U.S. patent application number 10/440341 was filed with the patent office on 2003-09-25 for raf protein kinase therapeutics.
Invention is credited to App, Harald, Rapp, Ulf R., Storm, Stephen M..
Application Number | 20030181413 10/440341 |
Document ID | / |
Family ID | 25011518 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030181413 |
Kind Code |
A1 |
Rapp, Ulf R. ; et
al. |
September 25, 2003 |
Raf protein kinase therapeutics
Abstract
It is a general object of this invention to provide a DNA
segment comprising a Raf gene in an antisense orientation
downstream of a promoter. It is a specific object of this invention
to provide a method of inhibiting Raf expression comprising
expressing an antisense Raf gene in a cell such that said Raf
expression is inhibited. It is a further object of the invention to
provide a method of inhibiting Raf kinase activity comprising
replacement of a serine or threonine amino acid within the Raf gene
for a non-phosphorylated amino acid.
Inventors: |
Rapp, Ulf R.; (Washington,
DC) ; App, Harald; (Frederick, MD) ; Storm,
Stephen M.; (Frederick, MD) |
Correspondence
Address: |
NEEDLE & ROSENBERG, P.C.
SUITE 1000
999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Family ID: |
25011518 |
Appl. No.: |
10/440341 |
Filed: |
May 15, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10440341 |
May 15, 2003 |
|
|
|
08207954 |
Mar 18, 1994 |
|
|
|
08207954 |
Mar 18, 1994 |
|
|
|
07748931 |
Aug 23, 1991 |
|
|
|
Current U.S.
Class: |
514/44A ;
435/320.1; 435/375; 435/6.1; 435/6.11 |
Current CPC
Class: |
A61K 38/00 20130101;
C12N 15/1135 20130101; C07K 14/82 20130101 |
Class at
Publication: |
514/44 ;
435/320.1; 435/375; 435/6 |
International
Class: |
A61K 048/00; C12Q
001/68; C12N 015/00 |
Claims
What is claimed is:
1. A construct comprising a DNA segment comprising a Raf gene in an
antisense orientation downstream of a promoter.
2. The construct according to claim 1 wherein said Raf gene is
Raf-1.
3. A method of inhibiting Raf expression comprising expressing an
antisense Raf gene in a cell under conditions such that said Raf
expression is inhibited.
4. The method according to claim 3, wherein said Raf gene is
Raf-1.
5. A method of inhibiting Raf kinase activity comprising: replacing
a codon within the Raf gene encoding a serine or threonine amino
acid for a codon encoding an amino acid not suceptable to
phosphorylation and transforming said gene into a cell such that
said Raf activity is inhibited.
6. The method according to claim 5, wherein said amino acid not
suceptable to phosphorylation is alanine.
7. A method of inhibiting Raf kinase activity comprising: modifying
Raf by replacing a serine or threonine amino acid within Raf for an
amino acid not suceptable to phosphorylation and delivering said
modified Raf to a cell such that said Raf expression is
inhibited.
8. The method according to claim 7, wherein said amino acid not
suceptable to phosphorylation is alanine.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates, in general, to methods of
inducing a therapeutic effect. In particular, the present invention
relates to therapeutic uses of Raf protein kinases.
[0003] 2. Background Information
[0004] Raf serine- and threonine-specific protein kinases are
cytosolic enzymes that stimulate cell growth in a variety of cell
systems (Rapp, U. R., et al. (1988) In The oncogene handbook; T.
Curran, E. P. Reddy, and A. Skalka (ed.) Elsevier Science
Publishers; The Netherlands, pp.213-253; Rapp, U. R., et al. (1988)
Cold Spring Harbor Sym. Quant. Biol. 53:173-184; Rapp, U. R., et
al. (1990) In: Curr. Top. Microbiol. Immunol. Potter and Melchers
(eds), Berlin, Springer-Verlag 166:129-139). Three isozymes have
been characterized: c-Raf (Raf-1) (Bonner, T. I., et al. (1986)
Nucleic Acids Res. 14:1009-1015). A-Raf (Beck, T. W., et al. (1987)
Nucleic Acids Res. 15:595-609), and B-Raf (Ikawa, S., et al. (1988)
Mol. Cell. Biol. 8:2651-2654; Sithanandam, G. et al. (1990)
Oncogene 5:1775). These enzymes differ in their expression in
various tissues. Raf-1 is expressed in all organs and in all cell
lines that have been examined, and A- and B-Raf are expressed in
urogenital and brain tissues, respectively (Storm, S. M. (1990)
Oncogene 5:345-351).
[0005] Raf genes are proto-oncogenes: they can initiate malignant
transforation of cells when expressed in specifically altered
forms. Genetic changes that lead to oncogenic activation generate a
constitutively active protein kinase by removal or interference
with an N-terminal negative regulatory domain of the protein
(Heidecker, G., et al. (1990) Mol. Cell. Biol. 10:2503-2512; Rapp,
U. R., et al. (1987) In Oncogenes and cancer S. A. Aaronson, J.
Bishop, T. Sugimura, M. Terada, K. Toyoshima, and P. K. Vogt (ed.)
Japan Scientific Press, Tokyo). Microinjection into NIH 3T3 cells
of oncogenically activated but not wild-type versions of the
Raf-protein prepared with Escherichia coli expression vectors
results in morphological transformation and stimulates DNA
synthesis (Rapp, U. R., et al. (1987) In Oncogenes and cancer; S.
