li)sicologr Lelrers, 51 (1990) 7-l 2

Elsevier

7

TOXLET 02293

Effects of activators and inhibitors of protein kinase C on two-stage transformation in BALB/3T3 cells

Mari Semba’ and Naomichi Inui2

s Laboratory qf Ceil Touicolog_y, Hatatzo Re.reurch institute, Food and Drug Safe@ Center. 729-S. Ochiai.

Hamno, Kana~a~~~ 2.57 and 2 P~~u~~lac~iical Re.~ea~c~~ Lab[}r~t~~i~s. Japan Tobacco Inc., 6-2. U~e~~~~~a.

Midori-ku. Yokokanra. Kanqawa 227 (Japan j

(Received 8 May 1989)

(Accepted 25 September 1989)

fG,r words: TPA; Protein kinase C; Two-stage transformation; BALB/3T3 cells

SUMMARY

To determine the relationship between protein kinase C and the promotion of carcinogenesis, we inves-

tigated the effects of activators and inhibitors of protein kinase C on two-stage transformation in BALB/

3T3 cells. Diacylglycerols, which are activators. and specific inhibitors. such as palmitoyl-AL-~rnitine

chloride (PC), I-(5-isoquinolinesulfonyl)-2-methyipiperazine dihydrochloride (H-7). and staurosporine

(ST) were used. Treatment with diacylglycerols enhanced focus formation in 3methylcholanthrene

(3MC)-initiated cells, but not as much as 12-O-tetradecanoylphorbol-13-acetate (TPA). PC and H-7 inhi-

bited TPA-enhanced transformation by 76 and 79%. respectively. ST, the most potent inhibitor of protein

kinase C. had a low inhibitory effect on transformation at non-toxic doses (33% inhibitions. The results

suggest that protein kinase C may play an important role in the process by which transformation is pro-

moted in BALB/3T3 cells.

INTRODUCTION

lo-~-Tetradecanoylphorboi-13-acetate (TPA) is the most potent mouse skin

tumor promoter among a series of phorbol esters isolated from croton oil [l]. The

exact biochemical mechanism by which TPA promotes tumors is not well under-

stood, but it is assumed that TPA acts via binding to its receptor 121, and available

evidence indicates that protein kinase C is a receptor for TPA [3]. Protein kinase C

forms part of the signal transducing sytem involving the turnover of inositol phos-

Address for correspondence: Mari Semba, Laboratory of Cell Toxicology. Hatano Research Institute,

Food and Drug Safety Center, 729-5, Ochiai, Hatano, Kanagawa 257, Japan.

0378-4274~90~~ 3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedi~l Division)

8

phohpid and is thought to be activated by diacylglycerol, which is produced as a by-

product of this turnover. TPA appears to interact at the same site as diacylglycerol

[4] and can substitute for the intracellular messenger diacylglycerol to activate pro-

tein kinase C [5].

Protein kinase C has not been proven to play an indispensable role in tumor pro-

motion. Inhibitors of protein kinase C, such as palmitoylcarnitine. have been shown

to inhibit skin tumor promotion by TPA [6]. Diacylglycerols are endogenous activa-

tors of protein kinase C and mimic many effects of TPA [7]. Recently, Verma [8]

showed that dioctanoylglycerol is a potent Stage II mouse skin tumor promoter.

However. there are no reports concerning activators and inhibitors of protein ki-

nase C in vitro. Previously, we reported that radical scavengers and inhibitors of

phospholipid metabolism inhibit TPA-enhanced transformation in BALB/3T? cells

[9-l 11. To determine the involvement of protein kinase C in TPA promotion, we eval-

uated the effects of activators and inhibitors of protein kinase C on two-stage trans-

formation in vitro.

MATERIALS AND METHODS

12-O-Tetradecanoylphorbol-13-acetate (TPA) diolein. 1 -oleoyl-2-acetylglycerol

(OAG), and parmitoyl-DL-carnitine chloride (PC) were purchased from Sigma Chemi-

cal Co. Ltd., St. Louis. MO; I-(5isoquinolinesulfonyl)-2-methylpiperazine dihydro-

chloride (H-7) was obtained from Seikagaku Kogyo Co. Ltd.. Tokyo; and stauro-

sporine (ST) came from Kyowa Medex Co. Ltd., Tokyo. 3-Methylcholanthrene

(3-MC) was purchased from Tokyo Chemicals Industry Co. Ltd.. Tokyo.

