Induction of Apoptosis in Melanoma Cell Lines by p53 and itsRelated Proteins

Toshiharu Yamashita,*² Takashi Tokino,² Hidefumi Tonoki,³ Tetsuya Moriuchi,³ Hai-Ying Jin,*Fusayuki Omori,* and Kowichi Jimbow**Department of Dermatology and ²Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University School of Medicine,

Sapporo, Japan; ³Division of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan

Melanoma cells rarely contain mutant p53 and hardlyundergo apoptosis by wild-type p53. By usingrecombinant adenoviruses that express p53 or p53-related p51A or p73b, we tested their apoptoticactivities in melanoma cells. Yeast functional assayrevealed a mutation of p53 at the 258th codon (AAA[K] instead of GAA [E]) in one cell line, 70W, out ofsix human melanoma cell lines analyzed (SK-mel-23,SK-mel-24, SK-mel-118, TXM18, 70W, and G361).Adenovirus-mediated transfer of p53, p51A, and/orp73b suppressed growth and induced apoptotic DNAfragmentation of SK-mel-23, SK-mel-118, and 70Wcells. Interestingly, p51A induced DNA fragmenta-tion in them more signi®cantly than p53 and p73b.By Western blotting we analyzed levels of apoptosis-related proteins in cells expressing p53 family mem-

bers. Apoptotic Bax and antiapoptotic Bcl-2 werenot signi®cantly upregulated or downregulated byexpression of p53, p51A, or p73b, except for p53-expressing 70W cells, which contained a largeramount of Bax protein than LacZ-expressing cells.Activation of caspase-3 was demonstrated only inp51A-expressing SK-mel-118 cells. We show herethat p51A can mediate apoptosis in both wild-typeand mutant p53-expressing melanoma cells more sig-ni®cantly than p53 and p73b. It is also suggested thatin melanoma cells (i) cellular target protein(s) otherthan Bcl-2 and Bax might be responsible for induc-tion of p51A-mediated apoptosis and (ii) caspase-3 isnot always involved in the apoptosis by p53 familymembers. Key words: recombinant adenovirus/Bax/cas-pase-3. J Invest Dermatol 117:914±919, 2001

More than 50% of human cancers contain mutatedp53 or lose functional p53 (Hollstein et al, 1991;Levine et al, 1994). Mice lacking functional p53can grow apparently normal but develop tumors(Donehower et al, 1992), suggesting that p53 has

an essential role in tumor suppression. Introduction or high-levelexpression of wild-type p53 in p53-de®cient cancer cells mightresult in cell cycle arrest or apoptosis (Yonish-Rouach et al, 1991;Chen et al, 1996; Hermeking et al, 1997). p53 has been shown to bea sequence-speci®c transactivator for promoters containing thep53-binding site (Bargonetti et al, 1991; El-Deiry et al, 1992; Funket al, 1992; Kern et al, 1992), and it is believed that many of thebiologic functions of p53 result from transcriptional activation oftarget genes (Kastan et al, 1992; El-Deiry et al, 1993; Pietenpol et al,1994). Although apoptosis-related cellular proteins including Baxand insulin-like growth factor binding protein 3 have been shownto be induced by p53 (Miyashita and Reed, 1995; Buckbinder et al,1995; Polyak et al, 1997), the mechanism of p53-mediatedapoptosis has not yet been elucidated.