A. Aaronson, J. Bishop, T. Sugimura, M. Terada, K. Toyoshima, and
P. K. Vogt (ed.) Japan Scientific Press, Tokyo; Smith, M. R., et
al. (1990) Mol. Cell. Biol. 10:3828-3833). Thus, activated Raf-1 is
an intracellular activator of cell growth. Raf-1 protein serine
kinase is a candidate downstream effector of mitogen signal
transduction, since Raf oncogenes overcome growth arrest resulting
from a block of cellular ras activity due either to a cellular
mutation (ras revertant cells) or microinjection of anti-ras
antibodies (Rapp, U. R., et al. (1988) In The Oncogene Handbook, T.
Curran, E. P. Reddy, and A. Skalka (ed.), Elsevier Science
Publishers; The Netherlands, pp.213-253; Smith, M. R., et al.
(1986) Nature (London) 320:540-543).
[0006] c-Ras function is required for transformation by a variety
of membrane-bound oncogenes and for growth stimulation by mitogens
contained in serum (Smith, M. R., et al. (1986) Nature (London)
320:540-543). Raf-1 protein serine kinase activity is regulated by
mitogens via phosphorylation (Morrison, D. K., et al. (1989) Cell
58:648-657), which also effects subcellular distribution (Olah, Z.,
et al. (1991) Exp. Brain Res.84:403; Rapp, U. R., et al. (1988)
Cold Spring Harbor Sym. Quant. Biol. 53:173-184).
[0007] Raf-1 activating growth factors include platelet-derived
growth factor (PDGF) (Morrison, D. K., et al. (1988) Proc. Natl.
Acad. Sci. USA 85:8855-8859), colony-stimulating factor
1(Baccarini, M., et al. (1990) EMBO J. 9:3649-3657), insulin
(Blackshear, P. J., et al. (1990) J. Biol. Chem. 265:12131-12134;
Kovacina, K. S., et al. (1990) J. Biol. Chem. 265:12115-12118),
epidermal growth factor (EGF) (Morrison, D. K., et al. (1988) Proc.
Natl. Acad. Sci. USA 85:8855-8859), interleukin 2 (Turner, B. C. et
al. (1991) Proc. Natl. Acad. Sci. USA 88:1227), and interleukin 3
and granulocyte-macrophage colony-stimulating factor (Carroll, M.
P., et al (1990) J. Biol. Chem. 265:19812-19817). Upon mitogen
treatment of cells, the transiently activated Raf-1 protein serine
kinase translocates to the perinuclear area and the nucleus (Olah,
Z., et al. (1991) Exp. Brain Res. 84:403; Rapp, U. R., et al.
(1988) Cold Spring Harbor Sym. Quant. Biol. 53:173-184). Cells
containing activated Raf are altered in their pattern of gene
expression (Heidecker, G., et al. (1989) In Genes and signal
transduction in multistage carcinogenesis, N. Colburn (ed.), Marcel
Dekker, Inc., New York. pp. 339-374), and Raf oncogenes activate
transcription from Ap-1/PEA3-dependent promoters in transient
transfection assays (Jamal, S., et al. (1990) Science 344:463-466;
Kaibuchi, K., et al. (1989) J. Biol. Chem. 264:20855-20858;
Wasylyk, C., et al. (1989) Mol. Cell. Biol. 9:2247-2250).
[0008] There are at least two independent pathways for Raf-1
activation by extracellular mitogens: one involving protein kinase
C (KC) and a second initiated by protein tyrosine kinases
(Blackshear, P. J. , et al. (1990) J. Biol. Chem. 265:12131-12134;
Kovacina, K. S., et al. (1990) J. Biol. Chem. 265:12115-12118;
Morrison, D. K., et al. (1988) Proc. Natl. Acad. Sci. USA
85:8855-8859; Siegel, J. N., et al. (1990) J. Biol. Chem.
265:18472-18480; Turner, B. C. et al. (1991) Proc. Natl. Acad. Sci.
USA 88:1227). In either case, activation involves Raf-1 protein
phosphorylation. Raf-1 phosphorylation may be a consequence of a
kinase cascade amplified by autophosphorylation or may be caused
entirely by autophosphorylation initiated by binding of a putative
activating ligand to the Raf-1 regulatory domain, analogous to PKC
activation by diacylglycerol (Nishizuka, Y. (1986) Science
233:305-312).
SUMMARY OF THE INVENTION
[0009] It is a general object of this invention to provide a
construct comprising a DNA segment comprising a Raf gene in an
antisense orientation downstream of a promoter.
[0010] It is a specific object of this invention to provide a
method of inhibiting Raf expression comprising expressing an
antisense Raf gene in a cell such that said Raf expression is
inhibited.
[0011] It is a further object of the invention to provide a method
of inhibiting Raf kinase activity comprising replacing a serine or
threonine amino acid within the Raf gene for an amino acid not
susceptible to phosphorylation.
[0012] Further objects and advantages of the present invention will
be clear from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. Schematic diagram of murine and human c-raf-1 cDNAs
and expression plasmids used. GMA and GMS contain Stu1 restriction
fragments of the mouse c-raf-1 cDNA. HCR an N-terminal HincII
fragment of the human cDNA. p301 consists of all the coding
sequence of a mutant human c-raf-1 cDNA. The lysine(375) to
tryptophan (K.fwdarw.W) mutation in the ATP-binding site is
indicated (Heidecker, G. et al. Molec. Cell. Biol. 10:2503-2512
(1990)). Restriction fragments were cloned in both sense and
antisense orientation. NIH/3T3 cells were transfected with sense
and antisense plasmids, and with the pMNC vector as control.
