MOLECULAR CARCINOGENESIS 8:290-298 (1993)

ras Protein p21 Processing Enzyme Farnesyltransferase in Chemical Carcinogen-Induced Murine Skin Tumors Rajesh Agarwal.' Sikandar G. Khan, Mohammad Athar, Syed 1. A. Zaidi, David R. Bickers, and Hasan Mukhtar

Department of Dermatology, Skin Diseases Research Center, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, Ohio

Farnesylation of ras protein p21 is crucial for the protein's membrane localization, which is essential for i ts cell-transforming activity, which in turn is thought to be critical for the ultimate induction of cancer. The cytosolic enzyme farnesyltransferase plays a major role in posttranslational modif icat ion of p21, but the level of farnesyltransferase activity in mammalian tumors and i t s relationship t o the processing of cytosolic p21 tha t leads to tumorigenesis are unknown. We report here tha t farnesyltransferase activity was significantly higher in chemical carcinogen-induced benign skin papillomas in SENCAR mice than in the epidermises of control animals. The enzyme is primarily epidermal in origin, and kinetic studies w i th cytosol from epidermis and papillo- mas showed tha t the reaction was linear with respect to time, substrate concentration, and protein content. Skin papillomas showed significantly elevated levels of both cytosolic and membrane-bound Ha-ras p21, whereas far lesser cytosolic and almost negligible amounts of membrane-bound p21 were present in the epidermis of control mice. There was a positive correlation between increased enzyme activity in papilloma cytosol and the processing of overexpressed cytosolic Ha-ras p21 for its localization to membrane.

Key words: Farnesyltransferase, farnesylation, ras oncogene, cutaneous carcinogenesis

o 1993 ~ i ~ e y - ~ i s s , Inc.

INTRODUCTION

The frequent detection of activated Ha-ras, Ki-ras, and N-ras oncogenes in human and animal neoplasms derived from different tissue types suggests that ras oncogenes make an important contribution to the multistep process of carcinogenesis [l-41. The three ras oncogenes encode a group of structurally related 21 -kDa proteins designated p21 [5,6], which are believed to function as molecular switches in signaling events of cell growth and differenti- ation [6,7]. Under normal physiological conditions, p21 ex- ists in the inactive GDP-bound conformation, and in most growth events only a small percentage of p21 is in the active form, viz the GTP-bound form [5,61. Increasing evi- dence suggests that conversion of the rasfamily of proto- oncogenes to their oncogenic forms by point mutation produces mutated forms of p21 with cell-transforming po- tential [2-71. Unlike normal p2 l, a high proportion of mu- tated p21 is found in the GTP-bound state because the point-mutated p21 protein is resistant to the action of the GTPase activating protein (GAP), so that GAP no longer promotes GTP hydrolysis [8]. This phenomenon is related to fa5 point mutations that activate the oncogenic poten- tial of ras and result in impaired GTPase activity or increased guanine nucleotide release by the mutated p21 [5,61. Sev- eral studies suggest that two key events are important for the cell-transforming function of mutated p2 1 : interaction with guanine nucleotides and association with the plasma membrane [5-101.

The mutated p21 transforms mammalian cells only when it is localized to the inner side of the plasma membrane [11-14].Themernbrane localization of p21 requiresat least three posttranslational modifications that mainly occur at

the carboxyl-terminal end of p21, which has a consensus motif Cys-Aaa-Aaa-Xaa (where Aaa is any aliphatic amino acid and Xaa is any amino acid) termed the CAAX box [ 15- 181. The major posttranslational modification of p21 re- quired for its membrane localization is the transfer of a farnesyl group to the cysteine residue of the CAAX box (farnesylation of p21), a reaction catalyzed by a specific cytosolic enzyme known as farnesyltransferase [lo, 1 5-20]. Studies of p21 farnesylation have emphasized the possibility that the p21-linked farnesyl group may program the inter- action between farnesylated p2 1 and specific attachment sites on the plasma membrane [5,10,15-201. The impor- tance of posttranslational processing in promoting the membrane association of p21 that is followed by cell- transforming activity has been verified by experimental mutagenesis of the p21 CAAX box [7]. Replacing the cys- teine or deleting the AAX residues in the C A M box of the oncogenic p21 produces mutant proteins that are not farnesylated (processed), are trapped in the cytosol, and do not localize to plasma membrane and are therefore com- pletely nontransforming [2 1-24].

