From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Antibody
Identifies
Against
Vimentin
J. Laster,
By Andrew
T Lymphoblastic
and Keratin
Thomas
J. Palker,
Leukemia-Associated
Intermediate
Filaments
Elizabeth
and
3-40
defines
on T lymphoblastic
Antibody
present
absent
on
normal
62:852.
proteins
1 983).
treated
with
35-
to
40-kd
leukemia
hematopoietic
Using
and
a
analysis
et
of
immunofluorescence
various
cytoskeletal
is
al. Blood
cytoskeletal
of
inhibitors.
that
cell
lines
in this
report
we have
demonstrated
that
antibody
3-40 also identifies
vimentin
intermediate
filaments
(IMF)
within
the
HSB-2
(T-Aii)
cell line as well as in normal
thymocytes.
peripheral blood
mononuclear
cells. and human
and rodent
fibro-
I
1983, Naito
et ab’ reported
the production
ofthe
munine
monocbonal
antibody
3-40,
produced
by immunizing
mice with the human
T lymphoblastic
leukemia
(T-ALL)
N
cell
line
MOLT-4.
By cytotoxicity,
immunofluorescence
strated
surface
with
surface
(IF)
reactivity
molecules
of normal
ing thymocytes.’
Reactivity
some non-T,
non-B
ALL
(AML)
cells
as well
malignant
tissues,
noma,
and ovarian
pitated
a 35,000-
labeled
MOLT-4
3-40
antibody
surface
While
cell
ofselect
thymus,
pancreatic
Antibody
3-40
to 40,000-dalton
of malignant
studying
antigen
includ-
surface
of
leukemia
normal
and
islets, melaimmunoprecifrom
surface-
et al’ therefore
postulated
that
an antigen
expressed
on the
transformed
T cells.
normal
thymocyte
and
in our
cells,
noted on the
myelogenous
sections
indirect
3-40
demoncell lines but not
hematopoietic
as in frozen
Naito
define
and
antibody
T-ALL
was also
and acute
including
carcinoma.
cells.
might
maturation
absorption,
assays,
with most
laboratory,
we
we show
intermediate
39,000-dalton
fibroblasts.
that
antibody
filaments
IMF-associated
noted
that
epitheliab
antibody
MATERIALS
3-40 binds to keratin
(IMF),
as well as
protein
AND
in epithebial
cells
and
to a
and
From the Department
of Medicine.
Division
of Rheumatology
and Immunology,
and the Department
ofMicrobiology
and Immunology. Duke University
School ofMedicine,
Durham,
NC, and the
Sloan-Kettering
Instituzefor
Cancer Research,
New York.
Supported
by grants
No. CA28936
and K0400695
from
the
Cancer
Institute.
Submitted
Jan 14, 1985; accepted
March 15, 1985.
Address
reprint requests to Dr Andrew J. Laster, Box 3258,
University.Medical
Center, Durham,
NC 27710.
©
I 985
by Grune
& Stratton,
0006-4971/85/6603-0026$03.00/0
642
Inc.
Duke
Ho,
lines.
was
Kazuyuki
Naito,
epithelial
Cross-reactivity
demonstrated
cell
lines.
fibroblast
cell
selectively
during
e
1985
in
human
we have shown
that
Iular IMF-associated
Bo
DuPont,
lines.
This
and
and
skin.
IMF-associated
on
the
transformation.
& Stratton,
and
keratin
human
subrodent
In addition.
defined
a 39-kd
intracelin HSB-2
cells.
epithelial
and
by Grune
carcinoma,
several
3-40
malignant
laryngeal
with
both
thymus.
antibody
protein
expressed
protein
surface
of
human
cell
line.
may
T
be
cells
Inc.
HSB-2
is a T-ALL
Thymocytes
were obtained
from fresh thymus by mechanical
dissociation,
collected in RPMI
1640 medium,
and isolated
by Hypaque-Ficoll
density
centnifugation
as previously
described.2
Peripheral
blood
mononuclear
cells were isolated by Hypaque-Ficoll
centnifugation
as
described.2
Human
skin was obtained
at postmortem
examination.
Cell lines were passed as previously
described.3
All cell lines were
maintained
in 5% CO2 in air, 37 #{176}C
in RPMI
1640 media (MA
Bioproducts,
Walkensville,
Md)
supplemented
to 15%
(vol/vol)
with
heat-inactivated
fetal calf serum (HIFCS).
Monocbonal
antibodies
AE-l and AE-3 against human keratins4’5
were the generous
gift of Dr Tung-Tien
Sun (New York University
School
of Medicine,
New York).
Antibody
43fE8
was kindly
provided
by Dr Arthur
Vogel (University
of Washington,
Seattle)
and reacts with vimentin and glial fibnillary acidic protein.6 A2BS is
an 1gM monoclonal
antibody
that recognizes
complex gangliosides
on endocrine
thymic epithelial
cells.2 Rabbit polyclonal
antisera
to
and
actin
were
purchased
from
Miles
Laboratories,
Inc.
