Vol.

167,

March

No.

30,

:3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1990

DETECTION

Masakazu

OF TRANSFORMING GROWTH FACTOR ALPHA HUMAN URINE AND PLASMA

Katohl,

Hirofumi

Mieko Laboratories Laboratories, 3Department

Received

Inagaki2p3,

Katsuura2

Kayoko

and Shigeaki

Tanaka2 Pharma Research Kawagoe, Saitama

of Hygiene and Public Health, Nippon Medical Sendagi, Bunkyo-ku, Tokyo 113, JAPAN 7,

IN

Kurosawa-Ohsawal,

for ICell Biology and 'Biochemistry, Hoechst Japan Limited, l-3-2 Minamidai, 350, JAPAN

December

1065-1072

School,

l-l-5

1989

A sensitive enzyme-linked immunosorbent assay (ELISA) system for human transforming growth factor alpha (TGFa) was developed in combination with polyclonal and monoclonal antibodies. Employing this assay s:ystem, we detected TGFU like activity in normal human plasma as well as in cancer patients' urine and plasma. These TGFa were analyzed by chromatography, inununoreactivity, and EGF-TGFa receptor binding assay and found to be identical to authentic human TGFa. The presence of TGFa circulating in normal adult plasma suggests a new role of TGFa in the human body. 01990 Academic Press, Inc.

Transforming polypeptide cell with

with

types. epidermal

and

induces

human

body found

is composed growth factor

fluids, in

the

mouse

Subsequently,

(4).

various

and/or detected placenta

disulfide

in

tumor malignant

been

milk, in

(5-61, tissues

cells.

saliva,

because

a potent

a

single-chain

mitogen

for

various

50 amino acids and shares 40% homology (EGF). TGFa binds to EGF-TGFa receptors

detected

fibroblasts

(TGFa),

is

of epithelial

conditioned

cells

alpha bonds,

of

urine,

TGFa has not

transformed of

It

three

factor

DNA synthesis

contrast, first

growth

medium and

platelets normal

was termed

(lo),

the and

urine

etc.

adult

of Moloney

TGFa was found in

EGF was found

the of

because

growth

culture

cancer

In

TGFa was

sarcoma

"sarcoma

in

most

(l-3).

tissues.

murine

in

virusfactor"

supernatants patients

TGFa like

(7-g),

activity

in normal embryonic tissues, mouse embryo (11-131, and (14), TGFa was considered to be the oncofetal counterpart

was human of

growth factor alpha; EGF, The abbreviations used are: TGFa, transforming epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; MeCN, liquid acetonitrile; TFA, trifluoroacetic acid; HPLC, high performance chromatography; NHK, normal rat kidney; PBS, phosphate-buffered saline; BSA, bovine serum albumin. 0006-291x/90 1065

$1.50

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

167,

No.

BIOCHEMICAL

3, 1990

Recently,

EGF .

(15-16),

however,

human

reported

indispensable. of

NRK 49F

cells

definition

of

from

and

EGF,

In order

specific In the

TGFa

and

early

in

bovine

(17),

TGFa.

system,

BIOPHYSICAL

untransformed

keratinocytes

to secrete

mammalian

AND

soft

(4).

additional

the

to clarify

transformation In were

this

have

regard,

quantitative

to

studies

Recently

we

immunosorbent sensitivity

been

employed

However,

this

assay

in human urine

of TGFa in the of

TGFa is

formation was

not

now assay

used

as

distinguish

TGFa by gel

the TGFa

filtration

during

through

development

studies

against

at

the

synthetic

TGFa from

EGF and

or

cell

mRNA level.

TGFU peptides are

used

also

for

on TGFa. have

assay (19).

were

performance liquid chromatography of TGFU. Therefore most data

antibodies

distinguish

role

and did

of

TGFa

obtained

cells (18)

of TGFa, a colony

characterization

specific

acknowledged

the

was

of

pituitary

analysis

agar

expression

COMMUNICATIONS

hepatocytes

quantitative

chromatography or reverse-phase high (HPLC) was required for identification concerning

anterior

and/or

studies

RESEARCH

established (ELISA)

Employing

a

sandwich-type

specific this

ELISA,

for

human

we examined

enzyme-linked TGFa the

with

nature

high of TGFa

and plasma.