Several studies have shown that farnesyltransferase ac- tivity is present in normal mammalian tissues [25-291; however, enzyme activity in mammalian tumors and its re-

'Corresponding author: Department of Dermatology, University Hos- pitals of Cleveland, Case Western Reserve University, 2074 Abington Road, Cleveland, OH 44106.

Abbreviations: GAF: GTPase activating protein; DMBA, 7.1 2-dimethyl- benz[a]anthracene; TPA, 1 2-0-tetradecanoylphorbol- 1 3-acetate; FPp. farnesylpyrophosphate; On, dithiothreitol; SDS, sodium dodecyl sul- fate; PAGE, polyacrylamide gel electrophoresis; TBST, Tris-bufferd sa- line, pH 8.0, with 0.05% Tween-20.

0 1993 WILEY-LISS, INC.

FARNESYLTRANSFERASE IN SKIN TUMORS 29 1

lationship to the processing of cytosolic p21 that leads to tumorigenesis are not known. In this study, we first as- sessed the levels of farnesyltransferase activity in the epidermises of untreated control mice and chemical carcinogen-induced benign skin papillomas in SENCAR mice and then correlated it with the processing (mem- brane association) of Ha-ras p21. Multistage skin chemical carcinogenesis is an ideal system for such studies since in this model, the Ha-ras oncogene has been shown to play an important role in tumor induction [I ,2,30-361. Furthermore, more than 90% of chemical carcinogen- induced papillomas and carcinomas in murine skin contain mutated Ha-rasoncogene [ I ,2,30-361.

MATERIALS AND METHODS

Induction of Benign Papillomas in Murine Skin

Six-week-old female SENCAR mice, obtained from the National Cancer Institute-Frederick Cancer Research Facil- i ty (Bethesda, MD) were used in a standard two-stage i nit ia t i o n - p rom o t io n pro toco I [ 3 71. Tu mor i n it ia t i o n was accomplished by a single topical application of 5 p g of 7,12-dimethylbenz[a]anthracene (DMBA) in 0.2 mL of acetone; 1 wk later tumor promotion with twice weekly top- ical applications of 1 p g of 12-0-tetradecanoylphorbol- 13-acetate (TPA) in 0.2 rnL of acetone was begun. After 20 wk on this protocol, all animals had developed tumors. These tumors, which were histologically verified as benign papillomas, were at identical stages of dysplasia.

Cytosolic and Membrane Fraction Preparations

To prepare cytosolic and membrane fraction from epi- dermis, untreated control SENCAR mice matched for age and sex with those bearing skin papillomas (and from the same lot) were shaved on their dorsal sides with electric clippers; Nair depilatory was then applied for 5 min to re- move the remaining hair. Next, the animals were killed by cervical dislocation, a 3 x 3-cm area of shaved skin was removed, and epidermis was separated from the whole skin by a brief heat treatment at 52°C for 30 s. To prepare cytosolic and membrane fractions from papillomas, one papilloma was removed from each of the papilloma-bearing animals. Part of the epidermis and papilloma tissue was used to prepare cytosolic and membrane fractions; the rest was used to detect cytosolic and membrane-bound Ha- ras p21.

Both epidermis and papilloma tissues were cut into small pieces, and a 10% (wiv) homogenate was prepared at 4°C in 0.1 M HEPES buffer, pH 7.4, containing 1 mM MgC12 and 1 m M dithiothreitol (DTT) using a Polytron tissue ho- mogenizer (Kinematica GmbH, Luzern, Switzerland). The homogenate was centrifuged at 1,000 x g for 20 min at 4°C. The 1,000 x g supernatant was then centrifuged at 100,000 x gfor 60 rnin at 4°C. The 100,000 x gsupernatant (the cytosolic fraction) was transferred into a separate vial and stored at - 170°C under liquid nitrogen. The pellet ob- tained from the 100,000 x gcentrifugation (the membrane fraction) was resuspended in the same buffer, sonicated to obtain a homogeneous suspension, and stored at - 170°C under liquid nitrogen. The protein concentration

was determined by the method of Bradford [381 using bo- vine serum albumin as the reference standard.

