Download Purification and NH2-Terminal Amino Acid

(CANCER RESEARCH 50, 1774-1778. March 15. I990|
Purification and NH2-Terminal Amino Acid Sequence of Guinea Pig Tumorsecreted Vascular Permeability Factor1
Donald R. Senger,2 Daniel T. Connolly, Livingston Van De Water, Joseph Feder, and Harold F. Dvorak
Department of Pathology. Beth Israel Hospital and Han'ard Medical School, and the Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts
02215 [D. R. S., L. V. D. H'., H. F. D./. and The Monsanto Company. St. Louis, Missouri 63167[D. T. C.. J. FJ
ABSTRACT
Rodent and human tumor cell lines secrete a potent vascular permea
bility factor (VPF) which causes a rapid and substantial increase in
microvascular permeability to plasma proteins without causing mast cell
degranulation, or endothelial cell damage or without exciting an inflam
matory cell infiltrate |D. R. Senger, S. J. Galli, A. M. Dvorak, C. A.
Perruzzi, V. S. Harvey, and H. F. Dvorak. Science (Wash. DC), 219:
983-985,1983; D. R. Senger, C. A. Perruzzi, J. Feder, and H. F. Dvorak.
Cancer Res., 46: 5629-5632, 1986|. VPF now has been purified to
homogeneity from guinea pig tumor cell culture medium; it is a V/,
34,000-43,000 protein, and a NH2-terminal amino acid sequence has
been derived. A synthetic peptide corresponding to amino acid residues
1-24 of the native protein was used to raise rabbit antibodies which bind
all of the vessel permeability-increasing activity secreted by guinea pig
tumor cells and which stain purified VPF on immunoblots. These findings
establish that this NHz-terminal amino acid sequence was derived from
the permeability factor. Homolog) searches found no identity or close
similarity between VPF Nil .-terminal sequence and database sequences,
indicating that VPF is distinct from other proteins for which sequence
data are available. In particular, no sequence similarity was found between
tumor-secreted VPF and other mediators of increased vessel permeability
including plasma and glandular kallikreins.
INTRODUCTION
Tumor blood vessels display increased permeability as com
pared with the vessels of normal tissues such that fluid and
plasma proteins accumulate in the interstitial space (1-7). Par
ticularly dramatic fluid accumulation occurs when tumors growin ascites form. Recent studies have shown that the majority of
hyperpermeable vessels are mature venules lined by a continu
ous endothelium (8), the type of vessel known to respond to
inflammatory mediators such as histamine. Therefore, any of
several inflammatory' mediators could contribute to the in
creased vessel permeability and fluid accumulation observed in
the vicinity of growing tumors. However, our prior studies with
guinea pig tumors growing in syngeneic hosts indicated that
tumor cells themselves alter host vascular permeability locally
and promote fluid accumulation by secreting a potent vascular
permeability factor with electrophoretic mobility corresponding
to M, 34,000-42,000 (9). VPF1 is readily detectable in tumor
ascites fluids but not in normal serum or plasma; and while it
is produced by a variety of rodent and human sarcomas (9, 10),
carcinomas (7,9, 10), and glioblastomas ( 11), it is not detectably
produced by normal fibroblasts or epithelial cells (7, 9, 10).
Received 1/30/89; revised 9/20/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by USPHS Grant CA 43967 (D.R.S.) awarded by
the National Cancer Institute. Department of Health and Human Services.
USPHS Grant GM 36812 (L.V.D.W.) awarded by the National Institute of
General Medical Sciences. Grant JFRA-198 (L.V.D.W.) from the American
Cancer Society, and by a grant from the Monsanto Company.
! To whom requests for reprints should be addressed, '/,•
Department of
Pathology. Beth Israel Hospital. 330 Brookline Avenue. Boston. MA 02215.
1The abbreviations used are: VPF, vascular permeability factor: DMEM,
Dulbecco's modified Eagle's medium: SDS, sodium dodecyl sulfate; PBS. phos
phate-buffered saline (0.15 M NaCI-0.01 M sodium phosphate. pH 7.3): HPLC,
high pressure liquid Chromatograph): i.d.. intradcrmally.
When injected into guinea pig skin, VPF causes a rapid (within
5 min) but transient (over by 30 min) increase in microvascular
permeability without causing endothelial cell damage or mast
cell degranulation, and its action is not blocked by antihistamines (9).
