U.S. patent application number 09/859000 was filed with the patent office on 2002-09-05 for rna probe for detecting c-fes mrna.
This patent application is currently assigned to The United States of America, Department of Health and Human Services. Invention is credited to Glazer, Robert I., Smithgall, Thomas E., Yu, Gang.
Application Number | 20020123049 09/859000 |
Document ID | / |
Family ID | 23396625 |
Filed Date | 2002-09-05 |
United States Patent
Application |
20020123049 |
Kind Code |
A1 |
Glazer, Robert I. ; et
al. |
September 5, 2002 |
RNA probe for detecting c-fes mRNA
Abstract
A recombinant plasmid and an RNA sequence expressed by said
plasmid are described. The RNA sequence hybridize specifically with
human c-fes mRNA.
Inventors: |
Glazer, Robert I.;
(Gaithersburg, MD) ; Smithgall, Thomas E.;
(Rockville, MD) ; Yu, Gang; (Bethesda,
MD) |
Correspondence
Address: |
GREGORY P. EINHORN
Fish & Richardson P.C.
Suite 500
4350 La Jolla Village Drive
San Diego
CA
92122
US
|
Assignee: |
The United States of America,
Department of Health and Human Services
|
Family ID: |
23396625 |
Appl. No.: |
09/859000 |
Filed: |
May 15, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09859000 |
May 15, 2001 |
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09262792 |
Mar 4, 1999 |
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6232069 |
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09262792 |
Mar 4, 1999 |
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08252136 |
May 31, 1994 |
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5879882 |
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08252136 |
May 31, 1994 |
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07954427 |
Sep 30, 1992 |
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07954427 |
Sep 30, 1992 |
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07355207 |
May 22, 1989 |
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Current U.S.
Class: |
435/6.12 ;
435/6.13; 536/23.1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C07K 14/82 20130101; C12Q 2600/158 20130101 |
Class at
Publication: |
435/6 ;
536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02 |
Claims
What is claimed is:
1. A kit for the detection of c-fes mRNA, comprising a container
containing an RNA sequence that hybridizes specifically with human
c-fes mRNA.
2. A method of detecting the prescence of c-fes mRNA, comprising
hybridizing RNA in a biological sample in which the prescence of
c-fes mRNA is to be ascertained, with an RNA sequence that
hybridizes specifically with human c-fes mRNA and determining the
occurrence of hybridization with said RNA sequence by conventional
methodologies, a positive hybridization reaction being indicative
of the presence of c-fes mRNA in said sample.
Description
[0001] The present invention is related generally to diagnostic
tests. More particularly, the present invention is related to an
RNA probe for detecting the presence of c-fes mRNA in biological
samples, such as human cell and tissue RNA preparations.
[0002] Expression of the c-fes oncogene is known to play a certain
functional role in myelopoiesis in henatopoietic cells (Smithgall
et al, 1988, J. Biol. Chem. 263, 15050-40 15055; Greer et al, 1988,
Mol. Cell. Biol., 8, 578-587). However, heretofore direct evidence
was lacking to prove that the expression of human c-fes gene
induced myeloid differentiation in cells. Furthermore, a specific
and sensitive assay to measure the level of c-fes mRNA in human
cells and tissues was also heretofore not available.
SUMMARY OF THE INVENTION
[0003] It is, therefore, an object of the present invention to
provide a kit for the detection of c-fes mRNA in biological samples
such as human cell and tissue RNA preparations.
[0004] It is a further object of the present invention to provide
an RNA probe for detecting the presence of c-fes mRNA in vitro or
in situ.
[0005] It is another object of the present invention to provide a
recombinant plasmid comprising exon 2 of the human c-fes genomic
sequence for the expression of the transcription product of the
c-fes oncogene in a suitable expression vector.
[0006] Other objects and advantages will become evident from the
following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] These and other objects, features and many of the attendant
advantages of the invention will be better understood upon a
reading of the following detailed description when considered in
connection with the accompanying drawings wherein:
[0008] FIG. 1 shows the various elements of the human c-fes genomic
clone.
[0009] FIG. 2 shows schematic construction of the recombinant
plasmid pcfes4ZKB.
[0010] FIG. 3 schematically shows various steps involved in the
RNase protection assay with the c-fes RNA probe in accordance with
the present invention.
[0011] FIG. 4 shows non-denaturing gel assay for p93.sup.c-fes
tyrosine kinase activity in colonies of K562 cells stably
transfected with pECE/fes. K562 cells were cotransfected with
pECE/fes and pSV2/neo as a selectable marker and G418 -resistant
colonies were selected and screened for p93.sup.c-fes tyrosine
kinase activity. Aliquots of membrane proteins (15 .mu.g) present
in 1.0% Triton X-100 cell extracts were assayed for tyrosine kinase
activity using the non-denaturing gel assay described in the
text.
