U.S. patent application number 11/696804 was filed with the patent office on 2007-08-09 for methods of screening apoptosis modulating compounds, compounds identified by said methods and use of said compounds as therapeutic agents.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Veronica Ayllon, Xavier Cayla, Aarne Fleischer, Alphonse GARCIA, Angelita Rebollo.
Application Number | 20070184435 11/696804 |
Document ID | / |
Family ID | 27741248 |
Filed Date | 2007-08-09 |
United States Patent
Application |
20070184435 |
Kind Code |
A1 |
GARCIA; Alphonse ; et
al. |
August 9, 2007 |
METHODS OF SCREENING APOPTOSIS MODULATING COMPOUNDS, COMPOUNDS
IDENTIFIED BY SAID METHODS AND USE OF SAID COMPOUNDS AS THERAPEUTIC
AGENTS
Abstract
A method of screening cellular polypeptides for pro-apoptotic or
anti-apoptotic activity in a cell of a particular cell-type, said
method comprising: (a) culturing cells of said particular cell-type
under non apoptotic conditions and culturing cells of said
particular cell-type under apoptotic conditions, and (b)
determining subcellular localisation of said cellular polypeptides
in the cultured cells, wherein a localization of a cellular
polypeptide in lipid rafts in cultured cells under non apoptotic
conditions and a segregation of said cellular polypeptide from
lipid rafts in cultured cells under apoptotic conditions is
indicative that said cellular polypeptide has a pro-apoptotic or an
anti-apoptotic activity in said particular cell-type.
Inventors: |
GARCIA; Alphonse; (Paris,
FR) ; Cayla; Xavier; (Bretigny-Sur-Orge, FR) ;
Rebollo; Angelita; (Madrid, ES) ; Ayllon;
Veronica; (Madrid, ES) ; Fleischer; Aarne;
(Madrid, ES) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
INSTITUT PASTEUR
Paris
FR
CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
Madrid
ES
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
Paris
FR
|
Family ID: |
27741248 |
Appl. No.: |
11/696804 |
Filed: |
April 5, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11140275 |
May 31, 2005 |
7217534 |
|
|
11696804 |
Apr 5, 2007 |
|
|
|
10934513 |
Sep 7, 2004 |
|
|
|
11140275 |
May 31, 2005 |
|
|
|
PCT/EP03/02921 |
Mar 7, 2003 |
|
|
|
10934513 |
Sep 7, 2004 |
|
|
|
Current U.S.
Class: |
435/5 ;
435/7.23 |
Current CPC
Class: |
A61P 31/00 20180101;
G01N 33/92 20130101; A61P 17/02 20180101; A61P 25/28 20180101; A61P
31/18 20180101; A61P 9/10 20180101; A61P 31/12 20180101; G01N
2510/00 20130101; G01N 33/505 20130101; G01N 33/5011 20130101; A61P
35/00 20180101; A61P 37/02 20180101; A61P 43/00 20180101; A61P
29/00 20180101 |
Class at
Publication: |
435/005 ;
435/007.23 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; G01N 33/574 20060101 G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 7, 2002 |
EP |
02290578.0 |
Claims
1-8. (canceled)
9. A method of screening compounds for their capacity to modulate
apoptosis in cells which produce pro- or anti-apoptotic
polypeptides which are located in lipid rafts when said cells are
cultured under non apoptotic conditions, said method comprising: a.
culturing said cells in a growth medium for maintaining non
apoptotic conditions; b. contacting said cultured cells with a
candidate compound; c. determining the level of one or several pro-
or anti-apoptotic polypeptides associated to lipid rafts ; and, d.
selecting the compound which interferes with the association of one
or several pro- or anti-apoptotic polypeptides with lipid rafts,
said compound having the capacity to modulate apoptosis.
10. A method of screening compounds for their capacity to promote
apoptosis in cells, said method comprising a. culturing mammalian
cells in a growth medium for maintaining non apoptotic conditions;
wherein said cells produce a pro-apoptotic protein which is located
in lipid rafts under non apoptotic conditions of said cells; b.
contacting said cultured cells with a candidate compound; and, c.
determining the absence or the presence of lipid rafts in said
cultured cells; d. in case of presence of lipid rafts, optionally
determining the level of pro-apoptotic protein located in the lipid
rafts, wherein the absence of lipid rafts in the plasma membrane
of, cells incubated with said candidate compound or if determined,
the reduced level of pro-apoptotic protein in the rafts is
indicative that said compound promotes apoptosis.
11. A method of screening compounds for their capacity to inhibit
or prevent apoptosis of cells, said method comprising a. culturing
cells in a growth medium for maintaining non apoptotic conditions;
wherein said cells produce a pro-apoptotic protein which is located
in lipid rafts under, non apoptotic conditions; b. contacting said
cells with a candidate compound; c. culturing cells under apoptotic
conditions; d. determining the absence or the presence of lipid
rafts; e. in the case of presence of lipid rafts, optionally
determining the level of pro-apoptotic protein located in the lipid
rafts; wherein the presence of lipid rafts in the plasma membranes
of cells incubated with said candidate compound and optionally the
maintained level of pro-apoptotic protein in the rafts is
indicative that said candidate compound inhibits or prevents
apoptosis.
12. The method according to claim 9, wherein a pro-apoptotic
protein located in lipid rafts under proliferative conditions is
the Bad protein.
13. The method of claim 12, wherein said cells which produce a Bad
protein are cells characteristic of the immune system, preferably T
cells.
14. The method according to claim 9, wherein the presence or the
absence of lipid rafts is visualized by confocal microscopy.
15. The method according to claim 9, wherein the presence or the
absence of lipid rafts is determined by the following steps i)
recovering the cultured cells incubated with said compound
candidate and resuspending said cells in a buffer appropriate for
subcellular fractionation, such as gradient sucrose buffer; ii)
ultracentrifugating the fractionated cells and; iii) recovering the
subcellular fraction which should contain lipid rafts; iv)
determining whether the recovered subcellular fraction contains
ganglioside and/or lipid raft associated molecule(s).
16. The method according to claim 15, wherein the presence or the
absence of lipid rafts is determined by the use of a marker which
specifically recognizes ganglioside or a raft-associated
molecule.
17. The method according to claim 16, wherein said marker is
selected among cholera toxin subunit B (CTx), anti-Bad antibody or
anti-Lck antibody.
18. The method of claim 9, wherein said cells are mammalian
cells.
19. The method of claim 9, wherein said non apoptotic conditions
are proliferative conditions.
20. The method of claim 9, wherein said growth medium comprises at
least a cytokine or a growth factor necessary for maintaining
proliferative growth conditions.
21. The method of claim 11, wherein the apoptotic conditions of
step c) are obtained by depriving the cells with said cytokine or
growth factor.
22. The method of claim 20, wherein a cytokine or growth factor
necessary for maintaining proliferative growth conditions is an
interleukin, preferably selected among IL-4, IL-2 or IL-9, or a
mixture thereof.
23. A use of a compound capable of modulating lipid rafts
formation, in the preparation of a medicine for the treatment of
disorders induced by or associated with a defective regulation of
cell death or of any specific pathology in which cell death may be
at least a part of the therapy.
24. The use of claim 23, wherein said defective regulation affects
cells which produce Bad protein.
25. The use of claim 24, wherein said defective regulation affects
cells of the immune system.
26. The use of any of claim 23, wherein said compound is capable of
disrupting lipid rafts and wherein said defective regulation of
cell death results in an abnormal decrease of cell death.
27. The use of claim 26, wherein said abnormal decrease of cell
death is related to cancer disease and especially to
lymphoproliferative cancers, infectious disease and especially
viral disease, inflammatory disease or auto-immune disease.
28. The use of any of claim 23, wherein a compound capable of
disrupting lipid rafts is methyl-.beta.-cyclodextrin or
filipin.
29. The use of claim 23, wherein said compound is capable of
reconstituting lipid rafts in the plasma membrane of cells and
wherein said defective regulation of apoptosis results in an
abnormal increase of cell death.
30. The use of claim 29, wherein a pathology resulting in an
abnormal increase of cell death is a disease associated to
senescence, neurodegenerative disease Alzheimer, AIDS, ischemic
cell death or wound-healing.
31. The use of claim 29, wherein a compound capable of
reconstituting lipid rafts is edelfosine.
32. An in vitro method for the detection of a defective regulation
of apoptosis, in a sample of cells of an individual, said method
comprising determining the presence or the absence of lipid rafts
in said cells, wherein the absence of said lipid rafts is
indicative of a defective regulation of apoptosis.
33. The method according to claim 32, wherein said cells are cells
of the immune system of an individual affected by a
lymphoproliferative disease.