Assay for Farnesyltransferase Activity

The farnesyltransferase assay used was based on the one described by Manne et al. [29], with slight modifications. Unless otherwise stated, each 1 0-pL reaction mixture con- tained 10 pg of cytosolic or membrane protein, 1 p g of purified recombinant Ha-ras ~ Z l g l Y - ’ ~ (a kind gift from Dr. M. Campa, Burroughs Wellcome Co., Research Triangle Park, NC), and 1 p L (0.2 pCi) of 10 pM [3Hlfarnesylpyrophos- phate (FPP) (20 Ciimmol; New England Nuclear, Boston, MA) in 0.1 M HEPES buffer, pH 7.4, containing 25 m M MgC12 and 10 mM DlT After incubation at 37°C for 1 h, the reaction was terminated by adding 10 pL of 2 x so- dium dodecyl sulfate (SDS)-polyacrylamide gel electropho- resis (PAGE) sample buffer (125 m M Tris-HCI, pH 6.8, containing 20% glycerol, 10% 2-mercaptoethanol, 0.1 YO bromophenol blue, and 5% SDS). The samples were boiled for 2 min and resolved on a 12.5% polyacrylamide gel, as described by Laemmli [39]. The radioactivity associated with the Ha-ras p21g1y-’2 protein was measured by cutting the approximately 22-kDa region out of the gel, solubilizing and destaining it in 1 mL of 30% hydrogen peroxide, and measuring the radioactivity in it with a Packard TriCarb 460 CD liquid scintillation counter (Packard Instrument Co., Inc., Meriden, CT).

Detection of Ha-ras p21 in Epidermis and Papillomas Homogenized in Detergent-Rich Lysis Buffer

To detect the levels of cytosolic and membrane-bound Ha-ras p21 in the epidermises of untreated control mice and chemical carcinogen--induced benign skin papillomas, the tissues were homogenized a t 4°C in lysis buffer (10 mM dibasic sodium phosphate, 150 mM NaCI, 0.1 YO SDS, 0.1% Triton X-100, 12 mM sodium deoxycholate, 1 mM sodium fluoride, 0.2% sodium azide, 2 mM phenylmethyl- sulfonyl fluoride, and 0.4 trypsin inhibitory unitsimL aprotonin, pH 7.2) for four strokes of 10 s each in 2 min using a Polytron tissue homogenizer, and a 10% (wiv) ho- mogenate was prepared. The homogenate was centrifuged at 15,000 x gfor 20 rnin at 4”C, and the supernatant was assayed for Ha-ras p21. In each case, 500 pg of protein from the 15,000 x g supernatant per mL of lysis buffer was mixed with 5 p L of an anti-v-Ha-ras (Ab-I) antibody (clone Y 13-259)-protein A-agarose bead complex (Oncogene Science, Uniondale, NY) and left at 4°C for 24 h. The im- munoprecipitate obtained was washed twice with phos- phate-buffered saline. The pellet was resuspended in 25 yL of 1 x SDS-PAGE sample buffer, boiled for 5 min, and centrifuged. The clear supernatant was subjected to SDS-PAGE on a 12.5% gel, and the resolved proteins were transferred onto a nitrocellulose membrane as described by Towbin et al. [40].

The nonspecific sites on the membrane were blocked by incubation at room temperature for 1 h with Tris- buffered saline, pH 8.0, with 0.05% Tween-20 (TBST) con- taining 3 % gelatin. After being washed twice with TBST for 10 min, the membrane was incubated at room tem- peraturefor 1 h with 5 pg of v-Ha-ras (Ab-1)-rat monoclonal

292 AGARWAL ETAL.

lgGl (clone Y 13-259; Oncogene Science) per mL of TBST containing 1% gelatin. The membrane was then washed twice again with TBST for 10 min and incubated at room temperature for 1 h with alkaline phosphatase-conjugated anti-rat IgG (Calbiochem, La Jolla, CA) diluted 1 :5,000 with TBST containing 1% gelatin. The membrane was then washed thrice extensively with TBST and incubated with alkaline-phosphatase substrate solution (0.1 M Tris-buffer solution, 5 pg/mL nitroblue tetrazolium, and 5 pg/mL 5-bromo-4-chloro-3-indolyl-phosphate (Kirkegaard & Perry Labs., Inc., Gaithersburg, MD) until color developed.

Detection of Ha-ras p21 in Cytosolic and Membrane Fractions Prepared From Epidermis and Papillomas

To detect Ha-ras p21 separately in cytosolic and mem- brane fractions, both fractions were prepared from epi- dermis and papillomas as detailed earlier. While the cytosolic fractions were used without further treatment, the mem- brane fractions were suspended in detergent-rich lysis buffer (as described earlier), ultrasonicated with two 10-s bursts at 4"C, and left overnight at 4°C to allow the membrane to solubilize. This solubilized, homogeneous membrane preparation was ultracentrifuged at 100,000 x gfor40 min atCC, andtheclear 100,000 xgsupernatantwasassayed for membrane-bound Ha-ras p21. No pellet was evident after this 100,000 x g run, showing that almost all the mem- brane fraction was solubilized in the detergent-rich lysis buffer. Ha-ras p2 1 was immunoprecipitated from 500 p g of protein from the cytosolic and soluble membrane frac- tions; the immunoprecipitate was subjected to SDS-PAGE followed by western blotting, and the Ha-ras p21 bands were visualized as described earlier.