Human native TGFa was prepared from the culture supernatant of a human melanoma cell line (20), as described previously SEKI (C24), (21) and was shown to be identical to recombinant human TGFa (Earth Chemical Co., JAPAN). Recombinant EGF was purchased from Wakunaga pharmaceuticals, JAPAN. Urine and plasma from cancer patients (gastric cancer, colon cancer, and hepatoma) were supplied by Dr. Imai (Saitama Medical School, Saitama, JAPAN). Urine and plasma from healthy donors were collected from 15 volunteers from our laboratory. Urine samples were pooled (1OOml from cancer patients and 1,OOOml from healthy donors) and centrifuged at 800xg for 10 min. Acetic acid was added to the supernatant at a final concentration of 1M and dialyzed against 1M acetic acid. Samples were concentrated by lyophilization and dissolved in 5ml of 1M acetic acid. Concentrated samples were stored at -2O'C prior to use. The pooled plasma of cancer patients (1Oml) and healthy donors (1OOm.l) was acidified with acetic acid at a final concentration of 3% and centrifuged to removed insoluble materials. Acidified supernatants were diluted with 0.1% trifluoroacetic acid (TFA) by lofold and applied to Sep-Pak Cls cartridges (Waters associates, U.S.A.). The cartridges were washed with 10% acetonitrile (MeCN) containing 0.1% TFA. TGFa was eluted with 40% MeCN containing 0.1% TFA. The eluate was lyophilized and dissolved in a small volume of phosphate-buffered saline (PBS, phosphate lOmM, pH7.2, NaCl 0.15M). Concentrated samples were stored at -20°C prior to assay.

(19).

ELISA for Briefly,

TGFa was 96-well

performed microtiter

according plates

1066

to the described method (Falcon, Becton-Dickinson,

Vol.

167,

No.

BIOCHEMICAL

3, 1990

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

U.S.A.) were coated with SOWl/well of lOug/ml of anti-C-terminal peptide [human TGFa (34-50)] rabbit polyclonal antibody in PBS for 1 hr at room temperature. Wells were washed with PBS three times and were blocked with 300pl/well of 1% bovine serum albumin (BSA) in PBS for 30 min. After the blocking solution was removed, samples or standard TGFa (O,O.l-long/ml) dissolved in 1% BSA in PBS were applied at 5O@/well and incubated for 1 hr. Following washing with PBS, 2pg/ml of antisynthetic human TGFa monoclonal antibodies was added to the wells at 50&l/well and incubated for 1 hr. Wells were washed with PBS three times, and then appropriately diluted peroxidase-labeled rabbit antibody against mouse immunoglobulins (Dakopatts, Denmark) in 1% BSA in PBS was added at 50pl/well and incubated for 1 hr. After the wells were washed extensively with PBS, calorimetric reaction was performed by adding SOpl/well of substrate solution for peroxidase (lmg/ml ophenylenediamine in O.lM Na citrate buffer (pH 5.0) containing 0.006% H202). The reaction was stopped by adding 50~1 of 2M HZS04 after incubation for 20 min. Finally, the absorbance at 490 nm was measured with an ELISA autoreader (Dynatech, U.S.A.). The amounts of immunoreactive material were expressed as equivalents of the natural TGFa purified from the culture supernatant of SEKI. Human EGF was measured by two-site sandwich of monoclonal antibodies as described previously EGF-Tvradio-receDtor

ELISA (22).

with

two

kinds

ama&!

Radio-receptor assay for EGF-TGFa was performed according to the method described previously using membrane fractions of A431 cells, human epidermoid carcinoma (23). lz51-TGFa (2x105Bq/pmole) as a tracer was prepared according to the chloramine-T method.

AND DISCUSSION TCFa

in

urine

Pooled

urine

carcinoma, under

and

donors the

both to

is

void

precursor

Also

a minor

volume

immunoreactive

conditions

used.

approximately Bio-Gel showed

P series various

acid.

in

urine,

peak

to

As shown

The

apparent

which gel

activity

molecular

was consistent

chromatography (24).

of

the

under

with

Fig.

