MOLECULAR CARCINOGENESIS 5:328-333 (1992)

Differential Induction of 12-OTetradecanoylphorbol- 13-Acetate Sequence Gene Expression in Murine Melanocytes and Melanoma Cells Gavin Brooks,' Martin W. Goss, and Ian R. Hart2 Biology of Metastasis Laboratory Imperial Cancer Research Fund, London, WCZA 3PX, United Kingdom

We previously showed that growth of the nontumorigenic, immortal murine melanocyte line Mel-ab correlates with the depletion o f protein kinase C (PKC), whereas quiescence is associated with elevated levels o f this enzyme (Brooks G, et al., Cancer Res 51: 3281-3288, 1991). Here we report responses that occur in these cells downstream o f PKC activation or downregulation. We examined induction o f 12-0-tetradecanoylphorbol- 13-acetate (TPA)-induciblesequence (TIS) gene expression in Mel-ab melanocytesand in theirtransformed coun- terparts, 616 melanoma cells. Exposure of quiescent Mel-ab cells t o the PKC-activating phorbol esters TPA or sapintoxin A at 81 nM for 2 h increased levels o f mRNA for six o f seven TI5 genes examined (twofold t o 80-fold increase in steady-state RNA levels for TIS 1, 7,8, 11, 21, and 28 (c-fos); TI5 10 expression was not affected). No induction of TIS gene expression was observed either in growing Mel-ab cells maintained in 324 n M phorbol 12,13-dibutyrate or in B16 cells previously unexposed to phorbol esters, in which normal PKC levels were endog- enouslydepressed. The CAMP-elevating agents choleratoxin (10 nM) and dibutyryl cyclic AMP (2.5 mM) increased levels of TI5 mRNA (with the exception of TIS 10) in both proliferating Mel-ab and B16 cells, suggesting that downregulation o f the PKC pathway is specific and not a consequence o f a general inhibition o f all signalling pathways. o 1992 ~ i ~ e y - ~ i s s , Inc.

Key words: Cell signalling, gene expression, melanoma, protein kinase C

INTRODUCTION

The growth of normal, untransformed melanocytes in vitro is dependent upon the continual presence of a biologically active phorbol ester, e.g., 12-0-tetradecanoyl- phorbol-13-acetate (TPA), in their culture medium [ I '21. Conversely, transformed melanoma cells proliferate well in the absence of these exogenous mitogens, and their growth may even be partially inhibited by chronic exposure to phorbol esters [1,21. Protein kinase C (PKC) comprises a family of closely related isoforms that serve as the main phorbol-ester receptor. Recently, this enzyme has been shown to play a pivotal role in regulating growth responses in melanocytic cells such that PKC downregulation ap- pears to be necessary for their proliferation in vitro 131.

To determine some of the consequences of PKC down- regulation in melanocytic cells, we monitored the induc- tion of expression of a series of TPA-inducible sequence (TIS) genes in these cells after exposure for 2 h to either tumor-promoting (TPA) or nonpromoting (sapintoxin A; SAP A) phorbol esters [41. The TIS genes are a family of primary response genes that were first described in Swiss 3T3 fibroblasts [5,61. Many of these genes were isolated either after serum stimulation of 3T3 fibroblasts (e.g., early growth response gene-I) or after nerve growth-factor stimu- lation of rat pheochromocytoma PCl2 cells (e.g., TIS 1) [reviewed in 61. They are superinducible in the presence of cycloheximide, and their expression can be induced with agents other than TPA, such as epidermal growth factor

(EGF) and fibroblast growth factor (FGF) [7]. Furthermore, EGF or FGF treatment of fibroblasts whose PKC levels have been downregulated by prolonged exposure to TPA can lead to expression of TIS genes, thus demonstrating that expression of such genes can be induced by both PKC- dependent and PKC-independent pathways [7] .

We report here that TIS gene expression was induced by treatment of the quiescent untransformed murine mel- anocyte line Mel-ab [8] with both tumor-promoting and nonpromoting phorbol esters. In contrast, proliferating Mel-ab cells in which PKC had been downregulated by con- tinuous exposure to phorbol 12,13-dibutyrate (PDB) [31 failed to display an induction of gene expression after expo- sure to phorbol esters. Interestingly, no induction of TIS gene expression was observed in cultured cells of the B16 melanoma exposed to these compounds; a line which displays constitutively downregulated levels of PKC [31. That CAMP-elevating agents were able to stimulate gene

'Current address: Cardiovascular Research, The Rayne Institute, St. Thomas' Hospital, London SEl 7EH. UK.

'Corresponding author: Biology of Metastasis Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WCZA 3PX. UK.