G418-resistant (400 .mu.g ml.sup.-1) colonies containing more than
50 cells were counted after three weeks. The pMNC vector served as
internal standard. The experiment was repeated three times (twice
for HCR) with different batches of plasmid preparations. Variations
between experiments were in the order of 10% but did not affect the
ratios between the different constructs shown. CR1-3, conserved
regions; ATP, ATP-binding domain; LTR, mouse Moloney virus long
terminal repeat; NEO, neomycin-resistance gene; CMV,
cytomegalovirus immediate early promoter.
[0014] FIG. 2. Morphological reversion of raf-transformed cells by
transfection with raf antisense and mutant constructs. a. p48
raf-transformed 208/F12 (Schultz, A. M. et al. Oncogene 2:187-193
(1988)) or b. v-raf-transformed F4 (Rapp, U. R., et al. Proc. Natl.
Acad. Sci. U.S.A. 80:4218-4222 (1983)) fibroblasts were transfected
with plasmids p301-1 (sense) and 301-2 (antisense) or GMS-7 (sense)
and GMS-8 (antisense), respectively, as well as with the pMNC
vector. Monolayer growth with minor irregularities and a decreased
ability to form soft agar colonies was categorized as partial
reversion. Flat clones showed no areas of overgrowth and did not
form colonies in soft agar. c. A representative analysis of Raf
protein expression in individual cell clones. t. Transformed; im.
intermediate; f. flat (clone GMS-8/2); f* (clone GMS-8/3).
[0015] FIG. 3. Mitogen responsiveness and proliferative capacity of
Raf-depleted cells. a. DNA synthesis induced by serum or TPA in
serum-starved cells is depicted as the number of nuclei
incorporating .sup.3H-thymidine. b. Long term growth curves GMS-7
is a pool of 10 clones transfected with sense DNA. GMS-8/2 and
GMS-8/3 are flat clones reverted with antisense DNA. a, , starved
cells; .quadrature., TPA-induced cells; .box-solid., SERUM-induced
cells. b, .quadrature., F4; , GMS-7b; O GMS-8/2; O GM-8/3.
[0016] FIG. 4. Time course of Raf-1 mobility shift upon growth
factor treatment. Cells (10.sup.7) expressing either wild-type
(HER14) or kinase-negative (DK721A) EGF-R were stimulated at
37.degree. C. with 40 nM EGF for the times indicated, lysed, and
subjected to immunoprecipitation with anti-SP63 polyclonal
antiserum. Immunoprecipitated proteins were separated by 7.5%
SDS-PAGE, transferred to nitro-cellulose, and probed with the same
antiserum. Immunoreactive proteins were detected with
.sup.125I-labeled protein A, and autoradiographs were exposed for
12 h. Each lane represents immunoprecipitates from 10.sup.7 cells.
Lanes: 2 through 5. HER14 cell; 7 and 8, DK721A cells: 9 and 10,
DK721A cells with competing SP63 peptide (10 .mu.g/ml); 1, 6, 9,
and 11, marker proteins of 97 and 67 kDa.
[0017] FIG. 5. Kinase acidity upon EGF treatment HER14 and DK721A
cells. Monolayer cultures of HER14 or K721A cells were incubated in
the presence or absence of 40 nM EGF for 10 min at 37.degree. C.
Lysates were centrifuged, and the resulting supernatants were
immunoprecipitated with Raf-1 antiserum. Immunocomplexes were
assayed for kinase activity using peptide (IVQQFGFQRRASDDGKLTD) as
substrate. In the absence of peptide, immune complex kinase assays
with unstimulated cells yielded .ltoreq.5% of counts observed in
the peptide assay with stimulated cells. No counts were
incorporated when a modified version of this peptide was used, in
which serine in position 12 was replaced by alanine, and position 5
retained the Raf-1-specific tyrosine.
[0018] FIG. 6. Association of Raf-1 with ligand-activated EGF-R in
HER14, DK721A, or A431 cells. Density-arrested and serum-starved
HER14, DK721A, or A431 cells were stimulated for 10 min with 40 nM
EGF at 37.degree. C. before lysis with RIPA buffer and
immunoprecipitation. (A) Immunoprecipitates from HER14 and DK721A
cells with anti-v-Raf 30K polycolonal antiserum or with a
monoclonal EGF-R antibody (108) were subjected to 7.5% SDS-PAGE and
transferred to nitrocellulose. EGF-R was detected by incubating
with a polyclonal antiserum (RK2) against the EGF receptor
(Margolis, B., et al. (1989) Cell 57:1101-1107), followed by
.sup.125I-labeled protein A labeling. Exposure times for
immunoblots were 3 days (lanes 1 through 4) or 1 day (lanes 5 and
6). (B and C) Immunoprecipitates from EGF-treated and control A431
cells with Raf-1 specific anti-v-Raf 30K antiserum or monoclonal
anti-EGF-R antibody 108 were blotted, and the blots were developed
sequentially with EGF-R antiserum RK2 (B) and anti-v-Raf 30K (C).
Exposure times were 3 days and 1 day for panels B and C,
respectively.
[0019] FIG. 7. Phosphoamino acid analysis of the immunoprecipitated
Raf-1 protein from EGF-treated and untreated HER14 cells, HER14
cells (10.sup.7) were phosphate starved for 16 h, labeled with 1
mCi of [.sup.32P]phosphate for 3 h at 37.degree. C., and treated
with 40 nM EGF for 10 min at 37.degree. C. Cells were lysed in RIPA
buffer and immunoprecipitated. Proteins were separated by 7.5%
SDS-PAGE, the Raf-1 bands were cut out of the gel, and the protein
was electroeluted. From the electroeluted Raf-1 protein 1,960 cpm
was recovered from the EGF-treated cells and 1,111 cpm was
recovered from the untreated cells. The proteins were hydrolyzed
for 2 h at 110.degree. C. in 6 N hydrocloric acid. Phosphoamino
acid analysis was performed at pH 1.8 as described by Cooer et al.
