Pergamon ft. Steroid Biochem. Molec. Biol. Vol. 55, No. 1, pp. 25-33, 1995

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Estrogen Induces c - H a - r a s Express ion Via Act ivat ion of Tyrosine Kinase in Uter ine

E n d o m e t r i a l Fibroblasts and Cancer Cells Jiro Fujimoto,* Satoshi Ichigo, Masashi Hori, Shigeo Morishita and

Teruhiko Tamaya Department of Obstetrics and Gynecology, Gifu University School of Medicine, 40 Tsukasa-Machi, Gifu City 500,

Japan

E n d o m e t r i a l f ibroblasts der ived f r o m uter ine e n d o m e t r i u m as controls and endomet r i a l cancer cells (Ishikawa and HHUA cells) were used to analyze the m a n n e r of induct ion of c-Ha-ras t r ansc r ip t s in endome t r i a l cancers, some of which are es t rogen-dependent in growth. Es t rogen increased c-Ha-ras expression and tyros ine kinase (TK) act ivi ty in f ibroblast and Ishikawa cells, but not in HHUA cells. P roges te rone d imin i shed c-Ha-ras expression and tyrosine kinase (TK) act ivi ty induced by es t radiol in the fibroblasts, but not in Ishikawa cells, which pers is tent ly overexpressed c-Ha-ras. In these cells, ep ide rma l growth fac tor (EGF) increased c-Ha-ras expression as did estradiol . P r e t r e a t m e n t with tyrphos t in , an inhib i tor of TK, abolished es t rogen- inducible overex- pression of c-Ha-ras. The combina t ion of both estradiol and EGF at m a x i m u m effective concen- t r a t ion exer ted no addi t ive or synergist ic effect on induct ion of c-Ha-ras expression. In conclusion, pers is tent ac t ivat ion of TK migh t lead to overexpression of c-Ha-ras in some endomet r i a l cancer cells under es t rogen p r e d o m i n a n t mil ieu, which migh t be associated with the t r a n s f o r m a t i o n or g rowth potent ial .

J. Steroid Biochem. Molec. Biol., Vol. 55, No. 1, pp. 25-33, 1995

INTRODUCTION

Estrogen is an important factor in supporting the growth, development, and differentiation of uterine endometrium from clinical evidences. Among estrogen effects on the endometrium, the growth of en- dometrium is critically related to estrogen status. Es- trogen induces the expression of genes such as c-los [1, 2], c-jun [3, 4], c-myc [5], c-Ha-ras [6, 7], and genes for EGF [8,9], EGF receptor [10-12] and estrogen receptor in mammalian uterus. Oncoproteins among estrogen-inducible proteins in human uterine endo- metrial cancers might be, at least in part, related to the transformation activity and the growth of endometrial neoplasia.

To determine the contribution made by ras tran- script to transformation or growth potential in endo- metrial cancers, we investigated the regulatory manner of one oncogene transcript, c-Ha-ras mRNA, under estrogen/progesterone predominant milieu in endo-

*Correspondence to J. Fujimoto. Received 7 Nov. 1994; accepted 24 Apr. 1995.

metrial cancer cells, such as Ishikawa [13] and HHUA cells [14], which are estrogen-dependent for growth. Endometrial fibroblasts derived from uterine en- dometrium, as a substitute for normal endometrial stroma, were used as a control. Moreover, they were investigated with reference to activation and regulation of protein kinases (tyrosine kinase, protein kinase A, C and calmodulin kinase).

MATERIALS AND METHODS

Chemicals

Estradiol-17fl, progesterone 8-(4-chlorophenyl- thio)-cAMP (CAMP), forskolin (FORS), cholera toxin (CT), 1-methyl-3-isobutyl-xanthine (MIX) and 12-O- tetradecanoylphorbol-13-acetate (TPA) were pur- chased from Sigma Chemical Co. (St Louis, MO, U.S.A.). One-(5-isoquinolinesulfonyl)-2-methylpiper- azine dihydrochloride (H-7) was obtained from Seika- gaku Corp. (Tokyo, Japan). Epidermal growth factor (EGF) was obtained from Austral Biologicals (San Ramon, CA, U.S.A.). Tyrphostin (TYR) was

25

26 Jiro Fujimoto et al.

purchased from Gibco BRL. (Gaithersburg, MD, U.S.A.). All other reagents were of experimental grade.

