J Am Soc Nephrol 9: 1873-1880,. 1998. Expression of. SET, an Inhibitor of Protein. Phosphatase. 2A, in. Renal Development and Wilms'. Tumor. SARA.
J Am Soc Nephrol
SET, Development
Expression
an Inhibitor and Wilms’
of
Renal
of Protein Tumor
Phosphatase
SARA G. CARLSON,* EUDORA ENG,* EUNG-GOOK ELIZABETH J. PERLMAN, TERRY D. COPELAND, BARBARA J. BALLERMANN* *Department
of Medicine, Maryland;
tChungbuk
Pathology,
The
Hopkins
National
The
Abstract.
component
human
gene
event
set
in a case
family
of major
serine/threonine
ulating
cell
sought
to define
cell
proliferation
ing
SET
was
The
human
gene,
of the set-can
set,
event in a case We independently and
set-a in the
39-kD the
Ser9
and
itor family
units,
(3). The
homologs
of
localizes
suggests
sequence
surrounding is phosphorylated
SET
was
at
these
to be a potent 2A
(PP2A)
serine/threonine
subunits
of PPA2,
A,
and
is also
and
PPA2
PPA2
inhib-
mRNA
and
regulates
B, and
encode
splicing
the
containing
and sub-
a 36-kD
different
of some
B subunit
a
isoforms
the B56f3
B
of these target
and B56
SET
participate
The
tyrosyl
during
and
protein
activity phosphatase
of
presence
protein
and
human
Finally, were adult
high
found
in
polycystic
neuronal
of PPA2
in the
differen-
can
also
display
of a recently
char-
(8),
although
in vivo
has
not
yet
demon-
that
inhibition
proliferation
and
activator
phosphatase
rat
expression
process
subunit
devel-
SET
kidney.
cell carcinoma, cell carcinoma.
in cell
renal
that
mature
in the
catalytic
tyrosyl
epithelial for SET
in developing
but not in renal or in transitional
activity
been
strated. Several
lines
of evidence
with
increased
associated
For example, antigen
in SV4O
inhibiting
This
inhibition
kinase
in adipocytes
in
the
G21M
transition
cdc25
results
in
an
of
small
complex,
of PP2A
prevents
kinases
ERK1
an
the tumor
the
cell
activa-
its actions has been
cycle,
(1 3) and inhibition accelerated
Fur-
promoter
inhibitor of PP2A, although for PP2A (I I , 12). PP2A
dephosphorylates acid
the SV4O PP2A
(I-MEKK),
In addition,
is
induc-
proliferation (9). an insulin-stimulated
kinase
(10).
of PP2A tumor
protein
in increased cell found to inhibit
acid is a potent entirely specific
okadaic
in the
its activity.
protein
tor of MEK
PP2A
cells,
B subunit
of the mitogen-activated
mitogen-activated
implicated
transformed
the
and MEK1 , resulting thermore, PPA2 was
okadaic are not
suggest cell
replaces
the inactivation a
findings
greater
of set
may
renal a role
demonstrating
levels
tion.
constitutes that
subunit
Distinct
specific
in cultured
was markedly reduced in starvation, contact inhibi-
differentiated,
tumor, disease
Maryland.
expression by serum by
much
of
Program.
with
in fully
tumor
cell proliferation consists of three
of genes
alternative
diversity.
to the nucleus,
including complex
a variable
C. A number
further
(4).
phosphatases
of cell functions, (5,6). The PPA2 subunit
and
than
Previous
extended
Department
Research
Consistent
than
PPA2
in
of Medicine,
Korea;
Frederick,
kidney
thereby
shown
phosphatase
subunit
were
acterized
to
lines
cells.
or differentiation.
opment
set,
in cells
cell
mRNA quiescent
phosphatase
a
Center,
set
tyrosyl
predominantly
that
endothelial
(1).
encodes
is phosphorylated
and rodent
primary
(7).
rat kidney
mRNA
set-a
human
isoforms
C (3).
SET
a 65-kD
(AUL)
Development
tiation
translocation
in neonatal
The
consensus
residues
catalytic
PPA2
expressed
and
of major
generates
highly
cDNA
ABL-Basic
Wilms’ kidney
as a component
leukemia
rat
encod-
in transformed
identified
which
large number differentiation
levels
by a somatic
two
of
mRNA
Maryland;
tion,
2A,
School
Baltimore,
expression
study
regulation
The
undifferentiated
in reg-
current
in the
produced
SET,
of protein
SET
originally
in culture
kinase
involved The
University Cheongju,
proliferation, cells rendered
SET was shown to phosphatase 2A, a
higher
termed
Recently,
in the
tumorigenesis.
(2).
Ser24
leuke-
expressed
was
were
of cells
protein
of
of nephrogenesis
phosphorylated by
role
isolated
protein
nucleus
the
a
undifferentiated
phosphatases differentiation.
as
a somatic
highly
gene
of acute that
-,
zone
by
Hopkins of Medicine,
and
1998
KIM,t and
School
Research
identified
Recently, of protein
at much
fusion
was
and
and
expressed
acute
set
morphogenesis. specific inhibitor
further
Institutions,
Cancer
produced
The Johns
University
Medical
originally
gene of
zone of nephron be a potent and
renal
was
kidney,
proliferation
of Nephrologv, National
Institute-Frederick
fusion
In the developing
mia.
Johns
Cancer
of the set-can
translocation
Division
Baltimore,
9: 1873-1880,
because
of PP2A
entry
into
by
mitosis
(14,15). It was Received
July 29, 1997. Accepted
Correspondence
cine,The Research
to Dr.
Barbara
March
18, 1998.