[0012] FIG. 5 shows the comparison of tyrosine kinase activity in
colony WS-1 with differentiated HL-60 cells. One percent Triton
X-100 extracts were prepared from either wild type K562 cells
("K562"), pSV2/neo-transfected K562 cells ("K562/neo"), colony WS-1
("K562/fes"), or HL-60 cells treated for 4 days with 1.6%
Me.sub.2SO, and p93.sup.c-fes tyrosine kinase was partially
purified by tyrosine-agarose chromatography. Eluates (3 .mu.g of
protein) were assayed for tyrosine kinase activity using the
non-denaturing gel assay as described in the text.
[0013] FIG. 6 shows the Southern blot analysis of colonies of K562
cells stably transfected with pECE/fes. DNA (10 .mu.g) was prepared
from wild type K562 cells ("K562") and selected colonies of cells
transfected with c-fes (designated as "WS-1, WS-5, WS-6, WD-1,
WD-2, WD-3, WD-4, and WD-7"), and digested with Eco RI. Plasmid p80
DNA ("p80") containing the 13.2 kb c-fes gene served as a control.
After electrophoresis in 1% agarose gels, Southern blots were
prepared and hybridized with a v-fes probe as described herein
below. Levels of c-fes integration relative to wild type K562 cells
were determined by laser densitometry of the 13.2 kb Eco RI
fragment. The endogenous K562 c-fes gene is not visible in the
exposure shown (12 h); determination of the c-fes gene in wild type
cells required longer autoradiographic exposure (>48 h; data not
shown).
[0014] FIG. 7 shows the Southern blot analysis of a restriction
digest of DNA prepared from colony WS-1. DNA (10 .mu.g) was
prepared from wild type K562 cells ("K562") and colony WS-1
("K562/fes") and digested with Eco RI and Xho I. Plasmid p80 DNA
("p80") containing the 13.2 kb c-fes gene served as a control.
Southern blots and hybridization were carried out as described in
the text.
[0015] FIG. 8 shows the RNase protection assay of parental and
clonal variants of K562 cells stably transfected with pECE/fes.
Poly-A RNA was selected from 250 .mu.g of total RNA prepared from
wild type HL-60 cells ("HL-60"), wild type K562 cells ("K562"), and
colonies WS-1 ("K562/WS-1"), WS-5 ("K562/WS-5") and WS-6
("K562/WS-6"). Solution hybridization was carried out with
10.degree. cpm of a .sup.oxP-labeled c-fes antisense RNA probe
containing the 222 bp sequence complementary to exon 2 of the human
c-fes gene. Following overnight incubation, the hybridization
reaction was digested with RNase and the protected dsRNA fragments
were resolved by electrophoresis on 6% polyacrylamide-urea gels,
and visualized by autoradiography.
[0016] FIG. 9 shows the immunoprecipitation of parental and clonal
variants of K562 cells stably transfected with pECE/fes. Cell
extracts were prepared from wild-type K562 cells. transfected
clones WS-1 and WS-5, and HL-60 cells labeled with [.sup.ooS
]Methionine, and p93.sup.c-fes was immunoprecipitated with an
anti-v-fes monoclonal antibody. Immunoprecipitates were analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography as
described in the text. The control lane shows precipitation of
HL-60 extracts in the absence of the monoclonal antibody.
[0017] FIG. 10 shows the growth curve of K562/fes clone WS-1,
K562/neo and parental K562 cells. Wild type K562 cells ("K562"),
pSV2/neo-transfected cells ( "K562/neo" ) and colony WS-1
("K562/fes") were grown for one week, and cells number was
determined at one day intervals with a Coulter particle counter.
Cell viability was greater than 95% as determined by trypan blue
exclusion.
[0018] FIG. 11 shows the photomicrographs of parental K562 cells
and K562/fes clone WS-1. Parental K562 cells (A,C,E) and
c-fes-transfected clone WS-1 (B,D,F) were tested for their response
to 2 day treatment with 10.sup.-7 M TPA (A,B), for their ability to
reduce NBT (C,D) or for their capacity to phagocytize sheep
erythrocytes (E,F).
DETAILED DESCRIPTION OF THE INVENTION
[0019] The above and various other objects and advantages of the
present invention are achieved by a specific RNA sequence which
hybridizes only with c-fes mRNA. said specific RNA sequence being
obtained from the expression of the recombinant plasmid pcfes4ZKB
in a suitable expression vector, such as E. coli, yeast, viruses
and other prokaryotic or eukaryotic vectors well known to one of
ordinary skill in the art.