Cell culture

Human endometria were obtained by endometrial biopsy from 6 patients (23-39 years old) with a regular menstrual cycle at the Department of Obstetrics and Gynecology, Gifu University School of Medicine, from March 1993 to December 1993. Informed consent was obtained from all patients. The endometrium was washed with phosphate buffer solution containing am- photelicin B (1.5 #g/ml) and kanamycin (60/~g/ml), and minced with a scalpel. The minced tissue, in Eagle's MEM (Nissui Pharm., Tokyo, Japan) with 0.25% trypsin, was blended for 20min and then pipetted to make a cell suspension. After addition of fetal bovine serum (FBS; Gibco BRL.) to the cell suspension to inactivate trypsin, the dispersed cells were recovered. The cells were cultured in MEM with amphotericin B and kanamycin overnight, and then the floating cells were discarded. After the 13th to 14th passage, spindle-shaped cells usually grew in a sheet- like manner, and occupied a whole square of the culture flask. Enriched collagen type I was detected in these spindle cells immunohistochemically, and thus they were considered to be fibroblasts from the en- dometrium.

Fibroblasts were cultured in 90% Eagle's MEM and 10% FBS. Afterwards, the culture was allowed to proceed in Eagle's MEM alone for 48 h, and then individual final concentrations of 10-12--10 -6 M estra- diol, 10-12-10-6M progesterone, 0 .5mM CAMP, 3 ~tM FORS, 50 nM CT, 0.5 mM MIX, 1 ~M TPA, 25 mM KC1, and 20/~M H-7 were settled in culture dishes.

Endometrial cancer cell lines of Ishikawa [13] and HHUA [14] were cultured in 90% RPMI 1640 (Nissui Pharm.) and 10% FBS, and in 85% Eagle's MEM and 15% FBS, respectively. The culture medium was changed to the same MEM without FBS 48 h before the addition of estrogen and progesterone to cells in culture dishes.

Reverse transcription-polymerase chain reaction (RT- PCR) to amplify c-Ha-ras transcripts

Total RNA was isolated from the cells by the acid guanidium thiocyanate-phenol-chloroform extraction method [15]. Total RNA (3/~g) was reverse transcribed with Moloney murine leukemia virus reverse transcrip- tase (MMLV-RTase, 200 units, Gibco BRL.) in 20mM Tris-HC1, pH8.4, 50mM KC1, 2 .5mM MgCI2, 0.1mg/ml bovine serum albumin. 10mM dithiothreitol (DTT), and 0.5 mM deoxynucleotides to generate cDNAs using random hexamers (50 ng, Gibco BRL.) at 37°C for 60min. The RT reaction was heated at 94°C for 5 min to inactivate MMLV- RTase.

Ten cycles of PCR for c-Ha-ras mRNA alone [16], consisting of 1 min at 94°C for denaturation, 1 min at 55°C for annealing, and 1 min at 72°C for extension, were carried out with reverse transcribed cDNAs and 0.1/~M specific primers using IWAKI thermal se- quencer TSR-300 (Iwaki Glass, Tokyo, Japan) with Vent DNA polymerase (New England Biolabs, Beverly, MA, U.S.A.) in 10mM KC1, 20mM Tris-HCl pH 8.8, 10 mM (NH4) 2 SO4, 2 mM MgSO4, 0.1% Triton X-100 and 0.15mM deoxynucleotides phosphates. Then an additional twenty cycles of PCR for c-Ha-ras mRNA and glyceraldehyde-3-phosphat- edehydrogenase (GAPDH, a housekeeping gene) mRNA [17] as an internal standard were performed in the same manner as described before in the same tube. The following oligodeoxynucleotides were synthesized and used as a specific primer in PCR: sense for c-Ha-ras : 5 ' -ATGACGGAATATAAGCTGGT-3 ' , antisense for c-Ha-ras: 5 ' - C T G T A C T G G T G G A T - GTCCTC-3 ' , sense for GAPDH: 5 ' - T G A A G G T C G - G A G T C A A C G G A T T T G G T - Y , and antisense for GAPDH: 5 ' - C A T G T G G G C C A T G A G G T C C A C - CAC-3'.