RESULTS

We first studied the in vitro farnesylation of purified re- combinant Ha-ras p21g'Y-'2 utilizing cytosolic and mem- brane fractions both from epidermises of control SENCAR mice and from benign skin papillomas induced by DMBA ini- tiation and TPA promotion. The experiment was performed with the cytosolic and membrane fractions from four epidermises and papilloma tissues; the autoradiograph from one typical experiment is shown here. As shown in Figure 1, only one protein band of about 22 kDa (based on the migration of molecular-size markers) was radioactive. With epidermal cytosol, the enzyme reaction was dependent on added p21; no band was observed otherwise (Figure IA, lane 1). In the papilloma cytosol, a distinct radioactive band was observed even without added p21 (Figure 15, lane 1). These results suggest that some endogenous p21 was present in the papilloma preparations that incorpo- rated radioactivity from [3H]FPF! resulting in farnesylation. Figure 1 also shows that enzyme activity was associated only with cytosol both in the epidermis and in the papillo- mas (Figure 1A and B, lane 2); no band was observed when a membrane fraction was used as the enzyme source (Fig- ure 1A and B, lane 3). Incorporation of radioactivity into p2 1 was an enzymatic process, since no band was observed in the absence of cytosol (Figure 1 A, lane 4). Figure 1 also shows that the incorporation of radioactivity was significantly higher in papilloma cytosol than in epidermal cytosol.

Figure 1. In vitro farnesylation of purified recombinant Ha- ras p21g'y-'2 using cytosolic and membrane fractions prepared from a representative epidermis and papilloma. After the elec- trophoresis of various reaction mixtures, the gel was fixed over- night in water/methanol/acetic acid (80:lO:lO. v/v/v), treated with Enlightening (New England Nuclear, Boston, MA), and dried with a gel drier. The dry gel was exposed at - 80°C to Kodak X-Omat film in the presence of intensifier screens for 48 h. The size of the radioactive (farnesylated) Ha-ras p21, indicated by an arrow, is based on molecular-size markers not shown here. (A) Epider- mis of an untreated control SENCAR mouse. Lane 1,cytosolicfrac- tion; lane 2, cytosolic fraction plus recombinant Ha-ras p21; lane 3, membrane fraction plus recombinant Ha-ras p21; lane 4, recombinant Ha-ras p21 alone. (B) Chemical carcinogen-in- duced benign skin papilloma from a SENCAR mouse. Lane 1, cytosolicfraction; lane 2, cytosolicfraction plus recombinant Ha- ras p21; lane 3, membrane fraction plus recombinant Ha-ras p21. All the lanes are from the same gel.

We also analyzed the intensity of each band shown in Figure 1A (lane 2) and Figure 1 B (lanes 1 and 2) using a Shimadzu CS-930 thin-layer chromatography scanner (Sci. Ins. Inc., Columbia, MD) with a DR-2 data recorder pro- grammed to normalize for the area. In this densitometric analysis, the intensity of the band in lane 2 of Figure 1B was significantly greater (1.61, an arbitrary unit based on the recorder printout) than that of the band in lane 2 of Figure 1A (0.66). The intensity of the band in lane 1 of Figure 1 B was significantly less (0.1 1) than that of the band in lane 2 in both Figures 1A and 1B. These data suggest that farnesyltransferase activity was substantially greater in papillomas than in the epidermisn: of untreated con- trol mice and that the darker band in lane 2 of Figure 1 B was not due to the additive effects of farnesylated recom- binant Ha-ras p21 and endogenous ras p21 from the tumor.

We next assessed the subcellular distribution of enzyme activity in both the epidermises and the papillomas. As shown in Table 1, the subcellular distribution of farnesyl- transferase activity was identical in both tissues. The max- imum enzyme activity was observed in the 100,000 x g supernatant (cytosolic) fraction, followed by the 9,000 x g and 1,000 x g supernatants. No enzyme activity was de- tected in the 100,000 x g membrane fraction. Thus, the enzyme activity responsible for farnesylation of ras p2 1 was present primarily in cytosol. We also evaluated the local- ization of the farnesyltransferase activity in the epidermises and dermises of untreated control SENCAR mice. As shown in Table 1, the enzyme activity was fivefold to sixfold higher in epidermis than in dermis, suggesting that farnesyltrans- ferase activity was primarily localized in the epidermal com- partment of the skin.