[0020] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned hereunder are incorporated herein by
reference. Unless mentioned otherwise, the techniques employed
herein are standard methodologies well known to one of ordinary
skill in the art. The materials, methods and examples are
illustrative only and not limiting.
MATERIALS AND METHODS
[0021] Materials--All radioisotopes were obtained from Du Pont-New
England Nuclear, Boston, Mass. Tyrosine-agarose, Me.sub.2SO, and
poly(glu,tyr).sub.412 were purchased from Sigma, St. Louis, Mo. The
v-fes probe (460 bp Pst I-Pst I fragment) was purchased from Oncor,
Gaithersburg, Md. Rabbit antisera to a recombinant c-fes peptide
was provided by Dr. Dennis J. Slamon, UCLA School of Medicine, Los
Angeles, Calif. Geneticin (G418) was purchased from Gibco, Grand
Island, N.Y. Plasmids p80 and pSV2/neo were obtained from the
American Type Culture Collection, Rockville, Md. The Mac-1
monoclonal antibody against the macrophage-specific differentiation
was obtained from Hybritech, San Diego, Calif. The monoclonal
antibody (Ab-1) directed against the fes transforming protein
common to both the Snyder-Theilen and Gardner strains of feline
sarcoma virus was purchased from Oncogene Sciences, Manhasset,
N.Y.
[0022] Cell Culture--HL-60, K562, and Cos-1 cells were obtained
from the American Type Culture Collection. HL-60 and K562 cells
were grown in RPMI-1640 medium supplemented with 10%
heat-inactivated fetal calf serum, 40 mM Hepes, PH 7.4, 1 mM sodium
pyruvate, nonessential amino acids, 100 units/ml penicillin, and
100 .mu.g/ml streptomycin. Cos-1 cells were grown in Dulbecco's
Modified Eagle's medium supplemented as above. All cells were
subcultured twice weekly, and maintained at a density of
10.sup.5-10.sup.6 cells/mi. HL-60 cells were treated with 1.6%
Me.sub.2SO for 4 days to induce granulocytic differentiation.
[0023] Preparation of Cell Extracts--Cells (0.5-1.0.times.10.sup.8)
were collected by centrifugation and washed twice in Hank's
balanced salt solution containing 20 mM EDTA without Mg.sup.2-or
Ca.sup.2-. The cell pellet was sonicated for 5 seconds in 0.5 ml of
50 mM Tris-HCl (pH 7.5) containing 2 mM EGTA, 10 mM DTT, 0.1%
Triton-X 100, 1 mM PMSF, 50 .mu.g/ml aprotinin, 200 .mu.g/ml
leupeptin and 400 .mu.g/ml soybean trypsin inhibitor, and
centrifuged at 15,000.times.g at 4.degree. C. for 10 min. The
supernatant was removed and the pellet was re-extracted with an
identical buffer containing 1% Triton X-100. Protein concentrations
were determined using a Coomassie blue-based reagent (Pierce
Chemical Co.) and BSA as a standard.
[0024] Non-denaturing Gel Assay for Tyrosine Kinase
Activity--Tyrosine kinase activity present in crude cell extracts
and column fractions was assayed by non-denaturing polyacrylamide
gel electrophoresis as described by Glazer et al (1987) Anal.
Biochem. 164, 214-220. Briefly, protein samples were subjected to
electrophoresis in 4.5% polyacrylamide mini-gels (Hoefer
Scientific) at 4.degree. C. Following electrophoresis, the gels
were incubated with Mg.sup.2-, Mn.sup.2-and [t-.sup.32P]ATP in the
presence and absence of poly(glu,tyr).sub.4:1, a synthetic polymer
substrate in which tyrosine acts as sole phosphate acceptor.
Following incubation at 37.degree. C. for 30 min, the gels were
washed extensively in 5% trichloroacetic acid containing 10 mM
sodium pyrophosphate, dried and kinase activity was quantitated by
autoradiography.
[0025] Tyrosine-agarose Chromatography, Immunoblotting, and
Immunoprecipitation--One percent Triton X-100 extracts were further
fractionated by tyrosine-agarose chromatography (Yu et al, 1987, J.
Biol. Chem. 262. 17543-17548). Extracts were applied to 1.5 ml
tyrosine-agarose columns and aliquots (10 .mu.g of protein) present
in the eluate were resolved on 7.5% SDS-polyacrylamide mini-gels
using them Laemmli buffer system (Laemmli, U. K., 1970, Nature 227,
680-685). Proteins were transferred to nitrocellulose membranes
using the Genie electrophoretic blotter (Idea Scientific).
