
parameters
Note: Following irradiation, B-lineage LPCs were assayed for clonogenic growth as described in Methods and Materials. The values for the mean % plating efficiency were 1.0 for FL112, 2.4 for FL114, 1.3 for REH, 1.7 for NALM-6, 15.3 for KM-3, 11.7 for HPBNULL, 14.0 for NAMALWA and 7.5 for RAJI. The radiation dose survival curves of B-lineage LPC cell lines were constructed by linear regression analysis. Results are presented as the values for Do, D, and n. All computations were performed using the Statworks Statistics Program for the Macintosh obtained from Cricket Software. CSC = Composite survival curve.
man B lymphocyte ontogeny. Table 1 details their immunophenotype, genotype, proliferative activity, and biochemical
features, including protein content, GSH level, GST activity, [pH]i, and [Ca2+]i. Of these eight cell lines, FL112,
Radiation sensitivity of B-lineage LF’C ??F. M. UCKUNet al.
1557
10 t
OO ID, 20
,
40
,
60
,
60
,
100
,
120
1
140
GST bnol/mWm~$
’ Radiation Dose kGy1 Fig. 1. Radiation sensitivity of B-lineage LF’C at discrete developmental stages. The graphs represent the composite radiation survival curves of B-lineage LPC showing survival of cells as a function of the radiation dose. Cells were irradiated, assayed for clonogenic growth and the radiation survival curves were gener-
ated by linear regression analysis as described in Materials and Methods. See Table 1 for details. Bars represent SE.
FL114, KM-3, and RAJI were analyzed in two independent experiments, REH and HPB-NULL were analyzed in three independent experiments, while NALM-6 and NAMALWA were analyzed in four independent experimerits. T-lineage LPC cell lines CEM and MOLT-3, which were used as control LPC cell lines, were analyzed each in three independent experiments. Throughout the study, each experiment was performed in six replicates. The Do, D,, and n values for the single dose radiation survival curves of each cell line are shown in Table 2. The composite survival curves (CSC) of six representative B-lineage LPC cell lines are depicted in Figure 1. Notably, the Do values showed a pronounced variation among the different cell lines corresponding to distinct stages of development, as determined by their composite immunophenotype and ge-
Fig. 2. Correlation of cellular radiation sensitivity with stage of maturation. The radiation sensitivity of (i.e., D, value of the single dose radiation survival curves) of B-lineage LPC was correlated on a continuous scale with stage of maturation, %S, GSH content and GST activity by single regression. CC = coefficient of correlation. P = p-value (i.e., statistical significance) of the correlation.
notype. FL112 and FL114 fetal liver pro-B cells (Stage 0 B-lineage LPC) with germline immunoglobulin heavy chain (IgH) genes but rearranged T-cell receptor gamma (T,) genes (mean Do = 80.3 cGy, Do of CSC = 67.7 cGy for FL112; mean Do = 50.2 cGy, Do of CSC = 52.0 cGy for FL114), REH ALL pre-pre-B cells (Stage I B-lineage LPC) with rearranged IgH and TCRy genes (mean Do = 66.1 cGy; Do of CSC = 65.0 cGy), and NALM-6 ALL pre-pre-B/pre-B cells (Stage II B-lineage LPC) (mean Do = 50.5 cGy; Do of CSC = 58.6 cGy) corresponding to the earliest three stages of human B-lymphocyte development were the most radiation sensitive B-lineage LPC populations. By comparison, KM-3 ALL pre-B (Stage III B-lineage LPC; mean Do = 194.7 cGy, Do of CSC = 182.9 cGy), HPB-NULL ALL pre-B (Stage IV B-lineage LPC; mean Do = 134.6 cGy, Do of CSC = 154.4 cGy), NAMALWAk4.B early B (Stage Va/b B-lineage LPC; mean Do = 144.0/165.5 cGy, Do of CSC = 144.4/169.1 cGy) cell lines corre-
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I. J. Radiation Oncology
0 Biology 0 Physics
sponding to the later stages of human B-lymphocyte development were much more radiation resistant. Overall, the mean Do value for pro-B (Stage 0 LPC), pre-pre-B (Stage I LPC), and pre-pre-B/pre-B (Stage II LPC) from a total of 11 independent experiments was significantly lower than the mean Do value for pre-B (Stage III and Stage IV LPC) and early B cells (Stage V LPC) from a total of 11 independent experiments (60.1 cGyk4.5 cGy vs 158.5 cGy 2 11.8 cGy, p = 0.0001). This temporal association of the radiation sensitivity of B-lineage LPC with discrete stages of maturation is further illustrated in Figure 2. We next asked whether the radiation sensitivity of B-lineage LPC correlates with any other biologic or biochemical parameters besides their developmental stage (Fig. 2). Notably, the percentage of S-phase cells (coefficient of correlation = 0.1, p = 0.5), cellular protein content (coefficient of correlation = 0.230; p=O.6), intracellular glutathione (GSH) level (coefficient of correlation = 0.175; p = 