34. The method of claim 32, wherein the presence or the absence of
lipid rafts is determined by detecting the presence or absence of a
pro-apoptotic or an anti-apoptotic protein which is known to be
located in lipid rafts under non apoptotic conditions of cells.
35. The method of claim 34, wherein a pro-apoptotic protein known
to be located in lipid rafts under non apoptotic conditions is a
Bad protein.
36. A use of a compound appropriate for detecting the presence of
lipid rafts, in the in vitro detection method according to claim
32.
37. The use of claim 36, wherein a compound appropriate for
detecting the presence of lipid rafts is a compound which
specifically recognizes Bad protein, Lck protein or ganglioside
GM1.
38. The use of claim 37, wherein said compound is selected among
cholera toxin subunit B (CTx), anti-Bad antibody and anti-Lck
antibody.
39. A use of a compound capable of modulating pro- or
anti-apoptotic protein rafts localization for the preparation of a
medicine for the treatment of disorders induced by or associated
with a defective regulation of cell death or of any specific
pathology in which cell death may be at least a part of the
therapy.
40. The use of claim 39, wherein said compound promote pro- or
anti-apoptotic protein segregation from lipid rafts.
41. The use of claim 39, wherein said compound promotes pro- or
anti-apoptotic protein localization in lipid rafts.
42. An in vitro method for the detection of a defective regulation
of apoptosis, in a sample of cells of an individual, said method
comprising determining the presence or the absence of lipid rafts
in said cells, wherein the presence of a great number of lipid
rafts compared to normal cells is indicative of a defect regulation
of apoptosis.
43. The method according to claim 10, wherein a pro-apoptotic
protein located in lipid rafts under proliferative conditions is
the Bad protein.
44. The method of claim 43, wherein said cells which produce a Bad
protein are cells characteristic of the immune system, preferably T
cells.
45. The method according to claim 10, wherein the presence or the
absence of lipid rafts is visualized by confocal microscopy.
46. The method according to claim 10, wherein the presence or the
absence of lipid rafts is determined by the following steps i)
recovering the cultured cells incubated with said compound
candidate and resuspending said cells in a buffer appropriate for
subcellular fractionation, such as gradient sucrose buffer; ii)
ultracentrifugating the fractionated cells and; iii) recovering the
subcellular fraction which should contain lipid rafts; iv)
determining whether the recovered subcellular fraction contains
ganglioside and/or lipid raft associated molecule(s).
47. The method according to claim 45, wherein the presence or the
absence of lipid rafts is determined by the use of a marker which
specifically recognizes ganglioside or a raft-associated
molecule.
48. The method according to claim 47, wherein said marker is
selected among cholera toxin subunit B (CTx), anti-Bad antibody or
anti-Lck antibody.
49. The method of claim 10, wherein said cells are mammalian
cells.
50. The method of claim 10, wherein said non apoptotic conditions
are proliferative conditions.
51. The method of claim 10, wherein said growth medium comprises at
least a cytokine or a growth factor necessary for maintaining
proliferative growth conditions.
52. The method according to claim 11, wherein a pro-apoptotic
protein located in lipid rafts under proliferative conditions is
the Bad protein.
53. The method of claim 52, wherein said cells which produce a Bad
protein are cells characteristic of the immune system, preferably T
cells.
54. The method according to claim 11, wherein the presence or the
absence of lipid rafts is visualized by confocal microscopy.
55. The method according to claim 11, wherein the presence or the
absence of lipid rafts is determined by the following steps i)
recovering the cultured cells incubated with said compound
candidate and resuspending said cells in a buffer appropriate for
subcellular fractionation, such as gradient sucrose buffer; ii)
ultracentrifugating the fractionated cells and; iii) recovering the
subcellular fraction which should contain lipid rafts; iv)
determining whether the recovered subcellular fraction contains
ganglioside and/or lipid raft associated molecule(s).
56. The method according to claim 54, wherein the presence or the
absence of lipid rafts is determined by the use of a marker which
specifically recognizes ganglioside or a raft-associated
molecule.
57. The method according to claim 56, wherein said marker is
selected among cholera toxin subunit B (CTx), anti-Bad antibody or
anti-Lck antibody.
58. The method of claim 11, wherein said cells are mammalian
cells.
59. The method of claim 11, wherein said non apoptotic conditions
are proliferative conditions.
60. The method of claim 11, wherein said growth medium comprises at
least a cytokine or a growth factor necessary for maintaining
proliferative growth conditions.
Description
[0001] The invention relates to the modulation of apoptosis in
mammalian cells. More particularly, the invention provides methods
for identifying novel pro-apoptotic or anti-apoptotic cellular
polypeptides, methods of screening compounds which modulate
apoptosis, and method of detecting early events of the apoptotic
process.
[0002] Apoptosis or programmed cell death is an active process in
which cells induce their self-destruction in response to specific
cell death signals or in the absence of cell survival signals. This
active process is actually essential in the normal development and
homeostasis of multicellular organisms. It is opposed to necrosis
which is cell death occurring as a is result of severe injurious
changes in the environment.
[0003] Apoptosis of a cell can be characterized at least by [0004]
the rapid condensation of the cell with collapse of the nucleus but
preservation of membranes; or, [0005] cleavage of nuclear DNA at
the linker regions between nucleosomes to produce fragments which
can be easily visualized by agarose gel electrophoresis as a
characteristic ladder pattern.
[0006] Various pathologies occur due to a defective or aberrant
regulation of apoptosis in the affected cells of an organism. For
example, defects that result in a decreased level of apoptosis in a
tissue as compared to the normal level required to maintain the
steady-state of the tissue can promote an abnormal increase of the
amount of cells in a tissue. This has been observed in various
cancers, where the formation of tumors occurs because the cells are
not dying at their normal rate. Some DNA viruses such Epstein-Barr
virus, African swine fever virus and adenovirus, also inhibit or
modulate apoptosis, thereby repressing cell death and allowing the
host cell to continue reproducing the virus.
[0007] On the contrary, a defect resulting in an increase of cell
death in a tissue may be associated with degenerative disorders
wherein cells are dying at a higher rate than they regenerate. This
is observed in various disorders, such as AIDS, senescence, and
neurodegenerative diseases.
[0008] Compounds that modulate positively or negatively apoptosis
can provide means for the treatment or the prevention of these
disorders. As a consequence, the delineation of apoptotic pathways
provides targets for the development of therapeutic agents that can
be used to modulate the response of a cell to apoptotic or cell
survival signals.
[0009] Progresses have been made in identifying extracellular,
intracellular and cell surface molecules that regulate apoptosis.
Previous studies have focused on the identification of specific
cell death signals, (such as the deprivation of growth factors, the
FAS/TNF system, genotoxic agents, glucocorticoids . . . ), members
of the Bcl-2 family and ICE-type proteases. But critical steps in
apoptotic pathways remain to be identified.
[0010] Accordingly, there is still a need in identifying the
cellular mechanisms involved in apoptotic pathways, and target for
the development of therapeutic agents that can be used to modulate
cell apoptosis.
[0011] Among the different transducing agents, the Bcl-2 family
proteins act as an intracellular checkpoint in the apoptotic
pathway. The family is divided into two functional groups
(medecine/sciences 97;13:384-6): the proteins that suppress
cell-death (anti-apoptotic members such as Bcl-2, Bcl-x.sub.L,
Bcl-w, Bag-1, McI-1, A1) and the proteins that promote cell death
(pro-apoptotic members such as Bim, Nix, Hzk, Bax, Bak,
Bcl-x.sub.s, Bad, Bik). The Bcl-2 family has been defined by
sequence homology based upon specific conserved motifs termed
BCL-homology regions (BH1, BH2, BH3 and BH4 domains). BH1, BH2 and
BH3 domains have been shown to be important in homodimerization or
heterodimerization and in modulating apoptosis. Anti-apoptotic
molecules have a specific BH4 domain.
[0012] It has been proposed that the ratio of pro-apoptotic members
to anti-apoptotic members expressed in a cell determines whether
this cell will respond to an apoptotic signal. Indeed,
pro-apoptotic and anti-apoptotic members antagonized each other by
forming inactive heterodimers (Oltvai et al., 1993, Cell 74:
609-619), as a consequence only the balance may promote or prevent
a cell to undergo apoptosis.
[0013] More recently, it has been shown that phosphorylation of
Bcl-2 proteins can also modulate their activity. Indeed, different
anti-apoptotic pathways are likely to be activated by growth
factors, involving phosphatidylinositol 3 kinase (Pl3K), Akt kinase
and Ras activated kinases.