Ill. Rabbit
antithymosin
a, was the gift of Dr Allan
Goldstein
(George
Washington
University,
Washington,
DC) and
was absorbed
and used as described.2
P3X63-Ag8,
a munine
myeloma
cell line that secretes
IgG, and DMH
1.3.3.5, an 1gM
antibody
to human Ia-like antigen (kindly provided
by Dr Bernard
Amos,
Duke University)
were used as immunoglobulin
isotype
controls.
Immunofluorescence
assays.
Indirect
and direct IF staining
of
viable cells in suspension
and 4-z acetone-fixed
tissue sections were
performed
as previously
described
in detail.7 A saturating
titer of
Naperville,
monoclonal
METHODS
7,s,L,,s and cell lines.
Five cell lines were used to examine
in
detail the reactivity
pattern of antibody
3-40. Rat thymic epithelial
cells (1T26R2I)
and nat fibnoblasts
(IT4SR91)
were kindly provided
by Dr Tsunetoshi
Itoh (Dana-Farber
Cancer Institute,
Boston), and
human
foreskin
fibnoblasts,
by Dr Kay Singer (Duke
University,
Dunham, NC). HEp-2 is an epithelial
cell line derived from a human
National
cell
classes
tubulin
thymic
3-40 bound
not only to the surface
of T-ALL
cells, but also
reacted
in indirect
IF assays
with the cytoskebeton
of T-ALL
cells, normal
thymocytes,
epithebiab
cells, and fibroblasts.
In
this report
vimentin
Winifred
(3-40)
Cells
F. Haynes
blast
antigen
cells
(Naito
cells
immunoblot
indirect
surface
(T-Aii)
A. Harden,
Barton
Antigen
in Normal
antibody
ascites
was
used.
Cells
in
culture
(lines
1T26R21,
1T45R91,
HEp-2, and human foreskin
fibroblasts)
were
prepared
for indirect
IF on Lab tek slides (Miles
Laboratories).
Cells were plated at low density on either four- or eight-chamber
slides
and
grown
I2
to
18
hours
with
or
without
cytoskeletal
inhibitors.
The cells were washed
once in RPM!
1640 + 15%
HIFCS
and then fixed in acetone
(-20
#{176}C)
for ten minutes.
Some
IT26R21
and 1T45R91
cells were treated with 0.5% Triton X-lOO
for one to 30 minutes.
Monoclonal
antibody
3-40 was directly
fluonesceinated
(F/P
7.3) as previously
described.8
Double
IF
assays with directly fluoresceinated
antibody
3-40 and either A2B5
or rabbit antithymosin
a, were performed
as described.’9
1T26R21
and IT4SR91
cell lines were also plated on Lab tek
slides, and one of the following
cytoskeletal
inhibitors
was then
added for the specified
remainder
of a 24-hour
growth period: (a)
colcemid
20 zg/mL (GIBCO,
Grand Island, NY) in RPMI for 16
hours; (b) cytochalasin
B 10 g/mL
(Sigma Chemical
Co, St Louis)
for two hours; (c) vinblastine
sulfate
S x i0
mol/L
for two
hours-prepared
as a stock solution of lO_2 mol/L in spectral grade
dimethyl
sulfoxide
(Mallinckrodt,
Paris,
Ky) as previously
described)0
Slides were then washed in RPM! 1640 + 15% HIFCS
Blood,
Vol
66,
No 3
(September),
1985:
pp 642-648
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
LEUKEMIA-ASSOCIATED
ANTIGEN
(3-40)
643
and fixed in acetone (-20
#{176}C)
for ten minutes,
and indirect IF was
performed.
Both colcemid
and vinblastine
cause vimentin
IMFs to form
peninuclear
coils, while cytokeratins
retain their filamentous
pattenn)#{176}’3In addition,
microtubules
are disrupted,
forming
crystallike structures
when
cells are incubated
with vinblastine.’#{176}
Treatment
with cytochalasin
B results in the disruption
of actin
microfilaments,
recognized
by the presence of asterisk-like
particles
or retraction
processes when stained with antiactin.’4
Preparations
of cytoskeletal
extracts.
Cytoskeletal
extracts
of
IT4SR91,
IT26R21,
HSB-2 T-ALL
cells, human
thymic stroma,
and epidermis
were isolated as described
by Pruss et al.’4 Briefly,
cells on tissue were rinsed in phosphate-buffered
saline (PBS) at
room temperature
and then treated with a solution of 171 mmol/L
NaC1, 6 mmol/L
NaPO4
(pH 7.4), 3 mmol/L
KC1, 2 mmol/L
EDTA,
1 mmol/L
phenylmethylsulfonylfluonide
(PMSF)
and 1
mmol/L
o-phenanthroline.
Cells were centrifuged
at 8,000 g for ten
minutes
at 4 #{176}C.
Supernatant
was
decanted,
and
cells
on tissue
were
homogenized
in an ice-cold extraction
buffer of 171 mmol/L
NaCl,
600 mmol/L
KC1, 0.5% (wt/vol)
Triton
X-lOO, 6 mmol/L
Na
phosphate
(pH 7.4), 1 mmol/L
EDTA,
1 mmol/L
PMSF,
and 1
mmol/L
o-phenanthroline
and centrifuged
at 8,000 g for ten mmutes at 4 #{176}C.