BALB/3T3 A3 1 -l- 1 cloned cells were grown in minimum essential medium

(MEM), Nissui Pharmaceutical Co. Ltd.. Tokyo, supplemented with 10% fetal calf

serum (FCS, Bock Neck. Canada). Cells were plated on 60-mm dishes (10’ cells/dish)

and treated with the compounds on the following day. Ten days later the cells were

fixed and stained with Giemsa solution. Colonies containing more than 50 cells were

counted. The plating efficiency was expressed as the ratio to the control.

Trumformation u.ssu~~ Cells were plated onto 60-mm dishes ( lo4 cells/dish). The following day, and for

3 days thereafter, the cells were treated with 0.5 pug/ml of 3-MC as an initiator. Four

days after the 3-MC was removed, the cultures were exposed to both 0.1 /lg/ml of

TPA and the inhibitors simultaneously or just the activators singly for 3 weeks. The

cells were cultured in the normal medium for 3 successive weeks and transformed

foci were counted according to the criteria described by Reznikoff et al [ 121.

9

TABLE I

CYTOTOXICITY OF INHIBITORS OF PROTEIN KINASE C IN BALB/3T3 CELLS

Treatment Plating efficiency ($ of control)

PC (1 &ml) 96

PC (I 0 pgjml) 87

PC (25 pg!ml) 55

PC (50 pg/ml) 0

Ii-7 (I ,ug/ml) 96

H-7 (5 pg;ml) 50

H-7 (20 pug/ml) 0

ST (0.01 &ml) 99

ST (0.05 ng!ml) 75

ST (0.1 ng/mI) 39

ST (0.5 ng/ml) 0

RESULTS

Table I lists the cytotoxicity of inhibitors of protein kinase C. The ICY50 value of PC, H-7, and ST was 27 pg/ml, 5 /[g/ml, and 0.8 ng/ml, respectively. ST showed very strong cytotoxic activity. In contrast, TPA and diacylglycerols showed little cytotoxi- city in BALB/3T3 cells (data not shown).

Table II shows the effects of diacylglycerols on 3-MC-initiated cells. A single 3-MC treatment induced 0.2 foci per dish at a dose of 0.5 pg/ml. Subsequent treatment with TPA enhanced focus formation dose-dependently. with a maximum of 4.4 foci/dish obtained at a dose of 1 hg/ml. Two diacylglycerols (OAG and diolein) enhanced focus formation up to 1.3 foci/dish at a dose of 10 pg/ml. However, neither of these com- pounds is as strong as TPA.

The effect of inhibitors of protein kinase C on TPA-enhanced transformation was examined at non-toxic doses (Table III). As shown in Table III, PC, H-7 and ST sup- pressed TPA-enhanced transformation in a dose-dependent manner. The percent of inhibition for PC and H-7 was 76 and 79& respectively. while for ST, the most potent inhibitor of protein kinase C, it was 33% at a dose of 50 pg/ml at which some cytotoxi- city was observed.

DISCUSSION

We clarified the relationship between protein kinase C and promotion by TPA by first testing whether diacylglycerols enhance 3-MC-initiated transfo~ation, and then testing whether inhibitors of protein kinase C suppress TPA-promoted transfor- mation.

It has been shown that all diacylglycerols with one saturated fatty acid at either

T.4BLL: II

EFFECTS OF DIACYLGLYCEROLS ON 3.MC-INITIATED (‘ELLS

1st trcatmenl

11 hl so 3.MC

3.MC

3-hlC

3.MC

3.hlC

3.MC

3-MC

3.h(C‘

3.MC

3-MC

3-x4(’

2nd treatment

( jrg: ml 1

TPA((1.001 )

TPA(O.01)

TP.L\(O.I)

TPA( I)

OAG(0. I )

0.4G( I I

0.4G( IO)

dlolein(0. I ) dioleln( I ) diolein( IO)

No. ofdisheq

used

30 3u

IO

70

3X

IO

10

‘0

35

Ih 10 20

0

0.7 tl). I

0.8 +0.7** _ 1.5+0 2** - 3.3+0.2**

3.4;0.3**

I I *02** I. I 4 0.2** _ 1.3+0.2** -

o.h+o 2** - 0.9+0.7**

1.3+0.3**

Number of foci;dish k man value obtained from all the dishes tested in each expwment **Indicates

significant enhancement (F<O.Ol) relative to the negative control (treatment with 0.5 /cg:ml of3-MC).