In order to characterize this potentially important molecule
further, we have now purified VPF to homogeneity from large
volumes of serum-free tumor cell culture medium. We also
report the derivation of NH^-terminal amino acid sequence,
comparison of VPF sequence to those of previously described
mediators of increased vessel permeability, and production of
anti-VPF antibodies with a synthetic peptide based on VPF
NHj-terminal sequence.
MATERIALS AND METHODS
Materials
Reagents for electrophoresis were purchased from Bio-Rad; nitro
cellulose paper was from Schleicher and Schuell; i:5I-protein A (9 ^Ci/
¿ig)
was from Dupont-NEN. For chromatography. heparin-Sepharose
was purchased from Pharmacia, hydroxylapatite from Bio-Rad, the SP5PW (7.5 mm x 7.5 cm) and C,8-MBondapak (3.9 mm x 30 cm) HPLC
columns from Waters, and the C3 Ultrapore RSPC (4.6 mm x 7.5 cm)
column from Beckman.
Cell Culture and Serum-Free Culture Medium
Guinea pig line 10 tumor cells were grown in suspension in DMEM
containing 5% calf serum and 4.5 g/liter glucose. Cultures were initially
scaled up in Bélico
culture vessels to 12-liter volumes. The harvests
from two to four 12-liter vessels were used to start 100-liter Vibromixer
reactors (12) at an initial density of 1-2 x IO5cells/ml. For serum-free
culture medium, cells were harvested from the Vibromixer reactors,
washed three times in serum-free DMEM, and incubated in serum-free
DMEM at 1.0 x IO6cells/ml for 24 h.
Miles Vessel Permeability Assay
The Miles assay (13) was performed as described previously (9).
Increased vessel permeability was quantitated by spectrophotometric
measurement of the Evans blue dye extracted from skin test sites (14).
Polyacrylamide Gel Electrophoresis and Immunoblotting
SDS-polyacrylamide gel electrophoresis was performed according to
the method of Laemmli (15). Gels were silver stained with Gelcode
(Pierce Chemical Co.) according to the manufacturer's recommenda
tions. Immunoblotting was performed as described previously (16) and
bound antibody was detected with i;5I-protein A and autoradiography
(Kodak X-Omat AR film). Blots were stained with antiserum or preim
mune control serum diluted 1:200: '25I-protein A was used at a concen
tration of 0.6 ¿
Purification of VPF
VPF
graphie
activity
of each
PBS to
Step
was purified to homogeneity with the following Chromato
steps. At each stage of purification, eluted fractions with VPF
were identified with the Miles assay (see above); small aliquots
fraction were diluted as necessary1(in all cases >20-fold) with
bring them within the nonsaturating range of the assay.
1. Serum-free line 10 culture medium (80 liters) was passed
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Table I Purification of \'PF
VPF was purified from 80 liters of line 10 culture medium (containing 2.2 g total protein) with the following Chromatographie columns, according to the protocols
described in "Materials and Methods."
ofpooledactivity
inpooled
protein
VPF-containingfractions144
fractions(VPF
totalprotein'')3308958.33024.40087.500160.000Overall
iniii.n.!'
pooledfractions48.00043.00040.00039.00035.00032.000Total
in
activity"units
VPF
Step
column1
Chromatography
Heparin-Sepharose2
Hydroxylapatite3a
exchange3b SP-5PW HPLC cation
3a4
Repeat of Step
phase5
C3 HPLC reverse
C 18 H PLC reverse phaseTotal
mg48
mg4.8mg1.6mg400
„¿g200^gSpecific
purification:Purification(-fold)152.79.32.93.61.87,300C
°One VPF activity unit is defined here as the amount of VPF (6.4 ng purified VPF) equivalent to 1.0 (*gdisfamine in the Miles vessel permeability assay (13). The
Miles assay is not strictly quantitative: numbers here represent our best estimates.
* Specific activity of VPF in the starting material (line 10 culture medium) was 22 units/nig total protein (48.000 units of activity/2.2 g protein).
c VPF was purified to homogeneity as demonstrated by electrophoresis and highly sensitive silver staining (Fig. 1) and NH; terminal amino acid sequence analysis
[a single NH¡-terminal sequence was obtained (Table 2)].