Analysis of quantities of c-Ha-ras expressions by Southern blot in PCR products

PCR products were applied to 1.2% agarose gel, and electrophoresis was performed at 50-100 V. PCR prod- ucts were capillary-transferred to Immobilon transfer membrane (Millipore Corp., Bedford, MA, U.S.A.) for 16 h. The membrane was dried at 80°C for 30 min, and was UV-irradiated to fix PCR products tightly. PCR products on the membrane were prehybridized in 1 M NaC1, 50 mM Tris-HC1, pH 7.6, 1 o/o SDS, at 42°C for 1 h, and hybridized in the same solution with the biotinylated oligodeoxynucleotides probes synthesized from the sequences of c-Ha-ras between the specific individual primers at 65°C overnight. Specific bands hybridized with the biotinylated probes were detected with Plex Luminescent Kits (Millipore Corp., Bedford, MA, U.S.A.), and the membranes were exposed X-ray films at room temperature for 10 min. The quantifi- cation of Southern blot was carried out with Bio Image (Millipore Corp., Ann Arbor, MI, U.S.A.). The inten- sity of specific bands was normalized with that of GAPDH mRNA.

Tyrosine kinase (TK) activity assay

All steps were carried out at 4°C, except where indicated. The cells (10-20 mg) were homogenized in HG buffer (5mM Tris-HCl, pH 7.4, 5 m M NaC1, 1 mM CaC12, 2 mM ethyleneglycol-bis-[fl-aminoethyl ether]-N,N,N',N'-tetraacetic acid, 1 mM MgC12, 2 mM D T T , 25 #g/ml aprotinin, 25 #g/ml leupeptin) by a Polytron homogenizer (Kinematics, Luzern, Switzerland). This suspension was centrifuged in a microfuge at 12,000rpm for 3min to remove the nuclear pellet. The protein concentration of samples

Estrogen Induces c-Ha-ras Expression 27

was measured by the method of Bradford [18] to standardize T K activity. TK activity in samples was measured with 10/~Ci/sample of [y-32p]ATP (3000-6000 Ci/mmol) by the Protein Tyrosine Kinase Assay System (Gibco BRL.).

RESULTS

Dose-response curve of estradiol for effects on c-Ha-ras expression in endometrial fibroblasts and cancer cells

The expression c,f c-Ha-ras mRNA in endo- inertial fibroblasts and endometrial cancer cells were analyzed by RT-PCR-Southern blot (RT-PCR-SB) [Fig. I(A)]. The level of c-Ha-ras mRNA was stan- dardized with that of glyceraldehyde-3-phosphate de- hydrogenase (GAPDH) mRNA, and designated as an arbitrary unit (AU)/GAPDH mRNA.

The level of c-Ha-ras mRNA in endometrial fibro- blasts and Ishikawa .:ells significantly (P < 0.05) in- creased by treatment: with up to 10-8M estradiol for 12 h, but not in HHUA cells [Fig. I(B)]. Therefore, the concentration of estradiol required to achieve opti- mum c-Ha-ras expression appeared to be 10-SM [Fig. I(B)].

Dose-response curve of progesterone for effects on estra- diol-induced c-Ha-ras expression in endometrial fibro- blasts and cancer cells

The expression of c-Ha-ras mRNA was analyzed by RT-PCR-SB [Fig. 2(A)]. The estradiol-induced ex- pression of c-Ha-ras mRNA in endometrial fibroblasts was significantly (P < 0.05) diminished by treatment with up to 10-6M progesterone for 12 h, but not in Ishikawa nor in HHUA cells [Fig. 2(B)]. Therefore, the concentration of progesterone required to achieve maximum inhibition of c-Ha-ras expression appeared to be 10 -6 M, based on progesterone solubility in the culture medium.