Abbreviations: dbcAMF: dibutyryl cyclic adenosine monophosphate; DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediamine- tetraacetic acid; EGF, epidermal growth factor; FCS, fetal calf serum; FGF, fibroblast growth factor; PDB, phorbol 12.1 3-dibutyrate; PKC, protein kinase C; SAP A, sapintoxin A; SDS, sodium dodecyl sulfate; SSC, standard saline citrate; TIS. 12-0-tetradecanoylphorbol-13-ace- tate-inducible sequence; TPA, 1 2-Otetradecanoylphorbol-I 3-acetate.

0 1992 WILEY-LISS, INC.

TIS GENE EXPRESSION IN MELANOCYTICCELLS 329

expression in both proliferating Mel-ab and B16 cells dem- onstrates that the abrogation of TIS gene induction observed with phorbol-ester treatment of melanocytic cells was specific and not a consequence of generalized inter- ference with all intracellular signalling pathways.

MATERIALS AND METHODS

Reagents

PDB and TPA were purchased from Scientific Marketing Associates (London, UK). SAP A was isolated from the unripe seeds of Sapiurn indicurn [9] and 5. sebiferurn [ lo] . Cycloheximide, choleratoxin, and dibutyryl cyclic adeno- sine monophosphate (dbcAMP) were obtained from Sigma (Poole, UK). Fetal calf serum (FCS) was purchased from GIBCO (Paisley, Scotland, UK). All other chemicals were of the purest grade commercially available. Plasmids contain- ing TIS gene cDNA clones (TIS 1, TIS 7, TIS 8, TIS 10, TIS 11, and TIS 21) were generous gifts from Dr. H. R. Herschman (UCLA School of Medicine, Los Angeles, CA). TIS 28 (v-fos) was purchased from Oncor, Inc. (Gaithers- burg, MD).

Cell Culture

The nontumorigenic, immortal murine melanocyte line Mel-ab was cultured routinely, as described previously [81, in Dulbecco‘s modified Eagle‘s medium (DMEM) contain- ing 10% FCS and 324 nM PDB. Quiescent Mel-ab cells were obtained by refeeding cells that had been originally plated in phorbol-ester-containing medium with serum-free DMEM:Waymouth’s(Z: l ) f o r3 d [31. Swiss3T3fibroblasts and transformed murine B16.Fl melanoma cells were main- tained in DMEM supplemented with 10% FCS only [31. All cells were kept in a humidified atmosphere of 10% C02 and 90% air at 37°C.

RNA Extraction and Northern Analysis

Total cytoplasmic RNA was prepared according to a modi- fication of the method described by Maniatis et al. [lll. Briefly, cells grown in 1 75-cm2 Nunc tissue-culture flasks were washed twice with ice-cold phosphate-buffered saline and lysed in 10 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0, and 0.5% sodium dodecyl sulfate (SDS) on ice. The cell lysates were scraped into a sterile tube, the dishes were washed with 0.1 M NaOAc, pH 5.2, and 10 mM EDTA, pH 8.0, and the washings were combined with the lysates. Proteinase K was added to a final concentration of 10 pg/mL, and the mixture was incubated at 65°C for 1 h. The lysate was partitioned against water-saturated phe- nol with shaking for 2 min, and the phases were sepa- rated by centrifugation. The upper, aqueous layer was transferred to a sterile tube containing 1 . I0 mL 1 M Tris-CI, pH 8.0, and 450 pL 5 M NaCI, and the RNA was precipi- tated twice by the addition of 2 vol of ice-cold ethanol for 2 h at - 20°C. After centrifugation and removal of the ethanol, the RNA pellet was redissolved in double-distilled RNase-free water. Total cytoplasmic RNA was separated electrophoretically on 1 YO agarose/2.2 M formaldehyde gels, transferred to nitrocellulose filters, and hybridized to

various 32P-labeled, nick-translated cDNA probes. The fil- ters were washed in 2 x standard saline citrate (SSC) and 0.1 % SDS at 42°C for 15 min and then incubated for 15 min in 2 x SSC and 0.1 YO SDS at 65°C. The washed filters were exposed to Fuji RX x-ray film (Fuji Photo Film Co. Ltd., Tokyo, Japan) with intensifying screens at - 70°C.

The levels of RNA loaded on the gel were quantitated by reprobing the blots with a 32P-labeled p-actin house- keeping cDNA gene and scanning the exposed films densitometrically wi th an LKB densitometer (Milton Keynes, UK).