0.7), glutathione-S-transferase (GST) activity (coefficient of correlation = 0.107; p = 0.8), intracellular pH (coefficient of correlation = 0.415; p = 0.3), or free cytoplasmic calcium concentration (coefficient of correlation = 0.048; p = 0.9) did not correlate with the radiation sensitivity of the B-lineage LPC. Furthermore, there was no correlation between any of these parameters, in particular the %S value, GSH content, or GST activity, and the developmental stage of B-lineage LPC. Thus, the observed differences in Do values of B-lineage LPC corresponding to different stages of development most likely reflect inherent and probably maturation-linked differences in their intrinsic radiation sensitivity. In summary, we studied and compared the radiation
November
199 1, Volume 2 1, Number 6
sensitivity of human B-lineage LPC populations at discrete developmental stages of human B-lineage lymphopoiesis. While expanding our current knowledge of the radiobiologic features of human hematopoietic precursor cells (1, 6, 9, 10, 12, 17, 19, 25, 26, 27, 40, 41, 45, 57, 58), this investigation prompts the hypothesis that the radiation sensitivity of B-lineage LPC decreases during maturation in a developmentally programmed fashion. As discussed in several widely used textbooks of radiation biology, differentiated cells are in general much more radiation resistant than undifferentiated cells, and this influence of the degree of cellular differentiation on radiation sensitivity cannot be explained solely by differences in the mitotic activity of cells (7, 21, 36, 37, 42). According to the well-known but not well-understood rule of Bergonie and Tribondeau, the radiation sensitivity of cells vary inversely with their degree of differentiation (7, 21, 36, 37, 42). Our findings provide strong evidence that this rule also applies for human B-lineage LPC populations. To our knowledge, the present study represents the first report on the radiation sensitivity of B-lineage LPC and provides novel evidence for a temporal association between the radiation sensitivity of B-lineage LPC populations and the discrete developmental stages of human B-lymphocyte ontogeny they represent. Nevertheless, the results should be interpreted with due caution since the conclusions regarding the coordinate sequence of antigen expression or gene rearrangements during human B-lymphocyte ontogeny are obligatorily speculative. Leukemic phenotypes may be altered by clonal evolution during and/or after the malignant transformation of a given B-lineage LPC clone or a misprogramming of differentiation in leukemic B-lineage LPC (44).
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Radiation sensitivity of B-lineage LPC 0 F. M. UCKUNet al. 17. Greenberger, J. S.; Chang, J. S.; Balzuno, S.; Fulmer, S.; King, V. P.; Moloney, W. C. Effects of daily low dose gamma irradiation on growth and differentiation of human myeloblastic leukemia cells in diffusion chambers. Scan. J. Hematol. 27:355-364; 1981. 18. Griesinger, F.; Greenberg, J. M.; Kersen, J. H. T cell receptor gamma and delta rearrangements in hematologic malignancies. J. Clin. Invest. 84506516; 1989. 19. Grilli, G.; Nothdurft, W.; Fliedner, T. M. Radiation sensitivity of human erythropoietic and granulopoietic cells in the blood and in the bone marrow. Int. J. Radiat. Biol. 41:685687; 1982. 20. Habig, W. H.; Jakoby, W. B. Assays for the differentiation of glutathione S-transferases. Meth. Enzymol. 77:398405; 1981. 21. Hall, E. J. Sensitivity of tissues. In: Radiobiology for the radiobiologist. New York: Harper & Row; 1978: 197-202. 22. Hantel, A.; Hilton, J.; Russon, J. E.; Donehower, R. C.; Colvin, 0. M. Glutathione content, glutathione S-transferase profiles and melphalan sensitivity in murine hematopoietic cell lines. Proc. A.A.C.R. 29:297-298; 1988. 23. Hollander, Z.; Shah, V. 0.; Civin, C. I.; Loken, M. R. Assessment of proliferation during maturation of the B-lymphoid lineage in normal human bone marrow. Blood 71, 528-531; 1988. 24. Jakoby, W. B.; Habig, W. H. Glutathione transferase. In: Jakoby, W. B., ed. Enzymatic basis of detoxification, vol. 2. New York: Academic Press; 1980:63-94. 25. Kimler, B. F.; Park, C. H.; Yakar, D.; Mies, R. M. Lack of recovery from radiation induced damaged in human hematopoietic cells. Br. J. Cancer 49:221-225; 1984. 26. Kimler, B. F.; Park, C. H.; Yakar, D.; Mies, R. M. Radiation response of human normal and leukemic hematopoietic cell assays by in vitro colony formation. Int. J. Radiat. Oncol. Biol. Phys. 11:809-816; 1985. 27. Kinsella, T. J.; Mitchell, J. B.; McPherson, S.; Miser, J.; Triche, T.; Glatstain, E. In vitro radiation studies on Ewing’s sarcoma cell lines and human bone marrow: application to the clinical use of total body irradiation. Int. J. Radiat. Oncol. Biol. Phys. 10:1005-1011; 1984. 