The soluble fraction was concentrated
in Spectnaphon
2
membrane
tubing
(mol wt cut-off
12,000 to 14,000)
(Spectrum
Medical
Industries,
Los Angeles)
in Aquacide
II (Calbiochem,
La
Jolla, Calif). Cytoskeletab
pellets were washed twice with ice-cold
PBS, and the final pellet was resuspended
and boiled in sodium
dodecyl sulfate (SDS) sample buffer with 5% vol/vol 2-mencaptoethanol.
Immunoblot
analysis.
Western
blot analysis
of cell/tissue
cytoskeletal
preparations
and Tniton-X
soluble fractions
were performed according
to the techniques
ofTowbin
et al,’5 with modifications of Palker
et al.’6 Following
separation
on a 10% SDSpolyacnylamide
slab gel, electnophonetic
transfer
of proteins
to
nitrocellubose
sheets (Schleicher
& Schuell, Keene, NH) was carried
out with an electrode
buffer of 25 mmol/L
Tnis, 192 mmol/L
glycine, 20% (vol/vol)
methanol,
pH 8.3, for 60 minutes at 0.7 to 0.9
A. After protein
transfer,
nonspecific
protein
binding
sites were
blocked by 3% bovine serum albumin
(BSA, Sigma) in Tnis-buffened
saline (TBS, 0.9% NaCI, 10 mmol/L
Tnis, pH 7.4) (12 hours at
4 #{176}C).
Individual
lanes were incubated
(two hours at 23 #{176}C)
in 5 mL
of monoclonal
antibody ascites diluted 1:500 in TBS 3% BSA. Strips
were washed five times in TBS for five minutes each, incubated
(30
minutes at 23 #{176}C)
in 2 mL ofTBS
3% BSA containing
106 cpm/mL
of ‘I-labeled
sheep antimouse
IgG + M F(ab’)2
(Amensham,
Arlington
Heights,
Ill), washed six times in TBS, blotted dry, and
autoradiographed
for four to six hours at - 70 #{176}C
using Kodak
X-omat
film (Eastman
Kodak,
Rochester,
NY) and a Dupont
Lightning-Plus
intensifying
screen.
‘4C-labeled
mol wt markers
(Amensham)
that were transferred
from the gel to the nitnocellulose
served
as internal mol wt standards.
Radioiodination
and
immunoprecipitation
of
RESULTS
Components
of
3-40.
As previously
variety
of normal
particular
normal
tissues
reactive
with
antibody
reported,
antibody
3-40 reacted
with
human
interest
tissues
tested
to characterize
lobular
cells
septae
and
the
within
medulla.
Double
IF
fluoresceinated
cells
and
Reactivity
of
reactivity
lines.
(Fig
and
of Normal
(Fig
rodent
and
human
cell
of Antibody
thymic
cell
Reactive
3-40
With
Reactivity
epithelmum,
vessel
interlobular
cells in epidermis,
epidermal
tive), vessel
en-
septae,
and
reticular
keratinocytes
cells in
(faintly
reac-
endothelium
Basal epithelium,
Basal
epithelial
bodies
dermis,
enoid
and
Tissues
capsule,
Langerhan’s
scattered
reticular
cells in ad-
scattered
reticular
cells
stroma
epithelium,
tonsillar
node
A2B5
Antibody
Neuroendocrine
Hassall’s
Lymph
and
identi-
D) as webb.
Human
Monoclonal
dothelium,
Tonsil
were
fibroblast
Pattern
Adenoid
that
2C and
with
human
Tissue
Skin
2A)
a, (Fig
3-40
3-40
thymus
1)
directly
characterize
the reactivity
of antibody
cell types within
thymus,
we assayed
3-40
1 . Components
Postnatal
epithelial
(Fig
A2B5
pres-
that 3-40
and inter-
using
antibody
3-40k
antibody
in rodent
Table
were
thymosin
lines.
To further
3-40 with specific
cortex
performed
either
types
of
epithelium
on with rabbit
thymosin
a,
that all cortical
and medullary
thymic
that
contained
cell
be seen
capsule
), as well as with
I
were
with
thymic
We found
antibody.2
epitheliab
(Fig
subcapsular
a
1 ). It was
3-40k
it could
thymic
stains
3-40
fies endocrine
2B)
vessels
thymic
(Table
the
ent in thymus.
Morphologically,
reacted
with mesodermal-derived
Vessel
subset
Postnatal
spleen
Reticular
Postnatal
liver
Large
in
stroma
endothelium,
cells,
HSB-2
HSB-2 cells in suspension
were surface labeled with 1251 by
the lactoperoxidase
technique
as previously
described.’7
Nonviable
cells were separated
by Ficoll-Hypaque
centnifugation;
cell viability,
as determined
by trypan blue exclusion
assay, was >98%. Washed,
labeled cells were solubilized
in 1.5 mL of extraction
buffer (10
mmol/L
Tnis (pH 8.0), 10 mmol/L
iodoacetamide,
2 mmol/L
PMSF,
0.5% (wt/vol)
ovalbumin,
1% (wt/vol)
NP-40),
vontexed,
and incubated
15 minutes
on a rocker
platform
at 4 #{176}C.