(Cooper, A. A., et al. (1983) Methods in Enzymol. 99:387-402). The
Raf-1 protein showed a shift in mobility when part of the
electroeluted protein was rerun on 7.5% SDS-PAGE.
[0020] FIG. 8. Independence of EGF-medicated Raf-1 activation from
PKC. HER14 cells (10.sup.7) were incubated for 48 h with or without
200 ng TPA and stimulated with either 100 ng of TPA for 20 min at
37.degree. C. Calls were lysed in RIPA buffer, equal amounts of
protein were immunoprecipitated with anti-v-Raf 30-kDa antiserum
and electrophoresed, and the separated proteins were blotted onto
nitrocellulose. The blot was incubated with the same antibody and
then labeled with .sup.125I-labeled protein A.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates to the Raf protein kinase
inhibitors and methods of use thereof.
[0022] In one embodiment, the present invention relates to
antisense expression constructs comprising a Raf protein kinase
gene. Raf-1 function is inhibited by expressing c-raf-1 antisense
RNA or kinase-defective c-raf-1 mutants. Antisense RNA for c-raf-1
interferes with proliferation of normal NIH/3T3 cells and reverts
raf-transformed cells. In revertant cells, DNA replication induced
by serum or TPA is eliminated or reduced proportionately to the
reduction in Raf protein levels. Expression of a kinase-defective
Raf-1 mutant (craf301) or a regulatory domain fragment (HCR)
inhibits serum-induced NIH/3T3-cell proliferation and raf
transformation even more efficiently. Inhibition by antisense RNA
or craf301 blocks proliferation and transformation by Ki- and
Ha-ras oncogenes. Thus, raf functions as an essential signal
transducer downstream of serum growth factor receptors, protein
kinase C and ras.
[0023] In another embodiment, the present invention relates to a
method of inhibiting Raf expression comprising expressing an
antisense Raf gene (more specifically, Raf-1) in a cell such that
said Raf expression is inhibited.
[0024] In another embodiment, the present invention relates to
inhibitory peptides of Raf derived from Raf kinase specific
substrate sequences. Phosphorylation sites of Raf substrates can be
determined which are expected to yield consensus phosphorylation
site motifs for the various Raf isozymes. Studies which gave rise
to the present invention demonstrate that Raf is the subject of
autophosphorylation. In one preferred embodiment, consensus
substrate peptides are altered by introduction of alanine for
phosphorylation targets serine or threonine.
[0025] In another preferred embodiment, the present invention
relates to a method of inhibiting Raf kinase activity comprising
replacing a codon within the Raf gene encoding a serine or
threonine amino acid for a codon encoding an amino acid not
suceptable to phosphorylation and transforming said gene into a
cell such that said Raf activity is inhibited.
[0026] In yet another embodiment, the present invention relates to
a method of inhibiting Raf kinase activity comprising modifying Raf
by replacing a serine or threonine amino acid within Raf for an
amino acid not suceptable to phosphorylation and delivering said
modified Raf to a cell such that said Raf expression is
inhibited.
[0027] The present invention is described in further detail in the
following non-limiting examples (see Kolch W. et al (1991) Nature
349:426 and App et al. (1991) Molec. Cell. Biol.
11(2):913-919).
EXAMPLES
[0028] The following protocols and experimental details are
referenced in the Examples that follow:
[0029] Expression plasmid construction. pMNC digested with XhoI and
BamHl was blunt ended with T4 DNA polymerase. The mouse and human
cDNAs were cut with StuI or HincII, respectively, and appropriate
sized fragments were ligated with the pMNC vector. GMA contains
residues 1254-1426 and GMS 1427-1697 of the mouse cDNA, HCR 1-903
of the human c-raf-1 cDNA (Bonner et al. Nucleic Acids Res.
14:1009-1015 (1986)). The translation termination codon for HCR
sense is provided by vector sequences resulting in the addition of
nine amino acids. To construct p301-1 (sense orientation), an
EcoRI-Xbal fragment of p628 (Bonner et al. Nucleic Acids Res.
14:1009-1015 (1986)) encompassing the coding sequence of a human
c-raf-1 cDNA was cloned into BluescriptKS (Stratagene). Lysine(375)
was changed to tryptophan by site-directed mutagenesis resulting in
the creation of a unique BamHI site. This cDNA was transferred into
the Sacl-XhoI sites of pSVL (Pharmacia), then cloned into the
Xhol-BamHI sites of pMNC as an XhoI-BamHI (partial digest)
fragment. The corresponding antisense plasmid, p301-2, was
generated by cloning the blunt-ended c-raf 301 EcoRi-Xbal fragment
into blunt-ended pMNC.
[0030] Western analysis with PBB1. Cells were lysed in TBST (160 mM
NaCl, 20 mM Tris HCl, pH7.5, 2 mM EDTA, 1% Triton X-100, 1 mM
PMSF). Lysates were adjusted to equal protein concentrations
(Biorad protein assay kit). Raf proteins were precipitated with the
monoclonal antibody PBB1 and analyzed by western blotting with the
polyclonal serum #137 as described previously (Kolch, W. et al.