Time-course for effects of estradiol and~or progesterone on c-Ha-ras expression in endometrial fibroblasts and cancer cells

The expression of c-Ha-ras mRNA was analyzed by RT-PCR-SB [Fig. 3(A)]. The level of c-Ha-ras mRNA in endometrial fibroblasts and Ishikawa cells was significantly (P < 0.05) increased by treatment with estradiol, and reached a plateau in 12 h, but not in HHUA cells [Fig. 3(B)]. The estradiol-induced expression of c-Ha-ras mRNA was significantly

(A)

Fibroblast Ishikawa HHUA

E 2conc. (M) C I0-1210 -I° I0 -8 I0 -6 C I0-1210 -1° 10 -s 10 -6 C I0-1210 -I° 10 -s 10 -6

c-.a-,os 29o

Z ,',' 200

r~ 180

~.~ 160

"-d 140

~ 120 Z E 100

i 80

D (B)

-- ~ . ~ Ishikawa

_ ~ Fibroblast

I I I I 10-12 10-10 10 -8 10 -6

Concentrat ion (M)

Fig. 1. D o s e - r e s p o n s e c u r v e showing the effects of e s t r a d i o l o n c - H a - r a s e x p r e s s i o n in e n d o m e t r i a l f i b r o b l a s t s a n d c a n c e r ce l l s . E n d o m e t r i a l f i b r o b l a s t s , I s h i k a w a a n d H H U A c e l l s w e r e i n c u b a t e d with 10-12-10-6M e s t r a d i o l in Eagle's MEM medium for 12 h. Total R N A w a s i s o l a t e d f r o m t h e m , a n d R T - P C R - S B A w a s c a r r i e d out (A). The level of c -Ha- ras m R N A w a s s t a n d a r d i z e d with that of glyceraldehyde-3-phosphate d e h y d r o g e n - a s e ( G A P D H ) m R N A , a n d d e s i g n a t e d as a n a r b i t r a r y u n i t ( A U ) / G A P D H m R N A . D a t a a r e t h e m e a n _ SD of

6 determinations (B). Ez, estradiol-17~.

28

(A)

Fibroblast

P conc. (M) C 10 -1° 10 -8 10 -~

GAPDH (983 bp)

Jiro Fujimoto et al.

Ish ikawa

C 10 -1° 10 -8 10 -6

HHUA

C 10 -1° 10 -s 10 -6

< Z

E ) T

r~

o 80

< 40 -- Z

E 2 0 -

0 10 -10 0 8 :~ 1 10 -6

Concentra t ion (M)

HIIUA

Ishikawa

Fibroblast

I

Fig. 2. D o s e - r e s p o n s e c u r v e s h o w i n g t h e e f f ec t s of p r o g e s t e r o n e o n e s t r a d i o l - i n d u c e d c -H a- ras e x p r e s s i o n in

e n d o m e t r i a l f i b r o b l a s t s a n d c a n c e r cel ls . A l l ce l l s w e r e i n c u b a t e d w i t h 10 -8 M e s t r a d i o l a n d / o r 10-~°-10 -6 M

p r o g e s t e r o n e in E a g l e ' s M E M m e d i u m f o r 12 h. R T - P C R - S B A w a s c a r r i e d o u t (A). T h e l eve l o f c -Ha-ras m R N A w a s d e t e r m i n e d as d e s c r i b e d fo r Fig. 1. D a t a a r e t h e m e a n + SD o f 6 d e t e r m i n a t i o n s (B). P,

p r o g e s t e r o n e .

(P < 0.05) diminished by progesterone in the fibro- blasts, but not in Ishikawa nor H H U A cells [Fig. 3(B)].

Effects of estradiol, E G F and T Y R on c-Ha-ras ex- pression in endometrial fibroblasts and cancer cells

The expression of c-Ha-ras mRNA was analyzed by R T - P C R - S B [Fig. 4(A)]. The level of c-Ha-ras ex- pression in endometrial fibroblasts and Ishikawa cells significantly (P < 0.05) increased for 12 h with treat- ment with E G F and/or estradiol, but not in H H U A cells [Fig. 4(B)]. The combination of E G F and estra- diol exerted no additive or synergistic effect on c-Ha- ras expression in the former two cells [Fig. 4(B)]. 1 h pretreatment with T Y R significantly (P < 0.05) dimin- ished the increase induced by either estradiol or E G F in the fibroblasts and Ishikawa cells, but not in H H U A cells [Fig. 4(B)].