On the cancer

EGF in

same

acidic

hand, urine.

estimated

previous conditions

EGF-TGFa

EGF,

authentic under the

as

(SEKI),

was the

close

other

patients'

the line

1,

ELISA

large-form

TGFa fractions

1251-TGFCi in the 1067

in

human

this

weight with

Immunoreactive

healthy

by EGF

authentic

be determined. in

Rad,

EGF was observed

nature

only

(Bio

from

was detected

of immunoreactive The

P-30

Urine

to

colon

as described

to Bio-Gel

corresponding

remains

sources

competing

concentrated

of urinary TGFa was exactly from human melanoma cell

9,000

carcinoma,

same way as controls.

was detected

TGFa

volume purified

and

1M acetic

(Nos.20-23).

or aggregate,

(gastric

subjected

to be abundant

(Nos.46-51)

The elution human TGFa

from

in

in the

known

fractions

cases. the

then

chromatography

was processed

patients

was acidified

and Methods,

gel

EGF, which

10 cancer

hepatoma)

Mat.erials

U.S.A.)

in

from

to

be

report

on

of

TGFa

(Nos.39-45) radio-receptor

Vol.

167,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1 .o 0.6 0.6

0.1 0.0

F^

0.2

s ii 2

0.0 43K

z 2

0.4

zl E.

1.5

f

22K

t

12K

6K

t

f

s

.

4.0

5.0

3.0 1.0

I? p

4.0 3.0

2.0

I

2.0 0.5

1.0

1.0

t

0.0

0.0

0.0

10

20

30

40

50

60

70

Fractions Bio-Gel P-30 gel chromatography of urine from cancer patients (a) and healthy donors (b). Ten ml of concentrated urine was applied on a Bio-Gel P-30 column (25~45Omm) in l.OM acetic acid at the flow rate of 0.25ml/min. Fractions of 4ml were collected and subjected to TGFa (0) and EGF (0) ELISA. Arrows indicate eluting positions for molecular markers (43kd, ovalbumin; 22kd, cytochrome C; 12kd, soybean trypsin inhibitor; 6kd, insulin) and authentic human TGFa and human EGF.

.Lb-l

assay.

:n

ELISA,

which

ELISA

detected

(SEKI). be

was

In

our

different

patients' the

TGFa

by

TGFa

ELISA

was

found

to

will

be

reported

TCFa

in

pl&%na

Pooled

ruled its ELISA

in

various

by

out.

EGF

in

significantly

high

it

exists,

around

the

explained

TGFa

is

TGFa

in

confirmed

was

patients. tumor

of

ELISA

of were

urinary

the

presence TGFa

presence

tumor

void by

The by

donors

in

appears

supernatants.

ELISA.

healthy

of

line

be

the

proved,

types

TGFa

cell

detected

Since

absence

This

activity

be

TGFa

(24). if

possibly

cannot

by

melanoma

culture

binding can

was

,

melanoma

detected

TGFa

be

TGFU

the

detected

measured The

patients'

level details

elsewhere.

plasma as

and

of

by

in

which be

urine

usefulness

directly

cannot

report

produced

and

was

was

previous

receptor

activity

TGFa

urinary

shown)

EGF

like it

a TGFa

detected

not

large-form

but

cancer

(data

TGFa

unlikely,

pretreated

that EGF-TGFa

low

of

additional

from

large-form

from

was

large-form

human

the

preparation,

activity

no

large-form

different

volume

of

preparation,

Therefore,

to

and

our

described

from

cancer under

patients Materials

1068

or and

healthy Methods.

donors To

eliminate

was

Vol.

167,

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

0.3

0.25 0.20

0.2

0.15 0.10

0.1

0.05 0.0

TGFu

1 .o

t

t

EGF

2.5

2.5

2

2.0

2.0

2

1.5

1.5

1.5

0.5 0.0

1.0

1 .o

0.5

0.5

0.0

0.0 10

20

30

40

50

Fractions Bio-Gel P-30 gel chromatography of plasma from cancer patients (a) and healthy donors (b). One ml of concentrated plasma was applied on a Bio-Gel P-30 column (lOx3OOmm) in l.OM acetic acid at the flow rate of 0.25ml/min. Fractions of lml were collected and subjected to 'PGFa (0) and EGF (0) ELISA. Molecular markers were same as described in the legend for w.