J. Ballermann,
Associate
SET Professor
Johns Hopkins University School of Medicine, Room Building, 720 Rutland Avenue, Baltimore, MD 21205.
1046-6673/09010-
1873$03.00/0
Journal of the American Society of Nephrology Copyright 0 1998 by the American Society of Nephrology
of Medi-
954
Ross
originally
in AUL
oncoprotein
postulated
might CAN,
provided a nuclear level of set mRNA gested
that
during
nephron
SET
be or
related
have
morphogenesis
the
tumorigenic
to activation
alternatively,
localization expression may
that
signal during
that
of the
the
SET
putative
).
independent Given
the action
of
sequence
for CAN ( 1 The renal development
functions (2).
action
of
high sugCAN
of PPA2
1874
to inhibit
Journal
of the American
cell-cycle-
and
tumorigenic
potential
terest PPA2
to explore inhibitor
defined
the
cultured
cells
sibility
other
of
inhibitors,
expression
and
kidney may
be
and
and
it was
role of the proliferation.
mRNA
set
in developing
SET
signaling
PPA2
further the potential SET in renal cell and
J Am
of Nephrology
mitogen-induced
of
expression
that
Society
in-
A
//!!
endogenous We further
SET
protein
explored
induced
the
of
gas
in
the pos-
in renal
Soc Nephrol 9: 1873-1880, 1998
.
.
-
‘a
iE-2ikb
tumors.
*1.6kb
Materials Cell
and Methods
Culture
Conditions
Glomerular described plates
previously in RPMI
plemented
and
endothelial
were
and
I 3 j.g/ml
Rat
kidney
CA).
previously
(17)
mented
and
with
g/ml streptomycin. cell line (LLC-PK1), normal
rat
(NRK)
Type
in the medium and rodent
experiments
(National
shown
induced
to differentiate
described
previously
in Figure
into
medium
suppleand
purchased
lA were
IC.
from
kindly
for
3T3-Ll
preadipocytes
analysis,
chloride
samples
1% agarose
membranes
were
stimulation
as
cushions
were (20)
(19). For Northern
electrophoresed
and
(Schleicher
tissues was isolated by followed by centrifuga-
were
on denaturing
transferred
& Schuell,
Inc.,
(RNA) gels
to Nytran
Keene,
NH).
blot
Plus
connylon
Ethidium
bro-
mide staining confirmed RNA integrity and equivalent loading samples. Membranes were hybridized overnight at 65#{176}C in 0.5
sodium
phosphate
buffer,
mm, 7% sodium netetraacetic acid
at 65#{176}C using bovine
serum
IS-mm
washes
SDS,
and
Total
sulfate
(EDTA)
(2 1 ). Two
40 mM with
40 mM
and
sodium
and protein
leupeptin,
S mm
in
SDS
EDTA,
phosphate
followed
buffer
by three
containing
1%
or frozen
Analysis from
tissue
25 p.g/ml
of total protein buffer
and
1. Expression
analysis
tissue in TBS
samples buffer
by homog(50 mM
Tris,
1% TritonX-lOO, 1% deoxyglycol-bis(f3-aminoethyb ether) phenybmethylsulfonyb fluoride, aprotinin.
The
protein
for each sample electrophoresed
mRNA
expression
in cultured
in cultured
RNA (10 pg per sample) from different was examined: human cell lines included noma), HepG2 (liver), MG63 (osteosarcoma), noma).
Rodent
(rat liver), blast). the
cell lines
WEHI-3
Arrows
cultured was
thebial), thebial), tumor),
examined:
and NRK
sample)
position
MDCK
(rat,
and
(dog,
epithelial).
in proliferating from
kidney
incubated
sizes
mesangiai)
cells
of transcripts
Madin-Darby
3T3-Ll
growing
(mouse preadipocytes
ovary),
H35
myo-
recognized
were
kidney,
Total
cells
epi-
was
of set mRNA
RNA
(10 ig per
examined:
to confluency
RKM
and
serum
(P)
grown
in culture for replenishment
preadipocyte)
cells
confluent
then
for 24 h. WEHI-3 5
(Q) or with medium (P),
by
gbomerular endo(human, rhabdoid
analysis
cells.
grown
or 20%
myelomonocyte) cells medium replenishment
cells.
(mouse
canine
(bovine, G40l
quiescent
were
(Q)
serum
hamster
and Ml
(C) Northern
and
Northern
rodent
of set mRNA expression in per sample) from six renal cell
and quiescent
proliferating
in 0.5%
4 (P).
actively
(Chinese
LLC-PKI (pig, epithelial), GEN RKM (rat, glomerular mesangial),
expression (rat
indicate
CHO
(A)
and
human and rodent cell HeLa (cervical carciand A375-C6 (mela-
myelomonocyte),
eDNA. (B) Northern analysis renal cells. Total RNA ( I 0 g
set
lines
included
(mouse
cells.
human
were
days
on
harvested
contact-inhibited
as prea-
(Q), or adipocytes (D) that had been induced to differentiate by hormonal stimulation. Ethidium bromide staining confirmed RNA integrity and equivalent loading of samples (not shown).
dipocytes
concentra-
(BioRad Laboinstructions.
was boiled on
of set
of set mRNA
Total lines
day
using the BioRad Protein Assay CA) according to the manufacturer’s sample
albu-
Figure
(mouse without
Immunoblot was isolated
and
A total of 30 or 60 g
serum
ethylenediami-
at 65#{176}C.