Effects of estradiol, C A M P , F O R S , CT, M I X , T P A and KCl on c-Ha-ras expression in endometrial fibro- blasts and cancer cells

The expression of c-Ha-ras mRNA was analyzed by R T - P C R - S B [Fig. 5(A)]. The level of c-Ha-ras ex- pression was not affected by CAMP, FORS, CT, MIX, T P A and KC1, except for estradiol, in all three types of cells [Fig. 5(B)].

Effects of estradiol and~or progesterone on T K activity in endometrial fibroblasts and cancer cells

T K activity was significantly (P < 0.05) increased in endometrial fibroblasts and Ishikawa cells by treatment with estradiol for 2 h, and persisted to some extent, but transiently in H H U A cells for 6 h (Fig. 6). Th e estra- diol-induced T K activity was significantly (P < 0.05) diminished by progesterone in fibroblasts and H H U A cells, but not in Ishikawa cells (Fig. 6).

DISCUSSION

Estrogen induces c-Ha-ras expression in rodent uterus [6, 7]. In human uterine endometrium, the expression of c-Ha-ras is higher at the proliferative phase than at the secretory phase of the menstrual cycle [19]. Trea tment with estradiol dipropionate signifi- cantly elevated the expression of c-Ha-ras m RNA in the endometr ium of the human subject [19]. Endo- metrial fibroblasts derived from uterine endometr ium as a substitute for normal endometrial stroma were used as controls and endometrial cancer cells were used to analyze the induction manner of c-Ha-ras tran- scripts in endometrial cancers, some of which are estrogen-dependent in growth. Th e expression of c- Ha-ras in the fibroblasts was significantly increased by

Estrogen Induces c -Ha-ras Expression 29

GAPDH (983 bp) E2 c-Ha.-ras (290 bp)

(A)

Fibroblast Ishikawa HHUA

C 3 6 12 24 C 3 6 1224 C 3 6 1224

GAPDH (983 bp) E2+ P c-Ha--ras (290 bp)

D D t D Q g l D , / P l S l t ~ a l t ,

GAPDH (983 bp) I I D 4 1 P B O D ~ I I I l ~ D q l P a D P c-Ha--ras (290 bp) . . . . .,~ . , , m . , , I m ~ am ,,=

180 - -

( B )

160 - ~ E2

140

.2 + P

~ 1 O0

E 80 I I t ' ~ 1 8 0 - Ishikawa T

< E2+ P ~ 160 <

~ 140 --

~ 120

~, l oo - p k. I

I I ~l 180 tltlUA

160 i 140

120

100 E 2 + P P

80 0 10 2O

Time (h)

Fig. 3. T ime-course for effects of estradiol and /or progesterone on c -Ha-ras expression in endomet r i a l f ibroblasts and cancer cells. All cells were incubated with 10-8M estradiol and/or 10 - 6 M proges terone in Eagle 's MEM m e d i u m for var ious periods of t ime. R T - P C R - S B A was car r ied out (A). The level of c -H a- ras

m R N A was de te rmined as descr ibed for Fig. 1. Data are the mean 4- SD of 6 de te rmina t ions (B).

GAPDII (983 bp) Fibroblast

c - t t a - ras (290 bp)

(A)

i ii O

o

Ishikawa

GAPDIt (983 bp)

c -H a- ras (290 bp)

. . . . . . . . . . . . . . . O :

HItUA

GAPDH (983 bp)

c -H a- ras (290 bp)

E z

EGF

TYR

+ - - + - - + - -

- - + + - - - - +

_ - - _ + + +

200

I00

Z

E

200 -- .<

.<

"~ 100

Z

E

' t 200

100 L

0 n

E 2 EGF TYR

(B)

Fibroblast

lshikawa

HIBJA

+ - - + - - + - -

- - + + - - - - +

- - - - - - + + +

Fig. 4. Effects of estradiol , EGF and TYR of c - H a - r a s express ion in endomet r i a l f ibroblasts and cancer cells. All cells were incuba ted with 10 -s M estradiol , I ng /ml EGF, and 15 pM TYR in Eagle 's MEM m e d i u m for 12 h. R T - P C R - S B A was ca r r i ed out (A). The level o f c - H a - r a s mRNA was d e t e r m i n e d as descr ibed for Fig. 1. Data

are the mean _+ SD of 6 de t e rmina t ions (B). EGF, ep ide rma l growth factor; TYR, tyrphos t in .

