Microsoft PowerPoint

The Function and Movement of CD45 Molecule
in IL-6-induced Myeloma Cell Proliferation
Michio M. Kawano
Department of Bio-Signal Analysis,
Yamaguchi University
2005.4.11 10th International Myeloma Workshop

Structures of CD45

CD45 expression in Myeloma cells
·
Myeloma cells are CD45- in paraffin sections.
(Kurtin PJ, Hum Pathol 16:353, 1985)
·
Normal plasma cells are CD45+ by flow cytometry
(Leon WMM, Blood 76:1739, 1990)
·
Establishment of a CD45+ immature plasma cell line
(Hata H, Leukemia 8:1768, 1994)
·
Higher labeling index In CD45+ myeloma cells
(Joshua D, Br J Haematol 94:76, 1996)
·
CD45+ and CD45- cells of peripheral blood plasma cells
(Schneider U, Br J Haematol 97:56, 1997)
·
CD45 induction in myeloma cells by IL-6
(Mahmoud MS, Blood 92:3887, 1998)
·
MPC-1-CD49e- Immature myeloma cells include CD45+ proliferationg cells
(Fujii R, Br J Haematol 105:131, 1999)
·
Myeloma cell heterogeneity could not be distinguished by the level of CD45
expression
(controversial report)
( Rawstron AC, Br J haematology 113:252, 2001)
·
In vivo homing characteristics of CD45- and CD45+ myeloma cell s
(Asosingh K, Exp Hematol 29:77, 2001)
·
SRC family kinase activity associated with CD45 Is required for IL-6-induced
proliferation
(Ishikawa H,
Blood 99:2172, 2002)
·
CD45+ cells are proliferating cells in normal and malignant plasma cells
(Pellat-Deceunynck C and Bataille R,
Blood Cells Mol Dis 32:293,2004)
·
High secretion of VEGF in CD45- myeloma cells
(Asosingh K, Blood 103:3131, 2004)

All normal plasma cells express CD45RA, CD45RB not CD45RO,
but a few of myeloma cells express CD45RO, CD45RB, not CD45RA.
(2~20%)
CD45+ myeloma cells
can respond to IL-6.

Subpopulations of Myeloma cells
(heterogeneous expression of CD45)
Immature
Intermediate
Mature
CD45
MPC-1
MPC-1
CD45
MPC-1
MPC-1-CD45+
CD45
CD49e-
0.1 ~ 2%
MPC-1+CD45-
MPC-1+CD45+
CD49e-
CD49e-
CD49e
MPC-1+CD45+
30~ 90%
5 ~ 30%
CD49e+
Dominant
0.1~ 3%
MPC-1-CD45-
CD49e-
5 ~ 30%
CD45+ myeloma cells = CD45+ immature
and CD45+ mature cells

MPC-1-CD45+ immature cells are proliferating.
Ki-67+ (%)
% in the BM
MPC-1- (%)
MPC-1-
Pt
CD38+++ (%) CD45-
CD45+
MPC-1+
MPC-1-
CD45-
CD45+
1
48.4
14.1
0.5
1.2
8.3
4.9
19.5
2
36.0
9.2
2.2
1.6
6.5
3.7
15.2
3
24.7
4.6
0.6
0.8
4.9
3.9
10.6
4
29.4
1.0
0.1
1.5
6.2
4.0
12.7
5
36.9
23.9
1.0
1.0
6.9
4.7
13.8

CD45+
1~5%
60 ~ 90 %
proliferating
IL-6
low proliferation
CD45-
capable of
long survival
proliferating
5~30%
Immature
Intermediate & Mature
(MPC-1-CD49e-)
(MPC-1+CD49e-/+CD45-)

Biological Significance of CD45 expression
in Myeloma cells
1)
CD45+(MPC-1-) Immature myeloma cells respond
to IL-6 to proliferate.
2) IL-6 induces translocation of CD45RO but not
CD45RA to lipid rafts.
3) CD45+(MPC-1-) immature myeloma cells are
sensitivity to stress stimuli.
4) CD45+(MPC-1-) immature cells increase in the
progressive states.

CD45+ myeloma cell lines proliferate in response to IL-6
A
CD45-
CD45+
ILKM2
ILKM3
ILKM8
NOP-2
KMS-5
U266
U266
(CD45+) (CD45+) (CD45+) (CD45-) (CD45-)
CD45
10-1 100 101 102 103 10-1 100 101 102 103
10-1 100 101 102 103
10-1 100 101 102 103
10-1 100 101 102 103
10-1 100 101 102 103
10-1 100 101 102 103
B 1.6
-IL-6
+IL-6
1.2
450
OD 0.8
0.4
0
C
IL-6R
(CD126)
10-1 100 101 102 103
10-1 100 101 102 103
10-1 100 101 102 103
10-1 100 101 102 103
100 101 102 103
10-1
10-1 100 101 102 103
10-1 100 101 102 103

