Expression of the HST1 oncogene in human germ cell tumors

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages ]324-]329

September 30, 1988

EXPRESSION

OF

Teruhiko Kiyoshi

THE

HSTI

ONCOGENE

Yoshida,

Masakazu

Tsutsumi,

Miyagawa,

Shinichi

Teshima,

and M a s a a k i

National I-I,

IN HUMAN

Cancer

Tsukiji

CELL

TUMORS

Hiromi

Sakamoto,

Takashi

Sugimura,

Terada

Center

5-chome,

GERM

Research

Chuo-ku,

Institute,

Tokyo

104,

Japan

Received August 2, 1988

HSTI (or HSTFI in h u m a n g e n e n o m e n c l a t u r e ) is a t r a n s forming gene isolated from several cancerous and noncancerous c e l l s . T h e HSTI p r o t e i n is a h e p a r i n - b i n d i n g growth factor with s i g n i f i c a n t h o m o l o g y w i t h h u m a n f i b r o b l a s t g r o w t h f a c t o r s and the mouse Int-2 protein. Here, we report the identification of e x p r e s s i o n of HSTI in a h u m a n t e r a t o m a cell line and in 5 out of 9 s u r g i c a l l y r e s e c t e d h u m a n t e s t i c u l a r g e r m cell t u m o r s i n c l u d i n g seminomas and embryonal carcinomas. M o u s e HSTI h o m o l o g u e was e x p r e s s e d in a c e r t a i n s t a g e of m o u s e e m b r y o but not in p o s t n a t a l mice. ® 1988 A c a d e m i c Press, Inc. SUMMARY:

The gene

HSTI

nomenclature

stomach

cancer

(2). T h i s gene

in

HSTI has

HSTI

protein

some

11,

13).

The of

and

and

was

was

was

assigned

the name

HSTFI

originally

isolated

from

from

and

(FGFs),

genes

frequently

HST1-encoded

a recombinant

found

mouse

are

basic Int-2

located

coamplified

protein

1324

DNA

NIH3T3

cells

and

of

a

cells

in m a n y

and

(3-10).

sequences acidic

protein

at b a n d

vector

in h u m a n

to be a transforming

synthesized

baculovirus

0006-29 lX/88 $1.50 Copyright © 1988 ~ Aca~mic Press, b~c. All r@h~ ~ r ~ r o ~ c t i o n in a~' ~ r m iwserved.

mouse

and g e n o m i c

to h u m a n and

using

noncancerous

its c D N A

homology

INT2

assay

subsequently

cancerous

deduced

factors HSTI

(I),

gene

significant

human

which

by transfection

several

growth

use

gene,

q13

The

(11,

12)

fibroblast

(12).

Both

the

of the c h r o m o -

cancer

cells

in s i l k w o r m purified

(1,9,

cells

by

by h e p a r i n -

Vol. 155, No. 3, 1988

affinity human HST2

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

chromatography

genome

was

contains

a close

(10). T h e K S or K - f g f

formant

induced

identical We

by

to HSTI

report

teratoma

of

germ

human

expressed

DNA

cell

homologue

oncogene

of

mitogen,

of H S T I ,

cloned

a Kaposi's

like

from

sarcoma

FGFs

(14).

The

designated

as

an N I H 3 T 3 turned

out

transto b e

(8, 15).

here

immature

a potent

the

cell

expression line

tumors.

in a c e r t a i n

stage

and

of t h e

in 5 out

Mouse

HSTI

of

gene

in a h u m a n

9 surgical

homologue

of e m b r y o n i c

MATERIALSAND

HSTI

was

specimens

found

to be

development.