Estrogen Induces c-Ha-ras Expression

(A)

GAPDH (983 bp) Fibroblast

c - H a - r a s (290 bp) I / ~ m l l t m m m

31

Ishikawa

GAPDH (983 bp)

c - H a - r a s (290 bp)

HHUA GAPDH (983 bp)

c - H a - r a s (290 bp)

C E 2 CAMP FORS CT MIX TPA KCI

(B)

200 --

100

.¢ Z

2o."

<

• .~ 100

o

Z fl

, 200

Fibroblast

Ishikawa

HHUA

C E 2 CAMP FORS CT MIX TPA KCI

Fig. 5. Effects of es t radiol , CAMP, FORS, CT, MIX, TPA, and KCI on c - H a - r a s express ion of h u m a n endome t r i a l f ibroblas ts and cancer cells. All cells were incuba ted with 10 -8 M estradiol , 0.5 m M CAMP, 3/~M FORS, 50 nM CT, 0.5 m M MIX, 1/ iM TPA, and 25 m M KCI in Eagle 's M E M m e d i u m for 12 h. R T - P C R - S B A was ca r r i ed out (A). The level of c - H a - r a s mRNA was d e t e r m i n e d as descr ibed for Fig. 1. Data are the m e a n _ SD of 6 de t e rmina t i ons (B). CAMP, 8- (4-chlorophenyl - th io) -cAMP; FORS, forskolin; CT, cholera

toxin; MIX, 1-methyl -3- i sobuty l -xanth ine ; TPA, 12-O- te t radecanoylphorbol -13-ace ta te .

32 Jiro Fujimoto et al.

SO f Fibro~ast

~ol 30

20 10

t - o ,

G) 0 i._ 5o Q. 0") 4o.

E = 30"

.c_

E2

Ishikawa E2

E2+P

E 20 " ~

0

• ~ S0' HHUA

zo E2

lO

o

1 2 3 4 6 12

Time (hours)

Fig. 6. T ime-course for effects of estradiol and/or progesterone on TK activity of h u m a n endometr ia l f ibroblasts and cancer cells. All cells were incubated with 10 -s M estradiol and /o r 10 -6 M proges te rone in Eagle's MEM m e d i u m for various periods of t ime. TK activity in the cells was measured with 10 pCi / sample of [V-32P] ATP by the Tyrosine Kinase Assay Sys tem Kit (Gibco BRL. Gai thersburg , MD, U.S.A.). Data are the

m e a n +__ SD of 6 de te rmina t ions .

estradiol, while progesterone significantly diminished it. On the other hand, in Ishikawa cells, which are estrogen-dependent in growth, the expression induced by estrogen was not significantly diminished by pro- gesterone. In the control, c-Ha-ras expression is regu- lated positively by estrogen, and negatively by pro- gesterone in the physiological state.

c-Ha-ras gene encodes ras p21 (RAS) among low molecular guanosine-5'-triphosphate (GTP) binding proteins [20,21]. The exchange of guanosine-5'- diphosphate (GDP) to GTP in the complex with RAS by exogenous stimulants can be evaluated as a biologi- cal activity of RAS. The functionally activated RAS is related to cellular growth, differentiation, and trans- formation as follows. EGF and platelet derived growth factor (PDGF) increase the ratio of RAS-GTP to RAS-GDP in proliferative Swiss 3T3 cells [22, 23]. Macrophage-colony stimulating factor (M-CSF) and

insulin increase the ratio of RAS-GTP to RAS-GDP, overexpressing their corresponding receptor in those cells with growth [23, 24]. In NIH3T3 cells trans- formed with src, abl and erbB-2, the ratio of RAS-GTP to RAS-GDP increases without any stimulant [23, 25]. Therefore, RAS activated by TK, such as receptor- type T K (erbB-2 coding protein, and the receptors for EGF, PDGF, M-CSF and insulin), and non-receptor- type T K (coding proteins of src and abl), is considered to be a transducer for cellular growth. The persistent activation of RAS might contribute, in part, to cellular transformation. In addition, PKC in T cells activated by lectin or anti-CD antibody seems to inhibit GT- Pase-activating-protein activity, resulting in enhanced ratios of RAS-GTP to RAS-GDP [26].

