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Induction of Apoptosis in Melanoma Cell Lines by p53 and its Related Proteins

2001; Elsevier BV; Volume: 117; Issue: 4 Linguagem: Inglês

10.1046/j.0022-202x.2001.01464.x

1523-1747

Toshiharu Yamashita, Takashi Tokino, Hidefumi Tonoki, Tetsuya Moriuchi, Hai‐Ying Jin, Fusayuki Omori, Kowichi Jimbow,

Molecular Biology Techniques and Applications

Melanoma cells rarely contain mutant p53 and hardly undergo apoptosis by wild-type p53. By using recombinant adenoviruses that express p53 or p53-related p51A or p73β, we tested their apoptotic activities in melanoma cells. Yeast functional assay revealed a mutation of p53 at the 258th codon (AAA [K] instead of GAA [E]) in one cell line, 70W, out of six 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/or p73β suppressed growth and induced apoptotic DNA fragmentation of SK-mel-23, SK-mel-118, and 70W cells. Interestingly, p51A induced DNA fragmentation in them more significantly than p53 and p73β. By Western blotting we analyzed levels of apoptosis-related proteins in cells expressing p53 family members. Apoptotic Bax and antiapoptotic Bcl-2 were not significantly upregulated or downregulated by expression of p53, p51A, or p73β, except for p53-expressing 70W cells, which contained a larger amount of Bax protein than LacZ-expressing cells. Activation of caspase-3 was demonstrated only in p51A-expressing SK-mel-118 cells. We show here that p51A can mediate apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and p73β. It is also suggested that in melanoma cells (i) cellular target protein(s) other than Bcl-2 and Bax might be responsible for induction of p51A-mediated apoptosis and (ii) caspase-3 is not always involved in the apoptosis by p53 family members. Melanoma cells rarely contain mutant p53 and hardly undergo apoptosis by wild-type p53. By using recombinant adenoviruses that express p53 or p53-related p51A or p73β, we tested their apoptotic activities in melanoma cells. Yeast functional assay revealed a mutation of p53 at the 258th codon (AAA [K] instead of GAA [E]) in one cell line, 70W, out of six 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/or p73β suppressed growth and induced apoptotic DNA fragmentation of SK-mel-23, SK-mel-118, and 70W cells. Interestingly, p51A induced DNA fragmentation in them more significantly than p53 and p73β. By Western blotting we analyzed levels of apoptosis-related proteins in cells expressing p53 family members. Apoptotic Bax and antiapoptotic Bcl-2 were not significantly upregulated or downregulated by expression of p53, p51A, or p73β, except for p53-expressing 70W cells, which contained a larger amount of Bax protein than LacZ-expressing cells. Activation of caspase-3 was demonstrated only in p51A-expressing SK-mel-118 cells. We show here that p51A can mediate apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and p73β. It is also suggested that in melanoma cells (i) cellular target protein(s) other than Bcl-2 and Bax might be responsible for induction of p51A-mediated apoptosis and (ii) caspase-3 is not always involved in the apoptosis by p53 family members. adenovirus multiplicity of infection plaque-forming unit More than 50% of human cancers contain mutated p53 or lose functional p53 (Hollstein et al., 1991Hollstein M. Sidransky D. Vogelstein B. Harris C.C. p53 mutations in human cancers.Science. 1991; 253: 49-53Crossref PubMed Scopus (7454) Google Scholar;Levine et al., 1994Levine A.J. Perry M.E. Chang A. Silver A. Dittmer D. Wu M. Welsh D. The 1993 Walter Hubert Lecture. The role of the p53 tumour-suppressor gene in tumorigenesis.Br J Cancer. 1994; 69: 409-416Crossref PubMed Scopus (438) Google Scholar). 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Induction of the growth inhibitor IGF-binding protein 3 by p53.Nature. 1995; 377: 646-649Crossref PubMed Scopus (806) Google Scholar;Polyak et al., 1997Polyak K. Xia Y. Zweier J.L. Kinzler K.W. Vogelstein B. A model for p53-induced apoptosis.Nature. 