The Mcl-1 anti-apoptotic protein
2 isoforms by alternative splicing :
­ Mcl-1L : anti-apoptotic (350 aa)
­ Mcl-1S : pro-apoptotic (248 aa) (BH-3 only)
Anti-apoptotic function
Hematopoietic system +++
Mcl-1 ASO induced apoptosis in all myeloma cells
tested (including both cell lines and patients) Derenne et al
Blood 2002
Expression of Mcl-1 in normal and
malignant plasma cells
35
30
ratio
25
20
15
1
2
10
fluorescence
5
0 0
1
3
Mean
Normal MM MM
BM PC
Relapse
Wuillème-Toumi et al Leukemia in press 2005
Bim
·Bim/Bod was isolated by an expression screen for
either Bcl-2 or Mcl-1 binding proteins (1998 )
Three major isoforms: BimEL,BimL and BimS
·BimEL and BimL interact with dynein chain which
sequesters them to cytoskeleton-associated
complexes
· Bim has a pivotal role in cytokine regulation of
hematopoietic cell survival
· Bim is required for normal homeostasis in the
lympho-hematopoietic compartment
Bim is expressed on most myeloma cell
lines and addition of IL-6 induced a
decrease of Bim isoforms
3h
6h
-XG-6-SBN -MDN-LP1 -NAN-1-NAN-3
IL-6
-
+
-+
-BimEL
EL
-BimL
-BimS
S
-Actin
Actin
Apoptosis is induced by IL-6
starvation
cells
80
Control
6 h
8 h
iodide
60
3%
7%
11%
positive
40
2%
29%
58%
20
Propidium
4
0
4
4
100 101 10 2 103 10
10 101 102
3
10
10
100 101 102 103 10
annexin
0
of
Annexin V
6h
12h
%
Control
IL-6 starvation
AG490
Opposite regulation of Mcl-1 and
Bim after IL-6 privation or blockade
AG-490 treatment
IL-6 privation
time (h) 0
2
4
6
8 12
time (h)
03
6
Bim EL
EL
BimL
L
BimS
S
Mcl -1
Mcl--1
Bcl-2
Bcl-2
Actin
Actin
Down-regulation of Mcl-1 is the
result of Mcl-1cleavage by caspases
hours
0
2
7
24
24
AG-490
-
+
+
+
+
zVADfmk(100µM)
-
-
-
-
+
Mcl--1 L
Mcl--1 S
26 kDa Mcl-1 BH1-3
Mcl-1 and Bcl-2 are associated with
Bim
IP
Mcl-1
Bcl-x
Bcl-2
L
WB
Mcl-1
Bim
Bcl-xL Bim
Bim
-BimEL
-BimEL
-BimL
-BimL
-BimL
-BimS
-BimS
-BimS
Bim is not associated to Bax
and Mcl-1 is wealky associated to
Bax
IP
Bax
Bim
Mcl-1
MDN
WB
Bim
Bax
Bax
Bax
Bim is essentially located on
mitochondria and weakly associated to
dynein
IP
Mcl-1 Dynein
memb
WB
Bim
Bim
Mito Light Cytosol
-BimEL
-BimL
Ig L chain
-BimS
BimL -
BimS -
Eur J Immul 2005 Gomez-Bougie et al
All bim isoforms are associated to
either Mcl-1, Bcl-2 or dynein
IP supernatant
IPs
IP-
IPs
Control
IPs= anti-Mcl-1,
anti-Bcl-2, anti-
WB:Bim
Dynein
Only the Mcl-1/Bim interaction is
disrupted during apoptosis induction
starvation
starvation
starvation
IL-6
control
AG490
control
IL-6
control
AG490
control
IL-6
control
control
Bcl-2
Bcl-2
AG490
Bcl-2
Mcl-1
Mcl-1
Bcl-2
Input
Mcl-1
Mcl-1
IP
IP
IP
Input
IP
IP
Input
Input
IP
IP
IP
WB Bim
Eur J Immul 2004 Gomez-Bougie et al
Bim does not interact with cleaved
form of Mcl-1
control
AG490
control
AG490
Bim
Bim
Input
Input
IP
IP
Mcl--1 L
WB Mcl-1
26 kDa Mcl-1 BH1-3
Bax activation after IL-6 privation or
blockade
6 h
12 h
120
120
22 %
42 %
IL-6 privation
0
0
100 101 102 103 104
100 101 102 103 104
number
6 h
12 h
cell
120
120
46 %
75 %
AG490 treatment
Relative
0
0
100 101 102 103 104
100 101 102 103 104
Fluorescence Intensity
Microinjection of Bim-BH3 peptides
Bim peptide
M R P E I W I A Q E L R R I G D E F N A
Mutant Bim peptide
M R P E I W I A Q E A R R I G A E F N A
Bim BH-3 peptide induces apoptosis
in myeloma cells
Bim BH-3 mutant peptide
100
75
cells
Bim BH-3 peptide
50
apoptotic
25
of%
0
4hrs
18hrs
Bim BH-3 peptide
Bim BH-3 mutant peptide
Endogeneous associations in
myeloma cells
Bad
Mcl-1L
14q-3-3
Bim
Bcl-2
Bim
Dynein
LC8
microtubules
Bcl-xL
Bim
Bak
Bax
nucleus
Potential mechanism of action of
Bim BH-3 protein
IL-6 privation
Reduced Mcl-1
Bak
Cleaved Mcl-1
Bad
Mcl-1
BH1-3
14q-3-3
Bcl-2
Hit and run
Bim
Bim
mechanism
Dynein
Bax
LC8
microtubules
Mitochondria
Bcl-xL
Bax activation
nucleus
Cell death
MM and PC
group
· Patricia Gomez-Bougie
· Monique Clément
· Régis Bataille
· Philippe Juin
· Martine Amiot