Table 2 NH2-terminal residues derived from VPF
a
(pmol)83859«35846345643453727617
Cycle1234567891011i:131415If,1718192021222324ResidueAlaProMetAlaG
-«67
-«43
-«30
" n.d.. not determined.
Fig. 1. Electrophoretic analysis of purified VPF. VPF purified from serumfree line 10 culture medium, as described in "Materials and Methods," was
subjected to SDS-polyacrylamide gel clectrophoresis (acrylamide concentration.
10 g/100 ml) without reduction of disulfide bonds: protein was visualized with
silver staining. Lane a, 2 >jg purified VPF. Lane h. molecular weight markers.
Right ordinate, positions and molecular weights (in thousands) of three in the
region of interest: bovine serum albumin (A/, 67.000): ovalbumin (A/, 43.000);
bovine erythrocyte carbonic anhydrase (,\1, 30.000).
over a 2.0-liter heparin-Sepharose column; all permeability-increasing
activity bound. The column was washed with PBS and bound protein
was eluted with a 5-liter linear gradient ranging from PBS to 1.2 M
NaCl in 0.01 M sodium phosphate, pH 7.3. With large gradient volumc:column bed volume ratios, VPF eluted as a peak centered at
approximately 0.40 M NaCl; however, in this large scale system (with
a gradicntxolumn volume ratio of only 2.5), VPF eluted in the range
of 0.75-0.95 M NaCl.
Step 2. VPF-containing fractions from the heparin-Sepharose col
umn (approximately 1 liter in total) were diluted with 4 volumes of
0.01 M sodium phosphate, pH 7.0, and loaded onto a 200-ml hydroxylapatite column. Again, all permeability-increasing activity bound. The
column was washed with 0.01 M sodium phosphate, pH 7.0, and VPF
was then eluted with a gradient of 0.01 M-0.50 M sodium phosphate,
pH 7.0: VPF eluted at -0.25 M sodium phosphate.
Step 3. VPF-containing fractions from Step 2 were pooled and
equilibrated with 0.01 M sodium phosphate, pH 6.1. All VPF remained
soluble in this buffer, but ~30/c of the total protein precipitated. The
Table 3 Immunoadsorplion of VPF with antibody raised to synthetic peptide
(NH2-terminal residues 1-24)
IgG-coated Staphylococcus aureus was prepared, the Miles vessel permeability
assay was performed, and dye was extracted from skin test sites as described in
"Materials and Methods." Test substance (0.2 ml) was injected i.d.; the presence
of dye. above that observed at control (PBS) injection sites, indicates increased
vessel permeability. Data represent mean values ±SD from three experiments.
Substance injected i.d.
Amount of dye present
in skin test site
(mean (>ig)±SD|
PBS
Line 10 tumor cell
2.5 ±0.2
27.4 ±0.9
Line 10 CM
Adsorbed with S. aiireux coated wi(h prcimmunc
30.0 ±1.7
CM"
(control) IgG
Line 10 CM
Adsorbed with .s aureus coated with immune IgG
Line 10 CM
Adsorbed with S. aureus coated with immune IgCi
(first blocked with synthetic peptide)
" CM. culture medium.
2.2 ±0.6
27.6 ±2.6
soluble protein was loaded onto an SP-5PW HPLC cation exchange
column (see "Materials"), and all VPF activity bound. The loaded
column was subjected at l ml/min to a 60-ml linear gradient of 0.01 M
sodium phosphate, pH 6.1, to 1.0 M NaCl in 0.01 M sodium phosphate.
pH 6.1. VPF eluted as a peak centered at 0.66 M NaCl. Repetition of
this step yielded an additional 3-fold purification.
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ester (Sulfo-MBS Crosslinker; Pierce Chemical Co.) and stirred for 15
min at 4°C:(b) the hemocyanin-cross-linker complex was isolated from
free cross-linker on a Sephadex G-25 (coarse) column in 0.1 M potas
sium phosphate. pH 6.O. and added to 30 mg peptide (pH of mixture
adjusted to 6.5-7.0); (c) the mixture was stirred overnight at 4°C,
-«43
-«25.7
-«18.4
VI 2.3
Fig. 2. Immunoblot analysis of VPF stained wilh antibody raised to synthetic
peptide (NH¡-terminal residues 1-24). Purified VPF. as in Fig. 1, was subjected
to SDS-polyacrylamide gel electrophoresis (acrylamide concentration. 12.5 g/100
ml) followed by electrophoretic transfer to nitrocellulose and staining with anti
body to synthetic peptide and 12!I-protein A. Lane a, VPF with disulfide bonds
reduced with dithiothreitol: Lane h. VPF without reduction of disulfide bonds.