28. Knapp, W.; Rieber, P.; Dorken, B.; Schmidt, R. E.; Stein, H.; Borne, A. E. G. K. Towards a better definition of human leukocyte surface molecules. Immun. Today 10:253258; 1989. 29. Korsmeyer, S. J.; Arnold, A.; Bakhshi, A.; Ravetch, J. V.; Siebenlist, U.; Hieter, P. A.; Sharrow, S. 0.; LeBien, T. W .; Kersey, J. H.; Poplack, D.G.; Leder, P.; Waldmann, T. A. Immunoglobulin gene rearrangement and cell surface antigen expression in acute lymphocytic leukemias of T cell and B cell precursor origins. J. Clin. Invest. 71:301-313; 1983. 30. Korsmeyer, S. J.; Hieter, P.A.; Ravetch, J. V; Poplack, D. G.; Waldmann, T. A.; Leder, P. Developmental hierarchy of immunoglobulin gene rearrangements in human leukemic pre-B-cells. Proc. Natl. Acad. Sci. USA 78:709&7100; 1981. Cl--,HC03 ex31. Kurtz, I.; Golchini, K. Naf-independent change in Madin-Darby canine kidney cells. J. Biol. Chem. 262:45164520; 1987. 32. LeBien, T. W.; Kersey, J. H.; Nakazawa, S.; Minato, K.; Minowada, J. Analysis of human leukemitiymphoma cells lines with monoclonal antibodies BA-1, BA-2 and BA-3. Leuk. Res. 3:299-305; 1982. 33. Meister, A. Selective modification of glutathione metabolism. Science (Wash. DC) 220:472--177; 1983. 34. Min, W. S.; Song, C. W.; Uckun, F. M. Thermal sensitivity and thermal tolerance of human B-lineage acute lymphoblastic leukemia (ALL) cells. Int. J. Radiat. Oncol. Biol.
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56. Wang, A. L.; Tew, K. D. Increased glutathione-stransferase activity in a cell line with acquired resistance to nitrogen mustards. Cancer Treat. Rep. 69:677682; 1985. 57. Weichselbaum, R. R.; Greenberger, J. S.; Karpas, A.; Schmidt, A.; Little, J. B. Radiosensitivity of human hematopoietic cell lines of distinguishable biochemical and physiological stages of differentiation. Radiol. 139:485-487; 1981. 58. Weichselbaum, R. R.; Greenberger, J. S.; Schmidt, A.; Karpas, A.; Moloney, W. C.; Little, J. B. In vitro radiosensitivity of human leukemia cell lines. Radiol. 139:485-487; 1981. 59. Zola, H. The surface antigens of human B-lymphocytes. mun. Today 8:308-315; 1987.
Im-
Copyright
0360-3016/91 s3.ca + .oo 0 1991 Pqamon Press plc
0 Biology Original Contribution
RADIATION SENSITIVITY OF HUMAN B-LINEAGE LYMPHOID PRECURSOR CELLS FATIHM. UCKIJN, M.D., PH.D. ,lT2JAMESB. MITCHELL,PH.D., 3 VEDAT OBUZ, M.D. ,l CHAE HEONJOOPARK, M.D. ,l KEVIN WADDICK, M.S., ’ NORMANFRIEDMAN,M.S. ,3 LAHCENOUBAHA, M.S., ’ Woo SUNG MIN, M.D. ’ AND CHANGW. SONG, PH.D. ’ ‘Sectionsof Cancerand LeukemiaBiology and RadiationBiology, Departmentof TherapeuticRadiology-Radiation Oncology, ‘Division of Hematology-Oncology-Bone Marrow Transplantation, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455; and 3Experimental Phototherapy Section, Radiation Oncology Branch, National Cancer Institute, Bethesda, MD 20892
We studied the radiation sensitivity of eight immunophenotypicaily distinct B-lineage lymphoid precursor cell (LPC) lines of acute lymphohlastic leukemia (ALL) or fetal liver origin corresponding to discrete developmentai stages of human B-ceii ontogeny. The radiation sensitivity of B-lineage LPC showed a temporal association with the distinct stages of development. FL112 and FL114 fetal liver pro-B ceiis (Stage 0 B-Lineage LPC) with germline immunogiobuUn heavy chaht (&II) genes but rearranged T-cell receptor gamma (T,) genes (Do of FL112 = 80.3 cGy, Do of FL114 = 50.2 cGy), REH ALL pre-pre-B ceiis (Stage I B-lineage LPC) with rearranged IgH and T, genes (D, = 66.1 cGy), and NALM-6 ALL pre-pre-B/pre-B ceiis (Stage II B-lineage LPC) (Do = 50.5 cGy) corresponding to the earliest three stages of human B-lymphocyte development were the most radiation sensitive B-lineage LPC populations. By comparison, KM-3 ALL pre-B (Stage III B-lineage LPC) (D, = 194.7 cGy), IIPB-NULL ALL pre-B (Stage IV B-lineage LPC) (D, = 134.6 cGy), and s&M+ RAJIJNAMALWA early B (Stage Va/b B-lineage LPC) ceii lines (Do of RAJI = 144.0 cGy, Do of NAMALWA = 165.5 cGy) corresponding to the later stages of human B-lymphocyte development were much more radiation resistant. These resuits indicate that the radiation sensitivity of B-lineage LPC decreases during maturation within the B-lineage lymphoid precursor pathway. By comparison, the S-phase index (% of S-phase cells as determined by DNA flow cytometry) or proliferation index (% S + G,M), celluiar protein content, intracelluiar giutathione (GSH) level, glutathione-S-transferase (GST) activity, intracellular pH, or free cytoplasmic calcium concentration did not correlate with the radiation sensitivity of the B-Lineage LPC. Radiation sensitivity, Lymphoid precursor cell, B-lymphocyte ontogeny, B-lineage lymphopoiesis.