METHODS

RNA blot hybridization analysis: P r e p a r a t i o n and e l e c t r o p h o r e t i c s e p a r a t i o n of poly(A)tRNAs, their transfer onto nitrocellulose filters, and hybridization in a b u f f e r c o n t a i n i n g 50% formamide at 42°C w e r e p e r f o r m e d essentially as d e s c r i b e d (16). F i l t e r s w e r e w a s h e d in a b u f f e r c o n s i s t i n g of 0.1x SSC (Ix SSC is 0.15 M N a C I plus 0.015 M s o d i u m citrate) and 0.1% s o d i u m d o d e c y l sulfate at 65°C. O n l y p o l y ( A ) + R N A s w e r e a n a l y z e d , and their q u a n t i t y and q u a l i t y w e r e c h e c k e d by h y b r i d i z a t i o n w i t h a B - a c t i n probe. Germ cell tumors: Fresh testicular germ cell tumors were obtained at t h e t i m e of s u r g e r y at e i t h e r T s u k u b a University Hospital, Ibaragi, J a p a n , or N a t i o n a l Cancer Center Hospital, Tokyo, Japan. Mouse embryos: P r e g n a n t ICR m i c e w e r e p u r c h a s e d to o b t a i n 7day postcoitum (p.c.) c o n c e p t u s , which may include both embryos and extraembryonic membranes, 11-,14-, a n d 1 7 - d a y p.c. e m b r y o s proper, and n e w b o r n mice. Z e r o - d a y p.c. c o r r e s p o n d s to the day on which the seminal plug was found. Depending o n t h e s i z e of t h e embryos, the f o l l o w i n g d i s s e c t i o n s w e r e m a d e b e f o r e p r e p a r a t i o n of p o l y ( A ) + ~ R N A s a m p l e s : 1 1 - d a y p.c. e m b r y o s i n t o h e a d a n d b o d y portions, 1 4 - d a y p.c. e m b r y o s i n t o h e a d , b o d y , a n d l i v e r , 1 7 - d a y p.c. e m b r y o s and n e w b o r n m i c e into brain, lung w i t h heart, liver, colon, kidney, muscle, skin, and the r e m a i n i n g b o d y parts. P r o b e s : A 282 b a s e - p a i r S a c I - A v a I I f r a g m e n t of the p r o t e i n coding region (ORFI) of t h e HSTI c D N A (11), t e r m e d LGC, was employed as a p r o b e to h y b r i d i z e w i t h h u m a n RNAs. T h e L G C p r o b e l a c k s a 5' G C - r i c h r e g i o n of O R F I , a n d a l s o it d o e s n o t c r o s s hybridize with HST2 (data not shown). For the detection of t h e HSTI t r a n s c r i p t in m o u s e RNAs, a g e n o m i c f r a g m e n t of a m o u s e HSTI homologue w a s c l o n e d a n d u s e d as a p r o b e . T h i s is a 1 . ~ k i l o b a s e (kb) E c o R I - E c o R I f r a g m e n t d e s i g n a t e d as MI.8. SS6 is a h u m a n INT2 g e n o m i c f r a g m e n t w h i c h c o n t a i n s an e x o n (Gordon Peters, p e r s o n a l communication).

RESULTS

Figure detected

~

AND

demonstrates

b y t h e HSTI

specific

DISCUSSION

the

presence

probe

1325

LGC

of

HSTI

transcripts

in p o l y ( A ) + R N A s

(3 ~g)

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

2

3

4

5

6

7

8

9

10

11

12

28 S ] ~ -

18 8 ~ "

FIG. I. R N A b l o t a n a l y s i s of R N A s f r o m h u m a n g e r m cell t u m o r s and n o n c a n c e r o u s p o r t i o n s of the testes. E a c h lane contains 3 ~g of poly(A) + RNA. The filter was hybridized with an HSTI specific probe, LGC, and exposed for 5 days with intensifying screen. L a n e s I to 9 c o n t a i n e d R N A s p r e p a r e d f r o m s u r g i c a l specimens of testicular germ cell tumors TTI to TT9, respectively. The pathological diagnoseS are presented in Table I. Lanes 10 and 11 are n o n c a n c e r o u s p o r t i o n s of the t e s t e s in w h i c h t u m o r s TT3 and TT7 d e v e l o p e d , r e s p e c t i v e l y . L a n e 12 is an i m m a t u r e teratoma cell line NCC-IT. The positions of the 28S and 18S rRNAs as d e t e r m i n e d by e t h i d i u m - b r o m i d e s t a i n i n g are i n d i c a t e d by arrowheads.

obtained

from

tumors

of

and

a cell

in

tinal

the

of

HSTI

line,

a faint for

amount

the

5 germ

20

of

presence

cell

are This

HSTI tumor of

germ

diagnosed

as

with

or

26

(not

TT3,

year-old in t u m o r

shown).

3.0 k b

and

which

human

TT6,

the

1.7

also

had

kb

with

HSTI

4.4 a n d

the

be

transcripts.

specimen.

seminomas, without

and

the

seminomas.

1326

other The

variable

do

not

the

3 were

HSTI

among

know

the

Table

stages,

transcripts

S i x o u t of

band

could

in

examined.

of

message

we

tumors

sizes

of

although

HSTI

after

in all

4.8kb

clinical

The

3.0 k b

specimens,

diagnoses,

18).

present

highly

each

a medias-

the

appears

cells

TT9)

evident

transcripts

tumor

and

(17,

was

was

cell

TT7

In NCC-IT,

1.7 k b

germ

from

male TT7

3.0 kb t r a n s c r i p t but

of

established

of the detectable

cell

pure

a

TT2,

was

3.0 kb

pathological

or absence

testicular

carcinomas

days

only,

the

the

at

specimens

codes:

which of

tumors,

percentage

summarizes

band

one.

cell

in T T 3

The

actual

NCC-IT,

teratoma

major

5 germ

9 surgical

(sample

transcripts

the

detected

of

testis

exposure

being the

the

out

immature

presence long

5

and

I the

in the

9 cases

9

were

embryonal

transcripts

were

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I.