[0014] In particular, upon stimulation of cells with IL-3 and NGF,
the pro-apoptotic Bad protein (Bcl-x.sub.L/Bcl-2 Associated cell
Death regulator, Downward, 1999, Nature Cell Biol 1: 33-35) becomes
serine phosphorylated, resulting in association to 14-3-3 protein
(Hsu et aL, 1997, Mol Endocrinol 11: 1858-1867). It was proposed
that such interaction facilitates the translocation of
phosphorylated Bad from the mitochondrial membrane to cytosolic
compartments, sequestering it therein and thus, preventing further
interaction with other anti-apoptotic Bcl-2 members (U.S. Pat. No.
5,856,445).
[0015] It was further shown that association of 14-3-3 protein to
Bad is dependent upon serine 155 phosphorylation of Bad (WO
0110888, Apoptosis Technology Inc., 2001).
[0016] The results disclosed in the present invention indicate that
some pro- or anti-apoptotic proteins especially of the Bcl-2
family, are regulated through a newly identified subcellular
localization that is in lipid rafts formed in the plasma membrane.
This observation offers a way to a novel general mechanism of
regulation of cell apoptosis that may play a role in the regulation
of pro- or anti-apoptotic molecules in response to cell death or
cell survival signals.
[0017] Localization of proteins to distinct subcellular
compartments, including membranes, is a critical event in multiple
cellular pathways.
[0018] Plasma membranes of many cell types contain microdomains
commonly referred to as lipid rafts, which are biochemically
distinct from bulk plasma membranes (Brown and London, 1998, Annu
Rev Cell Dev Biol 14: 111-136). These domains consist of dynamic
assemblies of sphingolipids and cholesterol. More specifically, the
presence of saturated hydrocarbon chains in sphingolipids allows
for cholesterol to be tightly intercalated, leading to the presence
of distinct liquid-ordered phases, and thereby more fluid, lipid
bilayer. Lipid rafts can be isolated by subcellular fractionation
and density gradient ultracentrifugation according to methods
described in Hacki et al. (Oncogene 2000 19: 2286-2295) and Millan
et al. (Eur J Immunol 1998 28: 2675-3684). They can also be
visualized in intact cells by confocal microscopy using, for
example, fluorescently labelled cholera toxin subunit B (CTx) which
binds to the ganglioside GM1 (Harder et aL, 1998, J Cell Biol 141:
929-942). One key element of lipid rafts is that they can include
or exclude proteins to varying degrees.
[0019] In T cells, a number of proteins involved in signal
transduction such as IcK, Lat, copurify with lipid rafts isolated
on sucrose gradient. Disruption of rafts integrity by a variety of
methods inhibits early activation events, supporting a critical
role for these domains in the recruitment for signalling and thus,
in signal transduction from cell surface receptors. For example,
the antitumor ether lipid
1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3;
edelfosine) was shown to trigger apoptosis via translocation of Fas
to lipid rafts and subsequent Fas recruitment by lipid rafts
(Gajate and Mollinedo, Blood, 2001, 98: 3860-3863).
[0020] The present invention results from the discovery of a novel
mechanism of cellular regulation of the activity of pro or
anti-apoptotic molecules in a cell by translocation of these
molecules into lipid rafts under non apoptotic conditions such as
proliferative conditions or conditions where cells do not
divide.
[0021] Indeed, the inventors have surprisingly found that
interaction of a pro-apoptotic protein, such as the Bad protein,
with rafts is an active process regulated by cytokines or growth
factors. They have also shown that segregation of this molecule
from rafts in cytokine or growth factor deprived-cells is involved
in the induction of apoptosis and associated with raft
disorganization.
[0022] The invention thus provides methods for identifying cellular
polypeptides which have pro-apoptotic or anti-apoptotic activity in
a particular cell type.
[0023] The invention also provides means for screening apoptosis
modulating compounds which interfere with the newly identified
mechanism of apoptosis regulation. Candidate compounds in this
respect may either interfere by blocking, preventing or stimulating
translocation of one or several pro- or anti-apoptotic polypeptides
in lipid rafts under non apoptotic conditions. Candidate compounds
may in addition or alternatively interfere by disrupting or
reconstituting lipid rafts in a cell which normally produce pro- or
anti-apoptotic polypeptides located in lipid rafts under non
apoptotic conditions. According to another embodiment, candidate
compounds may in addition or alternatively interfere by segregation
of pro- or anti-apoptotic polypeptides from lipid rafts.
[0024] The invention also provides a compound capable of modulating
association of a pro- or anti-apoptotic polypeptide with lipid
rafts.
[0025] The invention also provides a compound capable of modulating
transfer of a pro- or anti-apoptotic polypeptide between a lipid
raft and another cellular localization.
[0026] The invention also provides for the use of compounds capable
of modulating lipid rafts formation or of modulating translocation
of pro- or anti-apoptotic proteins in rafts in the preparation of a
medicine for the prevention and/or treatment of disorders induced
by or associated with a defective regulation of cell death as well
as of specific pathologies in which the death of infected or
deregulated cells may be at least art of a therapy.
[0027] Among the several advantages of the present methods, it
should be noted that the apoptotic or non apoptotic state of a cell
can be determined according to the present methods in a relatively
short period of time by analysing lipid raft organization. In
particular, there is no need to quantify specific gene expression.
The methods of the invention are thus particularly appropriate for
routine high throughput screening of apoptosis modulating
compounds.
[0028] Furthermore, the invention provides methods for detecting
early events of the apoptotic process in a cell.
[0029] A first object of the invention is a method of screening
cellular polypeptides for pro-apoptotic or anti-apoptotic activity
in a cell of a particular cell-type, said method comprising: [0030]
a. culturing cells of said particular cell-type under non apoptotic
conditions and culturing cells of said particular cell-type under
apoptotic conditions; and, [0031] b. determining subcellular
localisation of said cellular polypeptides in the cultured
mammalian cells; wherein a localization of a cellular polypeptide
in lipid rafts in cells cultured under non apoptotic conditions and
a segregation of said cellular polypeptide from lipid rafts in
cells cultured under apoptotic conditions is indicative of the
pro-apoptotic or an anti-apoptotic activity of said cellular
polypeptide in said particular cell-type.
[0032] In a particular embodiment, said cultured cells are
mammalian cells.
[0033] In a preferred embodiment, the cells cultured under non
apoptotic conditions are cultured under proliferative conditions.
According to the methods of the invention, cells are considered to
be cultured "under non apoptotic conditions" when the proportion of
cells undergoing apoptotic process in the cell culture is
relatively stable in time and does not represent more than 10%,
preferably, more than 1% of the whole cell population (depending
upon the cell-type).
[0034] In a preferred embodiment, non apoptotic conditions are
proliferative conditions.
[0035] On the contrary, cells are considered to be cultured "under
apoptotic conditions" when the proportion of the cells undergoing
apoptotic process increases dramatically in time to reach, after a
certain period, especially for around 24 hours, from deprivation of
growth or proliferation factor or by use of an apoptotic factor,
more than 50% of the whole cell population.
[0036] Cells which have undergone apoptotic process can be
characterized, for example, by specific cleavage of nuclear DNA
which can be visualized on agarose gel electrophoresis.
[0037] As used herein, the term "cellular polypeptide" refers to
any polypeptide which is produced in a cell by gene expression. It
can be a polypeptide naturally encoded in said cell especially by a
native gene, or a polypeptide not naturally encoded in said cell,
meaning that the gene encoding said polypeptide or a coding
sequence derived from said identified gene has been recombined in
the genome of the cell to obtain expression. It can be a mutated
form of a naturally occurring polypeptide and more specifically, a
mutated form wherein the mutation is involved in abnormal
subcellular localisation of said polypeptide under proliferative
growth conditions.
[0038] As used herein, the term "lipid rafts" refers to dynamic
assemblies of sphingolipids and cholesterol in plasma membranes
forming microdomains with distinct liquid-ordered phases, said
microdomains stably retaining specific structures, such as
gangliosides or polypeptides such as Lck. Lipid rafts can be
biochemically isolated by subcellular fractionation and density
gradient ultracentrifugation according to the methods described in
Hacki et al. (Oncogene 2000 19: 2286-2295) and Millan et al. (Eur J
Immunol 1998 28: 2675-3684) and in the examples below. They can
also be visualized in intact cells by confocal microscopy using,
for example, fluorescently labelled cholera toxin subunit B (CTx)
which binds to the ganglioside GM1 which accumulate in lipid rafts
(Harder et al., 1998, J Cell Biol 141: 929-942). More specifically,
subcellular localization of a particular polypeptide in lipid rafts
is determined by double immunofluorescence using a labelled marker
detecting lipid rafts and another labelled marker detecting the
polypeptide to localize. It can also be determined by analysing the
presence of said polypeptide in subcellular fractions containing
lipid rafts.