Oncogene 6:713-720 (1990)).
[0031] Mitogen responsiveness and proliferative capacity assay.
10.sup.4 cells were plated on cover slips and serum-starved for 24
h before incubation with 20% fetal calf serum (Gibco) or 100 ng
ml.sup.-1 TPA (Sigma). 14 h after addition of mitogens, cells were
labelled with 1 .mu.Ciml.sup.-1 3H-thymidine for 5 h. Cells were
counter-stained with Giemsa and labelled nuclei were counted. For
long-term growth curves. 10.sup.5 cells were seeded in six-well
plates in DMEM medium supplemented with 10% FCS. Each day, one well
was trypsinized and counted with a Coulter cell counter. All
determinations were performed in triplicate.
[0032] Cell maintenance. NIH 3T3 clone 2.2 cells devoid of
endogenous EGF-R were transfected with wild-type (HER14) or
kinase-negative (DK721A) receptors as described previously
(Honegger, A. M., et al. (1987) Cell 51:199-209; Honegger, A. M.
(1987) Mol. Cell. Biol. 7:4567-4571; Margolis, B., et al. (1989)
Cell 57:1101-1107). In the case of the kinase-negative receptor
mutant, the putative ATP binding lysine was substituted by an
alanine (Honegger, A. M., et al. (1987) Cell 51:199-209; Honegger,
A. M. (1987) Mol. Cell. Biol. 7:4567-4571). Cells were maintained
in Dulbecco modified Eagle medium (DMEM) containing 10% (vol/vol)
calf serum.
[0033] Preparations of cytosolic cell extracts and
immunoprecipitations. Cells were grown in 75 cm.sup.2 flasks in
DMEM containing 10% calf serum until confluency and starved
overnight in 0.05% calf serum. Before lysis, cells were exposed to
40 nM EGF for 0 min at 37.degree. C. and rinsed three times in
phosphate-buffered saline. Control cells were not exposed to EGF.
Cells were lysed in TBST buffer (50 mM Tris hydrochloride (pH 7.3),
150 mM NaCl, 0.5% Triton X-100) or in RIPA buffer (50 mM Tris
hydrochloride (pH 7.3), 150 mM NaCl, 1% Triton X-100, 0.5%
desoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 1 mM
dithiothreitol, 0.2 mM sodium orthovanadate, 25 mM sodium fluoride,
10 mM sodium pyrophosphate, 25 mM glycerophosphate). Insoluble
material was removed by centrifugation at 4.degree. C.; for 30 min
at 12,000.times.g. Protein concentrations were determined by the
method of Bradford (Bradford, M. M. (1976) Anal. Bichem.
72:248-254). Immunoprecipitations were performed by incubating
lysates with polyclonal rabbit antiserum against the v-Raf 30-kDa
protein (Kolch, W., et al. (1988) Biochim. Biophys. Acta
949:233-239) or a polyclonal rabbit antiserum against a synthetic
peptide (SP63) corresponding to the last 12 carboxy-terminal amino
acids of the Raf-1 protein and protein A for 3 h at 4.degree.
C.
[0034] Western immunoblotting. The immunoprecipitates were resolved
by SDS-polyacrylamide gel electrophoresis (PAGE). The gels were
electroblotted on nitrocellulose, and the blots were blocked with
5% (wt/vol) gelatin in TBST buffer and incubated with polyclonal
antiserum against Raf-1 or EGF-R. After extensive washing with TBST
buffer, the blot was labeled with .sup.125J-staph protein A (Dupont
NEN). Nonbound .sup.125J-staph was removed by washing the blots
with TBST buffer, and the dried membrane was exposed to x-ray
film.
[0035] Immunocomplex kinase assay. Immunoprecipitates were washed
three times with cold RIPA buffer and twice with kinase buffer (50
mM Tris hydrochloride (pH 7.3), 150 mM NaCl, 12.5 mM MnCl.sub.2, 1
mM dithiothreitol, and 0.2% Tween 20). Immunocomplex kinase assays
were performed by incubating immunoprecipitates from 10.sup.6 cells
in 80 .mu.l of kinase buffer with 20 .mu.Ci of
[.gamma.-.sup.32P]ATP (10 mCi/ml) and 20 .mu.l of the Raf-1
substrate peptide (5 mg/ml) for 30 min at 25.degree. C. The
sequence of the Raf-1 substrate peptide is IVQQFGFQRRASDDGKLTD. A
control peptide had tyrosine in position 5, as does wild-type
Raf-1, and alanine in place of serine in position 12. The assay was
linear for at least 40 min. The phosphorylation reaction was
terminated by spotting 15-.mu.l aliquots of the assay mixture on a
2- by 2-cm Whatman P81 phosphocellulose filter. The filters were
washed four times for 30 min in 1% orthophosphoric acid and air
dried, and the amount of .sup.32P incorporated was determined by
the Cerenkov method. No differences were observed when counts were
compared between filters on which the whole reaction mix or only
the supernatant was spotted. Peptide phosphorylation in this assay
was verified by running the reaction products on 20% SDS gels.
[0036] Phosphoamino acid analysis. One-dimensional phosphoamino
acid analysis was carried out as described by Cooper et al.
(Cooper, A. A., et al. (1983) Methods in Enzymol. 99:387-402).
Phosphoamino acids were separated at pH 1.8 (6% formic acid and 15%
acetic acid) for 4 h at 750 V.