The maturation of human B-lineage lymphoid precursor cells into functional B-lymphocytes represents a developmentally programmed multistep process, which is accomparried by a cascade of somatic immunoglobulin and T-cell receptor gamma (y)/delta (6) gene rearrangements (15, 18, 29, 30), as well as a coordinated acquisition and loss of B-lineage differentiation antigens (4, 5, 11, 15, 16, 28, 32, 44, 46-48, 59). The current hypothetical models of human B-lymphocyte ontogeny are based on studies performed on leukemic B-lineage lymphoid precursors from B-lineage acute lymphoblastic leukemia (ALL) patients (3, 11, 15, 44). B-lineage ALL’s are thought to originate from developmental lesions in normal B-lineage lymphoid precursors cells (LPC) during early phases of ontogeny, which
allegedly lead to a maturational arrest at discrete stages of B-lineage lymphopoiesis (3, 15, 44). Therefore, immunophenotypically and genotypically distinct B-lineage ALL cell lines, which reflect normal B-lineage LPC phenotypes corresponding to sequential stages of early B-lymphocyte ontogeny, provide a unique opportunity for radiobiologic studies on subpopulations of B-lineage LPC in the human B-lineage lymphoid precursor cell compartment (44). In addition, EBV-transformed B-lineage LPC cell lines corresponding to the earliest stages of human B-lineage lymphopoiesis have been recently established from human fetal livers (44, 46, 47). Despite the wealth of information regarding the radiobiologic features of human and murine myeloid/erythroid precursor cell populations, very little information is available regarding the radiation sensitivity of human B-lineage
Reprint requests to: Fatih M. Uckun, M.D., Ph.D., Associate Professor, Department of Therapeutic Radiology-Radiation Oncology, Box 356 UMHC, Harvard Street at East River Road, Minneapolis, MN 55455. Supported in part by US Public Health Service Grants R29 CA 42111 and ROl CA-42633, and PO1 CA-21737.
from the National Cancer Institute, DHHS, and Special grants from the Minnesota Medical Foundation, Children’s Cancer Research Fund and Bone Marrow Transplant Research Fund, University of Minnesota. F. M. Uckun is a scholar of the Leukemia Society of America. Accepted for publication 29 March 1991.
INTRODUCTION
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LPC. The purpose of this study was to examine the radiation sensitivity of human B-lineage LPC at multiple developmental stages. To this end, we analyzed in detail the radiation response of eight immunophenotypically distinct B-lineage LPC cell lines of ALL or fetal liver origin reflecting discrete stages of maturation within the B-lineage lymphoid precursor pathway. Our findings provide unprecedented evidence that the radiation sensitivity of B-lineage LPC decreases during maturation in a developmentally programmed fashion. Stage 0 (Pro-B), Stage I (Pre-pre-B), and Stage II (Pre-pre-B/Pre-B) B-lineage LPC were more radiosensitive than Stage III/IV (Pre-B) or Stage V (Early B) B-lineage LPC. To our knowledge, the present study represents the first report on the radiation sensitivity of human B-lineage LPC and provides the first evidence for its temporal association with discrete developmental stages in human B-lymphocyte ontogeny.