[0039] In a particular embodiment, the method of the invention is
appropriate to screen a polypeptide, whose structure is known but
whose function is unknown, for a pro- or anti-apoptotic activity in
a particular cell type. In particular, the one skilled in the Art
can use the method of the invention to screen polypeptides which
are suspected to be involved in apoptosis regulation according to
specific features such as specific structural domains. The
screening of cellular polypeptides is however not lo necessarily
limited to cellular polypeptides of known structure.
[0040] Several pro-apoptotic proteins have been identified so far,
however, expression pattern of these proteins may vary depending
upon the cell type. The method of screening is thus also useful in
determining whether a putative cellular polypeptide, known to be
pro or anti-apoptotic in a certain cell-type is involved in
apoptosis modulation in another cell-type.
[0041] In a particular embodiment, the screened polypeptides belong
to the Bcl-2 family. As mentioned hereabove, the Bcl-2 family,
members are characterized by sequence homology based upon specific
conserved motifs termed BCL-homology regions (BH1, BH2, BH3 and BH4
domains). Accordingly, their subcellular localisation can be
determined by the use of a molecule which specifically recognizes a
BH domain. Such molecules encompass for example monoclonal
antibodies or polyclonal antibodies specifically recognizing a BH
domain. One example of a molecule that interacts with BH3 motif in
PP1a (Ayllon, et al. 2000. EMBO J.; 19: 2237-2246). Examples of
molecules that interact with BH4 motif are review in Admas, J. M.
and Cory, S. (1998) Science, 281, 1322). In a particular
embodiment, a molecule which specifically recognizes a BH4 domain
is used to screen preferably for anti-apoptotic molecules. In
another particular embodiment, a molecule which specifically
recognizes a BH3 domain is used to screen for pro- or
anti-apoptotic molecules. A combination of molecules recognizing
the different BH domains can also be used, for example to screen
for pro-apoptotic molecules which have only the BH3 domain.
[0042] The method of the invention is also appropriate to screen
novel polypeptides of the Bcl-2 family whose structure and function
are unknown at least in part, but which can be easily isolated
using molecular recognition of their BH domain(s). As a result, in
a preferred embodiment, said screened cellular polypeptides are
first isolated from biochemically isolated lipid rafts of said
mammalian cells cultured in proliferative conditions by the use of
a molecule which specifically recognizes a BH domain. More
specifically, the polypeptides present in the isolated lipid rafts
can be separated on a gel and analysed by Western Blot analysis
using an antibody which recognizes a BH domain or by similar
methods of protein analysis. The polypeptides recognized by a BH
domain can be isolated and antibodies which recognize each isolated
polypeptides can be produced according to usual methods well known
in the art. The subcellular localization of one or more of the
isolated polypeptides is then determined according to the method of
the invention using such specific antibodies to screen for
apoptotic activity.
[0043] The proteins which are associated with lipid rafts may have
a transmembrane domains or have undergone post-translational
modifications such as myristoylation. In another specific
embodiment, said screened cellular polypeptides are further
isolated by the use of a molecule which specifically recognizes a
mirystoylated polypeptide.
[0044] In a preferred embodiment, the method is carried out for
screening cellular polypeptides of a cell type characterized by the
production of Bad protein, i.e. Bad.sup.+cell type. According to
another preferred embodiment, the cells are characteristic of the
immune system, and most preferably are T cell lines.
[0045] Indeed, it is shown, in the examples below, that the
pro-apoptotic Bad protein is sequestered in lipid rafts of IL-4
stimulated T-cells and segregates from rafts in IL-4 deprived
T-cells.
[0046] More specifically, it is shown in the Example that Bad can
be co-purified with lipid-rafts by subcellular fractionation and
density gradient ultracentrifugation from cells under non-apoptotic
conditions, especially under proliferative conditions. These
results indicate that Bad is strongly associated with lipid rafts
in cells cultured under non apoptotic conditions such as
proliferative conditions.
[0047] Cellular polypeptides which physically interacts with Bad
protein in isolated lipid rafts of Bad.sup.+cells thus constitute
preferred putative polypeptides to screen for pro- or
anti-apoptotic activity. Accordingly, in a preferred embodiment,
the screened cellular polypeptides are isolated from isolated lipid
rafts of Bad.sup.+cells cultured under proliferative conditions and
are selected among the polypeptides which interact physically with
the Bad protein.
[0048] Naturally, the invention also pertains to the newly
identified cellular polypeptides having pro- or anti-apoptotic
activity and their use in providing means for modulating apoptosis
in cells, such as mammalian cells, expressing these
polypeptides.
[0049] It is another object of the invention to provide a method of
screening compounds for their capacity to modulate apoptosis in
cells, which produce pro- or anti-apoptotic polypeptides which are
located in lipid rafts when said cells are cultured under non
apoptotic conditions, said method comprising: [0050] a) culturing
said cells in a growth medium maintaining non apoptotic conditions;
[0051] b) contacting said cultured cells with a candidate compound;
[0052] c) determining the level of one or several pro- or
anti-apoptotic polypeptides associated to lipid rafts; [0053] d)
selecting the compound which interferes with the association of one
or several pro- or anti-apoptotic polypeptides with lipid rafts,
said compound having the capacity to modulate apoptosis.
[0054] A compound interferes with the association of a pro- or
anti-apoptotic polypeptide when it modifies said association,
including when it alters the chemical and/or the physical nature of
said association or when it provides or influences segregation of
pro- or anti-apoptotic polypeptides from lipid rafts, or when it
prevents said association, or also when it acts on and especially
promotes disruption of lipid rafts or more generally alter
constitution of lipid rafts.
[0055] The invention further relates to a method of screening
compounds for their capacity to promote apoptosis in cells, said
method comprising [0056] a) culturing cells in a growth medium
maintaining non apoptotic conditions; wherein said cells produce a
pro-apoptotic protein which is located in lipid rafts under non
apoptotic conditions of said cells; [0057] b) contacting said
cultured cells with a candidate compound; and, [0058] c)
determining the absence or the presence of lipid rafts in said
cultured cells; [0059] d) in case of presence of lipid rafts,
optionally determining the level of pro-apoptotic protein located
in the lipid rafts, wherein the absence of lipid rafts in the
plasma membrane of cells incubated with said candidate compound or
if determined, the reduced level of pro-apoptotic protein in the
rafts is indicative that said compound promotes apoptosis.
[0060] The invention also relates to a method of screening
compounds for their capacity to inhibit or prevent apoptosis of
cells, said method comprising: [0061] a) culturing cells in a
growth medium for maintaining non-apoptotic conditions; wherein
said cells produce a pro-apoptotic protein which is located in
lipid rafts under non apoptotic conditions; [0062] b) contacting
said cells with a candidate compound; [0063] c) culturing cells
under apoptotic conditions; and, [0064] d) determining the absence
or the presence of lipid rafts; [0065] e) in the case of presence
of lipid rafts, optionally determining the level of pro-apoptotic
protein located in the lipid rafts, wherein the presence of lipid
rafts in the plasma membranes of cells incubated with said
candidate compound and optionally the maintained level of
proapoptotic protein in the rafts is indicative that said candidate
compound inhibits or prevents apoptosis.
[0066] In a preferred embodiment, the cells are cultured in a
growth medium comprising at least a cytokine or a growth factor
necessary for maintaining proliferative growth conditions and step
c) of the method comprises depriving the cells of said cytokine or
growth factor necessary for maintaining proliferative growth
conditions.
[0067] As used herein, the term "compound" refers to inorganic or
organic chemical or biological compounds either natural (isolated)
or synthetic, and especially encompass nucleic acids, proteins,
polypeptides, peptides, glycopeptides, lipids, lipoproteins and
carbohydrates.
[0068] Any cells in-which pro or anti-apoptotic proteins may be
translocated in lipid rafts can be used in the methods of the
invention. In a preferred embodiment of the methods of the
invention, cells are mammalian cells.
[0069] Mammalian cells which are used in the methods of screening
compounds can be any mammalian cells whose cell survival can be
controlled by a specific cytokine or growth factor. In preferred
embodiments of the above methods, the cultured mammalian cells are
selected among those which produce the Bad protein as a
pro-apoptotic protein. More specifically, preferred mammalian cells
which produce a Bad protein are selected among cells characteristic
of the immune system, and more preferably among T cells.
[0070] As used herein, the term "cytokine or growth factor" refers
to any molecule which is necessary to be present in a growth medium
to prevent apoptotic process of a cultured cell and/or to promote
cell proliferation. Known cytokines include any interleukin. Known
growth factors include the fibroblast growth factors, bFGF, aFGF,
FGF6, the hepatocyte growth factors HGF/SF, the epidermis growth
factor, EGF and other characterized growth factors such as IGF-1,
PDGF, LIF, VEGF, SCF, TGFb, TNFa, NGF, BMP, neuregulin,
thrombopdietin and growth hormone. Growth factors according to the
invention can include also, progestagenes and derivatives thereof
(progesterone), oestrogens and derivative thereof (oestradiol),
androgenes (testosterone), mineralocorticoids and derivatives
thereof (aldosterone), LH, LH-RH, FSH et TSH hormones, T3, T4, and
retinoidic acid, calcitonine E2 and F2/alpha prostaglandins.