Identical lanes incubated with preimmune (control) serum instead of immune
serum showed no staining (not shown). Righi ordinate, positions of molecular
weight markers and their molecular weights (in thousands): ovalbumin (M,
43,000); n-chymotrypsinogen (M, 25.700); tf-lactoglobulin (M, 18,400); and cytochrome e (M, \ 2.300).
Step 4. VPF-containing fractions were pooled and dialyzed against
trifluoroacetic acid:water (0.1:100), and protein was loaded onto a C3
reverse-phase HPLC column equilibrated in the same solution. All VPF
activity bound, and protein was eluted at a flow rate of 1 ml/min with
a linear 60-ml gradient of H2O to acetonitrile (each containing 0.1 ml
trifluoroacetic acid/100 ml). VPF eluted as a peak centered at ~20 min
(acetonitrile:H2O = 33:67).
Step 5. VPF-containing fractions were pooled, diluted with 4 volumes
of trifluoroacetic acid:water (0.1:100), and applied to a C18 reversephase HPLC column; all VPF bound. Protein was eluted with an initial
gradient (0-20 min) of H2O to acetonitrile:H2O (40:60). A second
gradient (20-70 min) acetonitrile:H2O (40:60) to acetonitrile:H2O
(90:10) was then used to elute VPF. All gradient solutions contained
0.1 ml trifluoroacetic acid/100 ml, and flow rates were 1.0 ml/min.
VPF eluted at acetonitrile:H2O (45:55).
Protein Sequencing and Sequence Homology Searches
Sequencing was performed on VPF (purified as above) with an
Applied Biosystems gas-phase sequenator (Model 470A) using the
02CPTH program. HPLC was carried out with a Waters apparatus
(NOVA-PAK C,s column) to quantitate phenylthiohydantoin deriva
tives. Derived sequence was compared against the National Biomedicai
Research Foundation database using the FASTP program (17).
Preparation of Antisera to Synthetic Peptide: Immunoadsorption
Activity
of VPF
A synthetic peptide corresponding to residues 1-24 of purified VPF
(with cysteine added at position 25 for chemical coupling; see below)
was synthesized by the Peptide Synthesis Facility, The Children's
Hospital, Boston, MA. The composition of this peptide was confirmed
by both amino acid analysis and derivation of complete sequence as
described above. Synthetic peptides were coupled through the carboxyterminal cysteines to keyhole limpet hemocyanin as follows: (a) 12.4
mg hemocyanin in 2 ml of 0.01 M potassium phosphate, pH 7.0, was
added to 0.48 ml of 5 mg/ml /w-maleimidobenzoylsulfosuccinimide
quenched with 0.1 M ethanolamine for 60 min, dialyzed against PBS,
and frozen.
Rabbits were immunized with multiple i.d. and s.c. injections of
synthetic peptide-hemocyanin in complete Freund's adjuvant (total of
1-4 mg protein/animal). Animals were boosted with similar amounts
of immunogen in incomplete Freund's adjuvant 3 weeks later, and
antibody was found in 4 of 4 rabbits 4-6 weeks later.
Immunoadsorbent was prepared by mixing Staph A (IgGsorb; The
Enzyme Center, Maiden, MA) with immune or preimmune control
rabbit serum in the ratio of 1 ml serum (immune or preimmune control
to 0.5 ml packed Staph A. After a 30-min incubation, the Staph A was
washed 4 times with PBS and resuspended in 10 volumes of PBS.
Seven hundred /¿I
of the IgG-coated Staph A suspension was aliquoted
to individual tubes which were centrifuged, and the buffer above the
Staph A pellets was removed. The Staph A pellets were then resus
pended in I ml of line 10 culture medium (see above) and incubated
for 15 min at 25°C.After centrifugation, supernatants were assayed for
vessel permeability-increasing activity with the Miles assay. In order to
demonstrate the specificity of the adsorbing antibodies, some tubes
containing the immunoadsorbent were first incubated with 10 ^g of
synthetic peptide.