1997; 389: 300-305Crossref PubMed Scopus (2239) Google Scholar), the mechanism of p53-mediated apoptosis has not yet been elucidated. Recently, several proteins related to p53, i.e., p51A, p51B, p73α, and p73β, have been identified (Jost et al., 1997Jost C.A. Marin M.C. Kaelin Jr., W.G. p73 is a human p53-related protein that can induce apoptosis.Nature. 1997; 389: 191-194Crossref PubMed Scopus (901) Google Scholar;Kaghad et al., 1997Kaghad M. Bonnet H. Yang A. et al.Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers.Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1537) Google Scholar;Osada et al., 1998Osada M. Ohba M. Kawahara C. et al.Cloning and functional analysis of human p51, which structurally and functionally resembles p53.Nat Med. 1998; 4: 839-843Crossref PubMed Scopus (475) Google Scholar;Trink et al., 1998Trink B. Okami K. Wu W. Sriuranpong V. Jen J. Sidransky D. A new human p53 homologue.Nat Med. 1998; 4: 747Crossref PubMed Scopus (225) Google Scholar). The new members of the p53 family have significant structural homology in the conserved region of p53, including in the DNA-binding, transactivation, and oligomerization domains (Kaghad et al., 1997Kaghad M. Bonnet H. Yang A. et al.Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers.Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1537) Google Scholar;Osada et al., 1998Osada M. Ohba M. Kawahara C. et al.Cloning and functional analysis of human p51, which structurally and functionally resembles p53.Nat Med. 1998; 4: 839-843Crossref PubMed Scopus (475) Google Scholar). Among them, p51A and p51B, and p73α and p73β, are produced from different spliced transcripts of p51 and p73 loci, respectively, and are different from each other at the carboxy termini (Kaghad et al., 1997Kaghad M. Bonnet H. Yang A. et al.Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers.Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1537) Google Scholar;Osada et al., 1998Osada M. Ohba M. Kawahara C. et al.Cloning and functional analysis of human p51, which structurally and functionally resembles p53.Nat Med. 1998; 4: 839-843Crossref PubMed Scopus (475) Google Scholar). Although mutations of p51 and p73 genes have been infrequently found in human cancers including melanomas (Kaghad et al., 1997Kaghad M. Bonnet H. Yang A. et al.Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers.Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1537) Google Scholar;Osada et al., 1998Osada M. Ohba M. Kawahara C. et al.Cloning and functional analysis of human p51, which structurally and functionally resembles p53.Nat Med. 1998; 4: 839-843Crossref PubMed Scopus (475) Google Scholar;Schittek et al., 1999Schittek B. Sauer B. Garbe C. Lack of p73 mutations and late occurrence of p73 allelic deletions in melanoma tissues and cell lines.Int J Cancer. 1999; 82: 583-586Crossref PubMed Scopus (18) Google Scholar), both p51 and p73 can induce apoptosis in human cancer cells (Jost et al., 1997Jost C.A. Marin M.C. Kaelin Jr., W.G. p73 is a human p53-related protein that can induce apoptosis.Nature. 1997; 389: 191-194Crossref PubMed Scopus (901) Google Scholar;Osada et al., 1998Osada M. Ohba M. Kawahara C. et al.Cloning and functional analysis of human p51, which structurally and functionally resembles p53.Nat Med. 1998; 4: 839-843Crossref PubMed Scopus (475) Google Scholar;Ishida et al., 2000Ishida S. Yamashita T. Nakaya U. Tokino T. Adenovirus-mediated transfer of p53-related genes induces apoptosis of human cancer cells.Jpn J Cancer Res. 2000; 91: 174-180Crossref PubMed Scopus (63) Google Scholar). p51 (also known as p40, p63, p73L, and Ket) has been reported to be expressed primarily within the ectoderm and to be essential for epidermal and follicular formation (Mills et al., 1999Mills A.A. Zheng B. Wang X-J. Vogel H. Roop D.R. Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis.Nature. 1999; 398: 708-713Crossref PubMed Scopus (1704) Google Scholar;Yang et al., 1999Yang A. Schweitzer R. Sun D. et al.p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.Nature. 1999; 398: 714-718Crossref PubMed Scopus (1912) Google Scholar). Mutation of p53 is rarely detectable in primary human melanomas (Castresana et al., 1993Castresana J.S. Rubio M.P. Vazquez J.J. Idoate M. Sober A.J. Seizinger B.R. Barnhill R.L. Lack of allelic deletion and point mutation as mechanisms of p53 activation in human malignant melanoma.Int J Cancer. 