TABLE III

INHIBITORY EFFECTS OF INHIBITORS OF PROTEIN KINASE C ON TPA-ENHANCED

TRANSFORMATION

1st trcatmcnt

DTVlSO

3-MC

3-M<

2nd treatment

TPA

No. of No. of focl:dish p6 Inhibition

dishes used (mean + SE)

30 0

30 0.2*0.1

3X 3.3_+0.7 0

TPA + PC(0. I ,ug:ml)

TPA + PC( I ilg:‘rnl)

TPA + PC( IO /cg;‘ml)

30 ‘.I *0.3 3h**

Ih 1.7+0.1 a**

IX 0.x * 0.2 7h**

3.MC

!-MC

i-MC’

TPA + H-7(0.5 /~g:ml)

TPA + H-7( I /q+:ml)

TP.4 + H-7( 2 Icg:ml I

3-hIC TPA + ST( I pg:ml)

3-MC TPA + ST( IO pg:ml)

3-MC TPA + ST( 50 pg:ml)

70 3.3*0.2 0

IX 2.x +0.3 I5

32 7.2 +0.3 33** _

Number of foci’dish represents mean value obtained from number of dishes tested in each cxperlment.

**Indicates significant inhibition (P<O.Ol) relative to the positive control (treatment with 0.5 Lcg:ml 01

3-MC and 0 I ,ug:ml of TPA).

II

position 1 or 2 are able to activate protein kinase C [13]. For example, OAG, which

is a diacylglycerol with oleic acid at only one position, is more effective than diolein.

However, in our experiment the promoting effect of OAG on 3-MC-initiated trans-

formation was similar to that of diolein. On the other hand, Smart et al. [7] have

reported that OAG, but not diolein, increases ornithine decarboxylase activity, and

that diolein, but not OAG, stimulates DNA synthesis in mouse epidermis. They

showed that these compounds are less active than TPA and suggested that this is due

in part to the rapid metabolism of 1,2-diacylglycerols to inactive metabolites, such

as phosphatidic acid. It has been reported that, in a two-stage tumor promotion expe-

riment. TPA and dioctanoylglycerol activate protein kinase C when applied to mouse

skin, but that these compounds differ in their tumor-promoting properties; i.e., dioc-

tanoylglycerol is a Stage II mouse skin tumor promoter [14]. Repeated application

of OAG has been shown to enhance cell transformation in vitro [1.5]. Moreover,

OAG and TPA have been shown to have different effects on protein phosphorylation

and cell proliferation in a leukemia cell line [16]. The finding that diacylglycerols do

not enhance transformation as much as TPA is consistent with the above observa-

tions.

PC has been shown to inhibit TPA-induced ornithine decarboxylase and mouse

skin tumor promotion, but to fail to inhibit TPA-induced epidermal protein [17].

Nakadate et al. [18] reported that ST and H-7 inhibit both the catalytic fragment

of protein kinase C and the intact enzyme with similar potency but have no effect

on the binding domain. H-7 has also been shown to inhibit CAMP-dependent and

cGMP-dependent protein kinase [ 191. ST is uniquely potent as an inhibitor of protein

kinase C in vitro, being active at nanomolar concentrations rather than the micromo-

lar concentrations typical of other classes of inhibitors [18]. Moreover, it has been

reported that ST inhibits the induction of ornithine decarboxylase and tumor promo-

tion caused by TPA in in vivo experiments [20].

The present study demonstrated that activators of protein kinase C enhance trans-

formation in 3-MC-initiated cells and inhibitors of protein kinase C inhibit TPA-en-

hanced transformation in a dose-dependent manner. These findings indicate that pro-

tein kinase C may be essential to the promotion process in two-stage transformation.

ACKNOWLEDGEMENTS

We thank Drs. Y. Egashira and M. Yamada, Hatano Research Institute, Food

and Drug Safety Center for their encouragement and valuable suggestions.

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