of 0. 1 g of fresh
tion was determined ratories, Hercules,
1 mM
of M
washes were performed buffer containing 0.5%
I mM
pH 7.6, 150 mM NaCl) containing chobate, I mM EDTA, 1 mM ethylene N,N,N’,N’,-tetraacetic acid, 100 M 25 jig/mI
1% bovine and
30-mm phosphate
5% SDS,
Isolation
enization
(SDS),
sodium
albumin,
cellular
pH 8.0, containing
dodecyl
1 mM EDTA
Protein
PQD
PQ
PQ
( I 8).
cesium
RNA
taming
3T3-L1
WEH1-3
RKM
Baltimore, WEH1-3 and
the
by hormonal
protocol. Total RNA from in guanidine isothiocyanate
through
tion
C
the
provided
on Aging,
RNA
adipocytes
RNA
the
main-
RNA Isolation and Analysis Total RNA was isolated from cells in culture using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s homogenization
a
50
from
MD) and were
Institute the
San
as described
cell type.
in Figure
provided
I,
50 g/ml
penicillin,
were
for each
shown
ff
kidney cells, the pig kidney line 0401 (WT-G401), and
(Rockville,
recommended cell lines
1640 U/mI
cells
Collection,
by Dr. Nikki I. Holbrook MD). Dr. Holbrook also 3T3-Ll
50
B
sup-
Corp.,
isolated
in RPM!
as
VA)
(Clonetics
were
serum,
epithelial
Culture
Herndon,
extract
cells
maintained
kidney
gelatin-coated
50 U/mb penicillin,
brain
bovine
bovine on
Inc.,
Madin-Darby canine human tumor cell
kidney
American
serum,
bovine
fetal
from
maintained
mesangial
were
15%
isolated
(Mediatech,
with 20% fetal bovine
Diego,
human
and
medium
streptomycin,
tamed
were
(16)
1640
Treatments
cells
12%
for SDS-
polyacrybamide teins to PVDF
gels as described by Laemmli (22). membranes (Millipore, Burlington,
formed using a BioRad performed by incubation
tank
transfer
of membranes
Transfer of proMA) was per-
system.
lmmunobbotting
in TBS
buffer
containing
was 5%
J Am
Carnation
nonfat
0.05%
dry
Tween-20
4#{176}C, followed ies in fresh
buffer using
(Nestle
three
Co.,
Corp.,
Glendale,
St. Louis,
MO)
at room
5-mm
washes
temperature incubation peroxidase-linked
temperature
in buffer
in fresh antibody,
(Amersham
cDNA,
Anti-SET 3 to
16 and
described was used
SP-l
residues
44
previously at a dilution The same cell nuclear
chemicals
(St.
were
and
antisera
1 :4000. erating
probed San
Northern
polyclonal CA)
Cruz, CA) anti-mouse
analysis,
the
32P-dCTP
ing
(Life
Technologies,
in sample
oligonucleotide
1. Human
Gaithersburg,
probe
corresponding
sample
profiles
blots to
to set-a
were 185
b
rats were MA.
purchased
After
day
from
Charles
River
under
anesthesia,
exsanguination
from adult animals,
of Nephrology classifications
mRNA
set
I -day-old
1 7. Discarded
(Baltimore, for human
rodent
was
control
Abundance
To assess
Labelfor
rehybridized RNA
Com-
frozen
Labora-
neonates, human
kid-
and from
tissue
samples
MD). Case numbers, samples are provided
patient, and in Table 1.
Results
For
(2)
DNA To
Sample
a
Cruz
of I :4000.
Ml)).
Reagent deoxynucleotidyb
Laborato(Santa
Primers
Certified terminal
Technologies).
rats at gestational
Division histologic
blots
1: 3000. Horseradish secondary antibod-
eDNA
the Random
or transfer,
loading
rat
as
Additional
used at a dilution
length
using
fetal
residues
(Zymed
(Life
radiolabeled
1875
of adult and fetal normal kidney and tumors were obtained from the Johns Hopkins University Department of Surgical Pathology (Baltimore, MD). For renal cell carcinomas and Wilms’ tumors, normal appearing tissue from the same kidney was used as control. Frozen human tissue samples of normal and autosomal-dominant polycystic kidney disease (ADPKD) kidneys were kindly provided by Dr. Gregory 0. Germino, Johns Hopkins University School of Medicine,
purified SP- 1 at a dilution of
to WT-l
at dilutions of or anti-rabbit
full
with
variation
to PAX-2
using
Texas)
tories, Wilmington, neys were removed
IL).
respectively,
1 :4000.
Midland
Midland,
Tumors
and
pany,
Tissue Samples Sprague-Dawley
monoclonal anti-proliffrom Sigma Immuno-
antibody
were
secondary washes in
against
protein,
of
antibody
or polyclonal
radiobabeled System
SET
at a dilution
Life Science)
blot
generated
Development
OAAGACGATCAGATACCOT-3’, transferase
Probes
were
to 56 of the
MO)
Santa conjugated
ies (Amersham
SP-2
in Renal
milk,
Heights,
Oligonucleotide and
at
was performed and exposure to
Arlington
blots were reprobed with antigen (PCNA) antibody
Francisco,
Biotechnology; peroxidase-
of signals System
Science,
and
with antibod-
not containing
(23). For Western blots, affinity of I : 10,000 and SP-2 was used
Louis,
with
Life
CA) overnight
buffer con:aining and three 5-mm
not containing milk. Visualization the ECL Western Blotting Analysis
Antibodies,
Table
Food
Chemical
by a 2-h incubation
Hyperfilm-ECL
ries;
milk
(Sigma
buffer,
2-h room horseradish
an
SET Expression
Soc Nephrol 9: 1873-1880, 1998
transformed
using
total
RNA
rodent
cell
lines
(cervical
with
and
(5’-OOTTC-
in Cultured cell
isolated
carcinoma),
A375-C6
lines,
Northern
from
(Figure
lA). HepG2
(melanoma).