Glucocorticoids (natural or hemisynthetic, i.e. hydrocortisone,
dexamethasone, prednisolone or triamcinolone), can also be
used.
[0071] Cells characteristic of the immune system can be
advantageously cultured under stimulation with an interleukin for
maintaining proliferative growth conditions. In particular, IL-4,
IL-2 or IL-9 interleukin can be used in this context, or a mixture
thereof.
[0072] However, any available apoptosis model can be used to select
the cell type and the factors for non apoptotic conditions such as
the growth factor or cytokine, used in the methods. As used herein,
the term "apoptosis modef" comprises any teaching providing a way
to control cell apoptosis in a cell culture of a specific cell type
by the use of specific factors for non apoptotic conditions such as
a specific cytokine or growth factor or a mixture thereof. Such
apoptosis models are for example the control of IL-4 stimulated
T-cell lines, IL3 and hematopoietic progenitor, PC12 and CRH
(corticotropin-releasing hormone), HN9.10.
[0073] A candidate compound may modulate apoptosis by blocking or
preventing the association of said pro or anti-apoptotic
polypeptide with lipid rafts. In this context, the subcellular
localisation of said pro or anti-apoptotic polypeptide in lipid
rafts is no more observed in cells incubated with the compound when
compared to cells not incubated with the compound, or, at least,
lipid rafts subcellular localisation of said pro- or anti-apoptotic
polypeptide is significantly reduced when compared to cells not
incubated with the compound. It is another object of the invention
to provide compounds capable of modulating association of a pro- or
anti-apoptotic polypeptide with lipid rafts.
[0074] Some of these compounds may modulate apoptosis by preventing
the association of a pro- or anti-apoptotic polypeptide with lipid
rafts, by promoting segregation of a pro- or anti-apoptotic
polypeptide from lipid rafts or by promoting disruption of lipid
rafts.
[0075] Some of these compounds may modulate apoptosis by preventing
the segregation of a pro- or anti-apoptotic polypeptide from lipid
rafts, by promoting the association of a pro- or anti-apoptotic
polypeptide with lipid rafts or by promoting constitution of lipid
rafts.
[0076] It is another object of the invention to provide compounds
capable of modulating transfer of a pro- or anti-apoptotic
polypeptide between a lipid raft and another cellular
localization.
[0077] Some of these compounds may modulate apoptosis by preventing
transfer of a pro- or anti-apoptotic polypeptide from a cellular
localization, other than a lipid raft, to a lipid raft or from a
lipid raft to another cellular localization.
[0078] Some of these compounds may modulate apoptosis by promoting
transfer of a pro- or anti-apoptotic polypeptide from a cellular
localization, other that a lipid raft, to a lipid raft or from a
lipid raft to another cellular localization.
[0079] In a preferred embodiment, the compounds of the invention
modulating apoptosis are capable of modulating the association of a
pro-apoptotic protein of Bcl-2 family, especially Bad, with lipid
rafts or transfer of said protein between lipid rafts and another
cellular localization.
[0080] Some of said compounds may promote apoptosis in a
Bad-producing cell by preventing association of Bad with lipid
rafts, by promoting segregation of Bad from lipid rafts or by
promoting disruption of lipid rafts.
[0081] Some of said compounds may inhibit apoptosis in a
Bad-producing cell by promoting association of Bad with lipid
rafts, by preventing segregation of Bad from lipid rafts or by
promoting constitution of lipid rafts.
[0082] In a particular embodiment, a compound of the invention may
inhibit apoptosis of a Bad-producing cell by preventing transfer of
Bad to mitochondria after Bad segregation from lipid rafts. Such a
compound may interact with Bad by means of a motif similar to the
lipid raft motifs which allow association of Bad to lipid
rafts.
[0083] Lipid rafts subcellular localisation of said pro- or
anti-apoptotic polypeptide can be quantified by any quantitative
analysis methods available in the art. A significant reduction of
lipid rafts subcellular localisation is observed when the level of
pro- or anti-apoptotic polypeptide is reduced to at least 50%,
preferably 90% in cells incubated with the candidate compound or
compared to cells not incubated with the candidate compound.
[0084] A candidate compound may modulate apoptosis by blocking or
preventing the segregation of said pro- or anti-apoptotic
polypeptide from lipid rafts. In this context, the subcellular
localisation of said pro- or anti-apoptotic polypeptide in lipid
rafts of cells cultured under conditions promoting pro- or
anti-apoptotic protein segregation (e.g., apoptotic conditions for
pro-apoptotic proteins) remains observed in cells incubated with
the compound when compared to cells not incubated with the compound
and lipid rafts subcellular localisation of said pro- or
anti-apoptotic polypeptide is significantly maintained when
compared to cells not incubated with the compound.
[0085] Lipid rafts subcellular localisation of said pro- or
anti-apoptotic polypeptide can be quantified by any quantitative
analysis methods available in the art. A significant maintenance is
observed when the level of pro- or anti-apoptotic polypeptide is
maintained to at least 50%, preferably 90% in cells incubated with
the candidate compound or compared to cells not incubated with the
candidate compound or compared to cells not incubated with the
candidate compound.
[0086] By "determining the absence of lipid rafts", it is
understood that lipid rafts in cells incubated with the candidate
compound are not detected, or at least, are detected as traces, or
are detected in a significantly reduced level (i.e., minimum 20%
less) with the methods disclosed in the present invention as
compared with a culture of cells not incubated with the candidate
compound as a control. The proportion of lipid rafts in a cell can
be compared between the cell cultures using any quantitative
analysis methods available in the art. A significant reduction of
lipid rafts is observed when their proportion is reduced from 20%,
preferably is reduced to at least 50%, preferably 90% in cells
incubated with the candidate compound to the control. For example,
by confocal microscopy analysis, the profile of fluorescence can be
quantified by image analysis of cells incubated with the candidate
compound and cells not incubated with the candidate compound.
[0087] Similarly, by "determining the presence of lipid rafts", it
is understood that lipid rafts in cells incubated with the
candidate compound are detected in substantially the same amount as
compared with a culture of cells not incubated with the candidate
compound as a control.
[0088] Methods to isolate lipid rafts have been described below.
More specifically, it is possible to isolate lipid rafts from
mammalian cells, by cell fractionating over sucrose gradient and
immunoblofting subcellular fractions with markers specific for
rafts in order to identify rafts containing subcellular fractions.
The presence or the absence of lipid rafts can be thus determined
in a specific embodiment by the following steps [0089] i)
recovering the cultured cells incubated with said compound and
resuspending said cells in a buffer appropriate for subcellular
fractionation, such as gradient sucrose buffer; [0090] ii)
ultracentrifugating the fractionated cells; [0091] iii) recovering
the subcellular fraction which should contain lipid rafts; and,
[0092] iv) determining whether the recovered subcellular fraction
contains ganglioside and/or lipid raft associated molecule(s).
[0093] As used herein, "the subcellular fraction which should
contain lipid rafts" is the subcellular fraction corresponding to
the banded organelles of the gradient which contains lipid rafts in
a gradient obtained with cells cultured under non apoptotic
conditions. Naturally, in apoptotic conditions, the corresponding
cell fraction will contain much less lipid rafts. Lipid rafts and
lipid rafts subcellular localisation of pro- or anti-apoptotic
polypeptides can also be directly visualized in intact cells by
confocal microscopy using a molecular marker which specifically
binds to a raft-associated molecule or a ganglioside.
[0094] Such preferred molecular markers are, for example, the
cholera toxin subunit B (CTx) which specifically recognizes
ganglioside GM1, anti-Bad antibody or anti-Lck antibody. More
generally, any antibody directed to any cellular polypeptide newly
identified according to the method of the invention as described
above can be used as a molecular marker specific for raft.
[0095] The invention also concerns the compounds identified by the
methods of screening.
[0096] Such compounds identified by the above methods of the
invention are useful for the prevention and/or treatment of
disorders induced by or associated with a defect;He regulation of
cell death or of specific pathologies where death of infected or
deregulated cells may be at least part of a therapy.
[0097] The invention further provides a use of a compound capable
of modulating lipid rafts formation, in the preparation of a
medicine for the treatment of disorders induced by or associated
with a defective regulation of cell death.
[0098] In a preferred embodiment, said defective regulation affects
cells which produce Bad protein, more preferably, cells of the
immune system and most preferably T-cells.