T K activity in endometrial fibroblasts was increased by estradiol, and this activity was diminished by pro- gesterone. In Ishikawa cells, the activity was increased

Estrogen Induces c -Ha-ras Expression 33

by es t rogen, the increase o f wh ich was not s ignif icantly

d imin i shed by p roges te rone . P roges t e rone regu la t ion

m i g h t be lost in I sh ikawa cells. In H H U A cells, T K

act iv i ty was increased t rans ien t ly by estradiol , h o w -

ever, exer t ing no effect on induc t ion o f c - H a - r a s ex-

pression. T h e r e f o r e , pers i s ten t ac t iva t ion o f T K is

needed to induce c - H a - r a s express ion. In endomet r i a l

f ibroblasts and I sh ikawa cells, E G F , an ac t iva tor o f

T K , increased c - H a - r a s express ion as did estradiol .

P r e t r e a t m e n t wi th T Y R , an inh ib i to r o f T K , inh ib i t ed

the e s t rogen - induc ib l e ove rexpress ion o f c - H a - r a s .

T h e c o m b i n a t i o n o f bo th es t radiol and E G F at maxi -

m u m effective concen t ra t ion exer ted no addi t ive or

synergis t ic effect on incluct ion o f c - H a - r a s express ion.

T h e r e f o r e , es t rogen leads to pers i s ten t overexpress ion

o f c - H a - r a s via T K act iva t ion in t r ans fo rmed cells.

On the o ther hand, p ro te in kinase A ( P K A ) act iva-

tors ( C A M P , F O R S , C T , and M I X ) , a P K C act iva tor

( T P A ) , and a ca lmodu l in kinase ac t iva tor (KC1) did not

affect c - H a - r a s express ion in endomet r i a l f ibroblasts ,

I sh ikawa cells and H H U A cells. T h e r e f o r e , P K A , P K C

and ca lmodu l in kinase do not l ink di rec t ly to c - H a - r a s

express ion in these cells.

In conclus ion , pers i s ten t ac t iva t ion o f T K leads to

ove rexpress ion o f c - H a - r a s u n d e r es t rogen p r e d o m i -

nant mi l i eu in some endomet r i a l cancers, in wh ich

proges ta t iona l regula t ion m i g h t be lost. T h e s e migh t

be associated wi th the ma in t enance o f t r ans fo rming

activi ty.

R E F E R E N C E S

1. Loose-Mitchell D. S., Chiappetta C. and Stancel G. M.: Estro- gen regulation of c-fos messenger ribonucleic acid. Molec. Endocr. 2 (1988) 946-951.

2. Weisz A. and Bresciani F.: Estrogen induces expression of c-los and c-myc protooncogene in rat uterus. Molec. Endocr. 2 (1988) 816-824.

3. Weisz A., Cicatiello L., Persico E., Scalona M. and Bresciani F.: Estrogen stimulates transcription of c-jun protooncogene. Molec. Endocr. 4 (1990) 1041-1£150.

4. Webb D. K., Bruce C. M. and Khan S. A.: Estrogen induced expression of the c-jun proto-oncogene in the immature and mature rat uterus. Biochem. Biophys. Res. Commun. 168 (1990) 721-726.

5. Murphy L. J., Murphy L. C. and Fresen H. G.: Estrogen induction of N-myc and c-myc proto-oncogene expression in the rat uterus. Endocrinology 120 (1987) 1882-1888.

6. Travers M. T. and Knowler J. T.: Oestrogen-induced ex- pression of oncogenes in the immature rat uterus. F E B S Lett. 211 (1987) 27-30.

7. Cheng S. V. Y. and Polland J. W.: C-ras H and ornithine decarboxylase are induced by oestradiol- 17fl in the mouse uterine luminal epithelium independently of the proliferative status of the cell. F E B S Lett. 196 (1986) 309-314.

8. DiAugustine R. P., Petrusz P., Bell G. I., Brown C. F., Korach K. S., McLachlan J. A. and Teng C. T.: Influence of estrogen

on mouse uterine epidermal growth factor precursor protein and messenger ribonucleic acid. Endocrinology 122 (1988) 2355-2363.