Cells
of set mRNA
the expression
in diverse analysis
four
human
Human
cell
(liver), Rodent
cell lines
MG63 cell
human
was
lines
lines
and
included included
CHO
Age
RCC- 1
Renal
NR-l RCC-2
Normal kidney sample Renal cell carcinoma,
NR-2
Normal
RCC-3
Renal
NR-3
Normal
RCC-4
Renal
TCC-l NC-i
Ureter, Normal
kidney
sample
WT- 1
Wilms’
tumor,
predominantly
WN-l
Normal
kidney
sample
WT-2
Wilms’
tumor,
predominantly
WN-2
Normal
kidney
sample
WT-3
Wilms’
tumor,
predominantly
WN-3
Normal
kidney
sample
NP-l
Normal
kidney
with
NP-2
Normal
kidney
PKD-l
Polycystic
kidney,
end-stage
PKD-2
Polycystic
kidney,
end-stage
PKD-3 PKD-4
Pobycystic
kidney,
end-stage
Polycystic
kidney,
end-stage
Male
35 yr
NF-l
Normal
kidney,
fetal
Female
21
wkb
NF-2
Normal
kidney,
fetal
Female
22
wk”
Fuhrman Gestationab
Nuclear Age.
Grade.
cell carcinoma,
kidney
cell
papillary,
sample
from
carcinoma,
kidney
clear
sample
patient
and
from
cell carcinoma, transitional
Grade
IIIIIV
from patient RCC-l clear cell type, Grade
from from from microcysts
59 yr
Male
70 yr
Male
68 yr
Male
44 yr
Male
78 yr
Male
3 yr
Male
5 yr
Female
I yr
RCC-2
granular
cell type,
carcinoma, from
Male
cell
type,
Grade
IIIJIV
RCC-3
granular cell
II/IV
Grade
patient
WT-2
blastemal patient present
II
WT-l
epithelial patient
II
TCC-l
blastemab patient
Grade
four HeLa
(osteosarcoma),
Gender
Descriptio&’
and
performed
WT-3 Male
48
yr
Male
67
yr
Female
60
yr
Female
48
yr
Unknown
I 876
Journal
of the American
(Chinese hamster myebomonocyte),
Society
of Nephrology
ovary), H35 (rat liver), and M 1 (mouse myoblast).
J Am
WEHI-3 mRNA
set
(mouse was
cells were differentiation
growth arrested by either contact inhibition or by with hormone treatment (18). As shown in
expressed as three transcripts of approximately I .6, 2.2, and 3.0 kb in rodent cell lines, whereas only the 2.2- and 3.0-kb transcripts were observed in the human cell lines. In nonhuman
cent.
Thus,
cell
sion
is much
lines derived from the kidney (Figure IB), there set mRNA expression in Madin-Darby canine kidney cells, and no expression was observed in glomerular cells, LLC-PK 1 , or NRK epitheliab was strongly expressed in rat kidney
endothelial set mRNA and
in the human
mRNA proliferative
expression status of
set
formed
using
quiescent gial
total
cells.
cells
tumor-derived
and
line
in cultured cells the cells, Northern
RNA
isolated
Quiescence WEH-I
cell
from
was
cells
was weak epithelial capillary
Kidney
To determine
levels
starvation.
blot
analysis
RNA
than
mesan-
of expression these same
Mouse
3T3-Li
antisera
or
Protein
mRNA, than
was
in Developing
performed
from
2A).
in developing
mRNA
set rat
to examine
rat kidneys kidney,
against
different
with
the
of the
stages
was
lower
highest
SET protein using two
regions
mRNA
set
at different
abundance
in fetal (day 17) kidney. tissues were then examined
directed
the level of expresin quiescent cells.
Expression
isolated
(Figure
levels
levels in separate
SET
protein,
C
B
A
SET
of development
kidney
proliferating
and
in total
in adult
mRNA expression was obthe cells were made quies-
in cells expressing set greater in proliferating
Northern
with respect to the analysis was perin rat
IC, downregulation of set in all three cell types when
set mRNA
cells. However, mesangial cells .
rapidly
induced
by serum
G40l
Figure served
Soc Nephrol 9: 1873-1880. 1998
‘iii’!
ii, 66kD*-
!#{149}
.-3.Okb
sp.1 sP.1 3kDa
-2.2kb E-i.6
46kD*-
:
3OkDs-
kb
46kDsu
46kDa...
-
SP.2
SP2
.
3OkDs-
46kl.
-
PCNA
PCNA
kD*
e
--
3OkDa66kD*-
66kD.-
PAX.2 PAX.2
kDa
3OkDs6kDSp 66kDa-
wr.1
wr#{149}i kDa-
3OkDaFigure 2. Expression of set mRNA and SET protein in developing rat kidney was examined for set mRNA expression. The same blot
kidney. was
probed
(A) Total
RNA
for 185 rRNA
(30 p.g per sample) to control
from
for sample
fetal,
loading
neonatal,
and adult
and transfer.
Protein
extracts (60 g, upper three panels; 30 j.g, lower two panels) from (B) fetal, neonatal, and adult rat kidneys and (C) fetal and adult human kidneys were subjected to immunoblot analysis using SP-l or SP-2 antiserum (described in Materials and Methods section). The same blots were reprohed with monoclonal anti-proliferating cell nuclear antigen (anti-PCNA) antibody. Additional blots were probed with antibodies to
PAX-2 tumor pertinent
(fourth patients; samples
panels)
or WT-l
NP- I and
NR-2,
(from
the same
(bottom normal
blot)
panels). adult are
NF-l
kidney
placed
and NF-2, (see
side
Table
by side
normal I ). Original
in the final
fetal
kidney;
blots figures.