EXAMPLE 1
Antisense Experiments
[0037] Portions of c-raf-1 cDNAs were expressed in sense and
antisense orientation using the pMNC vector (FIG. 1). After
transfection into NIH/3T3 cells the number of neomycin-resistant
colonies was scored. Antisense constructs yielded roughly half the
number of colonies as did the corresponding sense construct or the
vector control, indicating that raf antisense RNA interferes with
viability and/or proliferation. As NIH/3T3 cells express no B-raf
and 10-fold less A-raf than Raf-1 (Storm, S. M. et al. Oncogene
5:345-351 (1990)), the effect can be ascribed to interference with
Raf-1. Antisense colonies were generally smaller and grew slower
than sense or vector control colonies. Ten out of ten antisense
colonies showed barely detectable levels of Raf-1 protein, whereas
levels in sense control clones were unchanged. An alternative
approach to Raf-1 inhibition used inactive mutants (Rapp, U. R. et
al. The Oncogene Handbook (eds. E. P. Reddy, A. M. Skalka and T.
Curran) 213-253 (Elsevier Science. The Netherlands, 1988);
Heidecker, G. et al. Molec. Cell. Biol. 10:2503-2512 (1990)). A
truncated Raf-1 protein (HCR) corresponding to conserved region 1
reduced colony numbers fourfold. A kinase-defective Raf-1 mutant
protein, craf301 (plasmid p301-1), was even more efficient,
decreasing colony yield about sevenfold (Heidecker, G. et al.
Molec. Cell. Biol. 10:2503-2512 (1990)). The surviving colonies
from these experiments could not be maintained as stable cell
lines. raf-transformed cell-lines were examined for morphological
reversion and inhibition of proliferation. p301 constructs were
transfected into 208-F12 fibroblasts which overexpress a
transforming mouse Raf-1 protein (Schultz, A. M. et al. Oncogene
2:187-193 (1988)). p301-2 caused partial or complete reversion of
the transformed phenotype in approximately half the transfectants.
Reversion correlated with loss of anchorage-independent growth.
Again, p301-1 was more efficient than p301-2 (FIG. 2a). These
clones were unstable, but cell-lines sufficiently stable for
biochemical analysis were obtained after pGMS transfection of v-raf
transformed cells, F4 (Rapp, U. R., et al. Proc. Natl. Acad. Sci.
U.S.A. 80:4218-4222 (1983)). Neither pMNC nor the control plasmid
GMS-7 was effective, whereas the antisense construct, GMS-8,
completely or partially reverted F4 (FIG. 2b). Reduction of raf
mRNA and protein levels correlated with the extent of reversion
(FIG. 2c). In one clone, GMS-8/3 (marked f* in FIG. 2c), raf
protein expression was undetectable. These cells grew extremely
slowly, arresting at 50-60% confluency, and eventually died.
[0038] To measure the effects of raf-protein depletion on the
mitogen response, the ability of serum and TPA to induce DNA
synthesis in serum-starved cells was determined (FIG. 3a). F4 and
GMS-7 cells synthesize DNA independently of mitogens. Constitutive
DNA synthesis was diminished in GMS-8/2, which retained an
inducible response similar to NIH/3T3 cells. GMS-8/3 was completely
blocked in constitutive and TPA-inducible DNA replication.
Serum-stimulation of GMS-8/3 was reduced seven-fold, and long-term
growth was also severely diminished (FIG. 3b).
[0039] v-Ki-ras-transformed NIH/3T3 cells were transfected with the
p301 plasmids (Table 1a). p301-1 and p301-2 reduced neomycin
(neo)-resistant colony yield to a similar degree as in NIH/3T3
cells (FIG. 1), suggesting that Raf-1 is required for proliferation
of raf-transformed cells. Morphological reversion of established
ras-transformed cells was less efficient than of raf-transformed
cells (FIG. 2). To test the effect of raf-inhibition on the
initiation of ras-transformation, a constant amount of v-Ha-ras
(pSV2neo/ras, Clanton, D. J. et al. Molec. Cell. Biol. 7:3092-3097
(1987)) plasmid was co-transfected with an equal or four-molar
amount of the p301 vectors (Table 1b). Although the neomycin
resistance of pMNC-based plasmids accounted for a background of
flat neo.cndot. colonies that presumably did not express
pSV2neo/ras, transfection with p301 vectors markedly increased the
number of morphological revertants at the expense of transformed
colonies. The inhibition was dose-dependent and almost complete at
four-molar excess of p301-1.
[0040] Thus, NIH/3T3 cells RAf-1 kinase functions downstream of
membrane receptors and ras proteins and is essential for
growth-induction by serum factors and protein kinase C. Membrane
receptor systems can now be examined individually for
Raf-1-dependence by inhibition with the blocking constructs
described herein. Furthermore, the proposed position of raf in the
communication pathway between cell membrane and nucleus makes raf
an attractive target for the design of novel antiproliferative
agents, especially as this data show that raf inhibition is
dominant over transformation by ras and by implication by other
non-nuclear oncogenes.
1TABLE I raf-inhibition blocks ras-mediated proliferation and
transformation a) v-Ki-ras cell trasfections Yield of nco.sup.1
Morphology of nco.sup.1 colonies Plasmids colonies flat
intermediate transformed pMNC.sup.1 100 .+-. 0% 0 .+-. 0% 0 .+-. 1%
100 .+-. 1% pMNC301-2.sup.2 61 .+-. 8% 2 .+-. 1% 15 .+-. 7% 83 .+-.