FARNESYLTRANSFERASE IN SKIN TUMORS 293

Table 1. Subcellular Distribution of Farnesyltransferase Activity in Epidermis and Papillomas and Localization

of Enzyme Activity in Normal Skin

Su bcel I u lar fraction

Epidermis

Enzyme activity*

5.51 2 0.49 8.35 f 0.72

11.09 2 1.01 Not detectable

8.92 ? 0.85 13.58 f 1.28 21.27 f 2.08 Not detectable

1,000 x g supernatant 9,000 x g supernatant 100,000 x g supernatant 100,000 x g membrane fraction

1,000 x g supernatant 9,000 x g supernatant 100,000 x g supernatant 100,000 x g membrane fraction

Papilloma

Localization of farnesyltransferase activity in normal skin

100,000 x g supernatant

100,000 x g supernatant from epidermis 11.67 & 1.21

from dermis 2.18 rfr 0.26

*Each assay was conducted in duplicate, and the values shown are the mean ? standard errors of three independent experiments. En- zyme activity is expressed as pmol [3H]farnesyl transferred to purified recombinant Ha-ras p2 1 QIy~'* h/mg protein.

We also studied the kinetics of farnesyltransferase ac- tivity in cytosolic fractions prepared from the epidermises of control mice and DMBA-TPA-induced benign skin pap- illomas. As shown in Figure 2A, the reaction was linear with time up to 60 min in both epidermis and papillomas. With purified recombinant Ha-ras ~219'Y- '~ and [3H]FPP, the rate of the reaction increased linearly with increasing substrate concentration, reaching saturation at 0.6 p g p21 and 0.5 p M [3H]FPP in both tissue preparations (Figures 2B and C, respectively). The enzyme activity was also lin- ear with respect to the amount of cytosolic protein in the reaction mixture (Figure 2D). The enzyme activity was found to be dependent on MgZf and DTT, was optimum at pH 7.4, and was not affected by brief heat treatment (30 s at 52°C. the procedure we used to isolate epidermis) (data not shown). These results suggest that the enzyme in both epidermis and papillomas was similar if not identical.

To compare the farnesyltransferase activity in the epi- dermis of control mice and chemical carcinogen-induced benign skin papillomas in SENCAR mice, enzyme activity was measured in 12 epidermis samples and 24 papillo- mas. Incorporation of radioactivity, as a [3H]farnesyl group from [3H]FPP, into recombinant Ha-ras ~21g'Y.'~ was mea- sured and was expressed as the specific farnesyltransferase activity in the tissue preparations used. The specific en- zyme activity was significantly higher in papilloma cytosol than in epidermis cytosol (Figure 3). In epidermis, the en- zymeactivitywas4.19-7.96(mean 6.03 * 0.34) pmol/h/mg protein, and in papillomas it was 9.20-23.04(mean 15.20 '. 0.83) pmollhlmg protein. The mean specific enzyme ac- tivity in papillomas was significantly different from the mean in epidermis (P < 0.001, Student's t-test).

Since even without added p21, papilloma cytosol showed evidence of catalytic activity that was probably due to en- dogenous p21 (Figure I B , lane l) , we also determined

whether the higher enzyme activity in the papilloma cyto- sol was due to excessive p21 substrate from endogenous and exogenous sources. In the absence of added p21, farnesyltransferase activity was not detected in epidermal cytosol and was 3.92 ? 0.29 pmol/h/mg protein in papil- loma cytosol. Enzyme activity in the absence of in vitro added p21 was subtracted from the specific enzyme ac- tivity data shown in Figure 3.

Since we used Nair (hair-removing cream that has a high concentration of calcium hydroxide) and treated the whole skin at 52°C for 30 s to isolate shaved normal epidermis, we also assessed whether these treatments caused any change or decrease in farnesyltransferase activity. In these studies (data not shown), exposure of papilloma tissues to similar treatments did not result in any change in farnesyltransferase activity, and epidermis obtained from electric clipper-shaved skin not treated with Nair had en- zyme activity similar to that of epidermis obtained from shaved and Nair-treated skin. Collectively, these results sug- gest that Nair application and brief heat treatment did not change or decrease specific farnesyltransferase activity.