RESULTS

Figure 1 depicts typical northern blots showing the response of melanocytic cells to treatment with either tumor-promoting (TPA) or nonpromoting (SAP A) phorbol esters in the presence of cycloheximide. In all cases, cyclo- heximide was added to the cultures 10 min before the addi- tion of the other agents and was used to superinduce expression of the genes, thus improving the sensitivity of the assay. Figure 1 A documents the induction of TIS 1 RNA and Figure 1B the induction of TIS 7 RNA 2 h after the addition of these compounds. Total RNA isolated from stim- ulated quiescent Swiss3T3 fibroblasts(1anesA-C)was used as a positive control and shows that both TPA (lane B) and SAP A (lane C) caused a substantial increase in the steady- state levels of TIS gene mRNA at that time. Similar treat- ment of proliferating B16 melanoma cells (lanes D-F) and proliferating Mel-ab cells (lanes J-L) evoked no detectable increase in TIS gene expression. In contrast, quiescent Mel-ab cells (lanes G-I) demonstrated an increase in TIS gene expression in response to both TPA and SAP A. The relative fold-induction of the various TIS genes is presented in Table 1 from comparative densitometric scans of levels of TIS RNA normalized to scans of p-actin RNA. From these results it is apparent that, of the seven TIS genes tested, only TIS 10 was not stimulated by the two phorbol esters in quiescent Mel-ab cells; TIS 10 expression, however, was induced in quiescent Swiss 3T3 fibroblasts when treated with these compounds (Table 1).

Control experiments with proliferating Swiss 3T3 fibro- blasts showed a similar degree of induction of TIS gene expression, suggesting that proliferation was not sufficient in itself to abrogate the increase in TIS steady-state RNA (data not shown). This conclusion was confirmed by the response of proliferating Mel-ab cells (Figure 2A) and pro- liferating B16 melanoma cells (Figure 2B) to CAMP-elevating agents. In the autoradiographs presented in Figure 2, blots were probed with 32P-labeled TIS 28 (c-fos) and TIS 1 cDNA probes, respectively. Similar results have been obtained for TIS 7 expression. Treatment of quiescent Mel-ab cells with CAMP-elevating agents induced these same TIS genes (data not shown). From Figure 2A it is apparent that whereas a 2-h treatment of proliferating Mel-ab cells with 81 nM TPA failed to increase c-fos expression (lane C), treatment with either 10 nM choleratoxin (lanes D and E) or 2.5 m M dbCAMP (lanes F and G) produced a substantial increase in steady-state levels of c-fos (TIS 28) mRNA. Similarly, the steady-state levels of TIS 1 mRNA were elevated in 816

330 BROOKS ETAL.

Swiss 3T3 A

3.3kb +

ACTIN +

B

1.8kb +

ACTIN 3

B16.Fl Quiescent Growing Mel-ab Mel-ab

A B C D E F G H I J K L

Swiss 3T3 B16.Fl Quiescent Growing Mel-ab Mel-ab

A B C D Figure 1. Autoradiograph of northern analysis showing

induction of (A) TIS 1 and (B) TIS 7 gene expression in cells treated with tumor-promoting and nonpromoting phorbol esters. Cells grown in 175-cm2 Nunc tissue-culture flasks were treated with 10 pg/mL cycloheximide (lanes A, D, G, and J), 10 pg/mL cyclo- heximide and 81 nM TPA (lanes B, E, H. and K), or 10 pg/rnL cyclo- heximide and 81 nM SAP A (lanes C, F, I, and L) for 2 h (cycloheximide was added to the cultures 10 min before add- ing the other agents). For both (A) and (B), the contents of lanes were as follows: lanes A-C, quiescent Swiss 3T3 fibroblasts (Swiss 3T3); IanesD-F, proliferating 816 melanoma cells(B16.Fl); lanes G-I, quiescent Mel-ab melanocytes (Quiescent Mel-ab); lanes J-L,

cells treated with 10 nM choleratoxin (Figure 2B, lane C) or 2.5 m M dbcAMP (lane D) for 2 h, whereas a similar treatment of these cells with 81 nM TPA failed to induce a response (lane B) greater than that observed in control cells treated with cycloheximide alone (lane A). In contrast, the TIS 10 gene was not inducible in melanocytic cells treated with CAMP-elevating agents, although it was induced in quiescent Swiss 3T3 fibroblast treated with these agents (data not shown).