7% pMNC301-1.sup.3 30 .+-. 7% 15 .+-. 3% 15 .+-. 5% 70 .+-. 6% b)
NIH/3T3 co-transfection with v-Ha-ras (pSV2nco/ras) and p301
Plasmids Morphology of nco.sup.1 colonies (ratio 1:1)
inhibition.sup.4 flat intermediate transformed ras + pMNC.sup.1 0
.+-. 3% 27 .+-. 3% 17 .+-. 5% 56 .+-. 3% ras + p301-2.sup.2 53 .+-.
4% 28 .+-. 1% 46 .+-. 9% 26 .+-. 7% ras + p301-1.sup.3 61 .+-. 3%
46 .+-. 4% 32 .+-. 7% 22 .+-. 4% Plasmids (ratio 1:4) ras +
pMNC.sup.1 0 .+-. 1% 33 .+-. 4% 23 .+-. 3% 44 .+-. 3% ras +
p301-2.sup.2 61 .+-. 5% 48 .+-. 5% 35 '5 7% 17 .+-. 4$ ras +
p301-1.sup.3 84 .+-. 4% 67 .+-. 4% 25 .+-. 6% 7 .+-. 3% Cells were
transfected and G418-resistant (400 .mu.g ml.sup.-1) colonies were
morphologically examined according to the criteria described in
FIG. 2 Percentages are calculated for two experiments with
.gtoreq.200 (a) or .gtoreq.400 (b) colonies per transfection.
.sup.1vector control .sup.2antisense orientation .sup.3sense
orientation .sup.4The efficiency of inhibition of ras
transformation is given as percentage reduction in the number of
transformed colonies.
EXAMPLE 2
Association and Kinase Activity of Raf-1 with the EGF Receptor
[0041] To determine whether EGF induces the shift in migration in
SDS gels that is typical for phosphorylation activation of Raf-1
protein kinase, lysates of treated and control cells were subjected
to immunoprecipitation and immunoblotting with Raf-1-specific
antiserum. NIH 3T3 cells lacking endogenous EGF-R but expressing
approximately 3.times.10.sup.5 human wild-type (HER14) EGF-R or
kinase-negative mutant K721A EGF-R were transferred to starvation
medium (0.05% calf serum) at early confluency and stimulated with
EGF at 40 nM for 0 to 10 min. The effect of EGF on Raf-1 mobility
is shown in FIG. 4. In the absence of EGF treatment, Raf-1 migrates
as a single polypetide of 72 kDa, corresponding to the expected
molecular mass of Raf-1 protein kinase (Bonner, T. I., et al.
(1986) Nucleic Acids Res. 14:1009-1015). The addition of EFG to
HER14 but not to K721A cells resulted in a small increase in
apparent mass of Raf-1 to 74 kDa. This shift first became
detectable by 5 min, when approximately 50% of Raf-1 protein was
affected, and continued to spread so that by 10 min the entire pool
of Raf-1 protein had been modified. The inability of EFG to induce
the Raf mobility shift in NIH 3T3 cells expressing the
kinase-negative mutant of EGF-R demonstrates that receptor
dimerization is not sufficient for Raf-1 modification, since the
point mutation in K721A does not affect this event (Ulrich, A., et
al. (1990) Cell 61:203-212). It therefore seemed likely that the
kinase activity of the EGF-R was important in mediating induction
of the mobility shift in Raf-1.
[0042] The increase in apparent molecular mass of Raf-1 protein
upon EGF treatment was due to phosphorylation, since incubation
with potato acid phosphatase completely reversed the gel
retardation. To evaluate the effect of EFG-stimulated raf-1 protein
phosphorylation on its serine- and threonine-specific protein
kinase activity, immune complex kinase assays were performed that
utilized a synthetic peptide (IVQQFGFQRRASDDGKLTD) or histone H1 as
a substrate. The peptide corresponds to a potential
autophosphorylation site in the Raf-1 kinase, which has been
altered by substitution of phenylalanine for tyrosine in position 7
so as to restrict it from tyrosine phosphorylation.
[0043] For kinase assays, lysates of HER14 and K721A cells were
prepared before and after stimulation with 40 nM EFG for 10 min.
Comparison of the levels of kinase activity in Raf-containing
immunoprecipitates showed a sixfold stimulation in HER14 cells upon
EFG treatment (FIG. 5). Similar data were obtained when histone H1
was used as a substrate. Consistent with the absence of the
EGF-induced mobility shift of Raf-1 in NIH 3T3 cells expressing the
kinase-negative mutant for of the EGF-R, no stimulation of Raf-1
protein kinase activity was observed in K721A cells (FIG. 5). When
Raf-1 kinase activity was assayed with a modified version of the
substrate peptide in which Ser-12 was replaced by alanine and Tyr-5
was retained, no counts were detected on the spotted filters. This
indicates that the kinase activity measured by the assay did not
include a contribution of a contaminating tyrosine kinase
activity.
[0044] Activity EGF-R associates with the candidate signal
transducing enzyme PLC.sub..gamma. (Margolis, B., et al. (1990)
Mol. Cell. Biol. 10:435-441; Margolis, B., et al. (1989) Cell
57:1101-1107; Meisenhelder, J., et al. (1989) Cell 57:1109-1112;
Wahl, M., et al. (1989) Proc. Natl. Acad. Sci. USA 86:1568-1572).
Similarly, Raf-1 was shown to coimmunoprecipitate with activated
PDGF-.beta. receptor in cell lines expressing high levels of
receptors (Morrison, D. K., et al. (1989) Cell 58:648-657). To
evaluate whether ligand-induced activation of Raf-1 protein kinase
by the EGF-R correlated with receptor association, two cell systems
were used: the NIH 3T3 cells expressing wild-type and mutant
receptors (FIG. 6A) and human A431 cells (FIG. 6B) expressing
approximately 2.times.10.sup.6 EGF-R per cell (15, 16).
