Article no. bjoc.1999.0572 The expression of p73 is incresed in lung cncer, independent of p53 gene ltertion Y Tokuchi 1, T Hshimoto 1, Y Kobyshi 2, M Hyshi 1, K Nishid 2, S Hyshi 1, K Imi 1, K Nkchi 1, Y Ishikw 3, K Nkgw 4, Y Kwkmi 5 nd E Tsuchiy 1,2 1 Sitm Cncer Center Reserch Institute, 818 Komuro, In, Sitm 362-0806, Jpn; 2 Deprtment of Clinicl Pthology, Sitm Cncer Center Hospitl, 818 Kumuro, In, Sitm 362-0806, Jpn; Deprtments of 3 Pthology nd 4 Respirtory Surgery, Cncer Institute, 1-37-1 Kmi-Ikebukuro, Toshimku, Tokyo 170-8455, Jpn; 5 First Deprtment of Medicine, School of Medicine, Hokkido University, Kit 15 Nishi 7, Spporo, 060-8636, Jpn Summry p73 gene, new p53 homologue, hs been identified: it supposedly cts s tumour suppressor gene in neuroblstom. To clrify whether p73 might be involved in lung crcinogenesis, we exmined p73 expression in resected lung cncer nd pired norml lung in 60 cses using semi-quntittive reverse trnscription-polymerse chin rection (RT-PCR). We lso exmined p73 gene sttus in three representtive cses using Southern blot, nd p53 gene ltertion in 49 cses using PCR-single-strnd conformtion polymorphism (PCR-SSCP) nd direct sequence. In 87% of the cses (52/60) p73 expression in tumour ws more thn twice s high s tht in pired norml lung tissues, nd the difference between p73 expression in tumour nd norml lung tissue ws significnt (P < 0.0001). However, Southern blot nlysis reveled tht none of the cses showed p73 gene mplifiction. Compred with clinicopthologicl chrcteristics, p73 expression correltes significntly with histologicl differences nd ge of ptient, independently (P < 0.05). Concerning p53 gene sttus, 43% (21/49) showed p53 gene ltertion, but there ws no correltion between p73 overexpression nd p53 gene ltertion. Our results suggest tht need for further functionl nlysis of the role of p73 in lung crcinogenesis. Keywords: lung cncer; p73; 1p36; p53; tumour suppressor gene p73, new cndidte tumour suppressor gene, ws recently identified t 1p36, the short rm of chromosome 1, by Kghd et l (1997). The homology between p73 nd p53 is extensive within most conserved p53 domins (Zmbetti et l, 1993; Ko et l, 1996; Kghd et l, 1997). Wild p53 works s so-clled security gurd to induce cell cycle rrest or poptosis in response to cellulr stresses on DNA dmge (Livingstone et l, 1992; Lowe et l, 1993; Dickmn, 1997), nd loss or inctivtion of p53 is thought to contribute to the development of 50% of ll humn cncers (Levine, 1997). Therefore, ccording to the known homology between p53 nd p73, p73 is lso expected to work s tumour suppressor gene. Deletions of 1p36 re common in neuroblstoms, nd extremely low level of p73 mrna hve been found in the mjority of neuroblstom cell lines (Cron et l, 1995; Kghrd et l, 1997), showing tht, t lest in neuroblstom, p73 pprently works s tumour supprressor gene in spite of the lck of definitive evidence. However, very recently Mi et l (1998) reported tht p73 might be overexpressed in lung cncer tissues in comprison with norml lung. Furthermore, p73 genomic muttion hs not been found even fter intensive reserch (Mi et l, 1998; Nomoto et l, 1998; Tkhshi et l, 1998). Accordingly, it is questionble whether p73 works