Recently, several proteins related to p53, i.e., p51A, p51B,p73a, and p73b, have been identi®ed (Jost et al, 1997; Kaghad et al,

1997; Osada et al, 1998; Trink et al, 1998). The new members ofthe p53 family have signi®cant structural homology in theconserved region of p53, including in the DNA-binding,transactivation, and oligomerization domains (Kaghad et al, 1997;Osada et al, 1998). Among them, p51A and p51B, and p73a andp73b, are produced from different spliced transcripts of p51 andp73 loci, respectively, and are different from each other at thecarboxy termini (Kaghad et al, 1997; Osada et al, 1998). Althoughmutations of p51 and p73 genes have been infrequently found inhuman cancers including melanomas (Kaghad et al, 1997; Osadaet al, 1998; Schittek et al, 1999), both p51 and p73 can induceapoptosis in human cancer cells (Jost et al, 1997; Osada et al, 1998;Ishida et al, 2000). p51 (also known as p40, p63, p73L, and Ket) hasbeen reported to be expressed primarily within the ectoderm and tobe essential for epidermal and follicular formation (Mills et al, 1999;Yang et al, 1999).

Mutation of p53 is rarely detectable in primary humanmelanomas (Castresana et al, 1993; Albino et al, 1994; Lubbe et al,1994; Papp et al, 1996). It remains unclear why, although themelanoma cells typically express excess amounts of wild-type p53,they are extremely radioresistant (Geara and Ang, 1996; Jenrette,1996). It is thus interesting to examine whether new members ofthe p53 family could induce apoptosis in melanoma cells. For thispurpose, we expressed p53 and its related p51A and p73b inmelanoma cells by using recombinant adenovirus and studiedinducibility of apoptosis. In this study, we analyzed (i) p53 of twopigmented (70W and G361) and three nonpigmented (SK-mel-24,SK-mel-118, and TXM18) melanoma cell lines by yeast functionalassay, (ii) growth inhibition and apoptosis induction by infection of

Manuscript received October 2, 2000; revised May 29, 2001; acceptedfor publication June 13, 2001.

Reprint requests to: Dr. Toshiharu Yamashita, Department ofDermatology, Sapporo Medical University School of Medicine, South 1,West 16, Chuo-ku, Sapporo 060, Japan. Email: yamasita@sapmed.ac.jp

Abbreviations: Ad, adenovirus; moi, multiplicity of infection; pfu,plaque-forming unit.

0022-202X/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc.

914

recombinant adenovirus, and (iii) apoptosis-related cellular proteinsby Western blotting.

MATERIALS AND METHODS

Cells and cell culture Human melanoma cell lines analyzed in thisstudy are shown in Table I. SK-mel-23, 70W, and G361 are pigmen-ted, and SK-mel-24, SK-mel-118, and TXM18 are nonpigmentedhuman melanoma cell lines. MRC5 is nontransformed human ®broblastsderived from an embryonic lung. HeLa and SiHa are cervical carcinomacell lines containing human papillomavirus type 18 (HPV18) andHPV16, respectively (Schwarz et al, 1985). 293 is an adenovirus type 5(Ad 5) DNA-transformed human embryonic kidney cell line used inconstruction and propagation of recombinant adenoviruses (Graham et al,1977). Melanoma cell lines were kindly supplied by Dr. A Houghton,Sloan Kettering Cancer Center, NY, and others were purchased fromATCC (Rockville, MD). Cells were cultured in Dulbecco's modi®edEagle's medium (DMEM) supplemented with 5% fetal bovine serum(FBS; Gibco BRL, Tokyo, Japan), penicillin G, and streptomycin.

RNA extraction and reverse transcription polymerase chainreaction (RT-PCR) Total cellular RNA was prepared from cellscultured in one or two 10 cm dishes with acid guanidinium thiocyanatephenol chloroform (Chomczynski and Sacchi, 1987). p53 cDNA wassynthesized as described previously (Takahashi et al, 2000) using Molonymurine leukemia virus reverse transcriptase (Gibco BRL) from 3 mg oftotal RNA in 20 ml of reverse transcriptase buffer containing 25 pmolp53-speci®c primer RT-1 (5¢-CGGGAGGTAGAC-3¢). The p53 cDNAwas PCR-ampli®ed in 20 ml of reaction mixture containing 2 ml ofreverse transcriptase reaction product and 1.25 plaque-forming units (Pfu)of DNA polymerase (Stratagene, La Jolla, CA). For the p53 functionalassay, primers P3 (5¢-ATTTGATGCTGTCCCCGGACGATATTG-AA(S)C-3¢, where (S) represents a phosphorothioate linkage) and P4 (5¢-ACCCTTTTTGGACTTCAGGTGGCTGGAGT(S)G-3¢) were used.PCR was run on a Thermal Cycler Model 2400 (Perkin-Elmer, Chiba,Japan) at 96°C for 1 min, then 35 cycles at 95°C for 40 s, 65°C for 70 s,and 78°C for 90 s, followed by 78°C for 2 min.