0.8

0.6

I 2

4.0

TGFa

J

1

EGF

4.0

6 n

3.0

0

8

0.4

E 2 T

2.0

F?

2 0.2

73

1.0

E 0.0

0.0 0

20

60

40

Retention

time

( min.)

Reverse-phase HPLC of plasma TGFa. Pooled fractions (Nos.34Fio. 38) of gel filtration were concentrated and applied to a Vydac Cls column (5pm particle size, 4.6x25Omm) . The mobile phase was 0.10% TFA and the mobile-phase modifier was MeCH3 containing 0.08% TFA. The concentration of M&H3 was increased linearly from 0% to 40% during 1 hr at the flow rate of O.Sml/min. Fractions of lml were collected and sub2ected to TGFU (0) and EGF (0) ELISA.

1069

Vol.

BIOCHEMICAL

167, No. 3, 1990 abundant

plasma

proteins

reverse-phase cartridge. through fraction and eluate

was analyzed

TGFa ELISA, also

in

in

cancer

donors'

When platelet indicating normal

plasma

far

analyzed

obtained those to

the

by

authentic

concentration in culture for isolation,

Fig.

series

between 4,

the of

dilutions

found (data

in

plasma

is

and high

and

O.O28ng/ml

plasma

derived

TGFa

limit. amount

at the

TGFa found

HPLC (Fig.

be

There

were TGFa and curves

superimposed

functionally to

in

on

identical The

vitro.

stimulate

cell

growth

with like

acid TGFB

(dilution) 104

103

102

10'

100

0

Authentic Innnunoreactivity

TGFa

(rig/ml)

(a) and EGF-TGFa Fia. (b) of plasma derived TGFa. HPLC fractions were dialyzed against PBS and then directly titration curve of plasma derived TGFa (0) corresponding curve of authentic TGFa (0).

1070

The

to that

TGFU was proved

enough

in

3).

TGFU and plasma

TGFa can

of

same level,

not shown), but as the sample was treated TGFa in plasma might exist in a latent form

Human

and

detection

with antibodies The titration

plasma

TGFU immunochemically

plasma

normal

supernatants.

shown.

plasma

Therefore,

in

to be identical

interaction are

but

a reasonable

urinary

By

plasma

TGFa and EGF.

SEKI culture

2).

patients' be

by reverse-phase

receptor

TGFa.

a disposable

(Fig.

the

TGFa remained of

properties

EGF-TGFU

of authentic

to

was below

TGFa was shown

from

In

cancer

estimated

while

of plasma-derived

examined. with

were

was further

in

in

or serum was prepared,

TGFa purified

to

chromatography

The EGF level

plasma

applied

The TGFa levels

localization

no differences

gel

only

plasma.

different

time

binding

not

was recovered

the

of authentic so

P-30

plasma

rich

platelets

was

proteins were recovered in the flowTGFa was eluted with 40% meCNj. The

by Bio-Gel

respectively.

retention

sample

Most retained

patients'

O.O5lng/ml EGF in

the

TGFa was detected

healthy

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

--ereceptor binding activity containing TGFa activity applied on each assay. was superimposed onto

The the

Vol.

167,

No.

BIOCHEMICAL

3, 1990

The presence

(25). concept

of

TGFa

endocrinological

of

TGFa in

being

an

role

in

the

AND

normal

body.

of

TGFa in

reports

on the

presence

(15-16),

normal

keratinocytes

the help which

distribution shares

the

the

role

plasma

contrary

into anterior normal

factor.

viva

in

to

suggests

bovine

and sensitive

of TGFa in

COMMUNICATIONS

and/or

growth

specific

is and

Taking

(17),

ubiquitous

of TGFa with

us to understand

RESEARCH

protein,

human

cells

TGFa may be a rather

adult

oncofetal

recent (18),

BIOPHYSICAL

the some

account

the

pituitary hepatocytes

Investigation assay connection

methods with

of will EGF,

same receptor.