[0099] When said defective regulation of cell death results in an
abnormal decrease of cell death, the used compound is preferably a
compound which is capable of disrupting lipid rafts, thereby
promoting apoptosis. Such compounds are for example,
methyl-.beta.-cyclodextrin or filipin. Examples of disorders
resulting in an abnormal decrease of cell death are cancer diseases
and especially lymphoproliferative cancers, infectious diseases and
especially viral diseases, inflammatory diseases or auto-immune
diseases.
[0100] Conversely, when defective regulation of apoptosis results
in an abnormal increase of cell death, the used compound is
preferably a compound capable of reconstituting lipid rafts in the
plasma membrane of cells, such as edelfosine thereby preventing
apoptosis. Examples of disorders resulting in an abnormal increase
of cell death is diseases associated to senescence,
neuro-degenerative diseases, including Alzheimer disease, ischemic
cell death, wound-healing or AIDS.
[0101] The invention provides new means to detect early events of
the apoptotic process. In particular, the invention enables to
identify the apoptotic state of a cell by determining the presence
or the absence of lipid rafts. Accordingly, another object of the
invention is an in vitro method for the detection of a defective
regulation of apoptosis, in a sample of cells of an individual,
said method comprising determining the presence or the absence of
lipid rafts in said cells, wherein the absence of said lipid rafts
is indicative of a defective regulation of apoptosis.
[0102] Examples of methods for determining the presence or absence
of lipid rafts in cells have already been described above. In a
preferred embodiment, the presence or the absence of lipid rafts is
determined by detecting the presence or absence of a pro-apoptotic
or an anti-apoptotic protein which is known to be located in lipid
rafts under proliferative growth conditions, such as the Bad
protein, or any other protein, and especially a cellular
polypeptide identified according to the method of the invention
exposed above.
[0103] In a specific embodiment, said isolated cells are cells
characteristic of the immune system of an individual affected by a
lymphoproliferative disease.
[0104] Naturally, the invention also concerns a use of a compound
appropriate for detecting the presence of lipid rafts, in the in
vitro detection method described above.
[0105] Examples of a compound appropriate for detecting the
presence of lipid rafts is a compound which specifically recognizes
Bad protein, Lck protein or ganglioside GM1. In a specific
embodiment, said compound used in the in vitro detection method is
selected among cholera toxin subunit B (CTx), anti-Bad antibody and
anti-Lck antibody.
LEGENDS TO THE FIGURES
[0106] FIG. 1. Effect of IL4 on association of Bad to 14-3-3
protein
[0107] Cytoplasmic extracts from 10.times.10.sup.6 IL-4-stimulated
or -deprived cells were immunoprecipitated with anti-Bad or
anti-Raf antibodies and blotted with anti-14-3-3, anti-Raf and
anti-Bad. Total extracts (lane T) were used as a positive control
of 14-3-3 and Rat expression. Similar results were obtained in
three independent experiments.
[0108] FIG. 2. Subcellular localization of Bad in IL4-stimulated or
-deprived cells.
[0109] A) Anti-Bad, anti-Lck (rafts), CTx-Biotin (GM1 ganglioside,
rafts), anti-caspase 3 (cytosol), anti-calnexin (endoplasmic
reticulum, ER) and anti-cytochrome C (mitochondria) immunoblot
analysis of subcellular fractions from IL4-stimulated or -deprived
cells. The fractions (1 to 4) were prepared by sucrose gradient
ultracentrfugation and tested for their purity using antibodies
against mitochondria, rafts, ER and cytosol. Nuclear fraction is
not shown in the blot (fraction 5). Protein loaded per well in each
gradient fraction corresponds to that of 5.times.106 cells. Total
extracts, 30 .mu.g of protein. Similar results were obtained in
three independent experiments. B) IL-4-stimulated or -deprived
cells were Triton X-100 extracted and fractionated in Optiprep
flotation gradient. Fractions were collected from the top to the
bottom of gradient and analyzed by western blot. Only the first,
insoluble proteins (I) and the last fraction, soluble proteins (S)
are shown. Similar results were obtained in two independent
experiments.
[0110] FIG. 3. Rafts localization of Bad in IL-4-stimulated
cells
[0111] A) IL-4-stimulated or -deprived cells were stained with
CTX-FITC and either anti-Lck or anti-Bad antibodies as indicated,
followed by Cy3-labeled secondary antibody and analyzed by confocal
microscopy.
[0112] Similar results were obtained in three independent
experiments. Single confocal sections show fluorescence in green
(FITC) and red (Cy3). B) IL-4-stimulated or -deprived cells were
stained with anti-Bad and anti-mitochondria antibodies, followed by
FITC- and Cy3-labeled secondary antibodies and analyzed as above.
Similar results were obtained in three independent experiments.
[0113] FIG. 4. Methyl-.beta.-cyclodextrin (M-.beta.-CD) treatment
abolishes association of Bad to rafts and induces apoptosis.
[0114] A) IL-4-stimulated cells were serum-starved for 30 min and
then treated with or without 10 mM M-.beta.-CD for 30 min at
370.degree. C. before incubation with CTx-FITC and anti-Bad or
anti-Lck antibodies, followed by Cy3-labeled secondary antibody.
Then, cells were analyzed by confocal microscopy.
[0115] Similar results were obtained in two independent
experiments. Single confocal sections show green (FITC) and red
Cy3) fluorescence. B) IL-4-stimulated cells were serum-starved for
30 min and then treated with or without 10 mM M-.beta.-CD for 30
min at 37.degree. C., then washed and transferred to complete
medium supplemented with IL-4. At different times, apoptosis was
measured. Sub G1 region of the fluorescence scale was used to
determine the percentage of cells present in the initial step of
apoptosis. Similar results were obtained in two independent
experiments. White bars, control cells; grey bars,
M-.beta.-CD-treated cells.
[0116] FIG. 5. Effect of IL-4 on serine phosphorylation of Bad.
[0117] A) Cytoplasmic extracts from IL-4-stimulated or -deprived
cells were immunoprecipitated with anti-Bad antibody and blotted
with anti-Bad serine 136, 112 and 155. As internal control, the
blot was developed with anti-Bad. Positive control for serine 112
and 136 phosphorylation, IL-2-stimulated 1; cells; positive control
for serine 155 phosphorylation of Bad, Bad-transfected COS cells
(C). B) Western blot from FIG. 2A was proved with anti-Bad serine
136 antibody. Molecular weight of the corresponding proteins is
shown.
EXAMPLES
1. Materials and Methods
1.1 Cells, Lymphokines and Reagents
[0118] TS1.alpha..beta.is a murine T cell line that can be
propagated independently in IL-2, IL-4 or IL-9 Cells were cultured
in RPMI-1640 as previously described (Pitton et al., 1993, Cytokine
5, 362-371). Murine rIL-4 or supernatant of a HeLa subline
transfected with PKCRIL-4.neo was used as a source of murine IL-4.
Fluorescein isothiocyanate (FITC-)-labeled cholera toxin (CTx) B
subunit, CTx-Biotin and methyl-p-cyclodextrin (M-.beta.-CD) were
obtained from Sigma-Aldrich (St. Louis, MO). Cy3- and
Cy2-conjugated secondary antibodies were purchased from Molecular
Probes (Eugene, OR). Anti-mitochondria serum (mito 2813, pyruvate
dehydrogenase) was a gift from Dr A. Serrano (CNB, Madrid,
Spain).
1.2 Immunoprecipitation and Western Blot
[0119] Cells (1.times.107) were IL-4-stimulated or -deprived and
lysed for 20 min at 4.degree. C. in lysis buffer (50 mM Tris-HCl pH
8, 1% Nonidet P-40, 1137 mM NaCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2,
10% glycerol and protease inhibitor mixture). Lysates were
immunoprecipitated with the corresponding antibody (Calbiochem
Transduction Laboratory). Protein A-Sepharose was added for 1 h at
4.degree. C. and, after washing, immunoprecipitates were separated
by SDS-PAGE. Alternatively, cells were lysed in Laemmli sample
buffer and protein extracts separated by SDS-PAGE, transferred to
nitrocellulose, blocked with 5% non fat dry milk in Tris-buffered
saline (TBS, 20 mM Tris HCl pH 7.5, 150 mM NaCl) and incubated with
primary antibody in TBS/0.5% non fat dry milk. Membranes were
washed with 0.05% Tween 20 in TBS and incubated with PO-conjugated
secondary antibody. After washing, proteins were developed using
the ECL system.