Plasmids The yeast expression vector pSS16 (Flaman et al, 1995) wasdigested with excess amounts of HindIII and StuI and electrophoresed ina 1% low-melting-temperature agarose gel (Sea Plaque agarose; FMC,Rockland, ME). The linearized plasmids were recovered from the gel,dephospholized with bovine intestinal alkaline phosphatase (Takara,Otsu, Japan), and puri®ed with a Wizard PCR prep kit (Promega,Madison, WI). A gap was created between codons 67 and 347.

Yeast p53 functional assay The yeast functional assay was performedaccording to the method of Kashiwazaki et al (1997). The yeast reporterstrain yIG397 (Flaman et al, 1995) was used throughout this study. Thestrain yIG397 contains an integrated plasmid with the ADE2 openreading frame under the control of a p53-responsive promoter. Whenthe strain is transformed with a plasmid encoding mutant p53, the cellsfail to express ADE2 (phosphoribosylaminoimidazole carboxylase, EC4.1.1.21) and form red colonies because of the accumulation of anoxidized, polymerized derivative of phosphoribosylaminoimidazole(Weisman et al, 1987). Yeast was cultured in 100 ml of YPD mediumsupplemented with 200 mg per ml of adenine until OD600 reached 0.8.The cells were pelleted, resuspended in 10 ml of LiOAc solutioncontaining 0.1 M lithium acetate, 10 mM Tris-HCl pH 8.0, and 1 mMethylenediamine tetraacetic acid (EDTA), pelleted again, and resuspended

in 500 ml of LiOAc solution. For each transformation, 50 ml of yeastsuspension was mixed with 1±5 ml of unpuri®ed p53 cDNA PCRproduct, 50±100 ng of linealized plasmid, 5 ml of sonicated single-stranded salmon sperm DNA (10 mg per ml) and 300 ml of lithiumacetate containing 40% PEG4000 (Kanto Chemical, Tokyo, Japan). Themixture was incubated at 30°C for 30 min and heat-shocked at 42°C for15 min. Yeast was then pelleted and resuspended with the mediumminus leucine plus adenine (5 mg per ml) and incubated for 48 h in a30°C humidi®ed chamber. More than 200 colonies were examined inthis assay.

Recovery of p53 plasmids from yeast and DNA sequencing Yeastwas digested with zymolyase (Seikagaku-Kogyo, Tokyo, Japan), and p53expression plasmids were extracted by the alkaline lysis method(QIAprep plasmid kit, Qiagen, Hilden, Germany) and transfected intoXL-1 blue Escherichia coli by electroporation. The plasmids wererecovered, puri®ed, and sequenced with a DyeDeoxy Terminator Kit(Perkin-Elmer, Urayasu, Japan) on an ABI 373 A automated sequencer(Nippon Applied Biosystem, Urayasu, Japan) under the conditions of themanufacturer's protocol using the following primers: P3seq, 5¢-ATT-TGATGCTGTCCCCGGACGATATTGAAC-3¢; P11seq, 5¢-TAC-TCCCCTGCCCTCAACAAGATG-3¢; P12seq, 5¢-TTGCGTGTG-GAGTATTTGGATGAC-3¢; P13seq, 5¢-GCCCATCCTCACCAT-CATCACACT-3¢.