CD45 expression and src activation are related to IL-6
-responsive proliferation of myeloma cell lines
Myeloma cell lines
CD45- U266CD45+ U266ILKM2 ILKM3 ILKM8 NOP-2 KMS-5
-
+
+
+
+
-
-
CD45 expression
++
++
+
+
+
+++
-
IL-6R expression
-
+
+
+
+
-
-
IL-6 response
STAT3 activation
+
+
+
+
+
+
-
ERK1/2 activation
+
+
+/-
+/-
+/-
+
-
Src activation
-
Lyn
Fyn
Fyn
Lyn
-
-
Lyn expression
+
+
+
+
+
+
+
Fyn expression
-
-
+
+
+/-
+
+/ -
Blk expression
+/ -
+/ -
+/ -
+/-
-
-
-
+++: markedly high, ++: high, +: detectable, +/-: weakly detectable, -: undetectable

In vivo growth of myeloma cell
lines or primary myeloma cells
ip
SCID-hIL6 Tg
injection of agarose gel(0.6%)
0.3% agarose H14(TAKARA)
+ 0.3% Sea-Plaque(TAKARA)
( providing a kind of scaffold )
and U-266 or Primary myeloma cells
8 ~ 12 W
harvest cells and mass from peritoneal cavity
stain the cells with FITC-CD38, PE-CD54 or-MPC-1,
PC5-CD45
analyze by cell sorter ( survival & proliferation

CD45+ U-266 cells changed to CD45- cells
in low concentration of IL-6 (SCID-hIL6(low producer) mice )
50000
45000
fluid
40000
35000
30000
peritoneal
total
25000
CD45+
the
20000
CD45-
in
15000
cells
10000
5000
viable
0
1x105 No.1
1x105 No.2
1x105 No.3

All normal plasma cells express CD45RA, CD45RB not CD45RO,
but a few of myeloma cells express CD45RO, CD45RB, not CD45RA.
CD45+ myeloma cells
can respond to IL-6.

Translocation of CD45RO,RB to lipid raft
Myeloma cells
A
A
B
B
C
B
C
wit B Or no B
h
Lipid raft
+ IL-6
P 396
lyn
lyn
507
P
(RA)
RB
CD45RO
(RA)
CD45RO,RB

Endogenous CD45RO and CD45RB translo
lipid rafts after IL-6 stimulation.

U266
U266
A
P
P
P
F
F
F
CD45RO
CD45RB
CD45RA
+
B
5
G
G
G
r
EGFP
-EGFP
-EGFP
-EGFP
4
e
-E
-E
-E
b
20
D
O
B
A
C
R
R
R
_ IL-6
6
P
5
5
5
6
F
4
4
4
Num
2
G
D
D
D
ll
+ IL-6
U
E
C
C
C
10
Ce
(kDa)
ev
207
ti
**
la
*
e
0
R
02
4
02
4
0
2
4
02
46
Culture Days
Fraction number
C
IL-6 1
2 3 4
5
6
7
8
9 10 11 12
+ IL-6
-IL-6
+
D
CD45RO
_
CD45RO
CTB
GFP
merge
CTB
merge
-TRITC
GFP
-TRITC
-EGFP
+
CTB
_
CD45RO
+
CD45RB
-EGFP
_
CD45RB
-EGFP
+
CTB
_
CD45RB
-EGFP
+
CD45RA
_
CD45RA
-EGFP
+
CTB
CD45RA
_
-EGFP
Transfected CD45RO, CD45RB, but not CD45RA moved to lipid rafts after IL-6 stimulation.
Transfected CD45RO and CD45RB are functional to augment the IL-6-Induced proliferation.

Translocated CD45RO functions as active phosphatase for Lyn.
Myeloma cells
A
A
B
B
C
B
C
wit B Or no B
h
IL-6
IL-6R
IL-6R
Lipid raft
+ IL-6
flotillin
flotillin
S
S
K(N )
PA
H
PA
H
3
G
G
inactive
3
P
Cs
S
S H
k
P H
Tyr
K(C )
C
396
2
2
P
p
Cs
K(C)
K(N )
Tyr
507
k
Lyn
C
inactive
Lyn active