METHODS AND MATERIALS Cell lines The following LPC cell lines were used in this study: FL1 12, pro-B; FL1 14, pro-B; REH, pre-pre-B; NALM-6, pre-pre-B/pre-B; KM3, pre-B; HPB-NULL, pre-B; RAJI, early B; NAMALWA, early B; CEM, pre-T; and MOLT-3, pre-T. FL1 12 and FL1 14 cell lines were established as previously described by EBV-transformation using LPC-enriched mononuclear cells from two fetal livers of 8 week and 14 week gestational age, respectively. NALM-6, KM3, HPB-NULL, CEM, and MOLT-3 cell lines were established using leukemic blasts from ALL patients, while RAJI and NAMALWA cell lines were established using malignant lymphoma cells from Burkitt’s lymphoma patients (29, 30, 32, 34). All cell lines were maintained at 37°C in a humidified 5% CO2 atmosphere in RPM1 1640 medium* supplemented with 10% (v/v) heat-inactivated fetal bovine serum?, 50 pg/mL streptomycin, 50 IU/mL penicillin, 2 mM L-glutamine, and 10 mM Hepes buffer. Irradiation of cells Cells were irradiated using a 13’Cs itradiatorS, as previous described (38, 45, 49, 50). In brief, cell suspensions (1 X lo5 cells/ml) in plastic tissue culture flasks4 were exposed to graded doses of gamma rays ranging from 25 cGy to 400 cGy at 109 cGy/min in a single exposure at 37°C. Irradiations were performed during a log phase and under aerobic conditions. Clonogenic assays The radiation responses of LPC cell lines were evaluated using clonogenic assays, as previously described in detail (34, 5 l-54). In brief, serial dilutions of the control and irradiated test cell suspensions were made, and six replicate samples from each dilution were cultured for 10 *Gibco Laboratories, Grand Island, NY. tHyclone Laboratories, Logan, UT.
November 1991, Volume 21, Number 6
days in 96-well flat-bottom tissue culture plates and assayed for clonogenic growth (51-54). Because this is a quanta1 assay, clones are not counted individually, but a judgment is made as to whether or not clonogenic growth has occurred in a particular well (34). The frequency of culture wells with clonogenic growth (wells containing clones with > 100 cells or diffusely growing cells covering >25% of the well) was used to estimate the number of clonogenic LPC in the original cell suspension by a modification of the Spearman Karber method (51-54). The single dose radiation survival curves were constructed by linear regression analysis, and the Do, D,, and n values were determined from the linear portion of the survival curves according to standard methods (45, 49, 50). Immunophenotyping The surface antigen profiles of the B-lineage LPC cell lines or the control cell lines were determined by immunofluorescence staining and multiparameter flow cytometry, as previously described (46-49). The following monoclonal antibodies (MoAb) were used as phycoerythrin (PE) or fluoresceinisothiocyanate (FITC) conjugates to detect the particular surface antigens on the target cell populations: G3.7 (anti-CD7), 24.1 (anti-CDlO), B43 (anti-CD19), BA-1 (anti-CD24), BU41 (anti-CD72), BU43 (anti-CD74),LN-1 (anti-CD75), CRIS-4 (anti-CD76), 2C3 (anti-IgM), gTA-4 (anti-IgD). The reactivity profiles of these MoAb with human lymphoid precursor cells at various developmental stages were detailed in a recent review article (44). Cell lines were scored as positive (+) for a given antigen if > 20% of cells reacted with the respective MoAb, positive/ negative ( 2 ) if lo-20% of cells reacted with the respective MoAb, and negative ( - ) if < 10% reacted with the respective MoAb. Southern blot hybridization analyses DNA extraction and Southern blot hybridization analyses were performed as previously described (18, 46, 47). The Cp probe is a 1.5 kb clone of the germline constant region gene (kindly provided by Dr. Brian Van Ness) hybridizing a 17 kb germline fragment in BamHl restricted DNA and a 1.3 kb germline fragment in ECOR 1 restricted DNA. The TCR B probe, JUR-B (kindly provided by Dr. Harry Orr) hybridizes to both TCR B constant region gene and TCR B J region and identifies a 10 kb germline EcoRl fragment. The TCR y probe (kindly provided by Dr. John H. Kersey) is a 0.7 kb HINDIII-EcoRI genomic fragment containing the Jy 1 gene identifying 1.5 kb, 3.2 kb germline EcoRI fragments, a 13 kb germline Bar&II fragment, and 2.1, 5.0 kb germline HINDIII fragments. Rearrangements of the IgH, TCR B, and TCR y genes were detected in EcoRI and/or Ban-HI digests of high molecular weight DNA from LPC cell lines using these different probes, as previously described in detail (18, 29, 30, 46, 47). $J.L. Shephard and Assoc., $Falcon, Becton Dickinson,
Glendale, CA, Model Mark I. Oxnard, CA.
Radiation sensitivity of B-lineage LPC 0 F. M.