1.3 Cell Cycle Analysis
[0120] A total of 2.times.10.sup.5 IL-4-stimulated cells treated
with or without M-.beta.-CD were washed, resuspended in PBS,
permeabilized with 0.1% Nonidet P-40 and stained with 50 .mu.g/ml
propidium iodide (PI). At different times, samples were analyzed
using a EpicsXL flow cytometer (Coulter, Hialeah, Fla.). Apoptosis
was measured as the percentage of cells in the sub-G.sub.1 region
of the fluorescence scale having an hypodiploid DNA content.
[0121] Cell cycle was also analyzed by annexin staining. A total of
2.times.10.sup.3 cells were washed with ice-cold PBS diluted in
ice-cold binding buffer and stained with annexin and propidium
iodide. Samples were maintained on ice for 10 min in the dark and
then analyzed by flow cytometry.
1.4 Subcellular Fractionation
[0122] Subcellular fractionation was performed as previously
described (Hacki et al., 2000, Oncogene 19, 2286-2295; Millan and
Alonso, 1998, Eur. J. Immunol. 28, 3675-3684). Briefly,
IL-4-stimulated or -deprived cells were washed in PBS and then
resuspended for 2 min in extraction buffer-STE (10 mM Hepes pH 7.4,
1 mM EDTA, 0.25 mM sucrose, 2 .mu.g/ml aprotinin, 10 .mu.g/ml
leupeptin, 1 mM PMSF, 1 .mu.g/ml pepstatin). The extract was
inspected under the microscope and more than 95% of the cells were
lysed. The homogenates were applied to a linear gradient sucrose
(0.73 to 1.9M) and ultracentrifuged at 20,000 g overnight. The
banded organelles were recovered by syringe, diluted with an equal
volume of 10 mM Hepes buffer and sedimented at the speed
appropriated for the respective organelles. The purity of the
organelles was determined by Western blot using antibodies against
specific markers: anti-cytochrome C for mitochondria, anti-Lck and
CTx-Biotin for rafts, anti-calnexin for endoplasmic reticulum (ER)
and anti-caspase 3 for cytosol. For preparation of cytosol, the
homogenate was precentrifuged at 750 g for 10 min to remove nuclei
and unbroken cells, followed by a centrifugation a 100,000 g for 1
h to clear off the membranes.
1.5 CTx-FITC Labeling
[0123] IL-4-stimulated or -deprived cells were fixed with 1%
paraformaldehyde for 5 min on ice, perneabilized and then incubated
with CTx-FITC (20 min, 6 .mu.g/ml) and anti-Bad antibody for 1 h in
PBS-BSA. Cy3-labeled secondary antibody was added and incubated for
1 h. Finally, and after several washing steps, cells were incubated
with methanol at -20.degree. C. for 10 min, mounted with
Vectpshield medium, and analyzed by confocal microscopy. The
program used for quantification of samples was Leica TSC NT version
1.5.451 (Leica, Lasertechnik, Heidelberg, Germany).
1.6 Cholesterol Depletion
[0124] IL-4-stimulated serum-deprived cells were treated for 30 min
at 37.degree. C. with 10 mM M-.beta.-CD, washed and then incubated
with CTx-FITC and anti-Bad or anti-Lck antibodies as above.
Secondary antibody was added and incubated for 1 h. Finally, cells
were incubated with methanol at -20.degree. C. for 10 min and
mounted as described above.
1.7 Triton X-100 Flotation
[0125] IL-4-stimulated or deprived cells were lysed in TXNE buffer
(50 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.2% Triton X-100)
containing protease inhibitor mixture. Detergent insoluble
membranes were isolated by ultracentrifugation (17,000 g, 4 h,
4.degree. C.) in 30-35% gradient of Optiprep as previously
described (Manes et al., 1999, EMBO J. 18, 6211-6220).
1.8 Isolation of Mitochondria and S-100 Fraction
[0126] Mitochondria were isolated using a modification of the
method described by Yang et al., 1997, Science 275: 1129. Briefly,
20.times.106 cells were IL-4 stimulated or deprived, harvested, and
washed with ice-cold PBS. Cell pellet was suspended in 5 vol.
icecold buffer A (20 Mm HEPES-KOH (pH 7.5), OmM KCl, 1,5 MM
MgCl.sub.2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and 250 mM
sucrose) supplemented with protease inhibitors. Cells were
disrupted in a Dounce homogenizer (Kontes, Vineland, NJ), the
nucleic were centrifuged (1.000.times.g, 10 min. 4.degree. C.), and
the supernatant was further centrifuged (1.000.times.g. 15 min.,
4.degree. C.). The resulting mitochondrial pellet was resuspended
in buffer A and stored at -80.degree. C. The supernatant was
centrifuged (100,000.times.g, 1 h, 4.degree. C.), and the resulting
S-100 fraction was stored at -80.degree. C.
2. RESULTS
2.1 Bad Associates with Lipid Rafts in IL-4-stimulated Cells
[0127] It has been shown that after IL-3-stimulation, Bad becomes
phosphorylated, resulting in association to 14-3-3 protein. More
recently, it has been shown that IL-2 induces Bad phosphorylation,
but not association with 14-3-3 protein (Ayllbn et al., 2001, J.
Immunol. 166, 7345-7352). FIG. 1 shows that neither
IL-4-stimulation nor IL-4-deprivation results in association of Bad
to 14-3-3 protein. As internal control, the interaction of Raf and
the 14-3-3 protein is shown (FIG. 1).
[0128] The subcellular distribution of Bad in IL-4-stimulated or
-deprived cells has been analyzed. IL-4-stimulated or -deprived
cells were lysed and fractionated over sucrose gradient. To
validate the gradient protocol, fractions (1 to 4) were
immunoblotted with markers for rafts (Lck and GM1 ganglioside),
mitochondria (cytochrome C), endoplasmic reticulum (calnexin) and
cytosol (caspase 3). Nuclear fraction (fraction 5) is not shown in
the blot because there is not Bad localization in the nucleus.
Rafts were detected by western blot in fraction 1 using antiLck
antibody and CTx-Biotin, which recognizes GM1 ganglioside (FIG.
2A). Most of Bad was found in rafts (fraction 1), although a very
small fraction was also present in mitochondria (fraction 4),
cytosol and endoplasmic reticulum (fraction 2). As internal
control, Bad was observed in total extracts of IL-4-stimulated
cells. Finally, it has been observed that the fraction of Bad that
is sequestered in lipid rafts is dephosphorylated.
[0129] It has been previously reported that Bcl-2 is expressed in
IL-2-stimulated cells and BCl-X.sub.L in IL-4 cultured cells
(Gomez, J. et al, 1998, Oncogene 17: 1233). When IL-4-maintained
cells are deprived of lymphokine, they undergo apoptosis. As early
as 4 h after IL-4 deprivation, .apprxeq.9% of the cells were
apoptotic, reaching 40% at 24 h, whereas control IL-4-stimulated
cells showed no significant level of apoptosis.
[0130] IL-4-deprivation induces disorganization of rafts (fraction
1), which are not detected using either anti-Lck antibody or
CTx-Biotin. The mitochondria marker, which also contains other
cellular structures with similar density, is observed in fraction 4
and most of the caspase 3 is cleaved, given a new protein of lower
molecular weight. More interesting, Bad is almost undetectable in
cytosol and rafts are only observed in fraction 4, which
corresponds to mitochondria and cellular structures with similar
density (FIG. 2A). This result strongly suggests an IL-4-dependent
association of Bad with rafts and translocation to mitochondria
upon IL-4-deprivation. Rafts were also isolated by Triton X-100
flotation gradient. As shown in FIG. 2B, Bad and Lck are detected
in the detergent insoluble fraction (I) of IL-4-stimulated cells,
which corresponds to lipid rafts. In IL-4-deprived cells, Bad and
Lck are detected in the fraction corresponding to soluble proteins
(S). It has been observed that post-translational myristoylation
targets Bad to rafts (data not shown).
[0131] The subcellular localization of Bad was also analyzed in
mitochondrial and cytosolic fractions of IL-4-stimulated or
deprived cells. Bad was detected in the mitochondrial fraction of
IL-4-stimulated cells. The amount of Bad associated with
mitochondra increased upon IL-4 deprivation. Traces of Bad were
detected in the cytosolic fraction of IL4 stimulated or Deprived
cells. The antiapoptotic molecule BCl-x.sub.L was weakly detected
in the mitochondrial fraction of IL-4-stimulated cells, increasing
after IL-4 deprivation. As an internal control of protein
fractionation, the blot was probed with anti-caspase 3 (cytosolic
marker), anti-mitochondria Mito 2813 (pyruvate dehydrogenase,
mitochondrial marker), and anti-calnexin to show the lack of
endoplasmic reticulum contamination in mitochondrial preparation.