Immunoreactive p93.sup.c-fes was visualized using antiserum to a
recombinant human c-fes peptide, and the Protoblot detection system
(Promega Biotec) as described by the manufacturer. For
immunoprecipitation, 5 .times.10.sup.7 cells were labeled by
incubation at 37.degree. C. for 18 h in 3 ml of methionine-free
Iscove's Modified Dulbeccol's Medium containing 5% fetal calf serum
and 200 .mu.Ci/ml [.sup.35S]-methionine (1,140 Ci/mmol). Cells were
then washed, lysed and subjected to immunoprecipitation with
biotinylated anti-v-fes monoclonal antibody (Veronese et al, 1982,
J. Virol. 43, 896-904) and streptavidin-agarose according to the
manufacturer's protocol. Following extensive washing, immune
complexes resolved by electrophoresis through 8% SDS-polyacrylamide
gels. Gels were treated with Fluoro-Hance (Research Products
International, Mount Prospect, Ill.) prior to autoradiography at
-80.degree. C.
[0026] Construction of the expression vector pECE/fes--An
SV40-based mammalian expression vector pECE (Ellis et al, 1986,
Cell 45, 721-732) was provided by Dr. William J. Rutter, University
of California, San Francisco. pECE was digested with Eco RI and
dephosphorylated with calf intestine alkaline phosphatase. The
entire human c-fes genomic sequence was isolated as a 13.2 kb Eco
RI fragment from the plasmid vector p80 (Trus et al, 1982, J. Biol.
Chem. 257, 2730-2733) and cloned into the expression vector PECE.
The orientation of c-fes was determined by Southern blots using the
v-fes probe and it was found to be in the correct orientation such
that transcription of the c-fes coding sequence is directed from
the SV40 early promoter. This recombinant plasmid is designated
pECE/fes.
[0027] Transfection of Cos-1 and K562 cells--Cos-1 cells
(5.times.10.sup.5 cells/100 mm plate) were transfected with 20
.mu.g of pECE/fes by the modified calcium phosphate precipitation
procedure described by Chen and Okayama (1987) Mol. Cell. Biol. 7,
2745-2752. For transient expression. analysis was performed 48 h
posttransfection. K562 cells were transfected by the protoplast
fusion technique (Yoakum, G. H., 1984, BioTechniques 2, 24-31).
Briefly, 100 ml of an overnight culture of E. coli transformed with
pECE/fes and pSV2/neo were centrifuged at 4000.times.g for 15 min.
The bacteria were incubated for 2 hr at room temperature
(22.degree.-24.degree. C.) with 3 ml of a freshly prepared lysozyme
solution (10 mg/ml in 20 mM Hepes, 20% sucrose, pH 7.1). The
incubation was stopped by adding 0.8 ml of 1.25 M CaCl.sub.2 and
the protoplast preparation was diluted to 10 ml with RPMI-1640.
K562 cells (5.times.10.sup.6 cells) were collected by
centrifugation and treated for 1.5 min with 2 ml of the protoplast
preparation and 1 ml of fresh 48% polyethylene glycol (mol wt
1000). The cells were then washed five times with RPMI-1640 medium
in a CO.sub.2 incubator with the medium changed daily for the first
two days. After 48 hr, cells were split and plated at 10.sup.3
cells per 100 mm plate containing RPMI-1640 medium supplemented
with 20% heat-inactivated fetal calf serum and 0.4% agarose
(SeaPlaque, FMC) and 2.5 mg/ml G418 (Gibco) for selection. After
about 14 days of incubation, colonies were selected and cultured in
RPMI-1640 medium with 0.2 mg/ml G418.
[0028] Southern Blot--High molecular weight DNA, prepared by the
Gross-Bellard method (Gross-Bellard et al, 1972, Eur. J. Biochem.
36, 32-39) was digested with either Eco RI or Xho I, separated in
0.8% agarose gels and transferred to nitrocellulose (Southern, E.
M., 1975, J. Mol. Appl . Genet. 1, 327-341). Hybridization was
carried out with the v-fes probe labeled with
[.alpha.-.sup.32P]dCTP by the random primer procedure (BRL) at
36.degree. C. for 16 hr in 50% formamide, 5X SSC, 0.5% SDS, 5X
Denhardt's solution and 100 .mu.g/ml denatured salmon sperm DNA.
Blots were washed with 0.1X SSC, 0.1SDS at 65.degree. C.