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UCKUN et al.
characterization of human B-lineage LPC cell lines
Table 1. Multiparameter
Genotype
Immunophenotype Cell line
Maturational stage
FL112 FL114 REH NALMd
CD7
CD10
CD19
Pro-B
_
+
+
Pro-B Pre-Pre-B
_
+
CD24
CD72
CD74
CD75
CD76
C&
IgM
+ +
_
_
_
_
_
_
_
+ +
+ +
_
_ _
_
RR
RG
RR
_
+
-
+
_
_
GG
GG
RR
+ *
+ +
+ +
_
_
DD
GG
RR
+
_
GG
GG
RG
_
+
+ •t
Pre-Pre-Bi Pre-B
_
+
+
* _
+
+
+
+
+
+
+
*
IgD
_
TY
TB
GIL
RR RR
GG GG
GG GR
KM-3
Pie-B
_
HPB-NULL NAMALWA
Pre-B Early B
_ _
+
+
+
+
+
+
+
-
+
-
GG
GG
DG
RAJI Controls
Early B
_
t
+
+
+
+
+
+
-
r
+
GG
GG
DG
CEM
he-T
+
_
_
_
-
_
_
_
RR
RR
GG
Pre-T
+
+ _
_
MOLT-3
_
_
_
_
_
RR
RR
GG
Proliferative
_
Biochemical
activity
%Go,,
IS
%G,M
Proliferation index J
Protein (mg/lO’ cells)
GHS level (*/mg)
features
GST activity (nmol/min/mg)
[PHI,
[Ca’+]i (Indo- 1 Ratio)
FL112
Pro-B
49.1
28.9
22.0
50.9
1.4
2.1
126.3
7.24
FL114
Pro-B
65.0
29.7
13.3
43.0
0.5
8.5
N.D
N.D
1.97
REH NALM-6
51.5 51.8
28.5 33.0
17.0 15.2
45.5 48.2
0.4 0.3
0.2 5.3
118.7 58.7
6.93 6.91
2.33 1.91
KM-3
Pre-he-B he-he-B/ pre-B Pre-B
52. I
32.6
15.3
47.9
0.4
5.8
112.4
7.13
1.88
HPB-NULL NAMALWA
Pre-B Early B
52.0 48.8
24.0 36.2
24.0 15.0
48.0 51.2
0.1 0.5
8.3 1.6
55.3 94.8
7.17 7.05
1.83 1.98
RAJI
1.98
1.39
Early B
49.2
31.1
19.7
50.8
0.3
12.7
3.2
7.05
Controls CEM
Pre-T
65.5
31.0
3.5
34.5
0.1
5.3
180.4
7.10
N.D
MOLT-3
Pre-T
73.7
23.7
2.6
26.3
0.4
2.2
N.D
7.40
N.D
Note: The multiparameter characterization of human B-lineage LPC cell lines was performed as de&bed in Materials and Methods. The biochemical parameters, except for the GST activity are presented as the mean values of multiple (3-4) measurements. The standard erors did not exceed 10% of the mean values. GST activity data were obtained by a single measurement. ND = Not determined; RR = both alleles rearranged; GG = both alleles in germline configuration; RG = Are allele rearranged, are allele in germline configuration; DG = One allele deleted are allele in germline configuration; DD = Both alleles deleted.
Analysis of cell cycle kinetics by quantitative DNA j7ow cytometry The UV excited dye, Hoechst 33342, was used to quan-
tify the DNA content of the B-lineage LPC populations, as previously reported (23). Quantitative DNA analysis was performed on a FACStar Plus flow cytometer equipped with Consort 40 computer using the COTFIT Program, which includes CELLCY, a cell cycle distribution function that fits DNA content histograms and calculates the percentages of cells in the Go,, , S, and GzM phases of the cell cycle, as originally described by Fox (12). Measurement of intracellular protein, GSH, GST activity, pH, and free calcium concentration The intracellular protein concentrations were determined
using the Bicinchoninic Acid Protein Assay Kit?. Bicinchoninic acid is a chromogenic reagent highly specific for Cu (I), which forms a purple complex with an absorbance at 562 nm that is directly proportional to the protein con*Becton Dickinson Immunocytometry
Systems,
tSigma Chemical Co., St. Louis, MO. SMolecular Probes, Eugene, OR.
San Jose, CA.
centration. GSH (8, 13, 22, 33, 35, 39, 55) determinations were performed using the GSH reductase procedure (43). The GST activity (2, 24, 56) was quantified by monitoring thioether formation between 1-chloro-2,4-dinitrobenzene and GSH at 340 nm and 25°C with the aid of a recording spectrophotometer, as previously described (20, 22). The intracellular pH in B-lineage LPC populations was determined using the fluorescent indicator 2’-7’-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and a fluorometer, as previously described (14, 31). Cytoplasmic free calcium concentration was measured with the dye Indo-ls and a flow cytometer,** as previously described (46-48). All of these analyses were performed on cells obtained during log phase. RESULTS AND DISCUSSION We studied the radiation responses of eight B-lineage LPC cell lines corresponding to five discrete stages of hu§Perkin-Elmer LS-5, Norwalk, CT. **FACStar Plus, Becton Dickinson, San Jose, CA.