WN-l
contained
and WN-3, numerous
normal
samples,
fetal
kidney
but for ease
from
Wilms’
of comparison,
J Am Soc Nephrol
designated
SP-
erated
against
9: 1873-1880,
1998
1 and
SP-l
peptides
(SP-2)
of SET,
44-56
residues
Figure
SP-2.
synthetic
and
39-kD
band
SP-2
antisera
to residues
3-16
respectively
2B, the affinity-purified
a distinct
SET Expression
SP-l
on
As
antibody
blots
of
gen-
(SP-
(23).
SET
Western
were
1) and
shown
fetal
1 7)
band
and
adult
SET
expression
kidney
and
neonatal
rat kidneys.
antibody
In human
recognized
predominant
and
protein
human
kidney
SET
compared
reprobed
with
status
of
adult
PCNA
these
with
the findings was
kidney.
antibody
tissues
Both
2,
B
protein
abundance
is significantly
ment than in mature To compare the
kidney
has
been
reported
rat
PCNA
the C).
greater
Figure
were protein
with
adult, mRNA
set
during
develop-
stages
of
of
SET
with
blots were probed PAX-2 and WT-
previously
subjected blot
clear
antigen
for
mice
renal
with two known 1 Similar to what
neonatal
kidney,
(Figure WT-
2B, I was
bottom
(24),
expression
parallels
kidneys
with
Because
renal in
tissue
In human adult
of PAX-2
but
expression
Expression epithelial
ADPKD,
from
in
four
different
3). In tissue
kidney,
SET
abundant
than
abundance neys when SET
was also compared
Protein
of protein carcinoma
in Figure
samples
was
renal
samples. cell
in
kidney of rat
I.
with
antibody
cells
continue
SET
protein
was
frorr
to proin
renal
ADPKD
was
end-stage
similar
in normal
was
4, the SET PCNA
carcinoma
PKD-2,
kidney.
Protein
or pobycystic
kidneys
using SP- 1 antiserum. The anti-proliferating cell nu-
NP- I and
and
PKD-4,
NP-2, normal polycystic
kidney kidney
1).
or
kidney.
human
slightly
less
PCNA
protein
in samples of ADPKD human renal tissue. Cell
and
used
for
that
protein
kid-
the
pediatric
expression sample,
in was
and
in all five
cell As
of set mRNA
when
were
fetal
of
same
patients.
the
using
the SP- 1 SET the
normal in
kidney.
tumors,
lated
in Wilms’
These
controls.
Hence,
SET
were
kidney
blot
analysis
was
PCNA
in contrast
expression
SA,
tumors
that
to the
appears
performed
in Figure
than
three
samples
SET
compared
expression
tumors
a
resembles
from
normal
Wilms’
SET
tumor,
isolated
and
in the
Wilms’
in human
kidney,
of Wilms’
and as shown
elevated
the
also
was
observed
findings
in
with
to be markedly
other
upregu-
tumor.
samples
compare
Western
kidney
greater
renal
tumor
antibody,
highly
adult
histologically
extracts
Wilms’
from
was
that
Protein
cases
was elevated with
in samples
neoplasm
kidney.
expression
compared
examined
Tumor
‘
were
also
the expression
probed
of SET
with
PAX-2
in Wilms’
and
tumors
WT-l
with
to
devel-
opmental markers. As shown in Figure 5 (third panel), similar to that seen in developing kidneys (Figure 2, B and C) several bands
are
kidney
2C), pies
detected
by the
However, in each seen
in all samples
no specific samples.
PAX-2
antibody
in Wilms’
tumor
at least one PAX-2 isoform is strongly sample. A faint, higher molecular weight
As
appears
PAX-2 with
expression of WT-l appear to be variable
to be a nonspecific
bands
human
are seen
fetal
in normal (Figure
5,
and
in normal
adult
tissues
band; mature (Figure
and Wilms’ tumors bottom panel).
sam-
tumor
four
normal
slightly
higher
in one
greater
in the
significantly
in Wilms
expression
embryonal
early
protein
analysis
Expression tissue
levels
samples. expressed
Transitional
immunoblot
abundance
observed
cell carcinoma sample compared with normal kidThus, in renal cell carcinoma, and possibly also in carcinoma, SET expression is not increased.
kidney
much normal
end-stage
extracts
fetal
with
Kidney
in Renal
to
PKD-3,
Protein
however,
similar
antibody.
,
Table
Because
separate
expression
extracts from four separate samples of renal and in one sample of transitional cell carcinoma.
shown tissue
in developing
of
not elevated with normal
Expression
Cell Carcinoma The SP- 1 SET
declines
stromal
protein
observed
not
expression Thus,
Polycystic
patients
(Figure that
samples, kidneys.
expression
examined ADPKD
in adult
of Wi’-
and
the
the opposite
expressed
and
increasing
(see
analysis monoclonal
with
(anti-PCNA) 1
in polycystic kidneys
of the
for WT- 1 , which was only slightly detectable
strongly
in fetal that
Protein
liferate
was
C). In rat kidneys,
seen was
panel).
variable
of SET
SET
was kidney,
but
protein normal
to immunobbot
transitional ney samples. transitional
protein
2, B and
expression in fetal
SET
human
reprobed
PKD-
samples
of
from
was
samples;
(fourth
pattern of detectable
g)
same
SET
Figure
(60
were
46- and 48-kD PAX-2 isoforms is highest in fetal rat and human kidney, with greatly diminished levels in neonatal rat kidney and undetectable levels in adult rat and human kidney panel,
3. Expression
extracts
proliferative
PCNA
3OkDa-
fetal
tissue.