The possible correlation of increased farnesyltransferase activity and the processing of cytosolic Ha-ras p21 that leads to the induction of tumorigenesis was assessed by deter- mining the levels of cytosolic (for unprocessed or non- farnesylated p21) and membrane-bound (for processed or farnesylated p21) Ha-ras p21 in each of the epidermal and benign skin papilloma samples assayed for farnesyltransferase activity. Several studies have shown that farnesylated p2 1, which associates with the plasma membrane, migrates faster by SDS-PAGE than does nonfarnesylated cytosolic p2 1 [17,18,22,23,41). Therefore, we homogenized the epidermis and papilloma samples in lysis buffer and immunoprecipitated both cytosolic and membrane-bound Ha-ras p21 with an anti-v-Ha-ras antibody. The immunoprecipitate was subjected to SDS-PAGE, the resolved proteins were transferred to a nitro- cellulose membrane, and the levels of both cytosolic and membrane-bound Ha-ras p21 were detected by probing the membrane with v-Ha-ras-rat monoclonal IgG,.

The results of these subcellular localization experiments are shown in Figure 4. In the approximately 22- to 22.5-kDa region, prominent single bands (in epidermis) or closely resolved doublets (in papillomas) were observed in each case (Figure 4). (The size estimate was based on the mo- lecular weight markers used in all the gels [not shown in Figure 4.1) As evident by one slower-migrating band, com- paratively less cytosolic Ha-ras p21 was found in each epi- dermal sample (Figure4, lanes 1-6). Although not evident in Figure 4 lanes 1-6, a very faint band of faster mobility in epidermal samples was also observed in the same re- gion of the blots, suggesting that there was a negligible amount of membrane-bound Ha-ras p21 in epidermis. In contrast, dark doublet bands, the slower one being cytosolic p21 and the faster one membrane-bound Ha-ras p21, were evident in each skin papilloma sample (Figure 4, lanes 7- 18), suggesting that there wassignificantly more of both forms of Ha-ras p21 in papillomas than in epidermis. The levels of cytosolic Ha-ras p2 1, however, were significantly higher than the levels of membrane-bound Ha-ras p21 in each papilloma sample (Figure 4, lanes 7-18). The same

294 AGARWAL ETAL.

C a c,

.- 2 n

F : 0

P E

20

15

10

5

0 30 60 90 120

Time (min)

25tC

FPP (pM)

Figure 2. Kinetics of farnesyltransferase activity in the cytosolic fractionsof epidermises (0) and papillomas (B). (A)Tirne course of enzyme activity. The 90-pL reaction mixture contained cytosolic fraction from epidermis or papilloma (90 pg protein), 9 pg of purified recombinant Ha-ras ~ 2 1 ~ ' ~ ' ~ . and 1 pM (1.8 KCi) [3H]FPP in 0.1 M HEPES buffer, pH 7.4, containing 25 mM MgCl2 and 10 rnM D'IT. The reaction was incubated a t 37°C. and a t the indi- cated times, 10-pL aliquots were removed and assayed. (B) Re- combinant Ha-ras p21 substrate saturation kinetics. Each 10-pL reaction mixture, containing 10 pg cytosolic fraction from epi-

pattern was observed for other epidermal and papilloma samples not shown in Figure 4

Several studies have shown that mutated p2 1, specifically p2 1 mutated at codon 61 in DMBA-TPA-induced skin tumors, migrates slightlyfaster than wild-type p21 in SDS-PAGE and IS recognized by the p21 antibody (clone Y 13-259) [42,43] Since we used DMBA-TPA-induced skin papillomas, we con- sidered it important to evaluate whether the faster migrat-

0 0.00 0.25 0.50 0.75 1.00

0.3

0.2 L

5 - E n

0.1

0.01 I I I I I

4 8 12 16 20

Enzyme (pg)

dermis or papilloma, 1 p M (0.2 pCi) [3H]FPP, and the indicated amount of p21 (p"), was incubated at 37°C for 60 min. (C) FPP substrate saturation kinetics. Each 10-pL reaction mixture, con- taining 10 pg cytosolic fraction from epidermis or papilloma, 1 pg recombinant Ha-ras p21, and the indicated amount of 13H]FPP, was incubated a t 37°C for 60 min. (D) Enzyme concentration curve. Each 10-pL reaction mixture, containing 1 pg of recombinant Ha-ras p21.1 KM (0.2 pCi) [3H]FPP, and the indicated amount of cytosolic protein from epidermis or papilloma, was incubated at 37°C for 60 rnin.

ing p21 in Figure 4 was membrane bound, mutated, or both. Therefore, we homogenized epidermis and papil- loma tissues in nondetergent buffer to avoid the solubili- zation of membrane fraction in detergent. The total tissue homogenate was fractionated into soluble cytosolic and membrane fractions, and the membrane pellet was then solubilized in detergent-rich lysis buffer to obtain homo- geneous soluble membrane fraction. Both cytosolic and

FARNESYLTRANSFERASE IN SKIN TUMORS 295

25 0 0 1

O E Epidermis Pap i I I omas

Figure 3. Farnesyltransferase activity in epidermis and pap- illomas. Each assay was done in duplicate, and the specific ac- tivity data shown are the means of the two values, corrected for the addition of p21.

soluble membrane fractions were then subjected to im- munoprecipitation, SDS-PAGE analysis, and western blot- ting under conditions absolutely identical to those used to obtain the data in Figure 4.