RESULTS AND DISCUSSION
Approximately 200 ¿ig
of purified VPF were derived from 80
liters of line 10 serum-free culture medium, and purification of
VPF from 80-liter starting volumes was performed four times.
The purification scheme is described in detail above and out
lined in Table 1. The purified protein consistently migrated on
SDS-polyacrylamide gels as a broad band corresponding to a
molecular weight of 34,000-43,000 (Fig. 1), in good agreement
with previous molecular weight estimates based on extraction
of VPF activity from gels (9). Sequence analyses (repeated three
times) of HPLC-purified VPF revealed a single NH2 terminus
and a consistent NH2-terminal amino acid sequence (Table 2).
To exclude the possibility that the vessel permeability-increas
ing activity in our purified preparations was attributable to an
undetected contaminating protein present in trace amounts, we
raised rabbit antisera to a synthetic peptide representing resi
dues 1-24 of the derived NH2-terminal sequence (see "Mate
rials and Methods"). All four rabbits immunized with this
peptide produced antibodies which bound all of the vessel
permeability-increasing activity in guinea pig line 10 tumor
culture medium (Table 3). Preimmune control sera from the
same rabbits were without effect, as were immune sera which
had been preabsorbed with synthetic peptide (Table 3). There
fore, it is certain that the NH2-terminal sequence presented in
Table 2 was derived from the vascular permeability factor.
On immunoblots stained with antisera to the VPF-synthetic
peptide, purified guinea pig VPF was visualized as a broad band
corresponding to A/r 34,000-43,000 (Fig. 2, Lane b), indicating
that proteins throughout this molecular weight range share
antigenic determinants present in NH2-terminal residues 1-24.
VPF reduced with dithiothreitol was visualized as several major
and minor bands ranging from M, 24,000 to M, 13,500 with
major bands at M, 22,000; M, 20,000; and M, 17,000 (Fig. 2,
Lane a). In addition, silver staining of purified VPF (as in Fig.
1), also demonstrated that reduced VPF consists of multiple
polypeptides in this same size range (not shown). Association
of chains with these different mobilities could account for the
broad size range of unreduced VPF. Consistent with the single
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B.
A.
a
cd
b
e
f
-«43
«43
-«30
Fig. 3. Immunoprecipitütion of "S-labeled VPK from line 10 cell culture medium. Line 10 cells were labeled for 4 h in medium containing one-tenth the normal
concentration of methionine. 5c'r calf serum, and 300 //Ci/mi i.-["S]methionine;
and equal aliquots(200»jl) of culture medium were subjected to immunoprecipitation
with Staph A and either preimmune (control) rabbit serum or anti-peptide (immune) rabbit serum as described in "Materials and Methods." A [autoradiogram of 10%
] animili (15) SDS-polyacrylamide gel; no reduction of disulfide bonds]. Lane a, precipitate with preimmunc (control) serum; Lane b, precipitate with immune (antipeptide) serum. Note presence in Lane h of broad band corresponding to M, 34.000-42.000 which is not present in the control lane (¡Jinea). All bands in Lane a are
nonspecifically bound to Staph A. and only the broad M, 34.000-42.000 band in Lane h represents antibody-specific precipitation. B (12.5rÃ- gels, reduction of
disulfide bonds where indicated). Lane a. precipitate with preimmune (control) serum; Lane b. precipitate with immune (anti-peptide) serum; no reduction of disulfide
bonds in Lanes a and b. Note antibody-dependent precipitation of a broad ,\tr 34.000-42,000 band in Lane b as in A (Lane b). luanes c (preimmune. control serum
precipitate) and d (immune, anti-peptide serum precipitate) correspond exactly to Lanes a and ft. respectively, except that Lanes <•
and d were reduced w ith dithiothrcitol
after precipitation and prior to electrophoresis. Note the absence of the broad A/r 34.000-42.000 band in Lane d and the presence instead of bands corresponding to
M, ~ 22,000, M, 20.000, and M, 17.000 which arc not in the control lane (Lane c). Labeled medium used for ¡Mnese and/was identical to that used for Lanes a-d,
except that it was reduced prior to immunoprecipitation
with dithiothrcitol ( 100 MIMI and alkylated ( IK-h dialysis against 10 rriM iodoacctic acid). Immunoprecipitates
of this medium were not reduced prior to electrophoresis. Note the presence in Lanc/(immunc,
anti-pcptide serum precipitate) of bands at M, ~ 22,000. M, 20.000.
and M, 17.000 which are not in Lane e (preimmune. control serum precipitate). Right ordinales, positions and molecular weights of markers in thousands: ovalbumin
(M, 43,000). bovine erylhrocyte carbonic anhydrase (M, 30.000), soybean trypsin inhibitor (M, 20.100). and bovine milk ir-lactalbumin (M, 14.400).