The
radiolabeled
suspension
was centrifuged
(15,000 g for 30 minutes),
and the supernatant
used for nadioimmunoprecipitation
(5 x 106
cpm per assay).
Staphylococcus
aureus
Cowan I strain (SaCI)
was used as an
immunoadsorbent
and radioimmunoprecipitation
was performed
as
cells.
previously
described.8
Because the isotype of antibody
3-40 is 1gM,
the soluble fraction was precleared
by successively
incubating
with
DMH 1.3.3.5 (control 1gM), SaCI preboaded
with rabbit antimouse
IgG (heavy and light chain specific)
(Cappel
Laboratories,
Cochnanville,
Pa), and unloaded
SaCI. The final soluble
fraction
was
incubated
overnight
with 5 ML of antibody
3-40 on DMH I .3.3.5 at
4 #{176}C
and then precipitated
with preloaded
SaCI. The immunoadsorbent was washed three times in PBS with 0.5% (wt/vol)
NP-40,
0.05% (wt/vol)
DOC, and 0.05% (wt/vol)
SDS, boiled for two
minutes in SDS sample buffer, and the soluble fraction analyzed
by
10% polyacnylamide
gel electnophoresis
(PAGE).
numerous
cells distributed
reticular
large
reticular
of lymphocytes
cells
throughout
scattered
spleen
among
negative
hepatocytes
Pancreas
Islets
and subset
of acinar
cells,
vessel
endo-
thelium
Fetal thymus
Same
Fetal
Reticular
liver
as postnatal
Fetal spleen
Vessel
Pituitary
Pituicytes
Brain
cortex
Medulla
Reactivity
Methods.
was
cells
endothelium
Filamentous
pattern
Filamentous
pattern
dot ermined
thymus
scattered
by indirect
throughout
liver
and stroma
IF as described
in Materials
and
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
644
LASTER
Fig 1 .
Indirect
IF reactivity
of antibody
3-40
on 4-es thick
human
thymus
tissue
sections.
Antibody
3-40
identifies
thymic
epithelial
cells in the subcapsular
cortex
(scc) and medulla
(m) but
not the cortex
(c). Macrophages
in the cortex
(arrowheads)
contain
autofluorescent
lipofuscin
granules.
Blood vessels
(arrow)
are also 3-4.0 reactive
(original
magnification
x400).
In indirect
IF assays,
epithebiab
previously
shown
AE-l
antibodies
tive, but
When
Similarly,
AE-l
rat
colcemid
pattern
changes
identified
analysis
of
by incubation
(Fig
antibody
were
1T45R91
noted
to
strated
retraction-bike
line
are negative
were
treated
characteristic
bodies
IT45R9I
IT45R91
the
and AE-3
fibroblasts
followed
coils
rescent
3-40
with
of vimentin
4).
with
by
antivimentin
tubulin
decorated
a crystal-like
cells treated
with vinblastine,
and
cells
(not
processes
shown).
3-40,
observed
B had
reactivity
the
is 4313E8
antibody
Cytochabasin
3-40
by I F with
not 4313E8
posi-
(not shown).
with
vinbbastine
were
no effect
IF
assay.
or
line, cobcemid,
on the pattern
vinblastine,
of 3-40
of keratin
characteristic
Cytoplasmic
cells, normal
mononuclear
also
thymocytes,
cells
(Fig
observed
and
on
the
5)
B-treated
thymic
However,
Biochemical
antigen.
identified
IMFs
immunoblots
from
the
To
or an
using
rodent
as expected
characterization
cellular
insoluble
lines,
in cytopreps
B had
pattern
HSB-2
of HSB-2
of peripheral
after
staining
from
the work
blood
with
of Naito
only on HSB-2
cells and
peripheral
lymphocytes,
(Table
2).
of the
determine
IMF-associated
the
cell
or cytochalasin
a reactivity
a subset
(Table
2)
et al,’ cell surface
IF was positive
not on normal
thymocytes,
normal
rodent
epithelial
cells, or fibrobbasts
3-40.
IF,
IMFs.
IF was
antibody
Anti-
rat
epithelial
no effect
peninu-
formation
in
antiactin
demonin the
of antibody
3-40
compared
with antibodies
on human thymus.
In double IF stains. using
antibody
3-40.
3-4O
thymic
epithelial
A2B5
(B)
and contained
thymosin
a1 (D)
x400).
A2B5
on fluoSimilar
antibody.
in cytochalasin
In contrast,
AL
a Tritonand human
D). We have
that the 1T26R2
I cell line reacts
and
AE-3
(antikeratin)
but
(antivimentin).3
clear
antibody
cytoskebetal
antigen
in both rodent
cells and fibroblasts
(Fig 3A through
X-resistant
Fig 2.
IF reactivity
and thymosin
a1
directly
fluoresceinated
cells (A and C) were
(original
magnification
ET
3-40-reactive
whether
protein,
we
cytoskeletal
T cells,
intra-
antibody
pellets
and
3-40
performed
human
derived
skin.