Recombinant adenovirus Recombinant adenoviruses that expressone of the human p53 family members, p53 (Ad-p53), p51A (Ad-p51A),and p73b (Ad-p73b), were described previously (Yamano et al, 1999;Ishida et al, 2000). Recombinant adenovirus expressing bacterialb-galactosidase, Ad-LacZ, was provided by Dr. M. Imperiale ofMichigan University. Each of the recombinant adenoviruses waspropagated in 293 cells and infected cell suspension was frozen andthawed twice; then the supernatant was aliquoted to serum tubes andstored at ±80°C until use. Concentration of virus stocks (4±10 3 108 pfuper ml) was determined by plaque formation in 293 cells.

b-galactosidase assay The relative ef®ciency of adenovirus infectionwas determined by X-gal (5-bromo-4-chloro-3-indolyl-b-D-galacto-pyranoside) staining of cells infected with Ad-LacZ. After cells had beenseeded and cultured for 24±48 h, they were infected with Ad-LacZ andcultured for 48 h. Then, after cells had been washed with phosphate-buffered saline (PBS) and ®xed with 2% formaldehyde and 0.2%glutaraldehyde for 5 min at 4°C, cells expressing b-galactosidase werevisualized after incubation at 37°C in the presence of X-gal. Infectivitywas evaluated as the ratio of the number of positively stained cells to thetotal number of cells.

Assay of growth inhibition by p53 family members Cells wereseeded at 2 3 105 in 6 cm dishes and cultured for 48 h. Cells were theninfected with recombinant adenovirus at a multiplicity of infection (moi)of 20 pfu per cell, incubated at 37°C for 60 min, and then cultured inDMEM with 1% FBS. The number of cells on the sixth day wascounted with a hemocytometer.

Detection of apoptotic DNA fragmentation Five 3 105 cells in6 cm dishes were infected with recombinant adenovirus at an moi of 20pfu per cell. After cells had been cultured for 48 h, adherent and ¯oatingcells were collected and resuspended in 400 ml of 5 mM Tris-HCl(pH 8.0), 10 mM EDTA, and 0.5% Triton X-100. After centrifugationat 16,000g for 20 min, the supernatant was transferred to a freshEppendorf tube and incubated with 100 mg per ml of RNase A for 1 hand then with 200 mg per ml of proteinase K and 1% sodium dodecylsulfate (SDS) for 2 h at 50°C. After the solution was extracted withphenol saturated with Tris-EDTA buffer, DNA was precipitated withethanol and ®nally dissolved in 30±50 ml of Tris-EDTA buffer. DNAwas electrophoresed by 1.5% agarose gel electrophoresis and visualizedby ethidium bromide staining.

For ¯uorescence-activated cell sorter analysis, adherent and ¯oatingcells were collected together, washed in ice-cold PBS, and ®xed in1.0 ml of 75% cold ethanol. Then, cells were rehydrated in cold PBSand treated with RNase A (50 mg per ml) at 37°C for 30 min. Afterincubation, cells were rinsed twice in ice-cold PBS and resuspended in2.0 ml PBS with 50 mg per ml propidium iodine (Sigma Aldrich Japan,Tokyo, Japan) at 4°C for 2 h. The cells were analyzed in a FACScan cellsorter (Nippon Becton Dickinson, Tokyo, Japan). The sub G1, G1, andG2/M populations were quanti®ed using the Cell Quest program.

Western blot analysis Cells cultured in 6 cm dishes were lyzed in200±400 ml of lysis buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM TrispH 7.4, 0.5% SDS, and 1 mM phenylmethylsulfonyl ¯uoride). After

Table I. Result of yeast functional assay of p53

Cell lines

Rates of red colonies

Status of p53Exp I Exp II

SK-mel-23 (pigmented) n.t. n.t. wild typea

SK-mel-24 (nonpigmented) 6.2% n.t. wild typeSK-mel-118 (nonpigmented) 8.2% n.t. wild typeTXM18 (nonpigmented) 6.3% 6.4% wild type70W (pigmented) 99.5% 98.5% mutantb

G361 (pigmented) 7.5% n.t. wild type

aVolkenandt et al, 1991; Montano et al, 1994.bCodon 258: from GAA(E) to AAA(K).