some clinical samples. We We thank Dr. Y. Imai for providing also thank Drs. E. Konz, H. B. Maruyama, and T. Matsuishi for their support and encouragement during this work.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Elder, J. B., Williams, G., Lacey, E., and Gregory, H. (1978) Nature (London) a, 466-467 Oka, Y., and Orth, D. N. (1983) J. Clin. Invest. Z?., 249-259 Petrides, P. E., Hosang, M., Shooter, E., Esch, F. S., and Bo:hlen, P. (1985) FEBS letters LBz, 89-95 De Larco, J. E., and Todaro, G. J. (1978) Proc. Natl. Acad. Sci. USA z, 4001-4005 Smith, J. J., Derynck, R., and Korc, M. (1987) Proc. Natl. Acad. Sci. USA 84, 7567-7570 Derynck, R., Goeddel, D. V., Ullrich, A., Gutterman, J. U., Williams, R. D., Bringman, T. S., and Berger, W. H. (1987) Cancer Res. 92, 707-712 Stromberg, K., Hudgins, W. R., and Orth, D. N. (1987) Biochem. Biophys. Res. Commun. ;L49, 1059-1068 H-W., Tsai,J-H., Yeh, Y-C., Tsai,J-F., Chuang, L-Y., Yeh, Florine, D. L., and Tam, J. P. (1987) Cancer Res. 92, 896-901 Gregory, H., Thomas, C. E., Willshire, I. R., Young, J. A., Anderson, H., Baildam, A., and Howell, A. (1989) Br. J. Cancer 3, 605-609 Arteaga, C. L., Hanauske, A. R., Clark, G. M., Osborne, C. K., Hazarika, P., Pardue, R. L., Tio, F., and Von Hoff, D. D. (1988) Cancer Res. 48, 5023-5028 Twardzik, D. R., Ranchalis, J. R., and Todaro, G. J. (1982) Cancer Res. 42, 590-593 Matrisian, L. M., Pathak, M., and Magun, B. E. ( .982) Biochem. Biophys. Res. Commun. l.Ql, 761-769 Twardzik, D. R. (1985) Cancer Res. 45, 5413-5416 Stromberg, K., Pigott, D. A., Ranchalis, J. E., and Twardzik, D. R. (1982) Biochem. Biophys. Res. Commun. Le6, 354-361 Samsoondar, J., Kobrin, M. S., and Kudlow, J. E. (1986) J. Bio. Chem. 26;L, 14408-14413 Kobrin, M. S., Samsoondar, J., and Kudlow, J. E. (1986) J. Bio. Chem. Z& 14414-14419 Coffey Jr, R. J., Derynck, R., Wilcox, J. N., Bringman, T. S., Goustin, A, S., Moses, H. L., and Pittelkow, M. R. (1987) Nature (Lond.) m, 817-820 Proc. Natl. Acad. Sci. Mead, J. E., and Fausto, N. (1989) USA a, 1558-1562 Inagaki, H., Katoh, M., Kurosawa-Ohsawa, K., and Tanaka, S., (in press) J. Immun. Method 1071

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Kondo, Y., Sato, K., Ueyama, Y., and Ohsawa, N. (1981) Cancer Res. fi, 2912-2916 Marquardt, H., Hunkapiller M. W., Hood, L. E., Twardzik, D. R., De Larco, J. E., Stephenson, J. R., and Todaro, G. J. (1983) Proc. Natl. Acad. Sci. USA &Q, 4684-4688 Katsuura, M., and Tanaka, S. (1989) J. Biochem. (Tokyo) ULh, 87-92 Kimball, E. S., and Warren, T. C. (1984) Biochem. Byophys. Acta. m, 82-88 Sherwin, S. A., Twardzik, D. R., Bohn, W. H., Cockley, K. D., and Todaro G. J. (1983) Cancer Res. Q, 403-407 Pircher, R., Jullien, P., and Lawrence, D. A. (1986) Biochem. Biophys. Res. Commun. j&, 30-37

21. 22. 23. 24. 25.

3, 1990

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AND

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