1993; 55: 562-565Crossref PubMed Scopus (96) Google Scholar;Albino et al., 1994Albino A.P. Vidal M.J. McNutt N.S. et al.Mutation and expression of the p53 gene in human malignant melanoma.Melanoma Res. 1994; 4: 35-45Crossref PubMed Scopus (159) Google Scholar;Lubbe et al., 1994Lubbe J. Reichel M. Burg G. Kleihues P. Absence of p53 gene mutations in cutaneous melanoma.J Inv Dermatol. 1994; 102: 819-821Abstract Full Text PDF PubMed Scopus (102) Google Scholar;Papp et al., 1996Papp T. Jafari M. Schiffmann D. Lack of p53 mutations and loss of heterozygosity in non-cultured human melanocytic lesions.J Cancer Res Clin Oncol. 1996; 122: 541-548Crossref PubMed Scopus (55) Google Scholar). It remains unclear why, although the melanoma cells typically express excess amounts of wild-type p53, they are extremely radioresistant (Geara and Ang, 1996Geara F.B. Ang K.K. Radiation therapy for malignant melanoma.Surg Clin N Am. 1996; 76: 1383-1398Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar;Jenrette, 1996Jenrette J.M. Malignant melanoma: the role of radiation therapy revisited.Semin Oncol. 1996; 23: 759-762PubMed Google Scholar). It is thus interesting to examine whether new members of the p53 family could induce apoptosis in melanoma cells. For this purpose, we expressed p53 and its related p51A and p73β in melanoma cells by using recombinant adenovirus and studied inducibility of apoptosis. In this study, we analyzed (i) p53 of two pigmented (70W and G361) and three nonpigmented (SK-mel-24, SK-mel-118, and TXM18) melanoma cell lines by yeast functional assay, (ii) growth inhibition and apoptosis induction by infection of recombinant adenovirus, and (iii) apoptosis-related cellular proteins by Western blotting. Human melanoma cell lines analyzed in this study are shown in Table I. SK-mel-23, 70W, and G361 are pigmen ted, and SK-mel-24, SK-mel-118, and TXM18 are nonpigmented human melanoma cell lines. MRC5 is nontransformed human fibroblasts derived from an embryonic lung. HeLa and SiHa are cervical carcinoma cell lines containing human papillomavirus type 18 (HPV18) and HPV16, respectively (Schwarz et al., 1985Schwarz E. Freese U.K. Gissmann L. Mayer W. Roggenbuck B. Stremlau A. zur Hausen H. Structure and transcription of human papillomavirus sequences in cervical carcinoma cells.Nature. 1985; 314: 111-114Crossref PubMed Scopus (1123) Google Scholar). 293 is an adenovirus type 5 (Ad 5) DNA-transformed human embryonic kidney cell line used in construction and propagation of recombinant adenoviruses (Graham et al., 1977Graham F.L. Smiley J. Russell W.C. Nairu R. Characterization of a human cell line transformed by DNA from human adenovirus type 5.J General Virol. 1977; 36: 59-72Crossref PubMed Scopus (3504) Google Scholar). Melanoma cell lines were kindly supplied by Dr. A Houghton, Sloan Kettering Cancer Center, NY, and others were purchased from ATCC (Rockville, MD). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum (FBS; Gibco BRL, Tokyo, Japan), penicillin G, and streptomycin.Table IResult of yeast functional assay of p53Rates of red coloniesCell linesExp IExp IIStatus of p53SK-mel-23(pigmented)n.t.n.t.wild typeaVolkenandt et al, 1991Montano et al, 1994.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%mutantbCodon 258: from GAA(E) to AAA(K).G361(pigmented)7.5%n.t.wild typea Volkennandt et al., 1991Volkennandt M. Schlegel U. Nanus D.M. Albino A.P. Mutational analysis of the human p53 gene in malignant melanoma.Pigment Cell Res. 1991; 4: 35-40Crossref PubMed Scopus (91) Google ScholarMontano et al., 1994Montano X. Shamser M. Whitehead P. Dawson K. Newton J. Analysis of p53 in human cutaneous melanoma cell lines.Oncogene. 1994; 9: 1455-1459PubMed Google Scholarb Codon 258: from GAA(E) to AAA(K). Open table in a new tab Total cellular RNA was prepared from cells cultured in one or two 10 cm dishes with acid guanidinium thiocyanate phenol chloroform (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63167) Google Scholar). p53 cDNA was synthesized as described previously (Takahashi et al., 2000Takahashi M. Tonoki H. Tada M. et al.Distinct prognostic values of p53 mutations and loss of estrogen receptor and their cumulative effect in primary breast cancers.Int J Cancer (Pred Oncol. 