NH2-terminal sequence derived from HPLC-purified VPF (Ta
ble 2), electrophoretic transfer of reduced VPF from SDSpolyacrylamide gels onto glass filter paper (Whatman GF/F)
followed by sequence analyses (18) revealed that for all residues
determined (residues 1-9), the NH2 termini of bands at M, ~
24,000 and M, 20,000 are identical with that determined for
unreduced VPF (not shown). Although the data do not rule out
the possibility that unreduced VPF contains a polypeptide chain
which is blocked to NH2-terminal sequence analysis, a simple
hypothesis which is consistent with the data is that VPF is a
two chain protein consisting of variant disulfide-bonded peptides, all with identical NH2 termini.
To determine whether the heterogeneity revealed in Figs. 1
and 2 is representative of newly synthesized VPF or is a
consequence of degradation that may have occurred during 24h culture or multistep purification, line 10 cells were labeled
with L-['5S]methionine, and secreted proteins were subjected to
immunoprecipitation with antisera raised to synthetic peptide.
Labeled VPF from 4-h culture medium also migrated as a broad
band corresponding to M, 34.000-43,000 and. upon reduction,
appeared as three bands corresponding to M, 22,000, M,
20,000, and M, 17,000 (Fig. 3). Consistent with the presence
of identical NH2 termini, these same three species were also
immunoprecipitated with anti-peptide antibody when labeled
VPF was first reduced and alkylated (Fig. 3). Therefore, as with
purified VPF analyzed in Figs. 1 and 2, newly synthesized,
metabolically labeled VPF also displayed electrophoretic het
erogeneity, and upon reduction it appeared as at least three
discrete bands all of which individually reacted with antibodies
raised to the synthetic peptide. This heterogeneity is likely to
represent naturally occurring structural differences and not
merely artifactual degradation.
The NH2-terminal sequence of Table 2 was subjected to
computerized homology searches and found to be unrelated to
NH2-terminal or internal sequences of other proteins for which
sequence was available. Proteins specifically compared included
plasma kallikrein (19), the multiprotein family of glandular or
tissue kallikreins (20-24), and thrombin (25), proteins that
have been implicated previously in altering vessel permeability
(directly or indirectly). Unlike less well-characterized proteins
that have been claimed to increase vessel permeability (26, 27),
VPF satisfies two important criteria: (a) it is active in the
species from which it was derived (i.e., guinea pig VPF is active
in guinea pig skin); and (/>) its action is rapid (within 5 min)
and not attributable to endothelial cell damage or to initiation
of an inflammatory cell response (9). Moreover, VPF increases
vessel permeability at very low concentrations, displaying activ
ity in the Miles assay at concentrations less than 1 x IO"9 M.
Comparisons of purified VPF with histamine indicated that
approximately 8 ng (~2 x 10~" mol of VPF increased vessel
permeability comparable to 1.25 Mg(~1 x '0~8 mol) of hista
mine (i.e., on a molar basis, VPF was more than 50,000 times
as potent as histamine).
In summary, we have purified tumor-secreted VPF to ho
mogeneity and derived NH2-terminal amino acid sequence. A
synthetic peptide corresponding to amino acid residues 1-24
was synthesized and used to raise antisera which bound VPF
activity, thus conclusively demonstrating that this amino acid
sequence was derived from the permeability factor. This NH2terminal sequence is not similar to previously published se
quences, establishing that VPF is distinct from plasma and
tissue kallikreins (19-24). VPF is a potent mediator, active at
concentrations of < 10~9M. Although it is expressed by a variety
of malignant cells, the gene encoding this protein is very likely
expressed by some normal cell types as well, and we are now
investigating this possibility.