[0029] Cloning of genomic c-fes fragments for riboprobe
synthesis--A 461 bp Kpn I-Bgl II fragment of the human c-fes locus
(Roebroek et al, 1985, EMBO. J. 4, 2897-2903) containing exon 2 and
some 3' and 5' intron sequences, was cloned into the polylinker
region of pGEM-4Z (Promega Biotec). This vector contains the
bacteriophage T7 promoter immediately downstream from and in an
opposite orientation to the cloning site, allowing for preparation
of a c-fes riboprobe (antisense RNA transcript). This was
accomplished by linearization of the vector 5' to the c-fes insert
and incubation with T7 RNA polymerase, [.alpha.-.sup.32P]CTP and
unlabeled nucleoside triphosphates according to the manufacturer's
protocol. The resulting riboprobe is 498 nucleotides in length, as
it contains some sequences transcribed from the vector
template.
[0030] Poly-A.sup.+ RNA Isolation and RNase Protection Assay--Total
cellular RNA was prepared by guanidinium isothiocyanate extraction
of 10 .sup.5 cells followed by centrifugation through cesium
chloride (Chirgwin et al, 1979, Biochemistry 18, 5294-5298; Glisin
et al, 1973, Biochemistry 13, 2633-2641). Poly-A.sup.- RNA was
selected from 250 .mu.g total RNA by batch adsorption to oligo-dT
cellulose (New England Biolabs). The fraction eluting from oligo-dT
cellulose selection was hybridized with 10.sup.6 cpm of the
.sup.a2P-labeled c-fes riboprobe (see above). Following overnight
(about 12-16 hrs) incubation, the hybridization reaction was
digested with RNase, and the protected dsRNA fragments were
resolved by polyacrylamide-urea gel electrophoresis and visualized
by autoradiography.
[0031] Histochemical Assays--Lysozyme activity was measured
spectrophotometrically at 450 nm by the lysis of M.
lysodeikticus(Selsted et al, 1978, Infection and Immunity 20,
782-791). The ability of cells to reduce NBT to formazan was
assessed by the method described by Breitman et al (1980) Proc.
Natl. Acad. Sci. U.S.A. 77, 2936-2940. Fc receptors and
immunophagocytosis were determined using sheep erythrocytes coated
with anti-erythrocyte antibodies (Breitman et al, 1984, in Methods
for Serum-Free Culture of Neuronal and Lymphoid Cells. Alan R.
Liss, Inc., N.Y., 215-236). Expression of the macrophage-specific
differentiation marker, Mac-1, was examined by immunofluorescence
following treatment of cells for 2 days with 100 nM TPA (Ball et
al, 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 5374-5378).
EXAMPLE
Construction of Recombinant Plasmids and Riboprobe Synthesis
[0032] The plasmid vector p80, which contains the entire human
c-fes genomic sequence (Trus et al, supra), was s digested with Kpn
I and Xba I. The resulting 1175 bp fragment, which contains c-fes
exons 2 and 3, was inserted into the polylinker region of pGEM4Z
(Promega Biotec, Madison, Wis.) (FIG. 1). This recombinant plasmid
was named pcfes4ZKX. To prepare a template for riboprobe synthesis,
pcfes4ZKX was digested with Bgl II and Xba I, which removed c-fes
exon 3 and about two-thirds of intron 2. The terminal Bgl II and
Xba I sites were filled in with the Klenow fragment of DNA
polymerase, and the plasmid was re-circularized with T4 DNA ligase.
The resulting recombinant plasmid, pcfes4ZKB, contains c-fes exon 2
flanked by partial sequences of introns 1 and 2 (FIG. 2). The c-fes
insert is upstream from and in opposite orientation to the
bacteriophage T7 promoter. Prior to riboprobe synthesis, pcfes4ZKB
was digested to completion with Eco RI, which cuts the plasmid 5'
to the c-fes insert.
[0033] Riboprobe synthesis was conducted in a 20 .mu.l reaction
containing 40 mm Tris-HCl, pH 7.5, 6 mM MgCl.sub.2, 2 mM
spermidine, 10 mM NaCl, 10 mM DTT, 40 units RNasin, 0.5 mM ATP,
UTP, and GTP, 12 .mu.M CTP, 50 .mu.Ci [.alpha.-.sup.32P]CTP (800
Ci/mmol), and 1.0 .mu.g linearized template DNA (FIG. 3). Reactions
were initiated by adding 20 units of T7 RNA polymerase. incubated
at 37.degree. C. for 1 h. and terminated by the addition of 5 units
of RQ1 DNase (Promega). Following DNase treatment for 15 min at
37.degree. C., 2 .mu.g of carrier tRNA were added, the reaction
mixture was extracted with phenol-chloroform, and the labeled RNA
was precipitated with ethanol. The riboprobe was re-dissolved in
100 .mu.l water, and the amount of labeled CTP incorporated was
determined by TCA precipitation (typically 10.sup.5 to 10.sup.9
cpm/.mu.g RNA). The c-fes riboprobe synthesized in this manner is
498 nucleotides in length, as it contains some sequences
transcribed from the parent vector. Probes were prepared on the
same day they were to be used, and the best results were obtained
with fresh isotope. This procedure is a modification of the method
originally described by Melton et al (1984) Nucleic Acids Res. 12,
7035-7056.