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I. J. Radiation Oncology 0 Biology 0 Physics Table 2. Radiobiological
November 1991, Volume 21, Number 6
features of human B-lineage lymphoic cell lines Radiobiologic
Cell line
Experiment
FL1 12, Pro-B
FL114, Pro-B
REH, Pre-pre-B
no.
Pre-Pm-B/Pre-B
D, (ICY)
2 Mean csc 1 2 3 Mean f SE csc 1 2 3 4 Mean ? SE csc 1 2 Mean csc
92.6 67.9 80.3 67.7 44.7 55.6 50.2 52.0 77.4 62.0 59.0 66.1 e4.7 65.0 45.8 63.8 45.5 47.2 50.5k3.8 58.6 167.8 221.7 194.7 182.9 169.3 120.6 113.8 134.6? 14.5 154.4 204.5 201.3 132.7 123.3 165.5 29.4 169.1 168.3 119.7 144.0 144.4
0.0 -21.0 - 11.0 - 13.3 - 17.8 -31.3 - 24.6 - 27.2 3.1 15.7 - 16.2 0.927.7 7.6 37.2 33.7 - 1.9 49.2 29.5k9.5 23.1 28.1 -51.5 - 11.7 13.4 50.5 78.2 22.9 50.5 * 13.3 27.8 -39.3 -20.3 -24.1 50.1 - 8.4+ 17.3 -6.7 30.9 32.9 31.9 32.3
1.0 0.7 0.9 0.8 0.7 0.6 0.7 0.6 1.0 1.3 0.8 l.O?O.l 1.1 2.2 1.7 1.0 2.8 1.920.3 1.5 1.2 0.8 1.0 1.1 1.3 1.9 1.2 1.520.2 1.2 0.8 0.9 0.8 1.5 1.0+0.1 1.0 1.2 1.3 1.3 1.3
1 2 3 Mean t SE 1 2 3 Mean f SE
65.5 62.0 63.2 63.6r 1.0 51.9 43.2 47.9 47.7k2.5
-5.0 - 24.4 24.9 - 1.5 +- 14.3 - 15.7 1.5 16.3 0.7k9.2
0.9 0.7 1.5 l.OkO.2 0.7 1.0 1.4 l.OkO.2
1 2 Mean csc 1 2 Mean csc 1 3 Mean ? SE csc 1
; 4 Mean ? SE csc
1
KM-3, Pre-B
HPB-NULL,
Pre-B
NAMALWA,
Early B
RAJI, Early B
Controls CEM, Pre-T
MOLT-3, Pm-T
n
Do (cCy1
L
NALM-6,
parameters
Note: Following irradiation, B-lineage LPCs were assayed for clonogenic growth as described in Methods and Materials. The values for the mean % plating efficiency were 1.0 for FL112, 2.4 for FL114, 1.3 for REH, 1.7 for NALM-6, 15.3 for KM-3, 11.7 for HPBNULL, 14.0 for NAMALWA and 7.5 for RAJI. The radiation dose survival curves of B-lineage LPC cell lines were constructed by linear regression analysis. Results are presented as the values for Do, D, and n. All computations were performed using the Statworks Statistics Program for the Macintosh obtained from Cricket Software. CSC = Composite survival curve.
man B lymphocyte ontogeny. Table 1 details their immunophenotype, genotype, proliferative activity, and biochemical
features, including protein content, GSH level, GST activity, [pH]i, and [Ca2+]i. Of these eight cell lines, FL112,
Radiation sensitivity of B-lineage LF’C ??F. M. UCKUNet al.
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10 t
OO ID, 20
,
40
,
60
,
60
,
100
,
120
1
140
GST bnol/mWm~$
’ Radiation Dose kGy1 Fig. 1. Radiation sensitivity of B-lineage LF’C at discrete developmental stages. The graphs represent the composite radiation survival curves of B-lineage LPC showing survival of cells as a function of the radiation dose. Cells were irradiated, assayed for clonogenic growth and the radiation survival curves were gener-
ated by linear regression analysis as described in Materials and Methods. See Table 1 for details. Bars represent SE.
FL114, KM-3, and RAJI were analyzed in two independent experiments, REH and HPB-NULL were analyzed in three independent experiments, while NALM-6 and NAMALWA were analyzed in four independent experimerits. T-lineage LPC cell lines CEM and MOLT-3, which were used as control LPC cell lines, were analyzed each in three independent experiments. Throughout the study, each experiment was performed in six replicates. The Do, D,, and n values for the single dose radiation survival curves of each cell line are shown in Table 2. The composite survival curves (CSC) of six representative B-lineage LPC cell lines are depicted in Figure 1. Notably, the Do values showed a pronounced variation among the different cell lines corresponding to distinct stages of development, as determined by their composite immunophenotype and ge-
Fig. 2. Correlation of cellular radiation sensitivity with stage of maturation. The radiation sensitivity of (i.e., D, value of the single dose radiation survival curves) of B-lineage LPC was correlated on a continuous scale with stage of maturation, %S, GSH content and GST activity by single regression. CC = coefficient of correlation. P = p-value (i.e., statistical significance) of the correlation.