s tumour suppressor gene in lung cncer. To verify the role of p73 in lung crcinogenesis, we mesured the mount of p73 mrna expression in 86 primry lung cncer tissues nd pired Received 3 Sepember 1998 Revised 21 Jnury 1999 Accepted 27 Jnury 1999 Correspondence to: E Tsuchiy norml lung tissues by semi-quntittive reverse trnscriptionpolymerse chin rection (RT-PCR). We then nlysed the reltionship between the mount of p73 expression nd clinicopthologicl chrcteristics. Moreover, we exmined for p53 gene ltertion to clrify the reltionship between p73 mrna expression nd p53 gene sttus. MATERIALS AND METHODS Mterils nd histologicl clssifiction Surgiclly resected tumours nd pired corresponding norml tissues were obtined from 86 ptients with primry lung cncers t the Cncer Institute Hospitl (Tokyo, Jpn), from 1990 to 1993. All smples were quickly frozen in liquid nitrogen nd stored t 80 C until RNA ws extrcted. RNA ws estimted by β-ctin mrna expression mplified with RT-PCR, nd we excluded 26 cses becuse of the poor qulity of the extrcted RNA. Distribution of histologicl types of the 60 lung cncers were: 34 denocrcinoms, 19 squmous cell crcinoms, three lrge-cell crcinoms, two smll-cell crcinoms nd two denosqumous crcinoms (World Helth Orgniztion, 1981) (Tble 1). The medin ge of the 60 ptients ws 61 yers (rnge 44 80 yers). Forty-five of the 60 ptients were men, nd ll 15 femle ptients hd denocrcinom. The stge of ech tumour ws determined ccording to the TNM Clssifiction of Mlignnt Tumours defined by the Interntionl Union Aginst Cncer (UICC, 1992): 19 stge I, 11 stge II, 21 stge IIIA, eight stge IIIB nd one stge IV. To evlute the consumption of cigrettes smoked before lung cncer dignosis, smoking index ws used: 1623
1624 Y Tokuchi et l Tble 1 The reltionship mong chrcteristics of the ptients, the mount of p73 expression nd p53 gene sttus No. Sex Age Histology p-stge Smoking index b p73 in norml tissues c p73 in tumour p53 gene sttus d Exon Codon Bse chnge 48 M 72 Ad II 750 0.95 6.41 ND 52 M 65 Sq IIIA 900 1.78 27.88 ND 54 M 70 Sq IIIB 1250 2.54 6.14 Mutnt 5 149 175 deletion 74b 55 M 59 AS I 740 1.43 10.81 ND 57 M 66 L IIIB 1600 0.30 1.69 ND 60 M 68 Sq IIIA 940 0.34 8.14 Wild 62 M 80 Sq II 840 1.35 20.08 Wild 63 M 76 Sm IIIA 1250 4.08 58.06 ND 64 M 54 Sq IIIB 1350 1.04 3.93 Wild 65 M 60 Ad IIIA 800 0.28 0.84 Wild 66 W 51 Ad IIIB 0 0.41 0.42 Mutnt 7 245 GGC-AGC 68 W 44 Ad IIIB 0 0.69 1.58 Wild 69 M 58 Ad IIIA 1600 0.78 3.97 Mutnt 8 273 CGT-TGT 70 M 59 Sq IIIA 1200 0.72 9.62 Mutnt 5 175 CGC-CAC 85 M 58 Sq IIIA 1330 3.31 1.45 ND 87 M 65 Sq I 760 6.94 10.14 Mutnt 7 244 GGC-TGC 91 M 65 Ad I 2280 0.03 1.47 Wild 93 W 61 Ad I 310 0.42 2.44 Wild 94 M 78 Sq I 1020 0.78 29.20 Wild 97 M 54 Ad IIIA 960 0.22 0.48 Mutnt 6 198 GAA-TAA 98 M 67 Sm IIIA 940 0.05 1.88 ND 99 M 70 Sq I 1520 0.37 5.85 Wild 101 W 68 Ad IIIA 75 2.47 12.32 Mutnt 7 242 TGC-TAC 102 M 53 Sq I 234 0.24 2.12 Wild 106 M 51 AS IIIA 0 0.30 2.05 ND 111 M 54 Ad I 540 3.00 13.26 Wild 114 W 77 Ad I 0 0.70 2.72 Wild 117 W 59 Ad IIIA 0 1.51 5.33 Wild 121 M 64 Ad II 1320 0.38 3.32 Wild 122 W 74 Ad IIIA 240 0.88 3.33 Mutnt 6 209 AGA-TGA 126 M 66 L IIIA 460 0.53 4.63 ND 127 M 44 Ad I 340 0.06 0.66 Wild 130 W 55 Ad I 0 0.10 0.06 