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ACKNOWLEDGMENTS
We thank Carole Perruzzi, Ageliki Papadopoulos, and Debbie Heuvelman for expert technical assistance: Rick Nelson for performing
large-scale cell culture; Ned Siegel and Margaret Ehrhardt for sequence
analyses; Daniel Teñenfor sequence homology searches; and Jitka
Olander for preparing antisera.
Note Added in Proof
After this manuscript was submitted, others reported the purification
of a structurally similar protein secreted by bovine pituitary cells
(Ferrara, N. and Henzel. W. J. Pituitary follicular cells secrete a novel
heparin-binding growth factor specific for vascular endothelial cells.
Biochem. Biophys. Res. Commun., ¡61:851-858. 1989; Gospodarowicz. I).. Abraham, J. A., and Schilling. J. Isolation and characterization
of a vascular endothelial cell mitogen produced by pituitary-derived
folliculostellate cells. Proc. Nati. Acad. Sci. USA, 86: 7311-7315,
1989). This pituitary-derived protein stimulates endothelial cell growth
as does tumor-secreted VPF (Connolly, D. T., Heuvelman. D. M.,
Nelson. R., Olander, J. V., Eppley, B. L., Delfino. J. J.. Siegel, N. R.,
Leimgruber. R. M., and Feder, J. Tumor vascular permeability factor
stimulates endothelial cell growth and angiogenesis. J. Clin. Invest. 84:
1470-1478. 1989). Finally, predicted amino acid sequence for VPF
(derived from cDNA sequence) has recently been reported [Leung. D.
W., Cachianes, G., Kuang, W-J., Goeddel, D. V., and Ferrara, N.
Vascular endothelial growth factor is a secreted angiogenic mitogen.
Science (Wash. DC). 246: 1306-1309, 1989; Keck. P. J.. Hauser, S.
D., Krivi, G., Sanzo, K., Warren, T., Feder, J.. and Connolly, D. T.
Vascular permeability factor, an endothelial cell mitogen related to
PDGF. Science (Wash. DC), 246: 1309-1312, 1989].
REFERENCES
1. Underwood, J. C. E., and Carr. I. The ultrastructure and permeability
characteristics of the blood vessels of a transplantable rat sarcoma. J. Pathol..
107: 157-166, 1972.
2. Ackerman, N. B.. and Hechmer, P. A. Studies on the capillary- permeability
of experimental liver métastases.Surg. Gynecol. Obstet.. 146:884-888.1978.
3. Dvorak. H. F.. Harvey. V. S., and McDonagh. J. Quantitation of fibrinogen
influx and fibrin deposition and turnover in line 1 and line 10 guinea pig
carcinomas. Cancer Res., 44: 3348-3354. 1984.
4. Ccrlowski. L. E., and Jain. R. K. Microvascular permeability of normal and
neoplastic tissues. Microvasc. Res.. 31: 288-305. 1986.
5. Brown. L. F., Asch. B., Harvey. V. S.. Buchinski. B.. and Dvorak. H. F.
Fibrinogen influx and accumulation of cross-linked fibrin in mouse carcino
mas. Cancer Res.. 48: 1920-1925. 1988.
6. Heuser. L. S., and Miller, F. N. Differential macromolecular leakage from
the vasculature of tumors. Cancer (Phila.), 57/461-464, 1986.
7. Dvorak. H. F., Orenstein, N. S.. Carvalho. A. C, Churchill, W. H.. Dvorak,
A. M., Galli, S. J., Bitzer, A. M., Rypysc. J.. and Giovinco. P. Induction of
a fibrin-gel investment: an early event in line 10 hcpatocarcinoma growth
mediated by tumor-secreted products. J. Immunol.. 122: 166-174. 1979.
8. Dvorak. H. F., Nagy. J. A.. Dvorak. J. T., and Dvorak. A. M. Identification
and characterization of the blood vessels of solid tumors that are leaky to
circulating macromolecules. Am. J. Pathol., 133: 95-109. 1988.
9. Senger. D. R.. Galli. S. J.. Dvorak. A. M.. Perruzzi. C. A.. Harvey. V. S.,
and Dvorak. H. F. Tumor cells secrete a vascular permeability factor that
promotes accumulation of ascites fluid. Science (Wash. DC). 219: 983-985.
1983.
10. Senger. D. R.. Perruzzi, C. A., Feder. J., and Dvorak, H. F. A highly
conserved vascular permeability factor secreted by a variety of human and
rodent tumor cell lines. Cancer Res.. 46: 5629-5632. 1986.