notype. FL112 and FL114 fetal liver pro-B cells (Stage 0 B-lineage LPC) with germline immunoglobulin heavy chain (IgH) genes but rearranged T-cell receptor gamma (T,) genes (mean Do = 80.3 cGy, Do of CSC = 67.7 cGy for FL112; mean Do = 50.2 cGy, Do of CSC = 52.0 cGy for FL114), REH ALL pre-pre-B cells (Stage I B-lineage LPC) with rearranged IgH and TCRy genes (mean Do = 66.1 cGy; Do of CSC = 65.0 cGy), and NALM-6 ALL pre-pre-B/pre-B cells (Stage II B-lineage LPC) (mean Do = 50.5 cGy; Do of CSC = 58.6 cGy) corresponding to the earliest three stages of human B-lymphocyte development were the most radiation sensitive B-lineage LPC populations. By comparison, KM-3 ALL pre-B (Stage III B-lineage LPC; mean Do = 194.7 cGy, Do of CSC = 182.9 cGy), HPB-NULL ALL pre-B (Stage IV B-lineage LPC; mean Do = 134.6 cGy, Do of CSC = 154.4 cGy), NAMALWAk4.B early B (Stage Va/b B-lineage LPC; mean Do = 144.0/165.5 cGy, Do of CSC = 144.4/169.1 cGy) cell lines corre-
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sponding to the later stages of human B-lymphocyte development were much more radiation resistant. Overall, the mean Do value for pro-B (Stage 0 LPC), pre-pre-B (Stage I LPC), and pre-pre-B/pre-B (Stage II LPC) from a total of 11 independent experiments was significantly lower than the mean Do value for pre-B (Stage III and Stage IV LPC) and early B cells (Stage V LPC) from a total of 11 independent experiments (60.1 cGyk4.5 cGy vs 158.5 cGy 2 11.8 cGy, p = 0.0001). This temporal association of the radiation sensitivity of B-lineage LPC with discrete stages of maturation is further illustrated in Figure 2. We next asked whether the radiation sensitivity of B-lineage LPC correlates with any other biologic or biochemical parameters besides their developmental stage (Fig. 2). Notably, the percentage of S-phase cells (coefficient of correlation = 0.1, p = 0.5), cellular protein content (coefficient of correlation = 0.230; p=O.6), intracellular glutathione (GSH) level (coefficient of correlation = 0.175; p = 0.7), glutathione-S-transferase (GST) activity (coefficient of correlation = 0.107; p = 0.8), intracellular pH (coefficient of correlation = 0.415; p = 0.3), or free cytoplasmic calcium concentration (coefficient of correlation = 0.048; p = 0.9) did not correlate with the radiation sensitivity of the B-lineage LPC. Furthermore, there was no correlation between any of these parameters, in particular the %S value, GSH content, or GST activity, and the developmental stage of B-lineage LPC. Thus, the observed differences in Do values of B-lineage LPC corresponding to different stages of development most likely reflect inherent and probably maturation-linked differences in their intrinsic radiation sensitivity. In summary, we studied and compared the radiation
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sensitivity of human B-lineage LPC populations at discrete developmental stages of human B-lineage lymphopoiesis. While expanding our current knowledge of the radiobiologic features of human hematopoietic precursor cells (1, 6, 9, 10, 12, 17, 19, 25, 26, 27, 40, 41, 45, 57, 58), this investigation prompts the hypothesis that the radiation sensitivity of B-lineage LPC decreases during maturation in a developmentally programmed fashion. As discussed in several widely used textbooks of radiation biology, differentiated cells are in general much more radiation resistant than undifferentiated cells, and this influence of the degree of cellular differentiation on radiation sensitivity cannot be explained solely by differences in the mitotic activity of cells (7, 21, 36, 37, 42). According to the well-known but not well-understood rule of Bergonie and Tribondeau, the radiation sensitivity of cells vary inversely with their degree of differentiation (7, 21, 36, 37, 42). Our findings provide strong evidence that this rule also applies for human B-lineage LPC populations. To our knowledge, the present study represents the first report on the radiation sensitivity of B-lineage LPC and provides novel evidence for a temporal association between the radiation sensitivity of B-lineage LPC populations and the discrete developmental stages of human B-lymphocyte ontogeny they represent. Nevertheless, the results should be interpreted with due caution since the conclusions regarding the coordinate sequence of antigen expression or gene rearrangements during human B-lymphocyte ontogeny are obligatorily speculative. Leukemic phenotypes may be altered by clonal evolution during and/or after the malignant transformation of a given B-lineage LPC clone or a misprogramming of differentiation in leukemic B-lineage LPC (44).
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