expression
development, additional markers of development,
the
in human
abundance was greater in developing, compared renal tissue. Thus, in both rat and human kidney, and
also
in both
immunobbots
and
46kDa’-
SET
in developing
greater
to determine
(Figure
was
antibody
kDs-
of
the SP-l
This
SP-2
sp-1
(day
in extracts
2C),
1877
kidney
in fetal
detected
protein.
the
expression
with
highest
(Figure
by
Consistent protein
not
the 39-kD
recognized
kidney.
rat kidney,
was
kidney
only
band
was
and Tumors
46kDs-.
extracts, which has been previously shown to correspond to the SET protein (23). The SP-2 SET antibody also recognized the 39-kD band, and a second band at approximately 45 kD. The
39-kD
Development
i//il
I
in
recognized
rat
in Renal
kidney
Discussion The current SET,
study
an endogenous
sought
to define
inhibitor
further
of PPA2,
a potential
in the
role
regulation
for
of
1878
of the
Journal
renal
cell
levels
of
rodent studied,
cell
American
proliferation mRNA
set
Society
J Am
of Nephrology
and
tumorigenesis.
expressed
in
We
found
transformed
high
human
and
lines, but in the renal epitheliab and endothelial set mRNA expression was low or undetectable. findings that set mRNA is highly expressed during
vious
for
of set mRNA
and
not in renal in transitional This sion
SET
cell
focused cells.
cDNA
sequence. probably
set-a
end,
otherwise
mRNA
thelial these
set
was lines,
the
rhabdoid
tumor
of Wilms’ gial
were
mRNA. mouse
that
little
and
at passages
Transformed
rodent
as
three two
probe
PAX-2 3OkDa-
in human
previously
shown
to
66kDa-
of 3’ untranslated
WT.1
>
of
3OkD.
transformed
epi-
primary
not
augmented
, formerly
when to be
rat mesanabundant
cell
expressed
the
believed
primary
human
In
In contrast,
10 expressed
also
(1) found
et al.
or no set mRNA.
cultured
and
pre-adipocytes
5’ but are
only
of growth.
line G40l
(25),
66kDa-
at the
whereas
cultured
was
phase
cell
origin
a
with
expressed
Lindern
express
examined
3T3-Ll
von
expression
derived
was
splicing,
in a number
suggest
logarithmic
tumor
cells
3OkDa-
(1).
and
cells
mRNA
in the
PCNA
expres-
studies
diverge
lengths
ubiquitous
observations
set
were
levels
hybridizes
cDNA
set
size
et al. (23)
these
also
cell
with
to be
for
mRNA
in transcript
endothelial
cells,
cells
or
of alternative
rodent
hybridized
Adachi
and
but
disease
sequences
-
be due to the presence of varying sequences in the set mRNA species Whereas
tumor,
kidney
which
set-/3,
(2).
in
Differences
SET expression cell lines, our
in Wilms’
set mRNA
used
The set-a and as a consequence
species
high
polycystic
probe
encoding
transcripts
cells.
46kDa-
observed
on characterizing The
identical
distinct
adult
3OkDs-
carcinoma.
first
in cultured
full-length
expression
carcinoma, cell
study
protein
we
-
sP.1
porting
in development,
46kDs
lines, high
set
as well levels
as
of set
Figure
5.
extracts lower tissues
#{149})
1
“
(1
4’
from
SP4
the same
Wilms’
tumor
WT-
PCNA
crating
cell
nuclear
NR-3,
NC-!,
and
RCC-3, cell
and RCC-4,
carcinoma
from
extracts
in renal
Protein extracts (60 j.g) subjected to immunoblot
blot was reprobed antigen
NC-2,
(see
Table
monoclonal
samples; samples:
1).
cell
and
tran-
from human normal analysis using SP- I
antibody.
kidney
renal cell carcinoma ureter
with
(anti-PCNA) normal
subjected
panel).
Table
using
Original
by side
blots
pertinent
in the
final
Protein
to immunobbot
to
antibody.
samples
blots
subjected
(third
numerous
(from
WN-l, WT-3,
and
were
PAX-2
contained
analysis
with monoclonal
, WT-2,
1). Additional antibody
panel)
or
samples,
the same
but
blot)
are
figures.
suggesting
That
anti-prolifNR-l,
RCC-l, TCC-l,
NR-2, RCC-2,
transitional
set
is
preferentially
in vivo
expression cell-derived
was much greater in Wilms’ renal cell carcinomas.
render mRNA
is suggested
cells
and
by our
WEH1-3
in
expression findings
tumor
certain
of set
may
that
SET
protein
than
in epithelial
appear to relate, in part, to the in which set is expressed. In cells,
withdrawal
of serum
to
the cells quiescent resulted in a marked reduction of set expression, consistent with a robe of SET in cell proGrowth
arrest
or differentiation
PCNA activity.
in Wilms’ SET, cell
In
cells.
was
used
experiments
in tissues tumor
with
either
resulted
expressing
and in developing participates
Nevertheless,
contact
in reduced
In tissues,
Western
to assess shown
also
like PCNA, replication.
associated similarly
in 3T3-Ll (26,27)
greatest
to
cell-specific
in set expression state of the cells
expression
that
relatively
expressed
cell types. also occur
tion
The same
(see
of comparison, side
liferation.