VOL. 117, NO. 4 OCTOBER 2001 MELANOMA CELL APOPTOSIS BY p53 FAMILY MEMBERS 915

collection of cell lysates in Eppendorf tubes, they were disrupted usingBranson's sonicator for 10 s. The protein concentration in thesupernatants was determined using a BCA protein assay kit (Pierce,Rockford, IL). Samples containing 5.0 mg protein were denatured byheating at 94°C for 5 min in loading buffer (125 mM Tris pH 6.8, 4.0%SDS, 20% glycerol, 5% b-mercaptoethanol) and electrophoresed on 5%±20% SDS polyacrylamide gel (Ready Gel, Bio-Rad Laboratories, Tokyo,Japan). The differentiated proteins were transferred onto a nitrocellulosemembrane (Protran; Schleicher & Schuell, Dassel, Germany) anddetected with the following primary antibodies: DO-7 (Novocastra,Newcastle upon Tyne, U.K.) for p53, Waf1 (Novocastra) forp21Waf1/Cip1, anti-Bcl-2 (100) (Santa Cruz, Santa Cruz, CA) for Bcl-2,anti-Bax (BD PharMingen, San Diego, CA) for Bax, and anti-caspase-3(Upstate Biotechnology, Lake Placid, NY) for caspase-3. The speci®ccomplexes were visualized by enhanced chemiluminescence reagents(Amersham Pharmacia Biotech, Buckinghamshire, U.K.).

Detection of caspase-3 activity Caspase-3 activity was measured byusing a CPP32/caspase-3 Colorimetric Protease Assay Kit (MBL,Nagoya, Japan) according to the manufacturer's instructions. Cellsinfected with recombinant adenovirus were collected 48 h afterinfection. Five microliters of 4 mM DEVD-pNA substrate (200 mM®nal concentration) was added to 100 ml of solution containing 100 mgof cellular protein and incubated at 37°C for 2 h. Then, caspase-3-mediated cleaved chromophore p-nitroanilide (pNA) was measured witha microtiter plate reader at 405 nm.

RESULTS

Adenovirus-mediated gene transfer into melanoma celllines First, ef®ciencies of introduction and expression of anexogenous gene in human melanoma cell lines were tested usingb-galactosidase-expressing recombinant adenovirus Ad-LacZ.When cells were infected with Ad-LacZ at an moi of 20 pfu percell or more, almost maximum ef®ciency of gene expression wasobtained by our recombinant adenovirus. The rates ofb-galactosidase-expressing cells, including MRC5 ®broblasts, twocervical carcinoma cell lines (HeLa and SiHa), and six melanomacell lines (SK-mel-23, SK-mel-24, SK-mel-118, TXM18, 70W,and G361), were determined 48 h after infection of Ad-LacZ at themoi of 20 pfu per cell. All the cell lines tested showedapproximately 90% ef®ciency of b-galactosidase expression ormore except for SK-mel-23 (about 50%) (data not shown).

p53 status in melanoma cell lines In order to determinewhether p53 in the melanoma cell lines is wild type or mutant, totalcellular RNA was puri®ed from cultured melanoma cells andprocessed for yeast functional assay as described in Materials andMethods. RNA from SK-mel-24, SK-mel-118, TXM18, and G361produced white colonies > 90% (Table I), indicating that they

were expressing functionally wild-type p53. Meanwhile, RNAprepared from 70W cells generated red colonies > 98% (Table I),indicating that they were expressing functionally mutant p53. ThecDNA inserts were puri®ed from three different red coloniescontaining the p53 cDNA of 70W and were sequenced by thecycle sequencing method. As a result, all the p53 cDNA inserts of70W revealed the 258th codon of AAA (Lys) instead of GAA (Glu)of wild-type p53 (Table I).