2000; 89: 92-99Crossref PubMed Scopus (29) Google Scholar) using Molony murine leukemia virus reverse transcriptase (Gibco BRL) from 3 µg of total RNA in 20 µl of reverse transcriptase buffer containing 25 pmol p53-specific primer RT-1 (5′-CGGGAGGTAGAC-3′). The p53 cDNA was PCR-amplified in 20 µl of reaction mixture containing 2 µl of reverse transcriptase reaction product and 1.25 plaque-forming units (Pfu) of DNA polymerase (Stratagene, La Jolla, CA). For the p53 functional assay, 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. The yeast expression vector pSS16 (Flaman et al., 1995Flaman J-M. Frebourg T. Moreau V. et al.A simple p53 functional assay for screening cell lines, blood, and tumors.Proc Natl Acad Sci USA. 1995; 92: 3963-3967Crossref PubMed Scopus (429) Google Scholar) was digested with excess amounts of HindIII and StuI and electrophoresed in a 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 purified with a Wizard PCR prep kit (Promega, Madison, WI). A gap was created between codons 67 and 347. The yeast functional assay was performed according to the method ofKashiwazaki et al., 1997Kashiwazaki H. Tonoki H. Tada M. et al.High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay.Oncogene. 1997; 15: 2667-2674Crossref PubMed Scopus (83) Google Scholar. The yeast reporter strain yIG397 (Flaman et al., 1995Flaman J-M. Frebourg T. Moreau V. et al.A simple p53 functional assay for screening cell lines, blood, and tumors.Proc Natl Acad Sci USA. 1995; 92: 3963-3967Crossref PubMed Scopus (429) Google Scholar) was used throughout this study. The strain yIG397 contains an integrated plasmid with the ADE2 open reading frame under the control of a p53-responsive promoter. When the strain is transformed with a plasmid encoding mutant p53, the cells fail to express ADE2 (phosphoribosylaminoimidazole carboxylase, EC 4.1.1.21) and form red colonies because of the accumulation of an oxidized, polymerized derivative of phosphoribosylaminoimidazole (Weisman et al., 1987Weisman L.S. Bacallao R. Wickner W. Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle.J Cell Biol. 1987; 105: 1539-1547Crossref PubMed Scopus (159) Google Scholar). Yeast was cultured in 100 ml of YPD medium supplemented with 200 µg per ml of adenine until OD600 reached 0.8. The cells were pelleted, resuspended in 10 ml of LiOAc solution containing 0.1 M lithium acetate, 10 mM Tris-HCl pH 8.0, and 1 mM ethylenediamine tetraacetic acid (EDTA), pelleted again, and resuspended in 500 µl of LiOAc solution. For each transformation, 50 µl of yeast suspension was mixed with 1–5 µl of unpurified p53 cDNA PCR product, 50–100 ng of linealized plasmid, 5 µl of sonicated single-stranded salmon sperm DNA (10 mg per ml) and 300 µl of lithium acetate containing 40% PEG4000 (Kanto Chemical, Tokyo, Japan). The mixture was incubated at 30°C for 30 min and heat-shocked at 42°C for 15 min. Yeast was then pelleted and resuspended with the medium minus leucine plus adenine (5 µg per ml) and incubated for 48 h in a 30°C humidified chamber. More than 200 colonies were examined in this assay. Yeast was digested with zymolyase (Seikagaku-Kogyo, Tokyo, Japan), and p53 expression plasmids were extracted by the alkaline lysis method (QIAprep plasmid kit, Qiagen, Hilden, Germany) and transfected into XL-1 blue Escherichia coli by electroporation. The plasmids were recovered, purified, 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 the manufacturer'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 adenoviruses that express one of the human p53 family members, p53 (Ad-p53), p51A (Ad-p51A), and p73β (Ad-p73β), were described previously (Yamano et al., 1999Yamano S. Tokino T. Yasuda M. et al.Induction of transformation and p53-dependent apoptosis by adenovirus type 5 E4orf6/7 cDNA.J Virol. 