Fig 3.
Reactivity
of antibody
3-40
on both
human and rodent cell lines. Antibody
3-40
reacted
with keratin
lMFs in the 1T26R21
rat thymic
epithelial
cell line (A) and HEp-2 cells (B). (The reactivity
with spheroid
granules
seen in mitotic
HEp-2
cells has been previously
reported
for other antikeratin
antibodies27
and was also demonstrated
using antibody
AE-1 .) Antibody
3-40
also reacted
with vimentin
in the 1T45R91
rat fibroblast
cell line
(C) and in human foreskin
fibroblasts
(D) (original
magnification
x4.O0).
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
LEUKEMIA-ASSOCIATED
ANTIGEN
645
(3-40)
Table
2.
Summary
of Reactivity
of Antibody
Malignant
Surface
Cell Type
PB
mononuclear
1 rat fibroblasts
was
magnification
seen
with
colcemid
antibody
(20
to vimentin
g/mL
for
(43E8)
16
was
hours)
used
(20)
+
T-ALL
(#2)
leukemic
cells
+
(7)
+
+
(10)
+
+
(98)
+
fresh
T-ALL
cells
cell
line
was assayed
reactivity
reactivity
peripheral
was
by indirect
determined
by
IF on viable
indirect
IF on
cells.
lntnacyto-
acetone-fixed
cells.
blood.
immunoprecipitation
antibody
x400).
+
+
PB,
or
+
-
cells
plasmic
(original
-
leukemic
Surface
a control
+
(# 1 ) fresh
HSB-2
reactivity
+
-
T-ALL
S#{233}zaryleukemic
when
by IF
-
epithelial
cells
Fig 4.
Indirect
IF reactivity
of antibody
3-40 on rat fibroblasts
treated
with
vinblastine.
When
rat fibroblasts
were
incubated
with vinblastine
(5 x 10
mol/L
for two hours) and reacted
with
antibody
3-40. each cell demonstrated
a perinuclear
coil. Similar
and
Intracytoplasmic
Reactivity
(% of cells positive)
cells
1 rat thymic
1T45R9
to Normal
Reactivity
by IF
Thymocytes
1T26R2
3-40
Cell Types
of
3-40
251 surface
demonstrated
labeled
a 60-
HSB-2
to 63-kd
cells
with
antigen
(Fig
I 0).
Western
strated
blots of HSB-2
cytoskeletal
that antibody
3-40 identified
preparations
demona 55-kd
protein
that
commigrated
with the protein
recognized
cbonal antibody
to vimentin
and as well
6A).
Similar
results
were
In the rat epithelial
cell
line (Fig
obtained
Using
by a known
monoa 39-kd protein
(Fig
using
the
rat
line,
antibody
3-40
fibrobbast
3-40
recognized
epithelial
Furthermore,
fled
kd, 48.0
to 63-kd
50.0
kd,
45.0
kd,
and
skin,
fibroblasts,
T-ALL
also identified
a
rodent
kd,
7). In human
42.0
shown).
Because
Naito
et ab’ reported
that
antibody
3-40
identified
a 35- to 40-kd
membrane-associated
NP-40
soluble
antigen
in MOLT-4
cells,
immunoblots
were
performed
on the 6
mol/L
KCI Triton-X
soluble
fraction
of HSB-2
cells; antibody
9).
3-40
recognized
In contrast,
a protein
on SDS-PAGE
between
under
37 kd and
reducing
39 kd (Fig
conditions,
Fig 5.
Reactivity
of antibody
3-40 with
cytopreps
of
mononuclear
cells. Antibody
3-40 identifIed
a cytoplasmic filamentous
structure
showing
an organizing
center
and some perinuclear
coiling in HSB-2 cells (A), normal
thymocytes
(B). and a subset
of peripheral
blood mononuclear
cells (C). Similar
reactivity
was seen with an
antibody
to vimentin
(431E8).
The antikeratin
antibodies AE-1 and AE-3 were nonreactive.
and
immunobbot
3-40
analysis,
recognizes
we
cytoplasmic
have
IMFs
in
both normal
and malignant
cells. Antibody
3-40 identifies
a
family
of cytokeratins
found
in a rodent
epithelial
cell line,
human
skin, and human
thymus
and binds
to vimentin
in
cells.
of 56.5
(Fig
a
IF
antibody
major
keratin
bands
of 56 kd
thymus,
antibody
3-40 identi-
keratins
protein
with
56 kd that
In addi-
that
antibody
3-40 identified
two
and 50 kd (Fig 8). In human
kd (not
a 39-kd
reacted
43 and
antibodies.
indirect
shown
6B).
family
of cytoskeletal
proteins
between
were recognized
by known
antikeratin
tion,
DISCUSSION
and
surface
that antibody
in MOLT-4
antigen.
3-40
T-ALL
in nonmalignant
cytoskeletal
fibroblast
are
cell
in HSB-2
a 37- to 39-kd
recognized
IMFs
cells,
and thymocytes.