Induction of apoptosis by p53 family members In order toexamine whether growth suppression and/or apoptosis ofmelanoma cells was induced by p53 family proteins, they wereinfected with the recombinant adenovirus expressing p53 or itsrelated protein. All the p53 members suppressed growth ofmelanoma cell lines except for G361, growth of which wassuppressed by infection of the control adenovirus Ad-LacZ.TXM18 was only slightly suppressed by p53 family members. Onthe other hand, growth of SK-mel-23, SK-mel-24, SK-mel-118,and 70W cells was clearly suppressed by p53, p51A, and/or p73b.Figure 1 shows the relative numbers of viable cells of SK-mel-23,SK-mel-24, SK-mel-118, and 70W on the sixth day after virusinfection. It seems that, among the p53 family members, p51Apossesses a stronger cytotoxicity than other p53 members inmelanoma cells (Fig 1).

In order to determine whether the growth inhibition by p53family members was apoptosis or not, we analyzed cellular DNA byagarose gel electrophoresis and ¯ow cytometry. When DNA wasprepared from melanoma cells at 48 h after viral infection andelectrophoresed in 1.5% agarose gel, smeared and/or fragmentedDNAs were observed (Fig 2). p51A produced a clearer apoptoticDNA ladder in SK-mel-23, SK-mel-118, and 70W cells than p53or p73b did. FACScan analysis detected sub G1 fractions of virus-infected cells almost correlated with the DNA fragmentationobserved in agarose gel electrophoresis. Figure 3 shows a repre-sentative result of FACScan of SK-mel-118 cells. A large amount ofsub G1 fraction was seen in SK-mel-118 cells when they wereinfected with Ad-p51A. Interestingly, p53 and p73b hardly inducedapoptotic DNA fragmentation in SK-mel-118 cells (Figs 2, 3).

Expression of p21Waf1/Cip1 and Bcl family proteins One ofthe well-documented cellular proteins that is transactivated by p53and can mediate cell cycle arrest at G1 is p21Waf1/Cip1. In this study,however, Western blot analysis detected only small amounts ofp21Waf1/Cip1 in SK-mel-23, SK-mel-118, and 70W cells afterexpression of p53 and p51A (Fig 4). Levels of apoptosis-relatedBcl-2 and Bax proteins were also analyzed by Western blotting(Fig 4). Bcl-2 expression was detectable in SK-mel-23 and 70Wbut did not change before and after infection of the recombinantadenoviruses, whereas it was hardly detectable in SK-mel-118 cells(Fig 4). On the other hand, Bax protein was slightly induced in

Figure 1. Growth inhibition of melanoma cells by p53 and itsrelated proteins. Cells were infected with Ad-LacZ, Ad-p53, Ad-p51A, and Ad-p73b at the moi of 20 pfu per cell and cultured for 6 d.The number of viable cells (unstained by trypan blue) were counted atthe sixth day after infection. Mean 6 SD was determined from threedishes per infection.

Figure 2. Detection of DNA fragmentation by agarose gelelectrophoresis. Cells were infected with recombinant adenovirus (20pfu per cell) and cultured for 48 h, and then cells including ¯oating andadherent ones were collected. Fragmented DNA was extracted frominfected cells as described in Materials and Methods and electrophoresedon 1.5% agarose gels.

916 YAMASHITA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

70W cells infected with Ad-p53, but a comparable amount of Baxwas observed in SK-mel-118 cells (Fig 4). Thus, it seems that Baxmight not be involved in the process of p51A-mediated apoptosisof SK-mel-23 and SK-mel-118 cells.