[0034] A deposit of the recombinant plasmid pcfes4ZKB has been made
at the ATCC, Rockville, Md., on May 19, 1989, under the accession
number 40610. The deposit shall be viably maintained, replaced if
it becomes non-viable during the life of the patent, for a period
of 30 years from the date of the deposit, or for 5 years from the
last date of request for a sample of the deposit, whichever is
longer, and upon issuance of the patent made available to the
public without restriction in accordance with the provisions of the
law. The Commissioner of Patents and Trademarks, upon request,
shall have access to the deposit.
RESULTS
Transfection of Cos-1 cells with pECE/fes
[0035] The 13.2 kb Eco RI fragment identified previously as the
human c-fes gene (Trus et al, supra: Roebroek et al, supra) was
cloned into the SV40-based mammalian expression vector pECE (Ellis
et al, supra) and designated pECE/fes. To test this construct,
Cos-1 cells were transfected by calcium phosphate precipitation and
48 hr later, Triton X-100 extracts of cellular proteins were
analyzed for immunoreactive p93.sup.c-fes and for tyrosine kinase
activity. Cos-1 cells transfected with pECE/fes expressed a 93 kDa
protein which was specifically recognized on Western blots by the
c-fes polyclonal antibody (results not shown). Extracts prepared
from Cos-1 cells transfected with pECE/fes expressed a single
species of tyrosine kinase activity that was present in the 1.0%
Triton X-100 cell extract (results not shown). These results
indicated that Cos-1 cells are capable of expressing the genomic
DNA encoding c-fes and transcribing a functional gene product.
However, Cos-1 cells did not acquire characteristics of myeloid
cells as a result of c-fes transfection.
Co-transfection of K562 Cells with pECE/fes and DSV-2/neo
[0036] Since K562 cells do not express p93.sup.c-fes, they are an
ideal cell line for transfection experiments with pECE/fes. K562
cells were co-transfected with pECE/fes and pSV-2/neo by protoplast
fusion and were selected by cloning in soft agar containing 2.5
mg/ml G418. After 14 days in culture, G418-resistant colonies were
selected and amplified in RPMI-1640 medium. One percent Triton
X-100 cell extracts representing the membrane fraction of the cell
were prepared from G418-resistant colonies and were screened for
tyrosine kinase activity with the nondenaturing gel assay (FIG. 4).
Stably transfected colonies designated WS-1, WS-5, WS-6, and WD-7
had high levels of tyrosine kinase activity. Colony WS-1 expressed
a level of tyrosine kinase activity comparable to that present in
HL-60 cells treated with 1.6% Me.sub.2SO (FIG. 5), a treatment
which produces granulocytic differentiation (Zylber-Katz et al,
1985, Cancer Res. 45, 5159-5164). p93.sup.c-fes tyrosine kinase
activity was not present in either parental or
pSV-2/neo-transfected K562 cells (FIG. 5).
[0037] A Southern blot of the DNA prepared from several colonies of
stably transfected K562 cells indicated varying levels of
integration of the c-fes gene (FIG. 6). The most dramatic example
is seen in transfected clone WS-1, in which the level of the c-fes
gene is more than 30 times higher than that of the K562 wild-type
cells. The intensity of the hybridization signal was similar to the
level of tyrosine kinase activity expressed by the various clones
(FIG. 4). Digestion of WS-1 cell DNA with Eco RI and Xho I
generated the expected 13.2 kb and 4.4 kb fragments that were
identical to those present in p80 following hybridization with the
v-fes DNA probe (FIG. 7).
Analysis of c-fes transcript levels, mRNA processing, and
p93.sup.c-fesprotein synthesis in K562/fes clones
[0038] Steady-state levels of c-fes mRNA were determined in
transfected K562 clones using the RNase protection assay. The probe
used in this assay is an anti-sense RNA transcript 498 nucleotides
in length containing the 222 nucleotide sequence complimentary to
c-fes exon 2. The remainder of the probe Us made up of 5' and 3'
sequences complimentary to c-fes introns 2 and 3, and 37
nucleotides transcribed from the vector template. Poly-A.sup.- RNA
was prepared from K562/fes clones WS-1, WS-5, and WS-6 and
hybridized to the c-fes riboprobe overnight. Following RNase
digestion, polyacrylamide/urea gel electrophoresis revealed a major
protected fragment 222 nucleotides in length in each of the
transfected clones, which corresponds to c-fes exon 2 (FIG. 8). The
intensity of this band is proportional to the level of c-fes
genomic integration (FIG. 6), p93.sup.c-fes protein levels (see
below). Note that an identical protected fragment is present
following the RNase protection assay of poly-A.sup.31 RNA from
HL-60 cells, a cell line which normally expresses p93.sup.c-fes. By
contrast, no protected fragments were observed following the RNase
protection assay of untransfected K562 cells.