39-kd
cytoskeletal-associated
and
T-ALL
Tniton-X
Naito
recognizes
cells. Cell
lines
Antibody
3-40
protein
in
cells,
soluble
HSB-2
antigen
et al’ have
T-ALL
antibody
previously
a 35- to 40-kd surface
surface
reactivity
was
3-40
and
a 60shown
protein
not seen
cells.
a family
of relatively
proteins
found
Although
there
tin [mesenchyme],
are
in
insoluble
most
five classes
(keratins
desmin
[muscle],
a-type
vertebrate
[epithebiab],
neurofilaments
fibrous
cells.’820
vimen[neu-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
646
ET AL
LASTER
HSB-2
1145R91
O
Mrxl
TceIIs
69
Mr x
-
46
:
.y
.
30
-
:
14.3
E
ii
14.3
ABC
A BC
I
A B C DE
Fig 6.
Immunoblot
analysis
of antibody
3-40 reactivity
with
proteins
from
the HSB-2
and 1T45R91
cell lines.
Cytoskeletal
proteins
were extracted
from the HSB-2 T-cell line (A) or IT45R91
rat thymic
fibroblasts
(B) as previously
described.14
Samples
were
reduced
in 5% 2-mercaptoethanol
with sample
buffer,
separated
by 10% SDS-PAGE.
and electrophoretically
transferred
to nitrocellulose.
Mol wt markers
are indicated
in the center.
Lane A.
antibody
43E8
(antivimentin);
lane
B.
P3 x 63
(negative
C. antibody
3-40. Antibody
3-40 identified
a 55-kd
protein
in
both
HSB-2
and 1T45R91
cells that commigrated
with a protein
recognized
by the antibody
to vimentin;
antibody
3-40 also recognized a 39-kd
protein
in both cell lines as well as a 46-kd
protein
also recognized
by 43E8.
and glial
all share
a-helical
segments.
reacting
with
previously
keratin
fibrillary
certain
Not
more
acidic
proteins
[gbial
amino
acid
sequences
surprisingly,
than
described.’5’22
and
vimentin
one
The
monocbonal
class
binding
of
IMF
in this
the
antibodies
have
3-40
report.
been
with
More-
over,
similar
indirect
IF of antibody
to gbial fibrilbary
acidic
tions,
November
1984);
remains
to be demonstrated.
islets,
creatic
melanomas
have
also
with
a pattern
observaother
IMFs
keratin
or vimentin
IMFs,
report
of Naito
et al,’ who
reactivity
on frozen
sections
therenoted
of thymus,
pan-
skin, ovarian
stroma,
fetal
testis,
and
and ovarian,
breast,
and colonic
cancers.
noted
antibody
containing
epithelial
vimentin-containing
3-40
reactivity
organs
(thymus,
mesenchymal-derived
tissue stroma,
reported
that
macrophages).
resting
T cells
HSB-2
iO-3
Mrx
3-40 on brain
shows
protein
(unpublished
cross-reactivity
to recognize
the prior
3-40
antibody
connective
previously
1T26R21
69
Fig 8.
Immunoblot
analysis
of antibody
3-40
reactivity
with
keratins
from
human
epidermis.
Keratins
were
extracted
from
human
epidermis
reduced
in 5% 2-mercaptoethanol
with sample
buffer.
separated
by 1 0% SDS-PAGE.
and electrophoretically
transferred
to nitrocellulose.
Mol wt markers
are indicated
on the
left. Lane A. antibody
AE-1 ; lane B. antibody
AE-3; lane C, P3x63
negative
control;
lanes D and E. antibodies
to the 50- and 56.5-kd
human
keratins
(RTE-22
and RTE-23.
respectively);
lane F. antibody 3-40. Antibody 3-40 identifIes
the 50- and 56.5-kd
keratins
in
human skin.
The ability
fore, explains
cells]),2’
within
of antibody
is demonstrated
F
control);
lane
rons],
they
-
soluble
with
skin,
both
some
We
keratin-
pancreas)
and
tissue
(vessels,
Dellagi
et a123 have
and phytohemaggbu-
I cells
fraction
-
Mr
46
x103
69-
1
30-
46
-
30
-
14.3-
14.3AB
ABC
CD
Fig 7.
Immunoblot
analysis
of antibody
3-40
reactivity
with
keratins
from the 1T26R21
cell line. Cytoskeletal
proteins
were
extracted
from the 1T26R21
cell line. reduced
in 5% 2-mercaptoethanol
with sample
buffer.
separated
by 10% SDS-PAGE.
and
electrophoretically
transferred
to nitrocellulose.
Mol wt markers
are indicated
on the left. Lane A. antibody
AE-1 (antikeratin);
lane
B. antibody
AE-3 (antikeratin);
lane C. P3 x 63 negative
control;
lane D. antibody
3-40.
Antibody
3-40
identified
rodent
keratins
of
56 kd. 45 kd. and 43 kd. and as well identified
a 39-kd protein
not
recognized
by the other antikeratin
antibodies.
Fig 9.