CD45+ cells are sensitive to a set of apoptotic stimulation
through mitochondria mediated pathway
A
serum deprivation
B
thapsigargin (THG)
C hydrogen peroxide (H O ) Dultraviolet (UV) irradiation
2
2
CD45-
CD45+
CD45-
CD45+
CD45-
CD45+
CD45-
CD45+
0
viable cells
86%
62%
35%
75%
60%
90%
day
12%
40%
FS
non-viable
FS
PI
dead
dead
cells
9%
8.8%
28%
57%
cells
cells
20%
35%
SS
t
Annexin V
SS
5
t
n
t
88%
68%
n
live cells
n
80%
48%
day
live cells
cou
cou
34%
cou
30%
60%
40%
FS
cell
10%
28%
cell
cell
SS
DiOC
DiOC
6
6
DiOC6
E
arsenic trioxide (As O )
FG
2
3
CD45+
CD45-
control
CD45-
control
CD45-
CD45+
CD45+
56%
43%
IFN-
TNF-
IFN-
FS
TRAIL
melphalan
38%
47%
Fas antibody
DEX
SS
t
doxorubicin
n
osmotic stress
ATRA
cou
56%
38%
heat shock
thalidomide
cell
0
5
10
15
20
25
30
010
20
30
40
non-viable cells (%)
non-viable cells (%)
DiOC6

Effects of apoptotic stimuli on CD45- and CD45+ U266
cells
CD45- U266
CD45+ U266
Serum Depletion
+
++
Oxidative stress (H2O2)
+
++
UV Irradiation
+
++
Heat Shock
+
++
Hypertonic Condition
+
+
Melphalan
+
++
Doxorubicin
+
+
Dexamethason
-
-
IFN-
+
++
IFN-
-
-
PMA
+
+
Ionomycin
+
+
Fas Antibody
-
-
Trail
-
-
Thalidomide
-
-
TG (Thapsigargin)
+
++
Tunicamycin
-
-
ATRA
-
-
As2O3
+
++
-:Resistant. +:Sensitive. ++ More sensitive

Enhanced cytochrome C or AIF release and
caspase 3 activation in CD45+ U266 cells
UV Irradiation (600 J/m2)
0
6
9
12
0
6
9
12
Incubation time
(hrs)
C
Cytochrome
C
C
-Actin
CD45-
CD45+
Cytosolic fraction
Serum Depletion
pro-caspase 3
024h
AIF
-actin
cleaved caspase 3
CD45- CD45+ CD45- CD45+
-actin
CD45- CD45+ CD45- CD45+
0
12h

The genes of increased expression
in CD45+ U-266 cells
(subtraction & microarray)
B 23F H om o sapiensC G I-120 protein m RN A(LO C 51644)
B 25F H om o sapiens Ras-G TPase-activating-like protein(IQG AP1)
(H om o sapiens IQ-motif containing G TPase-activating protein 1)
B 26F H um an Valtage-dependent anion channelisoform 1(VD AC 1)
B 48F H om o sapiens similar to C dk5 and Ablenzym e substrate(LO C 91767)
B 96F H om o sapiens m alate dehydrogenase 1 ,NAD ((M DH1)
B 97F H om o sapiens heat shock 60KD protein 1(H SPD 1)
B 98F H om o sapiens heat shock 90KD protein 1,beta(H SPC B )
D 24
H om o sapiens splicing factor 3b,subunit 1
X6
H um an sapiens cD N A FLJ11948 fis
X19
H um an sapiens cD N A FLJ10772 fis
X99
New
SSX4
CD86

)4
VDAC1
10
CD45- U266
A
B
CD45+
(x
CD45+ U266
45-
45+
probe
ers 25
mb
VDAC-1
u 20
CD45-
ny
G3PDH
probe
p 15
· P=0.004
co 10
** P=0.03
VDAC1 was identified differentially
5
expressed in CD45+ cells
Relative
0
C
U266
AMO1
U266
vdac1
bcl-xL
bak
bax
mcl-1
G3PDH
CD45- CD45+
CD45lo CD45hi Mock CD45
Expression of VDAC1 gene was increased
RB
in CD45+ cells.
VDAC1
*
*
*
Hsp60
2.2
E
3.5
*
CD45+ myeloma cell lines expressed more VDAC1.
2
H2O2
*
3
%)
THG
1.8
CD45-
2.5
control
cells
CD45+
1.6
of
2
mock
VDAC1
1.4
D
Ratio
(apoptotic
CD45
-
+
-
+
1.5
1.2
VDAC1
1
1
Cox IV
mock
VDAC1
mock-
VDAc1
VDAC1 transfected cells
VDAC1 transfectants were more sensitive
to H2O2 and THG.

MPC-1-CD45+ immature myeloma cells are increased
in the progressive state
)
25.00
BMMNC
of
20.00
(%
cells
15.00
mature
10.00
im
5.00
0.00
MPC-1-CD45+
progressive
stable
progressive
stable
(n= 31)
(n= 15)
(n= 7)
(n= 22)
MM
MGUS

CD45+ immature myeloma cells (MPC-1-CD45+
cells) are proliferating in response to IL-6 in the BM,
but the amount of IL-6 is limited so that CD45+
cells are converted to CD45- cells which are more
stable to the environmental stress.
Loss of CD19
Pax-5(-)
Growth
IL-6 a limited amount of IL-6
CD45
in the bone marrow
Myeloma
Stable without IL-6
but not proliferate
CD45-