1999; 73: 10095-10103Crossref PubMed Google Scholar;Ishida et al., 2000Ishida S. Yamashita T. Nakaya U. Tokino T. Adenovirus-mediated transfer of p53-related genes induces apoptosis of human cancer cells.Jpn J Cancer Res. 2000; 91: 174-180Crossref PubMed Scopus (63) Google Scholar). Recombinant adenovirus expressing bacterial β-galactosidase, Ad-LacZ, was provided by Dr. M. Imperiale of Michigan University. Each of the recombinant adenoviruses was propagated in 293 cells and infected cell suspension was frozen and thawed twice; then the supernatant was aliquoted to serum tubes and stored at -80°C until use. Concentration of virus stocks (4–10 × 108 pfu per ml) was determined by plaque formation in 293 cells. The relative efficiency of adenovirus infection was determined by X-gal (5-bromo-4-chloro-3-indolyl-β-D-galacto pyranoside) staining of cells infected with Ad-LacZ. After cells had been seeded and cultured for 24–48 h, they were infected with Ad-LacZ and cultured for 48 h. Then, after cells had been washed with phosphate-buffered saline (PBS) and fixed with 2% formaldehyde and 0.2% glutaraldehyde for 5 min at 4°C, cells expressing β-galactosidase were visualized after incubation at 37°C in the presence of X-gal. Infectivity was evaluated as the ratio of the number of positively stained cells to the total number of cells. Cells were seeded at 2 × 105 in 6 cm dishes and cultured for 48 h. Cells were then infected with recombinant adenovirus at a multiplicity of infection (moi) of 20 pfu per cell, incubated at 37°C for 60 min, and then cultured in DMEM with 1% FBS. The number of cells on the sixth day was counted with a hemocytometer. Five × 105 cells in 6 cm dishes were infected with recombinant adenovirus at an moi of 20 pfu per cell. After cells had been cultured for 48 h, adherent and floating cells were collected and resuspended in 400 µl of 5 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 0.5% Triton X-100. After centrifugation at 16,000g for 20 min, the supernatant was transferred to a fresh Eppendorf tube and incubated with 100 µg per ml of RNase A for 1 h and then with 200 µg per ml of proteinase K and 1% sodium dodecyl sulfate (SDS) for 2 h at 50°C. After the solution was extracted with phenol saturated with Tris-EDTA buffer, DNA was precipitated with ethanol and finally dissolved in 30–50 µl of Tris-EDTA buffer. DNA was electrophoresed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. For fluorescence-activated cell sorter analysis, adherent and floating cells were collected together, washed in ice-cold PBS, and fixed in 1.0 ml of 75% cold ethanol. Then, cells were rehydrated in cold PBS and treated with RNase A (50 µg per ml) at 37°C for 30 min. After incubation, cells were rinsed twice in ice-cold PBS and resuspended in 2.0 ml PBS with 50 µg per ml propidium iodine (Sigma Aldrich Japan, Tokyo, Japan) at 4°C for 2 h. The cells were analyzed in a FACScan cell sorter (Nippon Becton Dickinson, Tokyo, Japan). The sub G1, G1, and G2/M populations were quantified using the Cell Quest program. Cells cultured in 6 cm dishes were lyzed in 200–400 µl of lysis buffer (10 mM KCl, 1.5 mM MgCl2, 10 mM Tris pH 7.4, 0.5% SDS, and 1 mM phenylmethylsulfonyl fluoride). After collection of cell lysates in Eppendorf tubes, they were disrupted using Branson's sonicator for 10 s. The protein concentration in the supernatants was determined using a BCA protein assay kit (Pierce, Rockford, IL). Samples containing 5.0 µg protein were denatured by heating at 94°C for 5 min in loading buffer (125 mM Tris pH 6.8, 4.0% SDS, 20% glycerol, 5% β-mercaptoethanol) and electrophoresed on 5%-20% SDS polyacrylamide gel (Ready Gel, Bio-Rad Laboratories, Tokyo, Japan). The differentiated proteins were transferred onto a nitrocellulose membrane (Protran; Schleicher & Schuell, Dassel, Germany) and detected with the following primary antibodies: DO-7 (Novocastra, Newcastle upon Tyne, U.K.) for p53, Waf1 (Novocastra) for p21Waf1/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 specific complexes were visualized by enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Caspase-3 activity was measured by using a CPP32/caspase-3 Colorimetric Protease Assay Kit (MBL, Nagoya, Japan) according to the manufacturer's instructions. Cells infected with recombinant adenovirus were collected 48 h after infection. Five microliters of 