Differences of Leukemic Stem Cells of Acute Myeloid Leukemias Induced byHigh-Dose-Rate and Low-Dose-Rate Gamma-ray Irradiations
Tokuhisa HIROUCHI, Satoshi TANAKA, Ignacia Braga-TANAKA, Kazuaki ICHINOHE,
Yoichi OGHISO, Kimio TANAKA
Department of Radiobiology
Abstract
It is important to clarify how mice exposed to high-dose γ-rays at a low-dose rate (LDR) have asignificantly higher leukemia incidence, although LDR γ-ray irradiation has very few DNA double strandbreaks. Then, this study focused on clarifying the leukemic stem cells of LDR radiation-induced leukemias,by comparing them with those of high-dose-rate (HDR) or middle-dose-rate (MDR) γ-ray-induced
leukemias. The LDR, MDR or HDR γ-ray-induced leukemias were obtained from mice which were exposed
to 8,000 mGy at 20 mGy/22h/day, 4,000 mGy at 400 mGy/22h/day and 3,000 mGy at 890 mGy/min,respectively. Leukemic stem cells from γ-ray-induced leukemias at three dose rates were identified byintravenous transplantation

to syngeneic mice of 100 cells each of 7 populations corresponding to different

hematopoietic differentiation stages. The leukemic stem cells from HDR and MDR γ-ray-induced leukemias
had hemizygous deletions of chromosome 2 around PU.1 allele and both showed identical CD antigen
profiles to those of normal common myeloid progenitor and similar gene expression profiles to those of
normal common myeloid progenitor. In contrast, leukemic stem cells from LDR or a part of the MDR
γ-ray-induced leukemias with intact PU.1 allele did not reveal similar profiles for CD-antigen and gene
expression to those of normal common myeloid progenitor, but frequently resembled those of common
lymphoid progenitor cells, granulocytes and monocytes. These results showed a possibility that murineleukemias induced by LDR γ-ray irradiation might be independent of directly induced DNA damages byradiation and the origin of their leukemic stem cells was in lymphoid lineages cells, which was in contrast tothat of HDR γ-ray leukemogenesis.

Table 1

Similarities of gene expression profiles of rAML stem cells with those of HSC, MPP, CMPand CLP

CD antigen profilea
LSK-LSK+CMP

CLP

Gr-1posCD45R/B220posa

Population with highest similarity with rAML stem cellb
Common myeloid progenitor
Common myeloid progenitor
Multipotent progenitor
Multipotent progenitor
Multipotent progenitor
Common myeloid progenitor
Common myeloid progenitor
Common myeloid progenitor
Common myeloid progenitor
Common myeloid progenitor
Common myeloid progenitor
Common lymphoid progenitor
Multipotent progenitor
Multipotent progenitor

400 mGy/day-4
1.0 Gy/min-9
1.0 Gy/min-10

Common lymphoid progenitor
Common myeloid progenitor
Common myeloid progenitor

0.91
0.95
0.93

20 mGy/day-120 mGy/day-3
400 mGy/day-3
1.0 Gy/min-10

Common myeloid progenitor
Common lymphoid progenitor
Common lymphoid progenitor
Hematopoietic stem cell

0.83
0.97
0.88
0.55

CD antigen profile of rAML stem cell.

follows:linb

AML sample
1.0 Gy/min-2
1.0 Gy/min-3
1.0 Gy/min-9
0 mGy/day-2
1.0 Gy/min-10
1.0 Gy/min-1
1.0 Gy/min-2
1.0 Gy/min-3
1.0 Gy/min-6
1.0 Gy/min-9
1.0 Gy/min-10
0 mGy/day-7
20 mGy/day-1
20 mGy/day-3

neg

Sca-1

LSK-,
neg

c-kit

pos

linCD34

neg

pos

Sca-1

posc-kit; CLP, lin

neg

pos

Confidence Measurec
0.63
0.94
0.95
0.95
0.83
0.97
0.89
0.98
0.97
0.93
0.91
0.66
0.65
0.95

Each abbreviation represents specific CD antigen profiles asCD34 n e g ;Sca-1

posc-kit

LSK+,
neg

CD34

pos

lin n e g Sca-1 p o s c-kit p o s CD34 p o s ;; Gr-1pos; and CD45R/B220

pos.