DISCUSSION

Recently, we demonstrated that downregulation of cytosolic PKC correlates with in vitro proliferation of both untransformed and transformed murine cells of the melano-

E F G H I J K L proliferating Mel-ab cells (Growing Mel-ab). Culture conditions are described in the text. Total RNA was prepared from treated cells as described in Materials and Methods. Samples (10 pg per lane) were loaded onto a 1 % agarose/2.2 M formaldehyde slab gel and electrophoresed at 25 V for 16 h. After being stained with 10 pg/mL ethidium bromide, the RNA was transferred to a nylon membrane (Hybond-N) and the blotwas hybridized with either (A) TIS 1 or (B) TIS 7 52P-labeled nick-translated cDNA probes as described in Materials and Methods. The degree of induction of gene expression was quantitated by densitometric scanning and comparison with the response obtained with the p-actin housekeeping gene.

cyte lineage [31. In the study presented here, we investi- gated responses that occur downstream of activation of the PKC signal transduction pathway in these cell types. The expression of certain early response genes, known as TIS genes, is induced in quiescent or proliferating Swiss 3T3 fibroblasts after a transient exposure t o the PKC- activating phorbol ester TPA [5,61. In this study we compared the effects of various PKC-activating and CAMP- elevating agents as inducers of TIS gene expression in the untransformed murine melanocyte line Mel-ab [81 and the sy ngeneic tumorigenic, transformed meia no ma line B16.Fl [3].

Both tumor-promoting (TPA) and nonpromoting (SAP A) phorbol esters [41 induced expression of TIS genes in

TIS GENE EXPRESSION IN MELANOCYTIC CELLS 33 I

Table 1. Fold-Induction of TIS Gene mRNA Expression in Quiescent Mel-ab Melanocytes Treated With Tumor-

Promoting or Nonpromoting Phorbol Esters

TIS gene Treatment* Fold-induction'

1 CHWTPA 2.42 1 CHWSAP A 2.32 7 CHWTPA 6.33 7 CHWSAP A 4.67 8 CHWTPA 84.9 8 C HWSAP A 49.7

10 CHWTPA Not induced 10 CHWSAP A Not induced 11 C HWTPA 2.40 11 CHWSAP A 1.89 21 CHWTPA 3.83 21 CHWSAP A 1.74 28 (C-fO5) CHWTPA 3.21 28 (C-fO5) CHWSAP A 2.98 1 o* CHWTPA 31.3 1 O* CHNSAP A 18.0

*CHX, cycloheximide. 'Fold-increase over expression in cycloheximide-treated cells in which fold-induction equals 1. Northern blots of total RNA prepared from con- fluent and quiescent Mel-ab cells 131 treated with either 81 nM TPA or 81 nM SAP A in the presence of 10 pg/mL cycloheximide for 2 h were probed with various 32P-labeled, nick-translated TIS gene cDNA probes as described in Materials and Methods. The data shown were calculated from densitometrically scanned autoradiographs of north- ern blots comparing the intensity of the TIS gene RNA band with the intensity of the p-actin RNA band area. *Induction of TIS 10 mRNA in Swiss 3T3 fibroblasts.

quiescent and proliferating Swiss 3T3 fibroblasts and in qui- escent Mel-ab melanocytes, suggesting that induction of these genes is not a consequence of the tumor-promoting capacity of phorbol esters but correlates more closely with their PKC-activating capacities I l l . Thus, a 2-h exposure of quiescent Mel-ab cells to both agents resulted in the induc- tion of expression of six of seven TIS genes tested (Table I), although the degree of stimulated expression varied depending upon the gene under study.

Interestingly, the expression of TI5 10 was not induced in quiescent Mel-ab cells after a 2-h incubation with either phorbol esters or cAMP elevators, although TIS 10 was in- ducible in Swiss 3T3 fibroblasts treated with both classes of agent (Table 1). A similar result has been reported in rat PC 12 pheochromocytoma cells treated with TPA for 3 h [ 121. Indeed, expression of the TIS 10 gene has now been shown to be more highly cell-type restricted than the expression of other primary response genes. For exam- ple, in a panel of rodent cell lines exposed to TPA and cyclo- heximide, including lines derived from smooth muscle, lymphoid tissue, and bone, only Swiss 3T3 fibroblasts and Rat-I embryonic cells displayed induction of TIS 10 gene expression [ 131. It remains to be determined whether the demonstration that TIS 10 encodes a novel prostaglandin synthase cyclooxygenase homologue [ I 3) explains this cell- type restriction. Recently, the lack of TIS 8 expression in the immature B cell line WEHI-231 was attributed to hypermethylation of the TIS 8 gene [14]. However, our Southern analyses of restriction-enzyme-digested genomic

DNA have not revealed this to be a valid explanation for the lack of TIS 10 induction in cells of melanocyte origin (data not shown).