4 mM DEVD-pNA substrate (200 mM final concentration) was added to 100 µl of solution containing 100 µg of cellular protein and incubated at 37°C for 2 h. Then, caspase-3-mediated cleaved chromophore p-nitroanilide (pNA) was measured with a microtiter plate reader at 405 nm. First, efficiencies of introduction and expression of an exogenous gene in human melanoma cell lines were tested using β-galactosidase-expressing recombinant adenovirus Ad-LacZ. When cells were infected with Ad-LacZ at an moi of 20 pfu per cell or more, almost maximum efficiency of gene expression was obtained by our recombinant adenovirus. The rates of β-galactosidase-expressing cells, including MRC5 fibroblasts, two cervical carcinoma cell lines (HeLa and SiHa), and six melanoma cell lines (SK-mel-23, SK-mel-24, SK-mel-118, TXM18, 70W, and G361), were determined 48 h after infection of Ad-LacZ at the moi of 20 pfu per cell. All the cell lines tested showed approximately 90% efficiency of β-galactosidase expression or more except for SK-mel-23 (about 50%) (data not shown). In order to determine whether p53 in the melanoma cell lines is wild type or mutant, total cellular RNA was purified from cultured melanoma cells and processed for yeast functional assay as described in Materials and Methods. RNA from SK-mel-24, SK-mel-118, TXM18, and G361 produced white colonies > 90% Table I, indicating that they were expressing functionally wild-type p53. Meanwhile, RNA prepared from 70W cells generated red colonies > 98% Table I, indicating that they were expressing functionally mutant p53. The cDNA inserts were purified from three different red colonies containing the p53 cDNA of 70W and were sequenced by the cycle sequencing method. As a result, all the p53 cDNA inserts of 70W revealed the 258th codon of AAA (Lys) instead of GAA (Glu) of wild-type p53 Table I. In order to examine whether growth suppression and/or apoptosis of melanoma cells was induced by p53 family proteins, they were infected with the recombinant adenovirus expressing p53 or its related protein. All the p53 members suppressed growth of melanoma cell lines except for G361, growth of which was suppressed by infection of the control adenovirus Ad-LacZ. TXM18 was only slightly suppressed by p53 family members. On the other hand, growth of SK-mel-23, SK-mel-24, SK-mel-118, and 70W cells was clearly suppressed by p53, p51A, and/or p73β. 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 virus infection. It seems that, among the p53 family members, p51A possesses a stronger cytotoxicity than other p53 members in melanoma cells Figure 1. In order to determine whether the growth inhibition by p53 family members was apoptosis or not, we analyzed cellular DNA by agarose gel electrophoresis and flow cytometry. When DNA was prepared from melanoma cells at 48 h after viral infection and electrophoresed in 1.5% agarose gel, smeared and/or fragmented DNAs were observed Figure 2. p51A produced a clearer apoptotic DNA ladder in SK-mel-23, SK-mel-118, and 70W cells than p53 or p73β did. FACScan analysis detected sub G1 fractions of virus-infected cells almost correlated with the DNA fragmentation observed in agarose gel electrophoresis. Figure 3 shows a representative result of FACScan of SK-mel-118 cells. A large amount of sub G1 fraction was seen in SK-mel-118 cells when they were infected with Ad-p51A. Interestingly, p53 and p73β hardly induced apoptotic DNA fragmentation in SK-mel-118 cells (Figures 2, 3).Figure 3A representative result of cell cycle analysis of SK-mel-118 cells. Cells were infected with each virus at the moi of 20 pfu per cell for 48 h and subjected to flow cytometry.View Large Image Figure ViewerDownload (PPT) One of the well-documented cellular proteins that is transactivated by p53 and can mediate cell cycle arrest at G1 is p21Waf1/Cip1. In this study, however, Western blot analysis detected only small amounts of p21Waf1/Cip1 in SK-mel-23, SK-mel-118, and 70W cells after expression of p53 and p51A Figure 4. Levels of apoptosis-related Bcl-2 and Bax proteins were also analyzed by Western blotting Figure 4. Bcl-2 expression was detectable in SK-mel-23 and 70W but did not