3OkIa-
protein
were
tumors.
or 30 g of protein, and normal kidney
The same blots were reprobed
analysis
I (bottom
mesangial
46kD.-
of SET
patients
samples
to immunoblot
Differences proliferative
cell carcinoma. or tumors were
in Wilms’ panels; tumors
C’
,
3OkDs
4. Expression
protein
anti-proliferating cell nuclear antigen (anti-PCNA) WN-2, and WN-3, normal kidney samples; WT-l
mRNA,
Figure
SET
4T4)
46kDs-
sitional kidneys
of
SP- I antiserum.
using
placed w#{176}v fV
Expression
(60 j.g of protein, upper two two panels) from three Wilms’
for ease
antiserum.
4?
4
renal
were extended by demonstration of much greater expression in developing rat and human kidney differentiated, mature kidney. Finally, also supSET
,Ft
cells Pre-
development SET protein than in fully a role
Soc Nephrol 9: 1873-1880, 1998
the
here,
blot
level PCNA
high
Thus,
namely
it is possible
events the
for was
of SET,
in regulating
because
analysis
of proliferative expression
levels
kidney.
inhibimRNA
set
that
epithelial
lead cell
J Am
Soc Nephrol 9: 1873-1880. 1998
lines
and
when
the
they
growth, cell
endothelial
were
it seems
in the
rat
isoforms
pression
fetal
of inhibitor-i
detected
in the
in the
cortical
of nephron current
of
human
fetal,
and
mature,
differentiated
observations oping
(2), of
meobox
gene,
(!GF-!I)
(29-32).
nancy
and epithelial
in reference
patients
with
mutations
WAGR
(Wilms’
same
locus,
hence
tumor
suppressor
radic
cases
mann
and
netic defect leading to remainder
tumor
involves alterations overexpression of the
is unknown. beads
to the
IGF-!I,
and itself
(35-37).
of SET
in Wilms’
gene
component
set
similar
to that
overexpressed be
genetic
a component
mutations,
is
tumor
is located
9q24,
cluster
cases of Wilms’ restricted pattern in Wibms’
tumors,
we postulate
same
signaling
!GF-Ii,
events
that
I.
of AUL The
cell
expression
may
also
previous
of
controlling
cell
original
set
and SET
study
participate
in Wilms’
prolifera-
with
that
in this
in
study, analysis to determine
The
between
tumor
elevated
localization
in
suggested
findings
proliferation
dis-
the putative might
have
suggest
that
in the dysregu-
tumor.
was supported by National Institutes in part, by the National Cancer Institute,
and Human Services the recipient of National was
the
recipient The
that
under
work
contract
Research
of
with
Service
National
during
of Health Grant Department of
ABL.
5G.
Carlson
Award
DK09388.
Service
Award
Research
was done
Established provided
SET that
Lindem
M, van G: can,
the tenure
E. DK
of an American
Investigator Award to B.J. expert technical assistance.
leukemogenesis,
may
genes:
Ballermann.
by fusion
with
A,
myeloid
of its 3’ half Mo!
Ce!!
to Bio!
1992
Adachi
Y, Pavlakis
phosphorylation the
A, Hagemeijer
associated
of the set gene.
3.
GN, sites
transbocation
Copeland
of SET,
BJ: Spatially restricted expresrat kidney. Atz J Physio/ 266 35]: F155-F16l , 1994 TD:
a nuclear
breakpoint
Identification
of in vivo
phosphoprotein
encoded
undifferentiated
leuke-
in acute
mia. FEBS 4.
is
Lett 340: 231-235, 1994 Li M, Makkinje A, Damuni Z: The myeloid
protein
could convert
be activated
Characterization
Kim
by
I, Raap
oncogene
E-G, Choi ME, Ballermann sion of set mRNA in developing [Renal Fluid Electrolyte Physiol
and
SET
5, Wiegant
2.
the a
Baal
a putative
12: 3346-3355,
is not
that
von
Grosveld
involve-
in familial
and
of cell
different
tumor. Nevertheless, given that of set mRNA expression (2) is and
SET
participates
development.
translocation
potential.
not
References
ge-
binding
therefore,
involved
SET
in a case
Heart Association Veronica Fergusson
tumor
it is of note and,
can
are
ac-
normal
in Wilms’
levels
of this study together (4) and subcellular
nephron
of a somatic
5T32DK07712.
of PAX-2,
a possible
PAX-8,
the
are
1 lpl5, In the
DNA
of PAX-2,
of
Eng
WT1
the
locus (34).
repression
formation, at
that
during
process
SET
are
in the
deletions,
of Wilms’
In considering
Health
of spo-
tumor,
a zinc-finger
suggest
This research DK50764 and,
Beckwith-Wiede-
Wilms’
of the two-hit
in of
mutations
or mutations
although
a role
abnormality
Acknowledgments
was
allele
10%
mechanism
transcriptional
of the contiguous
some sporadic the spatially
for
in
at chro-
of the
at the contiguous the IGF-II gene
WT1
that
human
risk
(2),
lation
germline
WT1
with
SET
increased
surrounding
actiLons
involve
In patients
at increased
protein ment
.
an
polycystin
deletions
in ADPKD. In summary, the results work defining the function
tumorigenic
tumor
deletions the
represent
in which
somatic
However,
oncogene
malig-
located
normal
to
in culture.
to play
of nephro-
to
Approximately
also
of WT1
of cases,
formation
the
product.
of Wibms’
syndrome,
large
(38),
proliferation
genitourinary
gene,
or eliminating
II
stromal,
is due
suppressor
containing
gene
splicing
aniridia,
held
in
transitional
cells
tion
are char-
Wilms’
usually
the tumors,
reducing
or aberrant
tumor,
tumor
Within
found,
Familial
retardation)
1 lpl3.
tumors blastemal,
widely
epithelial
not appear
of
metanephric
of the zone
kidney.
in the WT1
mosome
Wiims’
reminiscent
developing
abnormalities,
the
33). containing
does
development
covery
from
of renal
was
expression
and
of
tumor.
to arise
to that
expression
cells
the end-stage ADPKD tissues examined in this of early ADPKD tissues would be necessary whether SET plays a role in the early epithelial
been
renal
epithelial
in
ho-
also
renal
reminiscent
factor
have
is a common
thought
cell elements
in the
another
from
in
finding
cell carcinoma, set
formation.