[0039] In addition to the major band of 222 nucleotides, K562/fes
clones WS-1 and WS-5 also exhibited a protected fragment of 460
nucleotides (FIG. 8), which corresponds to the size of the c-fes
genomic fragment contained within the probe (i.e., intron and exon
sequences). This indicates that a significant fraction of the c-fes
mRNA from transfected cells contains intron sequences. and suggests
that c-fes mRNA is less efficiently processed in the transfected
clones than in HL-60 cells. which do not exhibit this band. Minor
protected fragments approximately 320 and 370 nucleotides in length
are also visible in transfected clones WS-1 and WS-5, as well as in
HL-60 cells. These fragments may arise from alternate processing of
the primary c-fes transcript that occurs 5' to exon 2, as several
alternate splice acceptor sites have been proposed in intron 2 of
the c-fes genomic sequence (Roebroek et al, supra).
[0040] The results indicate that the translation of c-fes ARENA
into p93.sup.c-fes protein proceeds normally in transfected K562
cells. This conclusion is based on immunoprecipitation experiments
using an anti-v-fes monoclonal antibody. As shown in FIG. 9,
transfected K562 clones WS-1 and WS-5 express an immunoreactive 93
kDa protein not seen in the K562 wild type cells. Note that an
immunoreactive protein of identical electrophoretic mobility is
also seen in immunoprecipitates of HL-60 cells, which are enriched
in p93.sup.c-fes.
Phenotypic alterations in colonies of K562 cells transfected with
c-fes
[0041] Clones WS-1, WS-5 and WS-6 were selected for further study
of the changes in maturation which accompanied selection of these
cell lines. After 2-3 passages, WS-1 cells grew at a slower rate
than wild type K562 cells (FIG. 10), a property which may be
indicative of differentiation. In addition, all clones adhered
loosely to the culture flask, a property which was not seen with
parental or pSV-2/neo transfected cells (Table I). Most notable was
the response of WS-1 and WS-5 cells to TPA, a treatment which
produced approximately 50% macrophage-like cells (FIGS. 11A,B).
TPA-treatmenrt of transfected K562 cells also resulted in
expression of the macrophage-specific differentiation antigen
Mac-1(Springer et al, 1979, Eur. J. Immunol. 9, 301-306), whereas
TPA-treated wild type cells displayed almost no detectable Mac-1
imnunofluorescence (Table I) Several functional parameters which
are indicative of mature myeloid cells were also examined.
Erythrophagocytosis increased dramatically in clones WS-1 and WS-5
and to a lesser extent in WS-6 (Table I and FIGS. 11E, F). The
percentage of Fc receptor positive cells is high in K562 cells
(Koeffler et al, 1981, Cancer Res. 41, 919-926) but doubled in all
the clones (Table I). Several enzymatic features of mature myeloid
cells were also acquired. In parental K562 cells, lysozyme activity
was absent, but it was readily detected in all selected clones, and
all transfected cell lines demonstrated high levels of NBT
reduction.
[0042] Although transfected K562 cells express mature myeloid
characteristics and respond to TPA, they still retain the ability
to undergo erythroid differentiation in response to hemin.
Treatment of K562, K562/WS-1, and K562/WS-5 with 100 mM hemin for 5
days resulted in 64%, 49% and 63% benzidine-positive cells,
respectively.
[0043] In summary, the results presented herein clearly indicate
that the differentiation-associated 93 kDa tyrosine kinase activity
is the product of the human c-fes gene. Expression of p93.sup.c-fes
is found to be especially high in mature peripheral monocytes and
granulocytes, acute and chronic myelogenous leukemias and in
leukemia cell lines capable of myeloid differentiation such as K562
and Kg-1a, p93.sup.c-fes expression is either very low or absent.
These findings suggest that p93.sup.c-fes plays a definitive role
during the process of maturation of myeloid cells.