Immunoblot
analysis
of antibody
3-40 reactivity
with
soluble fraction
of HSB-2
T cells. The 0.6 mol/i
KCI Triton-X
soluble
fraction
obtained
during
the cytoskeletal
extraction
of
HSB-2 T cells was concentrated
tenfold.
boiled for two minutes in
5% 2-mercaptoethanol
sample
buffer.
separated
by 1 0% SDSPAGE,
and electrophoretically
transferred
to nitrocellulose.
Mol
wt markers
are indicated
on the left. Lane A. antibody
43E8
(antivimentin);
lane B. P3x63
negative
control;
lane C. antibody
3-40.
Antibody
3-40
identifies
a protein
band between
37 and 39
the
kd.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
LEUKEMIA-ASSOCIATED
ANTIGEN
647
(3-40)
x1O
Mr
30-
14.3
-
AB
Fig I 0.
Radioimmunoprecipitation
of lactoperoxidase
surHSB-2 T cells using antibody
3-40.
HSB-2 T cells were
face-labeled
surface-labeled
with
cipitation
markers
control;
antibody
tinin-
or concanavalin
In this
T-ALL
sles
and
using lactoperoxidase.
1251
Radioimmunopre-
was
performed
using
the soluble
fraction.
Mol wt
are indicated
on the left. Lane A. DMH 1 .3.3.5 (negative
an 1gM directed
against
an la-like
antigen);
lane B.
3-4.0. Antibody
3-40
identifies
a 60- to 63-kd antigen.
A-stimulated
report
we have
cells and normal
T cells
demonstrated
thymocytes.
contain
as
well
Monoclonab
antibodies
to double-stranded
virus phosphoprotein,25
herpes
simplex
the Thy I surface
antigen26
have been
react
with
vimentin
IMFs.
cross-reacting
T-ALL
cells.
antigens
Using
(43fE8),
have
viable
we
T-ALL
observations,
exist on the
3-40
and
recently
cells
now
from
one
DNA,24
meavirus protein,25
shown
to cross-
extend
this
surface
antigen
antibody
to
shown
positive
of three
in
list
surface
patients
of
found on
vimentin
IF
of
(unpublished
November
1984).
Whether
vimentin
IMFs
surface
of T-ALL
cells, or whether
antibodies
43$E8
both
vimentin
currently
being
both
recognize
and
surface
investigated.
Furthermore,
protein
We
to the 3-40
a monoclonab
vimentin.
vimentin
whether
of normal
and
determinants
antigens
on
common
T-ALL
cells
remains
to
be
to
is
the
HSB-2
39-kd
cells,
determined.
cytoskeletal-associated
the
37- to 39-kd
HSB-2
Possibly
the
3-40
cells
may
be
T cells during
antigen
selectively
malignant
transformation.
ACKNOWLEDGMENT
Joyce Loweny,
assistance.
14. Pruss
filaments
monoclonal
found
intracellularly
in normal
expressed
on the surface
of human
We thank
for secretarial
12. Fnanke
WW, Schmid
E, Osbonn
M, Weben
K: Different
intermediate-sized
filaments
distinguished
by immunofluonescence
microscopy.
Proc Natl Acad Sci USA 75:5034,
1978
13. Hynes
RO, Destree
AT: lOnm
transformed
cells. Cell 13:151, 1978
soluble
antigen
and the 35- to 40-kd
MOLT-4
membraneassociated
antigen
all identified
by antibody
3-40 are identicab
2. Haynes
BF, Shimizu
K, Eisenbarth
GS: Identification
of
human
rodent thymic epithelium
using tetanus
toxin and monoclonal antibody
A2B5. i Clin Invest 71:9, 1983
3. Laster AJ, Itoh T, Palker TJ, Haynes
BF: Human
thymic
epithelial
cells contain
kenatins
of differentiated
cornified
epithehum. Clin Res 33:381A,
1985
4. Woodcock-Mitchell
J, Eichner
R, Nelson
WG, Sun T-T:
Immunolocalization
of keratin
polypeptides
in human
epidermis
using monocbonal
antibodies.
i Cell Biol 95:580, 1982
5. Sun T-T, Eichner
R, Nelson
WG, Tseng SCG, Weiss RA,
Janvinen
M, Woodcock-Mitchell
J: Keratin
classes:
Molecular
markers
for different
types of epithelial
differentiation.
J Invest
Dermatol
8I:I09s,
1983
6. Gown AM, Vogel AM: Monocbonal
antibodies
to human
intermediate
filament
proteins.
II. Distribution
of filament
proteins
in normal human tissues. Am i Path 114:309,
1984
7. Haynes
BF, Hensley
LL, Jegasothy
BV: Differentiation
of
human T lymphocytes.
II. Phenotypic
difference
in skin and blood
malignant
T cells in cutaneous
T cell lymphoma.
J Invest Denmatol
78:323, 1982
8. Haynes BF, Mann DL, Hemlen ME, Schroer
JA, Shelhamen
JA, Eisenbanth
GS, Thomas
CA, Mostowski
HS, Stramingen
JL,
Fauci AS: Characterization
of a monoclonal
antibody
which defines
an immunonegulatory
T-cell subset for immunoglobulin
synthesis
in
man Proc Natl Acad Sci USA 77:2914.