(29,30).
growth
!GF-Il
in Wilms’
heterogeneous,
genesis
and
(nephroblastoma)
(reviewed
acteristically
for
as insulin-like
expressed
children,
expression
Thus,
tumor
greater
this
allele
previous
PAX-2
transitional
tissue.
SET
now
tissue,
renal
similar
elevated
significantly
normal
in
The
pattern
was
of set may
in Wibms’
cell carcinoma,
cystic
nephro-
transcription
transcription
participate
by
in devel-
protein
gene
observed
PAX-8,
our
observed
that
also
as well
PAX-2,
in young
tract
is
PAX-8,
tumor
blastema
with
in renal
is quite
nephron
than
expression
with
I 879
during
IGF-II
companied
expression kidney
as the previously here
(2).
expression
and
that
may
protein
derived
ADPKD,
to our
kidney
neonatal
of set mRNA
expression
to be highly
Wilms’
and
to that of the homeobox
pattern
shown
rat
cells
and
compared
ADPKD
Also,
in the region
protein
in
carcinomas
cells
is similar
abundance
or
ex-
stem
SET
cells
PAX-2
is possible
by WT1
tumors
not the case
of PP- 1,
mesenchymal
SET
we demonstrate
mRNA
inhibitor
are consistent
levels
As well
SET is similar
addition,
observation
fetal
kidney
rat kidney.
In
greater
rat
of high
mRNA
kidney.
in developing
in
Wilms’
the
distribution of PP- 1 mRNA uniform expression of PP2-A
This
much
1 (PP-i)
hybridization
, it
by WT1
Tumors
and
epithelial
because
be regulated
formation. Whereas
expression
Development
to early
Furthermore,
is repressed also
phosphatase
in situ
set mRNA
morphogenesis
of
in Renal
blastema
genesis.
of cell
lineages. examined
protein
metanephric
of greater
findings
(28)
metanephric
even
phase
in the regulation
cell
Using
region.
findings
all,
et a!.
rat
mRNA
set
(I- 1), an endogenous
only
outer
previous
This
not
kidney.
throughout
express logarithmic
is involved
but
found changes in the in fetal rat kidney and
isoforms
set
that SET
in many,
developing
not the
work by Svennilson and the serine/threonine
authors
was
did
during
likely
proliferation
Recent of PP2-A
cells
examined
SET Expression
5.
SET
is a potent
inhibitor
of
protein
J Bio! Chem 271: 1 1059-1 1062, 1996 Mumby MC, Walter 0: Protein serine/threonine
leukemia-associated phosphatase phosphatases:
2A.
1880
Journal
Structure,
of the American
regulation,
73: 673-699,
6.
Wera
McCright
BA:
protein
phosphatase
Cayla
2A
5, Virshup
(PP2A)
cytoplasm.
Hoof
685,
B56 family
subunits
that target (‘hem
Sontag
E, Fedorov
Mumby
M: The
protein
phosphatase cell
24.
M, Waelkens
E, Vandekerckhove
cloning, expresthat activates the phosphatase 2A. J Bio!
C.
of SV4O
2A stimulates (‘el!
proliferation.
with
the MAP
kinase
pathway
and
75:
from
M,
antigen
887-897,
P. Shirazi
distinct
Cobb
tumor 1993
TAJ: Insulin activates a novel protein kinase kinase kinase that is
D,
small
P. Haystead
and
Gregory
Robbins
CMM,
A, Fadden
C, Dent
(‘hem
269:
acid,
A: Inhibitory
on protein
effect
of a marine-sponge
Biochem
phosphatases.
J 256:
toxin, 283-290,
TAJ,
Sim
ATR,
Carling
D, Honnor
RC,
Tsukitani
I 3.
78-81,
Clarke tion
cdc25-C
regulation 14.
I, Draetta
by
during
0, Karsenti
a type-2A
protein
the cell cycle
Ce!!4: 397-411, Goris I, Hermann
15.
Felix
E: DephosphorylaSpecific
egg extracts.
Mo! Bio/
in Xenopus P. Ozon
R, Merlevede
Cohen
P. Karsenti
by a type
2A
!aevis
E: Cdc2Hl
kinase
in the Xenopus
from
the effects
I 7.
Choi
morphogenesis
Ballermann
and associated forming growth 21150, Carlson
Regulation
Ce!!
Bio!
LG,
from
Bio!ogv,
Dibner
Church
Sei USA
GM,
Battey
cells.
edited
Gilbert
mutant trans270: 21 144-
M, Holbrook
by glucose
gaddl53
NJ:
de36.
1993
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MD,
Co.,
34.
AE, MacDonald RJ, Rutter WI: Isolation ribonucleic acid from sources enriched in
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1st ed.,
Publishing
gene
Bernier
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Biochemistr-v
RNA
ID,
IF:
by Davis Inc., W:
81: 1991-1995,
LG,
and
New
analysis
of
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Elsevier
Genomic
1984
sequencing.
37.
in human
Nat!
Bradbury
EM,
nuclear
antigen
complexes.
Lehnert
G, Laestadius
A: Evidence
during
early
P. Re GO,
fetal
kidney
N, Jung
BE:
CDK/ Gi 220:
Res
iT,
A, da Cruz
e Silva
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