Serum-starved cells were stimulated with 40 nM EGF for 10 min. and
lysates from cells were immunoprecipitated with Raf-1 or EGF-R
specific antibodies. After separation by SDS-PAGE and transfer to
nitrocellulose, immunoblotting was performed with either anti-EGF-R
or anti-Raf-1 antibodies. EGF-R is present in anti-Raf-1 antibody
immunoprecipitates from EGF-treated cells (FIG. 6). The
coprecipitating EGF-R in HER14 cells has a decreased mobility on
PAGE, compared with that of the EGF-R from untreated controls (FIG.
6A, lanes 4 and 6); this decreased mobility was previously
demonstrated to be due to ligand-induced autophosphorylation
(Margolis, B., et al. (1989) Cell 57:1101-1107). Cells expressing
the kinase-negative mutant receptor KD712A did not show the
mobility shift in the EGF-R upon EGF treatment and lacked EGF
stimulation of EGF-R Raf-1 coimmunoprecipitation. A small amount of
unshifted EGF-R was detected in Raf-1 immunoprocipitates from all
cells; this EGF-R could be reduced by preclearing with preimmune
serum. EGF-R can be coprecipitated in lysates from EGF-treated A431
cells, whereas there is not EGF-R present in immunoprecipitates
from untreated cells (FIG. 6). Sequential reprobing of the Western
blot with polyclonal Raf-1 rabbit antiserum (FIG. 6B) indicates
that a small fraction (.about.1%) of the EGF-R associates with
shifted Raf-1. Furthermore, the blot demonstrates that the
EGF-R-Raf-1 association was not due to unequal loading of the gel
with Raf-1 immunoprecipitates. Estimates from three independent
experiments indicate that the fraction of immunoprecipitable EGF-R
protein that is present in Raf-1 antibody precipitates from
EGF-treated HER14 or A431 cells is on the order of 1.0%.
[0045] Considering the observed association of Raf-1 protein with
activated EGF-R as well as the EGF-induced mobility shift of Raf-1,
it might be expected that the receptor-associated fraction of Raf-1
was phosphorylated on tyrosine. The immunoblots from experiments in
FIG. 6 were therefore reprobed with antiphosphotyrosine antibodies.
The antibodies readily detected EFG-induced tyrosine
phosphorylation of the EGF-R, PLC.tau., GAP, and other unknown
substrates (Ulrich, A., et al. (1990) Cell 61:203-212), but no
tyrosine phosphorylated bands in the size range of Raf-1 protein
were detected. The experiment was scaled up to examine the presence
of tyrosine-phosphorylated Raf-1 protein in anti-Raf or anti-EGF-R
antibody immunoprecipiates from 10.sup.6 HER14 cells per lane;
again, tyrosine phosphorylation of Raf-1 could not be detected.
Consistent with the absence of anti-phosphotyrosine
antibody-reactive Raf-1 protein, phosphoamino acid analysis of
Raf-1 from EGF-treated cells did not reveal any phosphotyrosine
(FIG. 7). For this experiment, 10.sup.7 HER14 cells were labeled
with .sup.32Pi, and the Raf-1 proteins were immunoprecipitated with
anti-v-Raf 30-kDa polyclonal antiserum and subjected to SDS-PAGE.
Phospholabeled Raf-1 protein was excised from the gel,
electroeluted, and hydrolyzed in 6 N HCl. The only labeled
phosphoamino acid detectable was phosphoserine; thus it can be
concluded that EGF induced an increase in serine phosphorylation of
c-Raf (FIG. 7). When the same experiment was done with A431 cells,
trace amounts of phosphotyrosine were detected that were
independent of EGF treatment. The lower limit for detection of
phosphotyrosine in Raf-1 in these experiments was on the order of
1% of phosphoserine.
[0046] The absence of tyrosine phosphorylation of Raf-1 protein in
response to EGF in HER14 cells raises the possibility that serine
protein kinase(s) acts as an intermediate in a kinase cascade
connecting the stimulated EGF-R to activation of Raf-1 kinase. One
candidate for this role is PKC, since this enzyme has previously
been shown, upon treatment of cells with
tetradecanoylphorbol-13-acetate (TPA), to trigger Raf-1
phosphorylation and kinase activation (Morrison, D. K., et al.
(1988) Proc. Natl. Acad. Sci. USA 85:8855-8859; Siegel, J. N., et
al. (1990) J. Biol. Chem. 265:18472-18480). It was therefore
examined whether EGF induction of the Raf-1 mobility shift was
dependent on the presence of PKC (FIG. 8). HER14 cells were
pretreated with 200 ng of TPA for 72 h for complete downregulation
of PKC and then tested for their ability to respond to EGF with
Raf-1 retardation. The PKC down-regulation by pretreatment with TPA
was effective in eliminating the TPA-induced Raf-1 retardation. In
contrast, EGF-induced Raf-1 mobility shift was not blocked by
down-regulation of PKC.
[0047] All publications mentioned hereinabove are hereby
incorporated in their entirety by reference. In particular, Kolch W
et al (1991) Nature 349:426-428 and App H et al (1991) Molecular
and Cellular Biology 11(2):913-919 are hereby incorporated in their
entirety by reference.
[0048] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be
appreciated by one skilled in the art from a reading of this
disclosure that various changes in form and detail can be made
without departing from the true scope of the invention and appended
claims.
Sequence CWU 1
1
* * * * *