change before and after infection of the recombinant adenoviruses, whereas it was hardly detectable in SK-mel-118 cells Figure 4. On the other hand, Bax protein was slightly induced in 70W cells infected with Ad-p53, but a comparable amount of Bax was observed in SK-mel-118 cells Figure 4. Thus, it seems that Bax might not be involved in the process of p51A-mediated apoptosis of SK-mel-23 and SK-mel-118 cells. Caspase-3 is cleaved and converted to an activated form in the final step of the activation cascade of caspase family proteins. We examined whether caspase-3 was activated in the melanoma cell apoptosis mediated by p53 family members. Colonic cancer cell line SW480 undergoes clear apoptosis by anti-Fas (CD95) antibody. In the process of Fas-mediated apoptosis of the SW480 cells, the level of caspase-3 is gradually decreased and caspase-3-specific cleaved product becomes detectable (data not shown). 70W cells undergo apoptosis by p53 family members Figure 2. The caspase-3 band of 70W cells was hardly detectable by Western blotting, however Figure 5, and its biochemical activation was undetectable even after the viral infection Figure 6. In SK-mel-23 cells, caspase-3 seems to be cleaved for activation as its band was decreased in Western blotting Figure 5; however, caspase-3 activation was not detected by DEVD-pNA assay Figure 6. Among cell lines that showed apoptosis by p53 family members, only SK-mel-118 cells infected with Ad-p51A produced caspase-3-activated product Figure 6. Thus, caspase-3 activation was not always required for the apoptotic process of melanoma cells by p53 family members.Figure 6Detection of caspase-3 activity in apoptotic melanoma cells. Caspase-3 activity was detected by CPP32/caspase-3 Colorimetric Protease Assay Kit (MBL, Nagoya, Japan) based on the recognition of DEVD-pNA by caspase-3. Cleaved pNA was detected by microplate reader at 405 nm. Mean ± SD was determined from three dishes per infection.View Large Image Figure ViewerDownload (PPT) We constructed recombinant adenoviruses that express one of the p53 family members, p53, p51A, p51B, p73α, and p73β, by human cytomegalovirus early promoter (Yamano et al., 1999Yamano S. Tokino T. Yasuda M. et al.Induction of transformation and p53-dependent apoptosis by adenovirus type 5 E4orf6/7 cDNA.J Virol. 1999; 73: 10095-10103Crossref PubMed Google Scholar;Ishida et al., 2000Ishida S. Yamashita T. Nakaya U. Tokino T. Adenovirus-mediated transfer of p53-related genes induces apoptosis of human cancer cells.Jpn J Cancer Res. 2000; 91: 174-180Crossref PubMed Scopus (63) Google Scholar). Similarly to the results observed in SaOS2 and H1229 cells (Ishida et al., 2000Ishida S. Yamashita T. Nakaya U. Tokino T. Adenovirus-mediated transfer of p53-related genes induces apoptosis of human cancer cells.Jpn J Cancer Res. 2000; 91: 174-180Crossref PubMed Scopus (63) Google Scholar), p51A and p73β induced apoptosis in melanoma cell lines more efficiently than p51B and p73α, respectively (data not shown). Therefore, we examined and compared the growth inhibitory and apoptotic activity of p53, p51A, and p73β in human melanoma cell lines. As p53 typically induces apoptosis in p53-mutated or -deficient cancer cell lines, we first analyzed p53 status in five melanoma cell lines by yeast functional assay. The 258th codon, at which p53 of 70W cells is mutated, is not a hot spot of the p53 mutation of human cancers, but this mutant is deficient for transcriptional transactivation from the ribosomal gene cluster sequence that is contained in the yeast functional assay. This suggests that 70W cells do not express functional p53. Melanoma cells were shown to express wild-type p53 of which Ser-376 is constitutively dephosphorylated, and it fails to interact with 14-3-3 protein in response to DNA damage (Satyamoorthy et al., 2000Satyamoorthy K. Chehab N.H. Waterman M.J.F. Lien M.C. El-Deiry W.S. Herlyn M. Halazonetis T.D. Aberrant regulation and function of wild-type p53 in radioresistant melanoma cells.Cell Growth Differ. 