ND 135 M 49 Sq IIIA 300 0.46 4.02 Mutnt 6 196 CGA-CCA 136 M 50 Ad I 1600 0.30 6.51 Wild 137 M 68 Ad IIIA 0 0.11 0.35 Wild 141 M 59 Sq I 780 0.53 1.45 Wild 142 M 70 Ad I 561 0.55 2.79 Wild 143 M 54 Ad IIIA 1440 3.18 12.21 Wild 146 M 76 Sq II 1250 1.51 15.88 Mutnt 5 144 CAG-CCG 147 W 68 Ad I 0 0.40 0.19 Wild 150 M 63 Ad IV 600 0.17 0.54 Wild 153 W 50 Ad II 0 1.00 1.06 Wild 157 M 49 Ad II 20 1.21 2.16 Mutnt 7 238 TGT-AGT 159 M 51 Sq II 1240 0.12 5.64 Mutnt 10 5 junction gat-tgat 160 M 50 Ad IIIB 2310 0.62 3.23 Mutnt 6 189 CCC-G_C 168 W 51 Ad II 0 0.88 3.62 Wild 174 M 72 Ad I 510 2.00 2.59 Mutnt 8 273 CGT-CAT 178 M 50 Ad I 1750 0.61 4.89 Wild 179 M 70 Sq IIIA 1590 2.27 9.40 Mutnt 6 195 ATC-ACC 188 M 53 Sq II 1320 0.90 4.77 Mutnt 8 271 GAG-TAG 193 M 68 Sq IIIA 1440 1.33 12.47 Mutnt 7 245 GGC-GTC 196 M 61 Ad II 3680 1.20 3.74 Wild 200 M 71 Sq IIIB 1020 1.56 5.65 Mutnt 4 103 TAC-TAG 201 W 51 Ad I 0 0.10 2.44 Wild 202 W 70 Ad IIIA 0 0.65 4.13 Wild 203 W 67 Ad IIIB 0 0.74 5.65 Mutnt 5 132 AAG-AGG 205 W 49 Ad I 0 0.44 1.39 Mutnt 6 213 CGA-TGA 206 M 49 L II 1015 2.18 15.74 ND 208 M 71 Ad IIIA 960 1.72 10.75 Mutnt 5 157 GTC-TTC Ad, Adenocrcinom; Sq, Squmous cell crcinom; Sm, Smll cell crcinom; L, Lrge cell crcinom; AS, Adenosqumous crcinom. b Smoking index is defined s cigrette consumption per dy multiplied by smoking yers. c p73 expression is estimted by dividing by β-ctin expression. d ND, not done.
p73 overexpression in lung cncer 1625 cigrette consumption per dy multiplied by smoking yers. Hevy smokers were defined s those with smoking indices over 400. Preprtion of RNA nd expression of p73 mrna using RT-PCR All tissue smples were frozen in liquid nitrogen immeditely fter surgery nd subjected to isoltion of RNA s previously reported (Tokuchi et l, 1999). RNAs were prepred from 0.1 0.2 g of humn primry lung cncers nd pired non-cncerous prenchym ccording to the method of Chomczynski nd Scchi (1987), which ws quntified using UV spectrometry t 260 nm. The RNAs (1.0 µg) in 40 µl rection volume were reverse trnscribed to synthesize cdna using Tkr RNA PCR kit (Tkr, Tokyo) with 2.5-µM rndom hexmers t 42 C. The oligonucleotides used in PCR mplifiction were s follows: P1 (sense strnd in exons 4 nd 5 of the p73 gene), GAC GTA CTC CCC GCT CTT GA; P2 (ntisense strnd in exon 7 of the p73 gene), TGG CTC ATA GGG CAC CAC GA; P3 (sense strnd in exon 7 of the p73 gene), ATT CAC CAC CAT CCT GTA CA; P4 (ntisense strnd in exon 9 of the p73 gene), GCT GCT GCT GCT GCC GAT AG; P5 (sense strnd of the β-ctin), CAA GAG ATG GCC ACG GCT GCT; P6 (ntisense strnd of the β-ctin), TCC TTC TGC ATC CTG TCG GCA. PCR mplifiction of the p73 cdna ws performed using 1 µl of the cdna, 0.2-µM primers P1 nd P2 in 25-µl mixtures consisting of 1 U Tq DNA polymerse (Boehringer Mnnheim), 0.05 MegBequerel (MBq) of [α- 32 P]dCTP, 10 mm Tris HCI (ph 8.3), 50 mm potssium chloride, 1.5 mm mgnesium chloride nd 40 µm deoxynucleotide triphosphtes (datp, dttp, dgtp nd dctp). The PCR rection comprised 35 cycles with denturing t 95 C for 30 s, nd nneling nd extension t 72 C for 20 s in ech cycle, using GeneAmp PCR System 9600 (Perkin-Elmer Corp.). Ten microlitres of the mplified product ws subjected to 5% polycrylmide