[0044] The K562 leukemia cell line provided a convenient model to
study the function of the human c-fes gene and its role in myeloid
differentiation. This cell line does not express p93.sup.c-fes and
cannot be induced to differentiate along the granulocyte/monocyte
pathway by a variety of differentiating agents (Koeffler et al,
supra). Therefore, this cell line was utilized herein for
transfection with the human c-fes gene in order to identify the
role of c-fes in the differentiation process. It was observed that
K562 cells transfected with the c-fes gene expressed an active
p93.sup.c-fes tyrosine kinase which coincided with the expression
of phenotypic markers indicative of a more differentiated cell type
such as increased phagocytosis, Fc receptors, NBT reduction and
lysozyme activity. . The latter activity in clone WS-1 was
comparable to levels found in mature leukocytes. This clonal cell
line also responded dramatically to the phorbol ester, TPA.
resulting in its morphologic transformation to a macrophage-like
cell and expression of the macrophage surface antigen, Mac-1. Thus,
these results demonstrate that an active c-fes gene is imperative
for the ultimate expression of the mature myeloid phenotype.
[0045] For the purpose of routine assays for the detection of c-fes
mRNA, non-radioactive riboprobes are easily prepared as
follows.
Nonradioactive RNA probe synthesis
[0046] Two procedure can be employed to prepare nonradioactive RNA
probes. The first procedure will utilize a 20 .mu.l reaction
containing 40 mM Tris-HCl, pH 7.5, 6 mM MgCl.sub.2, 2 mM
spermidine, 10 mM NaCl, 10 mM DTT, 40 units RNasin. 0.5 mM ATP,
CTP, and GTP, 0.5 mM
5(N-[N-biotinyl-.epsilon.-aminocaproyl]-3-aminoallyl)-uridine 5'-
triphosphate, and 1.0 .mu.g linearized template DNA. Reactions are
initiated by adding 20 units of T7 RNA polymerase, incubated at
37.degree. C. for 1 h, and terminated by the addition of units of
RQ1 DNase (Promega). Following DNase treatment for 15 min at
37.degree. C., 2 .mu.g of carrier tRNA is added, the reaction
mixture is extracted with phenol/chloroform, and the labeled RNA is
precipitated with ethanol.
[0047] The second method will employ the same reaction mixture
except that 0.5 mM UTP Is substituted for
5-(N-[N-biotinyl-.epsilon.-aminocaproyl]-3-- aminoallyl)-uridine
5'-triphosphate. Following precipitation with ethanol as described
above, the RNA is reacted with Photoprobe Biotin (Vector Labs,
Burlingame, Calif.), a photoactivatable form of biotin which
covalently labels the RNA probe.
[0048] In both instances, the biotinylated RNA probe used in the
RNase protection assay is detected with a
strepavidin-immunoglobulin-alkaline phosphatase conjugate utilizing
NBT and BCIP for color detection (Oncor, Gaithersburg, Md.).
However, other methods of color detection can, of course, also be
employed as will be suggested to one of ordinary skill in the
art.
[0049] A kit for the detection of c-fes mRNA comprises a container
containing the riboprobe of the present invention, either prepared
fresh or cryopreserved.
[0050] A method for the detection of c-fes mRNA in situ or in vitro
comprises reacting a cell or tissue preparation with the
radioactive or non-radioactive riboprobe of the present invention
and determining the degree of hybridization by standard
methodologies well known to one of ordinary skill in the art. Such
methodologies include radiolabeled, immuno-histochemical,
fluorescence measurement and the like.
[0051] Of course, the present invention now makes it possible to
induce myelopoiesis in immature myeloid cells by introducing
genomic c-fes gene in immature myeloid cells in which myeloid
differentiation is desired.
[0052] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended
claims.
1TABLE I Phenotype of parental K562 cells and colonies transfected
with c-fes Differentiation Marker.sup.a HL-60 K562 K562/neo WS-1
WS-5 WS-6 percent positive Phagocytosis 42.sup.b 0 1 65 56 12 Fc
receptors 68.sup.b 52 48 94 85 81 NBT reduction 77.sup.b 1 3 64 43
38 Lysozyme n.d..sup.d 0 0 4.6 3.6 4.5 activity.sup.c Adherence
0.sup.b 0 0 80 80 65 Response to 10.sup.-7 M TPA: Adherence 75 0 0
50 56 n.d. Mac-1 88 2 3 70 73 18 .sup.aThe values for phagocytosis,
NBT reduction and lysozyme activity are the average of duplicate
determinations. Other values represent a single determination.
.sup.bHL-60 cells were treated for 4 days with 1.25% Me.sub.2SO.
.sup.c.mu.g of lysozyme/10.sup.8 cell. Normal leukocytes range from
3.6-8.4. .sup.dn.d., not determined.
* * * * *