1980
9. Haynes
BF, Scearce
RM, Lobach
DF, Hensley
LL: Phenotypic characterization
and ontogeny
of mesodenmal-denived
and
endocrine
epithelial
components
of the human thymus microenvinonment. i Exp Med 159:1 149, 1984
10. Weber
K, Bibning T, Osborn
M: Specific
visualization
of
tubulin-containing
structures
in tissue culture cells by immunofluorescence.
Cytoplasmic
microtububes,
vinblastine-induced
paracrystals and mitotic figures. Exp Cell Res 95:111, 1975
1 1 . Ishikawa
H, Bischoff R, Holtzen H: Mitosis and intermediate
sized filaments
in developing
skeletal
muscle.
J Cell Biol 38:538,
I 968
Kim McClammy,
and LaVerne
Johnson
REFERENCES
1. Naito K, Knowles RW, Real FX, Monishima
Y, Kawashima
K, Dupont
B: Analysis
of two new leukemia-associated
antigens
detected
on human
T-cell acute
lymphoblastic
leukemia
using
monocbonal
antibodies.
Blood 62:852, 1983
RM,
share
Minsky
a
antibody.
common
Cell
R, Raff
MC:
antigenic
27:419,
filaments
All classes
determinant
in normal
and
of intermediate
defined
by
a
1981
I 5. Towbin H, Staehelin
T, Gordon J: Electrophoretic
transfer of
proteins
from polyacrylamide
gels to nitrocellubose
sheets.
Procedunes and some complications.
Proc Natl Acad Sci USA 76:4350,
1979
16. Palker Ti, Scearce
RM, Miller
DP, Gallo RM, Haynes BF: Monoclonal
cell beukemia-lymphoma
virus (HTLV)
ExpMed
159:1117,
SE, Popovic
M, Bolognesi
antibodies
against human T
p24 internal core protein. J
1984
17. Jones PP: Analysis
of radiolabeled
lymphocyte
proteins
by
one- and two-dimensional
polyacnybamide
gel electnophonesis,
in
Mishell BB, Shiigi SM (eds): Selected
Methods
in Cellular
Immunology. San Francisco,
WH Freeman,
1980, p 402
18. Lazanides
E, Gnanger BL, Gand DL, O’Connor
CM, Breckler
J, Price M, Danto SI: Desmin
and vimentin-containing
filaments
and their role in the assembly
of the Z disk in muscle cells. Cold
Spring Harbor Symposia
on Quantitative
Biology. Organization
of
the Cytoplasm
46:351, 1982
19. Andenton
gous structures.
BH: Intermediate
filaments:
A family
J Muscle Res Cell Motil 2: 141 , 1981
of homolo-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
LASTER
648
20. Weben K, Osborn M: Cytoskeleton:
Definition,
structure
and
gene regulation.
Pathol Res Pnact 175:128, 1982
2 1 . Osborn M: Intermediate
filaments
as histologic
markers:
An
overview. J Invest Dermatol
8l:lO4s,
1983
22. Dellagi K, Brouet JC, Perrcau
J, Paulin D: Human
monoclonal
1gM with autoantibody
activity
against
intermediate
filaments. Proc NatI Acad Sci USA 79:446, 1982
23. Dellagi K, Vainchenker
W, Vinci G, Paulin D, Brouet JC:
Alteration
of vimentin
intermediate
filament
expression
during
differentiation
of human hemopoietic
cells. EMBO J 2:1509, 1983
24. Andr#{233}-Schwartz i, Datta 5K, Shoenfeld
Y, Isenberg
DA,
Stollar D, Schwartz
RS: Binding of cytoskeletal
proteins
by monoclonal anti-DNA
lupus autoantibodies.
Clin Immunol
Immunopathol 31:261,
1984
ET AL
25. Fujinami
RS, Olstone
MBA, Wroblewska
Z, Frankel
ME,
Koprowski
H: Molecular
mimicry
in virus infection:
Cross-reaction
of measles virus phosphopnotein
or of herpes simplex virus protein
with human
intermediate
filaments.
Proc Natl Acad Sci USA
80:2346,
1983
26. Dubbecco
R, Unger
M, Bologna
M, Battifora
H, Syka P.
Okada
5: Cross reactivity
between
Thy 1 and a component
of
intermediate
filaments
demonstrated
using a monoclonal
antibody.
Nature 292:772,
1981
27. Geiger B, Kreis TE, Gigi 0, Schmid E, Mittnacht
5, Jorcano
JL, van Bassewitz
DB, Franke
WW: Dynamic
rearrangements
of
cytokeratins
in living cells, in Levine AJ, Vande Woude GF, Topp
WC, Watson JD (eds): Cancer Cells: The Transformed
Phenotype.
Cold Spring Harbor Laboratory,
January
1984, p 201
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1985 66: 642-648
Antibody against T lymphoblastic leukemia-associated antigen (3-40)
identifies vimentin and keratin intermediate filaments in normal cells
AJ Laster, TJ Palker, EA Harden, W Ho, K Naito, B DuPont and BF Haynes
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