As shown in Figure 5, the levels of Ha-ras p21 in both cytosolic and membrane fractions were significantly higher in all the papilloma samples than in normal epidermis (sim- ilar results were observed for other samples not shown in the figure). Furthermore, at approximately 22- to 22.5-kDa, only one prominent band was evident in each sample of normal epidermis or papilloma tissue. That the cvtosolic

and membrane fractions each had the clear single band for cytosolic and membrane-bound Ha-ras p2 1, respectively, further supports the data shown in Figure 4, which show that when detergent-rich lysis buffer was used for tissue lysis, it also solubilized the membrane fraction and resulted in two closely resolved bands, the slower one being cytosolic p21 and the other one membrane-bound Ha-ras p21, as shown previously by other investigators [17,18, 22,23,41].

Another possibility-that the membrane-associated Ha- ras p21 we observed in skin papillomas also had a codon 61 mutation-cannot be ruled out. The strongest support for this possibility was the presence of a comparatively small amount of membrane-bound Ha-ras p21 in normal epi- dermal samples (Figures 4 and 5B), which must be wild- type p21 and therefore involved in the normal growth and cell signaling pathway. However, being nonmutated, it would be in GDP-bound form and therefore even though associated with membrane would not possess cell-trans- forming potential [5]. On the other hand, the significantly higher levels of membrane-bound Ha-ras p21 in DMBA- TPA-induced skin papillomas (Figures 4 and 5B) may be the consequence of overexpressed cytosolic codon 61 - mutated Ha-ras p21 produced by mutation in the Ha-ras oncogene (A-T transversion at base 182) by DMBA [30, 33.351. The overexpressed, mutated Ha-ras p21, which ex- ists in the GTP-bound form [2-71, is localized to the plasma membrane by farnesyltransferase as a necessary step for its cell transforming potential [44-461. The results shown in Figures 4 and 5 are consistent with enhanced farnesyl- transferaseactivity in chemical carcinogen-induced benign skin papillomas and suggest that papilloma formation may be a consequence of enhanced farnesyltransferase activ-

Figure 4. Subcellular distribution of cytosolic and membrane- bound Ha-ras p21 in epidermises and papillomas homogenized in detergent-rich lysis buffer. Lanes 1-6, samples from the epi- dermis of untreated control SENCAR mice; lanes 7-18, samples from chemical carcinogen-induced benign skin papillomas in

SENCAR mice. The sizes of cytosolic Ha-ras p21 (c-p21) and membrane-bound Ha-ras p21 (m-p21) shown in the figure are based on the migration of molecular-size markers used as standards.

296 AGAR WAL ETA L.

Figure 5. Subcellular distribution of Ha-ras p21 in cytosolic and membrane fractions from epidermis and papillomas. (A) Western blot analysis of cytosolic fractions, showing the levels of unprocessed (soluble or nonfarnesylated) Ha-ras p21. Lanes 1-3, samples from the epidermis of untreated control SENCAR mice; Ianes4-8,samplesfrom chemical carcinogen-induced be-

ity that enhances membrane localization of overexpressed cytosolic mutated Ha-ras p21