Total extracts (late T) were used as a positive control of calnexin
expression. Finally, the association of Bad with some Bcl-2 family
members was explored. Coimmunoprecipitation experiments of
cytoplasmic proteins under IL-4 stimulation or deprivation
conditions using specific antibodies were performed. Bad was
detected by Western blot in anti-BcI-x.sub.L immunoprecipitates of
IL-4 stimulated cells, decreasing throughout the starvation period
analyzed. Probing the membrane with anti-Bcl-x.sub.L antibodies
showed similar levels in all analyzed conditions.
[0132] Bad association to rafts in IL-4-stimulated cells was also
analyzed in intact cells by confocal microscopy (FIG. 3A).
IL-4-stimulated or -deprived cells were incubated with the raft
marker cholera toxin B subunit (CTx-FITC) before secondary
labelling with anti-Bad or anti-Lck antibody. Double
immunofluorescence analysis with anti-Bad and CTx-FITC showed raft
localization of Bad in the surface of IL-4-stimulated cells. In
marked contrast, a disorganization of rafts in IL-4-deprved cells
was observed and consequently, no rafts localization of Bad in
IL-4-deprived cells (FIG. 3A). Double immunofluorescence analysis
with anti-Lck and CTx-FITC was used as a positive control of
localization of Lck in membrane rafts of IL-4-stimulated cells. Lck
associated with rafts was not detected in IL-4-deprived cells (FIG.
3A).
[0133] The profile of green and red fluorescence colocalization was
analyzed using the quantification software of Leica (TCS NT; Leica,
Rockleigh, N.J.). A high number of green and red colocalization
peaks was observed in the membrane of IL-4 stimulated cells stained
with CTx-Lck or CTx-Bad. On the contrary, the level of
colocalization of green and red fluorescence was strongly reduced
in IL-4-deprived cells.
[0134] This result suggests that Bad is preferentially localized in
lipid rafts in IL-4-stimulated cells and segregates from plasma
membrane in IL-4-deprived cells.
[0135] Similar results of colocalization of Bad with lipid rafts
were observed using freshly isolated thymocytes from mice.
[0136] Bad association with mitochondria in IL-4-deprived cells was
also analyzed in intact cells by confocal microscopy (FIG. 3B).
Double immunofluorescence analysis with anti-Bad and
anti-mitochondria antibodies shows weak association of Bad to
mitochondria in IL-4-stimulated cells while there is a high
fraction of Bad associated to mitochondria in IL-4-deprived cells
(FIG. 3B). This separation of Bad from rafts correlates with its
translocation to mitochondria in IL-4-deprived cells, as shown by
cellular fractionation and confocal microscopy (FIG. 2A and
2B).
[0137] The profile of green and red fluorescence colocalization was
also analyzed using quantification software (Leica) and showed
moderate green and red colocalization peaks in IL-4-stimulated
cells stained with anti-Bad and anti-mitochondria Abs. The level of
colocalization of both fluorescences strongly increased in IL-4
deprived cells.
2.2 Association of Bad to Lipid Rafts is Required for Prevention of
Apoptosis
[0138] Depletion of cellular cholesterol impairs the ability of
glycosyl phosphatidylinositol (GPI)-anchored proteins to associate
with lipid rafts. To examine whether there is a similar requirement
of cholesterol for the association of Bad with rafts,
IL-4-stimulated cells were treated for 30 min with or without 10 mM
methyl-.beta.-cyclodextrin (M-.beta.-CD) in serum-free medium to
deplete cellular cholesterol. Cells were then incubated with
CTx-FITC and labeled with anti-Bad or anti-Lck antibodies. Serum
depletion alone weakly disrupt the association of Lck or Bad to
lipid rafts (FIG. 4A). However, M-.beta.-CD treatment causes a
severe disruption of raft formation and association of Lck and Bad
with rafts in IL-4-stimulated cells (FIG. 4A). This result
indicates that disruption of raft formation by cholesterol
depletion induces segregation of Bad and Lck from rafts in
IL-4-stimulated cells.
[0139] Given that exclusion of Bad from rafts was also observed in
apoptotic IL-4-deprived cells (FIG. 3A), it was analyzed whether
Bad association to rafts and its integrity was necessary for
prevention of apoptosis. For this purpose, IL-4-stimulated cells
were treated for 30 min with or without M-.beta.-CD in serum-free
medium, then washed, resuspended in IL-4-supplemented complete
medium and analyzed for induction of apoptosis at different times
(FIG. 4B). M-.beta.-CD treated cells showed stronger level of
apoptosis compared with control non treated cells, reaching the
highest level 5 hours after M-.beta.-CD treatment. Eight hours upon
treatment, the amount of apoptotic cells detected in treated and
non treated cells were similar because addition of serum restores
the lipid composition of the membrane.
[0140] This result suggests that segregation of Bad from rafts is
involved in the induction of apoptosis. Posttranslational
modifications of Bad such as phosphorylation, and its role in Bad
localization in rafts or mitochondria was further analyzed. FIG. 5A
shows that IL-4 induces serine 136 phosphorylation of Bad, but not
serine 112 and 155. Moreover, IL-4-deprivation induces serine 136
dephosphorylation of Bad. Given that IL-4 induces serine 136
phosphorylation of Bad, western blot was reprobed from FIG. 2A with
anti-Bad serine 136 antibody. FIG. 5B shows that while most of Bad
is localized in rafts in IL-4-stimulated cells, only the weak
cytosolic fraction of Bad is serine 136 phosphorylated. In
IL-4-deprived cells, traces of serine 136 phosphorylation are
observed in cytosol and mitochondria. This result suggests that
dephosphorylated Bad is sequestered in rafts and IL-4-deprivation
induces segregation and translocation to mitochondra.
[0141] Subcellular localization of Bad enables to discover how Bad
function may be regulated by dynamic interaction with lipid rafts
or mitochondra.
[0142] The distinct Bad distribution and function is directly
related to IL-4-stimulation or -deprivation of the cells.
[0143] These data show that 14-3-3 protein does not control the
proapoptotic role of Bad, contrary to previous reports. On the
basis of this result, the subcellular distribution of Bad in
IL-4-stimulated or -deprived cells was analyzed. These results show
that different plasma membrane fractions can be separated using
subcellular fractionation sucrose ultracentrifugation gradient
because raft markers were successfully resolved from non-rafts
markers. Rafts and mitochondria were also isolated by Triton X-100
flotation gradient and differential centrifugations, respectively.
There are precedents for reversible raft association as has been
shown following the movement of single fluorescence lipid molecules
(Schutz et al., 2000, EMBO J. 19, 892-901). In addition, after
activation by ligand binding the epidermal growth factor migrates
out of rafts into bulk plasma membrane (Mineo et aL, 1999, J. Biol.
Chem. 274, 30636-30643). The association of proteins with lipid
rafts can be modulated because some proteins may be excluded from
rafts by association to other proteins (Field et al., 1995, Proc.
NatI. Acad. Sci. USA. 92, 9201-9205). Association of Bad with rafts
may be involved in steps leading to Bad inactivation, because rafts
do not constitute the final site of activation. IL-4-deprivation
induces segregation of Bad from rafts. This results suggests a two
steps apoptotic process: first, segregation of Bad from rafts, that
triggers apoptosis and second, disorganization of lipid rafts
during apoptotic process. This is strongly suggested by results
showing that disruption of cholesterol rich rafts prevents Bad
association and induces apoptosis in IL-4-stimulated
M-.beta.-CD-treated cells. Addition of fetal calf serum to
IL-4-supplemented medium restores the lipid components of the
plasma membrane, preventing progression of apoptosis.
[0144] Localization of proteins to distinct subcellular fractions
is an essential step in multiple signaling pathways, including
apoptosis. According to this, it has been shown that some signaling
molecules are sequestered in rafts. Cholesterol depletion disrupts
lipid rafts and modulates the activity of multiple signaling
pathways in T lymphocytes (Kabouridis et al., 2000, Eur. J. Immunol
30, 954-963). These results strongly suggest that in the absence of
association of Bad to 14-3-3 protein, Bad is sequestered in rafts,
avoiding a proapoptotic role and association with partners. IL-4
deprivation-induced segregation of Bad from rafts correlates with
translocation to mitochondria and induction of apoptosis.
Restriction of intermolecular interactions by sequestration in
lipid rafts has been also described from the .alpha.-chain of the
IL-2R, avoiding its association with the .beta.-and .gamma.-chains
of the IL-2R (Marmor, M; and M. Julius, 2001, Blood 98:1489). It is
interesting to notice that in IL-4-stimulated cells, most of
cellular Bad localizes in rafts in a dephosphorylated condition
while the weak cytosolic fraction is serine 136 phosphorylated.
These results show for the first time the sequestration of a
proapoptotic protein into lipid rafts as a mechanism that controls
the availability of said proapoptotic protein.
* * * * *