Cases of testicular germ cell tumors examined

Sample code

HSTI , expression

TTI TT2 TT3

pure seminoma pure seminoma mixture of: embryonal carcinoma (60%) immature teratoma (30%) yolk sac tumor (<10%) choliocarcinoma (<10%) pure seminoma, anaplastic pure seminoma embryonal carcinoma pure s e m i n o m a pure s e m i n o m a seminoma with embryonal carcinoma

-

+ ++

TT4 TT5 TT6 TT7 TT8 TT9

Stage

Pathological diagnosis

-

-

+ + -

+

A B C

A A B A A A

P r e s e n c e (+) or a b s e n c e (-) of HSTI t r a n s c r i p t s as d e t e r m i n e d by RNA blot analysis is indicated. ++ signifies intense expression. **Staging according to B o d e n and G i b b (21).

detected other

in

germ

portions

the

testes

in

testicular

was

the

no

which and

normal

observed

had

(data

rearrangement

not or

NCC-IT

cell

TT3

and

not

gene

tumors,

3

TT7

iden-

was

not

or n o r m a l

On Southern

amplification , TTI

3 of

embryonal

were

INT2

shown).

cells

in

noncancerous

tissues

The

9 germ

of

are

transcripts

tissues.

line,

from

TN7

cancerous

testicular

in D N A

and

HSTI

and

components

TN3

The

cell

seminomas,

some

which

examined

gross

6 pure

TT9).

from

NCC-IT

tissues

analysis,

of

respectively.

these

expressed

TT7)

tumors TT6

of

in

and

(TT3,

obtained,

tified

gene

(TT2

cell

carcinoma

were

2

of

or TT2

blot

the

HSTI

(data n o t

shown). Expression (Fig.

2).

head

and

cript. days

of

the

mouse

The

body

but

body

of

14-day

The

HSTI

message

not

HSTI head

p.c.

or

in n e w b o r n

mice

RNA

blot

analyses

failed

poly(A) +

RNAs

isolated

from

of

11-day

embryos

could

p.c.

homologue

not (data to

60

p.c.

had

detected

not

shown).

samples

also

HSTI

in embryos

t h e HSTI of

investigated

embryos

a 3.0 k b

be

identify

1327

was

and

transat

transcripts

cancerous

and

the

17

in

noncan-

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

2

3

4

5

6

7

8

9

10

11 12

13

14

28S

18S

FIG. 2. Expression of HSTI homologue in mouse embryos. Four to 5 ~g of poly(A) + RNAs was a n a l y z e d by h y b r i d i z a t i o n w i t h the MI.8 probe, a genomic fragment of a mouse HSTI homologue. Lanes: I, 7-day p.c. conceptus; 3 and 4, head and body of 11-day p.c. embryo, respectively; 5 through 7, head, body, and liver of 14day p.c. embryo, respectively; 8 through 15, brain, lung with heart, liver, colon, kidney, muscle, skin, and r e m a i n i n g body parts of the 17-day p.c. embryo, respectively. The p o s i t i o n s of the 28S and 18S rRNAs are indicated by arrowheads.

cerous

cell

thelial

cells,

leukemic

lines

cervix

We

showed

nic

germ

that

cell

including

members known

to be i n v o l v e d

Int-2 m e s s a g e

the HSTI members

of

HSTI

gene of

the

family,

of s o m e

germ

vein

endo-

person

and a

lung,

frequently

and a c e r t a i n was

basic

stage

liver,

and

embryos

solely

other Two

F G F a n d t h e Int-2 p r o t e i n ,

are

basic

FGF

acts

system

(20).

role

and that

It

embryo can

be

a meso-

cell

lines

preceding suggested

in e m b r y o g e n e s i s HSTI

as

(19), a n d t h e

in the t e r a t o c a r c i n o m a

transcript

embryo-

in m a n y

and n e w b o r n

in

mice.

s t a g e of the m o u s e

family,

expressed

of m o u s e

not detected

in t h e a m p h i b i a n

a specific

cell

a normal

stomach,

in e m b r y o g e n e s i s ;

HSTI

FGF

from

was

s t a g e of m o u s e

is p r e s e n t

plays

gene

expression

"morphogen"

the

of

umbilical

shown).

the p e r i - i m p l a n t a t i o n

appearance

ment

late

leukocytes

cells

but

including

cancers

the

tumor

of t h e F G F

derm-inducing

and

and

(data not

development,

cells

tissues,

fibroblasts,

patient,

uterine

human

and

is i n v o l v e d

the that

as do o t h e r in d e v e l o p -

tumors.

ACKNOWLEDGMENTS

W e t h a n k Drs. Y. S h i m o s a t o , T. K a k i z o e , T. Y a m a m o t o , Y. Mitsui, H. W a t a n a b e , T. O k a m o t o and Urology s t a f f of T s u k u b a U n i v e r s i t y H o s p i t a l a n d N a t i o n a l C a n c e r C e n t e r H o s p i t a l for p r o v i d i n g us cells, tissues, or R N A samples. T h i s w o r k was s u p p o r t e d

1328

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in part by a G r a n ~ i ~ A i d f r o m the M i n i s t r y of H e a l t h and W e l f a r e for a Comprehensive 10-Year Strategy for Cancer Control, Japan. REFERENCES

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