Detection of steady-state level and activated form ofcaspase-3 Caspase-3 is cleaved and converted to an activatedform in the ®nal step of the activation cascade of caspase familyproteins. We examined whether caspase-3 was activated in themelanoma cell apoptosis mediated by p53 family members. Coloniccancer cell line SW480 undergoes clear apoptosis by anti-Fas(CD95) antibody.

In the process of Fas-mediated apoptosis of the SW480 cells, thelevel of caspase-3 is gradually decreased and caspase-3-speci®ccleaved product becomes detectable (data not shown). 70W cellsundergo apoptosis by p53 family members (Fig 2). The caspase-3band of 70W cells was hardly detectable by Western blotting,however (Fig 5), and its biochemical activation was undetectableeven after the viral infection (Fig 6). In SK-mel-23 cells, caspase-3seems to be cleaved for activation as its band was decreased inWestern blotting (Fig 5); however, caspase-3 activation was notdetected by DEVD-pNA assay (Fig 6). Among cell lines thatshowed apoptosis by p53 family members, only SK-mel-118 cellsinfected with Ad-p51A produced caspase-3-activated product(Fig 6). Thus, caspase-3 activation was not always required forthe apoptotic process of melanoma cells by p53 family members.

DISCUSSION

We constructed recombinant adenoviruses that express one of thep53 family members, p53, p51A, p51B, p73a, and p73b, by humancytomegalovirus early promoter (Yamano et al, 1999; Ishida et al,2000). Similarly to the results observed in SaOS2 and H1229 cells(Ishida et al, 2000), p51A and p73b induced apoptosis in melanomacell lines more ef®ciently than p51B and p73a, respectively (datanot shown). Therefore, we examined and compared the growthinhibitory and apoptotic activity of p53, p51A, and p73b in humanmelanoma cell lines.

As p53 typically induces apoptosis in p53-mutated or -de®cientcancer cell lines, we ®rst analyzed p53 status in ®ve melanoma celllines by yeast functional assay. The 258th codon, at which p53 of70W cells is mutated, is not a hot spot of the p53 mutation ofhuman cancers, but this mutant is de®cient for transcriptionaltransactivation from the ribosomal gene cluster sequence that iscontained in the yeast functional assay. This suggests that 70W cellsdo not express functional p53.

Melanoma cells were shown to express wild-type p53 of whichSer-376 is constitutively dephosphorylated, and it fails to interactwith 14-3-3 protein in response to DNA damage (Satyamoorthy

et al, 2000). These melanoma cells do not undergo cell cycle arrestor apoptosis, even if high levels of ectopic wild-type p53 areexpressed. We showed here, however, that at least one of the p53family members, p51A, was able to induce apoptosis in both wild-type p53-expressing SK-mel-23 and SK-mel-118 and mutant p53-expressing 70W cells. In addition, p51A induced clear andsigni®cant apoptosis in SK-mel-118 cells that were resistant toapoptosis by p53 and p73b. SK-mel-23 cells expressed adenovirus-mediated b-galactosidase with a lower ef®ciency than other celllines tested, but still ef®ciently undergo cell death or apoptosis byp53 family proteins. Among melanoma cell lines analyzed, SK-mel-23 cells produce larger amounts of both wild-type p53 (Figs 4, 5)and cytotoxic melanin pigment than nonpigmented SK-mel-118

Figure 3. A representative result of cell cycleanalysis of SK-mel-118 cells. Cells wereinfected with each virus at the moi of 20 pfu percell for 48 h and subjected to ¯ow cytometry.

Figure 4. Western blot analysis of melanoma cells infected withrecombinant adenoviruses. Cells infected with recombinantadenovirus (20 pfu per cell) and cultured for 48 h were lyzed and 5.0 mgof protein was electrophoresed in 5%±20% SDS polyacrylamide gel.Antibodies against p53, p21, Bcl-2, and Bax were reacted and speci®cbands were detected by chemiluminescence (Amersham).