DISCUSSION

Since in chemical carcinogen-induced benign skin pap- illomas the farnesyltransferase activity and the levels of both cytosolic and membrane-bound Ha-ras p21 were sig- nificantly higher than in control epidermis, it is reasonable to assume that the activity and levels may be directly cor- related with tumorigenesis. The farnesyltransferase activ- i ty and the levels of cytosolic and membrane-bound Ha-ras p21 were not increased in DMBA-initiated SENCAR mouse epidermis examined up to 7 d after DMBA treatment (data not shown), the period during which point mutation of Ha-ras codon 61 has been shown to occur in murine skin [47]. Similarly, compared to wild type no change in enzyme activity or levels of Ha-ras p21 was observed in the epidermis of transgenic mice (obtained from Charles River Labora- tories, Inc., Wilmington, MA) carrying an activated v-Ha-ras oncogene fused to the promoter of the mouse embryonic d i k e 5-globin gene [48] (data not shown). Repeated topi- cal application of TPA to the skin of these mice has been shown to result in multiple papillomas, some of which pro- gress to squamous cell carcinomas and, more frequently, to underlying sarcomas 1481. These findings suggest that the v-Ha-ras transgene abrogates the initiation step in mouse skin tumorigenesis and that exposure to a carcino- gen is not required for tumor induction in this mouse model [48]. Twice weekly application of the skin tumor promoter TPA to the skin of DMBA-initiated SENCAR mice (after 7 d of initiation) and to transgenic mice with an activated v-Ha- ras oncogene also had not changed the enzyme activity and the levels of cytosolic and membrane-bound Ha-ras p2 1 up to 10 d (data not shown). These observations sug- gest that farnesyltransferase is not involved in tumor initi- ation and in the early phases of tumor promotion in the two-stage initiation-promotion protocol. Furthermore, these observations also suggest that mutation in the Ha-ras oncogene alone (viz, tumor initiation) and early stage of

nign skin papillomas in SENCAR mice. (6) Western blot analysis of membrane fractions showing the levels of processed (mem- brane-bound or farnesylated) Ha-ras p21. Lanes 1-3, epidermis; lanes 4-8, papillomas. The size of Ha-ras p21 shown in the fig- ure is based on the migration of molecular-size markers used as standards.

tumor promotion are not sufficient for the overexpression of mutated Ha-ras p21 and its membrane association, which our results indicate leads to tumor formation. This assumption is further supported by a number of studies showing that tumor initiation alone in murine skin with DMBA or other initiating agents, though an irreversible step that leads to initiated or mutated cells in skin, does not result in the appearance of a tumor during the life of the mice; nor do the early phases of tumor promotion [1,49,50].

The significant increase in farnesyltransferase activity in papillomas (Figure 3) with no significant change in initi- ated or initiated plus early-promoted skin suggests that the enzyme plays an important role in the later phases of tu- mor promotion, during which tumor formation becomes inevitable [ 1,49,50], and in the progression of papillomas to carcinomas. Similarly, the enhanced levels of cytosolic and membrane-bound Ha-ras p21 in papillomas (Figures 4 and 5) with no change in initiated or initiated-plus-early- promoted skin suggest that multiple applications of the tumor promoter result in overproduction of mutated cytosolic Ha-ras p21 during the later phases of tumor pro- motion of initiated skin carrying mutated Ha-rasoncogenes [47]. Localization of this overexpressed, mutated Ha-ras p21 to the membrane requires enhanced farnesyltransferase activity by some unknown mechanism. With papilloma for- mation as an end point, our data strongly support the hy- pothesis that farnesyltransferase plays an important role in the processing of ras protein p21 during tumorigene- sis, thereby leading to tumor formation.

Recently Reiss et al. [25,26] purified the farnesyltransferase enzyme from rat brain and showed it to be a dimer of non- identical subunits (49 and 46 kDa), designated CY and p, respectively. Reiss and coworkers have also generated an- tisera for both subunits [25,26]. Furthermore, the cDNAs for both farnesyltransferase subunits have been cloned and sequenced [51,52]. To understand the mechanism for the increased farnesyltransferase activity in papillomas we ob- served, it is important to assess whether the enzyme was changed at the transcriptional or translational levels or

FARNESYLTRANSFERASE IN SKIN TUMORS 297

whether only enzyme activity was changed. Further stud- ies are planned to answer these questions, using the pep- tide antisera for western blotting and the 01 and p subunit cDNAs for northern blot analysis. Since we have examined a population of skin papillomas induced by DMBA initia- tion and TPA promotion, and such papillomas are known to contain an activated Ha-rasoncogene with a point mu- tation in codon 61 [2,30-361, it is also important to assess the role of farnesyltransferase in those papillomas that lack an activated ras oncogene. Such studies will further strengthen the conclusions drawn in this study.

ACKNOWLEDGMENTS

This work is supported by U.S. Public Health Service Grants ES-I900 and P-30-AR-39750. RA is a recipient of an Upjohn Company Foundation Career Development Award through the Dermatology Foundation. SGK is sup- ported by a postdoctoral fellowship from the National In- stitutes of Health under Training Grant T-AR-07569. We thank Dr. Douglas R. Lowy (Chief, Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD) for his helpful discussions during the course of this study.

Received April 15, 1993; revised July 12, 1993; accepted July 21, 1993.

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