9. Huet-Hudson Y. M., Chakraboory C., De S. K., Suzuki Y., Andrews G. W. and Dey S. K.: Estrogen regulates the synthesis of epidermal growth factor in mouse uterine epithelial ceils. Molec. Endocr. 4 (1990) 510-523.

10. Mukku V. A. and Stancel G. M.: Regulation of epidermal growth factor receptor by estrogen, ft. Biol. Chem. 260 (1985) 9820-9824.

11. Lingham R. B., Stancel G. M. and Loose-Mitchell D. S.: Estrogen regulation of epidermal growth factor receptor messen- ger ribonucleic acid. Molec. Endocr. 2 (1988) 230-235.

12. Reynolds R. K.: Regulation of epidermal growth factor and insulin-like growth factor I receptors by estradiol and progester- one in normal and neoplastic endometrial cell cultures. Gynec. Oncol. 38 (1990) 396-406.

13. Nishida M., Kasahara K., Kaneko M. and Iwasaki H.: Establish- ment of a new human endometrial adenocarcinoma cell line, Ishikawa ceils, containing estrogen and progesterone receptors. Acta Obstet. Gynec. ffpn. 37 (1985) 1103-1110.

14. Ishiwata I. and Ishiwata C.: Effect of estradiol-17fl and pro- gesterone on cell proliferation and differentiation of the human endometrial carcinoma cell line (HHUA) in vitro. Asia-Oceania J. Obstet. Gynaec. 10 (1984) 531-537.

15. Chomczynski P. and Sacchi N.: Single-step method of RNA isolation by acid guanidium thiocyanate--phenol-chloroform ex- traction. Analyt. Biochem. 162 (1987) 156-159.

16. Capon D., Chen E. Y., Levinson A. D., Seeburg P. H. and Goeddel D. V.: Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature 302 (1983) 33-37.

17. Arcari P., Martinelli R. and Salvatore F.: The complete sequence of a full length cDNA for human liver glyceraldehyde-3-phos- phate dehydrogenase: evidence for multiple mRNA species. Nucl. Acids Res. 12 (1984) 9179-9189.

18. Bradford M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72 (1976) 315-323.

19. Fujimoto J., Hori M., Ichigo S., Nishigaki M. and Tamaya T.: Tissue difference in expression of mRNA of Ha-ras, c-myc, fos and jun among human uterine endometrium, myometrium and leiomyoma under the estrogen/progesterone effects. Tumor Biol. 15 (1994) 311-317.

20. Barbacid M.: ras GENES. A. Rev. Biochem. 56 (1987) 779-827. 21. Yamamoto T., Yamamoto K., Kikuchi A. and Takai Y.: Small

molecular weight GTP-binding proteins and signal transduction. Clin. Chim. Acta 185 (1989) 347-356.

22. Satoh T., Endo M., Nakafuku M., Nakamura S. and Kazireo Y.: Platelet-derived growth factor stimulates formation of active p21r~GTP complex in Swiss mouse 3T3 ceils. Proc. Natn. Acad. Sci. U.S.A. 87 (1990) 5993-5997.

23. Gibbs J. B., Marshall M. S., Scolnick E. M., Dixon R. A. and Vogel U. S.: Modulation of guanine nucleotides bound to ras in NIH3T3 cells by oncogenes, growth factors, and the GTPase activating protein (GAP). J. Biol. Chem. 265 (1990) 20,437-20,442.

24. Burgering B. M. T., Medema R. H., Maassen J. A., van de Wetering M. L., van der Eb A. J., McCormick F. and Bos J. L.: Insulin stimulation of gene expression mediated by p21ras activation. EMBO ft. 10 (1991) 1103-1109.

25. Satoh T., Endo M., Nakafuku M., Akiyama T., Yamamoto T. and Kaziro Y.: Accumulation of p2Va~GTP in response to stimulation with epidermal growth factor and oncogene products with tyrosine kinase activity. Proc. Natn. Acad. Sci. U.S.A. 87 (1990) 7926-7929.

26. Downward J., Graves J. D., Warne P. H., Rayter S. and Cantrell D. A.: Stimulation of p2V ~ upon T-cell activation. Nature 346 (1990) 719-723.