Population with highest similarities with rAML stem cell computed by the Run Prediction algorithm ofGeneSpring.
cCMP,

Confidence Measure scored by the Run Prediction algorithm gives reliability of the results.

Table 2

Alterations of expression levels of 23 genes by allelic copy number aberration had
potentials to contribute to cell proliferation and anti-apoptosis in rAML stem cells.

Chromosome Cytoband
chr2
chr6

qH2
qA2

qA3.1
qA3.3
qB2.3
qC1
qD1
qE1
qE3
qF1
qF3

qG1
aGeneChr. aberrationa

Rbl1
Cav2
Cav1
Met
Wnt2
Wnt16
LepCul1
Il12rb2
Il23r
Tgfa
Wnt7a
Cav3
Raf1

Amp (%)
0%
33%
33%
33%
33%
33%
33%
33%
33%
33%
33%
33%
33%
33%

Del (%)
33%0%0%
0%
0%
0%
0%
0%
0%
0%
0%0%0%0%

Cacna2d4
Wnt5b
Ntf3
Fgf6
Fgf23
Csda
Lrp6
Cdkn1b

33%
33%
33%
33%
33%
33%
33%
33%

0%
0%
0%
0%
0%
0%
0%
0%

Ratio to normal
CMPb (p value)
0.5 (0.16)
3.0 (0.21)
3.0 (0.30)
2.3 (0.12)
3.8 (0.18)
2.2 (0.38)
2.8 (0.26)
1.1 (0.83)
1.8 (0.55)
3.0 (0.23)
2.5 (0.32)
1.8 (0.54)
1.5 (0.71)
1.4 (0.40)
2.2 (0.41)
1.8 (0.27)
2.5 (0.29)
2.1 (0.43)
2.4 (0.39)
1.3 (0.53)
1.9 (0.06)
1.8 (0.32)

Effect of mutated gene on CMP-like rAML stem cellc

Enhancement ofMyc expressiond

Cell proliferation (TGF-β)
Up-regulation
Anti-apoptosis (Focal adhesion)
Anti-apoptosis (Focal adhesion)
Cell proliferation (Focal adhesion), Anti-apoptosis (Focal adhesion)
Cell proliferation (WNT)
Up-regulation
Cell proliferation (WNT)
Up-regulation
Cell proliferation (JAK-STAT), Anti-apoptosis (JAK-STAT)
Up-regulation
Cell proliferation (Cell cycle, TGF-β), Anti-apoptosis (TGF-β)
Up-regulation
Cell proliferation (JAK-STAT), Anti-apoptosis (JAK-STAT)
Up-regulation
Cell proliferation (JAK-STAT), Anti-apoptosis (JAK-STAT)
Up-regulation
Cell proliferation (ErbB), Anti-apoptosis (ErbB)
Cell proliferation (WNT)
Up-regulation
Anti-apoptosis (Focal adhesion)
Cell proliferation (MAPK, ErbB, Focal adhesion, VEGF),
Anti-apoptosis (ErbB, Focal adhesion)
Cell proliferation (MAPK)
Cell proliferation (WNT)
Up-regulation
Cell proliferation (MAPK)
Cell proliferation (MAPK)
Cell proliferation (MAPK)
Cell proliferation (Tight junction), Anti-apoptosis (Tight junction)
Cell proliferation (WNT)
Up-regulation
Cell proliferation (ErbB, Cell cycle)

The frequency of chromosomal copy number aberration of the gene in 6 rAMLs (rAML-1, 2, 3, 6, 9, and 10).

rAMLs: radiation-induced acute myeloid leukamias
bThe ratio of the averaged expression level of the gene in the 6 CMP-like rAMLs to that in 5 CMPs.

Pvalues are shown in parentheses (Student's t-test).
cEffect of the gene on CMP-like PU.1 d e l / m u t rAML logically predicted by referring to the KEGG pathway

database (http://www.genome.jp/kegg/pathway.html).
CMP-like PU.1

del/mut

The pathway through which the gene affected therAML is abbreviated within parentheses as follows: JAK-STAT (Jak-Stat signalingpathway); MAPK (MAPK signaling pathway); TGF-β (TGF-beta signaling pathway); focal adhesion (focaladhesion); WNT (Wnt signaling pathway); cell cycle (cell cycle); ErbB (ErbB signaling pathway); and VEGF(VEGF signaling pathway)
dPrediction of the effect of the gene on enhancement of Myc expression by referring to the KEGG pathway

database.