11. Bruce. J. N., Criscuolo. G. R.. Merrill. M. J.. Moquin. R. R.. Blacklock, J.
B.. and Oldfield. E. H. Vascular permeability induced by protein product of
malignant brain tumors: inhibition by dexamethasone. J. Neurosurg.. 67:
880-884. 1987.
12. Tolbert. W. R., Feder. J.. and Kimes. R. C. Large-scale rotating filter
perfusion system for high-density growth of mammalian suspension cultures.
In Vitro (Rockville), 77:885-890. 1981.
13. Miles. A. A., and Miles. E. M. Vascular reactions to histamine. hislamineliberator. and leukotaxine in the skin of guinea pigs. J. Physio). (Lond.). 118:
228-257, 1952.
14. I dak.i, K.. Takeuchi. V. and Mova!. H. Z. Simple method for quantitation
of enhanced vascular permeability. Proc. Soc. Exp. Biol. Med., 133: 13841387, 1970.
15. Laemmli, U. K. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature (Lond.). 227: 680-685. 1970.
16. Towbin. H.. Staehelin. T.. and Gordon. J. Electrophorctic transfer of proteins
from polvacrylamide gels to nitrocellulose sheets: procedure and some appli
cations. Proc. Nati. Acad. Sci. USA. 76:4350-4354. 1979.
17. Lipman. D. J.. and Pearson, W. R. Rapid and sensitive protein similarity
searches. Science (Wash. DC). 227: 1435-1441. 1985.
18. Aebcrsold. R. H.. Teplow, D. B.. Hood, L. E., and Kent. S. B. H. Electroblotting onto activated glass. High efficiency preparation of proteins from
analytical sodium dodecyl sulfate polyacrylamide gels for direct sequence
analysis. J. Biol. Chem. 261: 4229-4238. 1986.
19. Chung. D. W.. Fujikawa. K.. McMullen. B. A., and Davie, E. W. Human
plasma prekallikrein. a zymogen to a serine protease that contains four
tandem repeats. Biochemistry. 25: 2410-2417, 1986.
20. Dunbar, J. C.. and Bradshaw. R. A. Amino acid sequence of guinea pig
prostate kallikrein. Biochemistry'. 26: 3471-3478, 1987.
21. Swift. G. H., Dagorn. J. C., Ashley. P. L.. Cummings. S. W.. and MacDonald.
R. J. Rat pancreatic kallikrein mRNA: nucleotidc sequence and amino acid
sequence of the encoded preproenzyme. Proc. Nati. Acad. Sci. USA, 79:
7263-7267, 1982.
22. van Leeuwen, B. H., Evans, B. A., Tregear, G. W., and Richards, R. I. Mouse
glandular kallikrein genes. Identification, structure, and expression of the
renal kallikrein gene. J. Biol. Chem.. 261: 5529-5535, 1986.
23. Ashley. P. L., and MacDonald. R. J. Kallikrein-related mRNAs of the rat
submaxillary gland: nucleotide sequences of four distinct types including
tonin. Biochemistry. 24:4512-4520. 1985.
24. Fukushima. D.. Kitamura. N., and Nakanishi, S. Nucleotide sequence of
cloned cDNA for human pancreatic kallikrein. Biochemistry, 24:8037-8043.
1985.
25. Butkowski. R. J.. Elion. J., Downing. M. R., and Mann, K. G. Primary
structure of human prethrombin 2 and «-thrombin. J. Biol. Chem., 252:
4942-4957. 1977.
26. Ooyama. T., Kawamura, K., Orimo. H.. and Murukami. M. Identification
and characterization of a vascular permeability factor of renal origin. Lab.
Invest.. ¥0:615-621. 1979.
27. Lobb, R. R.. Key, M. E., Alderman. E. M., and Fett, J. W. Partial purification
and characterization of a vascular permeability factor secreted by a human
colon adenocarcinoma cell line. Int. J. Cancer, 36: 473-478, 1985.
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Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1990 American Association for Cancer Research.
Purification and NH2-Terminal Amino Acid Sequence of Guinea
Pig Tumor-secreted Vascular Permeability Factor
Donald R. Senger, Daniel T. Connolly, Livingston Van De Water, et al.
Cancer Res 1990;50:1774-1778.
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