Proliferating Mel-ab cells that were cultured in the con- tinuous presence of PDB, which downregulates PKC lev- els [31, exhibited no increase in levels of RNA for any TIS gene when treated with biologically active phorbol esters. Likewise, in B16 melanoma cells, which grow in the absence of exogenous PKC activators, there was a similar lack of TIS gene induction by this class of compounds. This result is consistent with the finding that these cells have rela- tively low levels of PKC and the proposal that this as-yet- uncharacterized downregulation is necessary for the growth of these transformed cells [3].

However, it appears that expression of early response gene mRNAs is differentially regulated in proliferating melanocytic cells. Thus, Yamanishi et al. [ I 51 recently reported increased steady-state levels of jun-B mRNA and decreased levels of cjun mRNA in proliferating human neo- natal melanocytes compared with quiescent cells. In addi- tion, they found that c-fos mRNA levels were greater in proliferating cells than in quiescent cultures. These latter observations are in direct contrast to those we report in this paper and may reflect species differences in the cells used in the two studies.

Previous studies [7,121 have documented the induction of TIS gene expression by agents other than TPA, such as FCS, EGF, FGF, and cAMP elevators, indicating that signal- ling pathways other than PKC also are involved in TIS gene regulation. Furthermore, induction of expression of these genes can occur in the absence of the PKC pathway. For example, Herschman and coworkers demonstrated in Swiss 3T3 fibroblasts depleted of PKC, by a 24-h pretreatment with TPA, that treatment with EGF or FGF could lead to induction of expression of these genes [7]. Possibly, the promoter region of these genes contains an AP2 site, which modulates PKC- and CAMP-mediated responses [ I 61, in addition to or instead of an API site, which mediates responses from the PKC pathway only [17,18]. The find- ing that cAMP-elevating agents can induce TIS gene expres- sion in proliferating Mel-ab and 816 cells [I91 (Figure 2) suggests that, in these cells, downregulation of the PKC pathway, whether induced or constitutive, is a specific event. Moreover, this result appears to rule out the possi- bility that it is simply the proliferation status of the cells that mediates the different response to signalling path- way stimulators. This conclusion is supported by the dem- onstration that TIS gene expression can also be induced in proliferating Swiss 3T3 fibroblasts [I61 (data not shown).

The role of TIS gene expression in melanocytic cell trans- formation remains unclear. That these genes are induc- ible in proliferating cells via PKC-independent pathways suggests that they are unlikely to be directly responsible for the transformation process. Our previous observation [3] that downregulation of PKC correlates with prolifera- tion of melanocytic cells suggests that the disruption of as-yet-unidentified downstream responses that are PKC specific may play an important role in the transformation of these cells.

332 BROOKS ETAL.

A

2.2kb +

ACTIN +

A B C D E F G

B

3.3kb +

ACTIN +

A Figure 2. Autoradiographs o f northern analysesshowing TIS

gene induction. (A) TIS 28 (c-fos) gene expression in proliferat- ing Met-ab melanocytes after treatment wi th PKC-activating or CAMP-elevating agents. Lane A, positive control o f Swiss 3T3 fibroblasts treated w i th cycloheximide and 81 nM SAP A for 2 h. Mel-ab cells received the following treatments: lane B, cyclo- heximide for 2 h; lane C, cycloheximide and 81 nM TPA for 2 h; lane D, cycloheximide and 10 nM choleratoxin for 1 h; lane E, cycloheximide and 10 nM choleratoxin for 2 h; lane F, cyclohex-

In summary, this paper describes for the first time the differential induction of certain TI5 genes in cells of mela- nocyte origin. This induction by phorbol esters correlates closely with the status of PKC activity, which appears to play a pivotal role in modulating the growth of cells of this lineage.

ACKNOWLEDGMENTS

We thank Dr. Susan F. Brooks for her advice and critical reading of this manuscript, Dr. Harvey Herschman for the kind gift of plasmids containing the TI5 gene cDNAs, Dr. Enrique Rozengurt for supplying the Swiss 3T3 fibroblasts, and Clare Middlemiss for secretarial assistance.

Received October 4, 1991; revised December 20, 1991; accepted January 23,1992.

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B C D imide and 2.5 m M dbCAMP for 1 h; lane G, cycloheximide and 2.5 mM dbCAMP for 2 h. (B) TIS 1 gene expression in proliferat- ing B16.Fl melanoma cells that received the following treat- ments: lane A, cyclohexirnide alone for 2 h; lane 8, cycloheximide and 81 nM TPA for 2 h; lane C, cyclohexirnide and 10 nM choleratoxin for 2 h; lane D, cycloheximide and 2.5 m M dbCAMP for 2 h. Culture conditions and treatments are detailed in the text, and the analysis was conducted as described in the legend t o Figure 1.

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