VOL. 117, NO. 4 OCTOBER 2001 MELANOMA CELL APOPTOSIS BY p53 FAMILY MEMBERS 917

and slightly pigmented 70W cells, and thus it is possible that a smallamount of additional p53 or its related protein could enhancesusceptibility of apoptosis.

It is estimated that the high level expression of Bcl-2 protein inmelanocytes is related to their long lifespan. Levels of antiapoptoticBcl-2 and apoptotic Bax proteins were variable among themelanoma cell lines we analyzed. SK-mel-23 cells, which under-went apoptosis by p53 and p51A, contained a smaller amount ofBax than SK-mel-118 and 70W cells. SK-mel-118 cells, whichwere resistant to apoptosis by p53 and p73b, contained hardlydetectable amounts of Bcl-2 protein. Thus, it is suggested that p53family members can induce apoptosis in melanoma cells thatcontain high levels of Bcl-2 and/or low levels of Bax proteins, orthat cellular protein(s) other than Bcl-2/Bax might be responsiblefor the sensitivity to p53-family-mediated apoptosis.

Expression of p51A induced severe cytotoxicity, clear DNAfragmentation, and activation of caspase-3 in SK-mel-118 cells. Ascaspase-3 activation was only demonstrated in p51A-infected SK-mel-118 cells, which showed a clearer DNA ladder than Ad-p51A-or Ad-p53-infected SK-mel-23 and 70W cells, it is suggested thattypical apoptotic DNA fragmentation is associated with caspase-3activation. The ladder-pattern fragmentation of cellular DNA,however, was also observed in SK-mel-23 and 70W cells infectedwith p53-family-expressing adenovirus (Fig 2). Of particular note,a steady-state level of caspase-3 is hardly detectable in 70W cells(Fig 5) and activation of caspase-3 was not demonstrated in themwith or without the virus infection (Fig 6). This suggests thatapoptotic DNA fragmentation not involving caspase-3-activatedDNase is responsible for p53-family-mediated apoptosis in SK-mel-23 and 70W cells.

The study using p51A knockout mice revealed that this p53-related protein is essential for formation of epidermal and adnexal

tissues (Mills et al, 1999; Yang et al, 1999). An N-terminaldefective, dominant-negative version of p51A was shown to beexpressed characteristically in the basal layer, outer sheath of thehair follicle, and in low-differentiated but not in highly differen-tiated squamous cell carcinoma cells (Parsa et al, 1999). Thus, itappears that p51A possesses important roles for growth anddifferentiation control of keratinocytes. Recently, it was reportedthat p51A not only accumulates and elicits its transactivatingfunction in response to DNA damage but also mediates erythroidcell differentiation (Katoh et al, 2000). Although it has not yet beentested whether or not p51A shows a growth-inhibitory effect innormal epidermal cells including keratinocytes, p51A was shownhere to possess strong apoptotic activity in some of the melanomacells. It is interesting to analyze the mechanism of p51A-mediatedapoptosis of SK-mel-118 cells, as neither p53 nor p73b inducesapoptosis in them. DNA microarray analysis or differential displaymight isolate cellular genes that are responsible for p51A-mediatedapoptosis.

This work was supported in part by Grants-in-Aid for Basic Research in

Dermatology from the Ministry of Education, Science, Sports, and Culture of Japan.

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Figure 6. Detection of caspase-3 activity in apoptotic melanomacells. Caspase-3 activity was detected by CPP32/caspase-3 ColorimetricProtease Assay Kit (MBL, Nagoya, Japan) based on the recognition ofDEVD-pNA by caspase-3. Cleaved pNA was detected by microplatereader at 405 nm. Mean 6 SD was determined from three dishes perinfection.

Figure 5. Detection of caspase-3 in melanoma cells. Cells infectedwith recombinant adenovirus (20 pfu per cell) were cultured for 0, 12,24, and 48 h and lyzed to prepare protein extracts. Electroblot on thenitrocellulose ®lter was incubated with caspase-3-speci®c antibody anddetected by chemiluminescence.

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