2000; 11: 467-474PubMed Google Scholar). These melanoma cells do not undergo cell cycle arrest or apoptosis, even if high levels of ectopic wild-type p53 are expressed. We showed here, however, that at least one of the p53 family 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 and significant apoptosis in SK-mel-118 cells that were resistant to apoptosis by p53 and p73β. SK-mel-23 cells expressed adenovirus-mediated β-galactosidase with a lower efficiency than other cell lines tested, but still efficiently undergo cell death or apoptosis by p53 family proteins. Among melanoma cell lines analyzed, SK-mel-23 cells produce larger amounts of both wild-type p53 (Figures 4, 5) and cytotoxic melanin pigment than nonpigmented SK-mel-118 and slightly pigmented 70W cells, and thus it is possible that a small amount of additional p53 or its related protein could enhance susceptibility of apoptosis. It is estimated that the high level expression of Bcl-2 protein in melanocytes is related to their long lifespan. Levels of antiapoptotic Bcl-2 and apoptotic Bax proteins were variable among the melanoma cell lines we analyzed. SK-mel-23 cells, which underwent apoptosis by p53 and p51A, contained a smaller amount of Bax than SK-mel-118 and 70W cells. SK-mel-118 cells, which were resistant to apoptosis by p53 and p73β, contained hardly detectable amounts of Bcl-2 protein. Thus, it is suggested that p53 family members can induce apoptosis in melanoma cells that contain high levels of Bcl-2 and/or low levels of Bax proteins, or that cellular protein(s) other than Bcl-2/Bax might be responsible for the sensitivity to p53-family-mediated apoptosis. Expression of p51A induced severe cytotoxicity, clear DNA fragmentation, and activation of caspase-3 in SK-mel-118 cells. As caspase-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 that typical apoptotic DNA fragmentation is associated with caspase-3 activation. The ladder-pattern fragmentation of cellular DNA, however, was also observed in SK-mel-23 and 70W cells infected with p53-family-expressing adenovirus Figure 2. Of particular note, a steady-state level of caspase-3 is hardly detectable in 70W cells Figure 5 and activation of caspase-3 was not demonstrated in them with or without the virus infection Figure 6. This suggests that apoptotic DNA fragmentation not involving caspase-3-activated DNase 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., 1999Mills A.A. Zheng B. Wang X-J. Vogel H. Roop D.R. Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis.Nature. 1999; 398: 708-713Crossref PubMed Scopus (1704) Google Scholar;Yang et al., 1999Yang A. Schweitzer R. Sun D. et al.p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.Nature. 1999; 398: 714-718Crossref PubMed Scopus (1912) Google Scholar). An N-terminal defective, dominant-negative version of p51A was shown to be expressed characteristically in the basal layer, outer sheath of the hair follicle, and in low-differentiated but not in highly differentiated squamous cell carcinoma cells (Parsa et al., 1999Parsa R. Yang A. McKeon F. Green H. Association of p63 with proliferative potential in normal and neoplastic human keratinocytes.J Inv Dermatol. 1999; 113: 1099-1105Crossref PubMed Scopus (381) Google Scholar). Thus, it appears that p51A possesses important roles for growth and differentiation control of keratinocytes. Recently, it was reported that p51A not only accumulates and elicits its transactivating function in response to DNA damage but also mediates erythroid cell differentiation (Katoh et al., 2000Katoh I. Aisaki K. Kurata S. Ikawa S. Ikawa Y. p51A (Tap63g), a p53 homolog, accumulates in response to DNA damage for cell regulation.Oncogene. 2000; 19: 3126-3130Crossref PubMed Scopus (85) Google Scholar). Although it has not yet been tested whether or not p51A shows a growth-inhibitory effect in normal epidermal cells including keratinocytes, p51A was shown here to possess strong apoptotic activity in some of the melanoma cells. It is interesting to analyze the mechanism of p51A-mediated apoptosis of SK-mel-118 cells, as neither p53 nor p73β induces apoptosis in them. DNA microarray analysis or differential display might isolate cellular genes that are responsible for p51A-mediated apoptosis. 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.