gel electrophoresis, nd the rdioctivity ws evluted with Bio-Imge nlyser BAS2000 (Fuji Film, Tokyo). The PCR product size using P1 nd P2 is 301 bses; using P3 nd P4, 439 bses. No expected PCR products were observed when RT ws not performed. To confirm the results with P1 nd P2, n dditionl PCR using P3 nd P4 ws lso performed: it comprised 40 cycles using GeneAmp PCR System 9600 (Perkin-Elmer Corp.) in the buffer described bove, with denturing t 95 C for 30 s, nd nneling nd extension t 70 C for 1 min in ech cycle. As control for RNA qulity, seprte portions of cdna were mplified using primers specific for humn β-ctin. PCR ws performed using 1 µl of cdna, 1 U Tq DNA polymerse (Boehringer Mnnheim), 0.5 µm P5 nd P6, 0.05 MBq of [α- 32 P]- dctp nd 0.15 mm mgnesium chloride in 25-µl incubtion buffers. The rection (95 C nd 63 C for 30 s, nd 72 C for 1 min) ws performed for 21 cycles. To quntify the mount of PCR products of p73 (using P1 nd P2) nd β-ctin, 18 mtched smples of lung tumours nd norml lung tissues were processed simultneously, nd the rdioctivity ws determined with BAS2000 (Fuji Film, Tokyo, Jpn). The mount of PCR product of ech smple ws expressed s vlue reltive to the verge rdioctivity of 18 norml lungs in ech ssy. Ech PCR nd electrophoresis procedure ws repeted twice, fter which we clculted the verge p73 nd β-ctin expression for ech smple. Finlly, we rrived t the reltive p73:β-ctin rtio, clculted by dividing the verge mount of p73 by tht of β-ctin, for ech smple. p73 genomic-southern blot hybridiztion Genomic DNAs from tumours nd pired norml lung tissues of three representtives cses were extrcted using stndrd methods s previously reported (Tsuchiy et l, 1992; Tokuchi et l, 1999). After ech 10-µg DNA were completely digested with HindIII, DNAs were subjected to 0.8% grose gel electrophoresis. Using vcuum blotter (Bio-Rd), DNA smples were rpidly trnsferred to the nylon membrne (Hybond-N; Amershm Jpn, Tokyo, Jpn) in 10 stndrd sline citrte (SSC), nd then cross-linked under UV light. RT-PCR products mplified with P1 nd P2 primers, which contined exon 5, 6 nd prt of exon 7 of p73 coding sequence, were cut from the gel nd purified using 0.45-µm centrifugl filter (Millipore), phenol chloroform extrction nd subsequent ethnol precipittion. After this, the purified DNAs were rdiolbelled with [α- 32 P]dCTP nd multiprime DNA lbelling systems kit (RPN. 1601Y, Amershm). Southern blot hybridiztion ws performed t 59 C in 5 SSC, 10 Denhrd s solution, 10 mm EDTA, 200 µg ml 1 slmon sperm DNA nd 1% sodium dodecyl sulphte (Smbrook et l, 1989). After sufficient wshing, the rdioctivity ws evluted with Bio-Imge nlyser BAS2000 (Fuji Film, Tokyo, Jpn). p53 muttion nlysis nd DNA sequencing In 49 of the 53 ptients with denocrcinom or squmous cell crcinom, genomic DNAs were obtined nd exmined for p53 ltertions (exons 4 8 nd 10) using PCR-single-strnd conformtion polymorphism (PCR-SSCP) (Kishimoto et l, 1992). Sequences of oligonucleotides used in PCR were s follows: exon 4 of p53, the sense primer, 5 -ACC TGG TCC TCT GAC TGC TCT TTT CA nd the ntisense primer, 5 -CCA GGC ATT GAA GTC TCA TGG AAG C; exon 5, 5 -TCT GTT CAC TTG TGC CCT GA nd 5 -GCC AGA CCT AAG AGC AAT CA; exon 6, 5 - GCT GGG GCT GGA GAG ACG AC nd 5 -GAC AAC CAC CCT TAA CCC CT; exon 7, 5 -CTT GCC ACA GGT CTC CCC AA nd 5 -GGT CAG CGG CAA GCA GAG GC; expn 8, 5 -TTA AAT GGG ACA GGT AGG AC nd 5 -GAT AAA AGT GAA TCT GAG GCA T; exon 10, 5 -TAT ACT TAC TTC TCC CCC TCC TCT nd 5 -ATG AGA ATG GAA TCC TAT GGC TTT. The 5 -end of ech primer ws lbelled with fluorescence: sense primer, 6-crboxyfluorescein, nd ntisense primer, 4,7,2,7 - tetrchloro-6-crboxyfluorescein (Jpn Bio Service Corp., Ask, Jpn). Genomic DNAs were extrcted from 49 tumours nd pired norml lung tissues (Tokuchi et l, 1999), nd subjected to PCR rection mixture contining 50 ng genomic DNA, 10 pm ech pir of primers, 0.2 mm deoxynucleotide triphosphtes (datp, dttp, dgtp nd dctp), 1.0 U Tq DNA polymerse (Boehringer Mnnheim), 10 mm Tris HCl (ph 8.3), 50 mm potssium chloride nd 1.5 mm mgnesium chloride. After initil denturtion (for 2 min t 94 C), 30 cycles PCR were crried out s follows: 94 C for 30 s, dequte nneling temperture for 30 s (exon 4, 69 C; exon 5, 53 C; exon 6, 63 C; exon 7, 64 C; exon 8, 59 C; nd exon 10, 63 C) nd 72 C for 30 s in GeneAmp PCR System 9600 (Perkin- Elmer Corp.). PCR products were dentured t 98 C for 5 min, pplied to 4% non-denturing polycrylmide gel with 10% glycerol, nd electrophoresed t 22 C using ABI PRISM TM 377 (Perkin-Elmer Corp.). SSCP dt were processed by GeneScn Anlysis 2.0.2 computer softwre (Perkin-Elmer Corp.). Where genomic DNAs extrcted from tumours showed different SSCP ptterns from corresponding norml lung tissues, both genomic
1626 Y Tokuchi et l A B p73 1 2 3 4 β-ctin 1 2 3 4 5 5 4 4 Reltive ctivity 3 2 Reltive ctivity 3 2 1 1 0 0 0.1 0.2 0.3 0.4 0.5 RNA (µg) 0 0 0.1 0.2 0.3 0.4 0.5 RNA (µg) Figure 1 Demonstrtion of reltive rdioctivity of p73 (A) nd β-ctin (B) mrna RT-PCR products. Vrious mounts of RNA of the tumour (cse No. 101) in 20-µl rection buffer were reverse-trnscribed (lne 1, 0.0625 µg; lne 2, 0.125 µg; lne 3, 0.25 µg; lne 4, 0.5 µg), nd ech 1 µl cdna ws mplified with rdiolbelled dctp by PCR with P1 nd P2, nd with P5 nd P6, s described in Mterils nd Methods. The dots on the grphs re the mens of duplicte exmintions DNAs were mplified with the primers in the presence of [α- 32 P]dCTP to elute the shifted DNA frgments for sequence nlysis. After PCR in the sme cycle condition, PCR products were electrophoresed in 5% non-denturing polycrylmide gel with 10% glycerol t the most suitble temperture (exon 4, 10 C; exon 5 7 nd 10, 25 C; exon 8, 15 C). After the gel ws vcuumdried nd exposed to X-ry film, both norml nd bnorml DNA frgments were eluted from the dried gel. Next, eluted DNAs were remplified by PCR using the sme primers, nd the remplified DNAs were sequenced using the drhodmine termintor cycle sequencing kit (Applied Biosystems Inc.) nd ABI PRISM TM 377 (Perkin-Elmer Corp.). Sttisticl nlysis All dt were nlysed using the Sttisticl Pckge for Socil Sciences (SPSS), nd comprison of reltive p73:β-ctin expression rtios in tumours nd in norml lung tissues ws crried out using the Mnn Whitney U-test. The ssocition between reltive p73:β-ctin expression rtio nd clinicopthologicl prmeters (ge, sex, smoking hbits, histologicl types nd pthologicl stging) ws estimted in 53 denocrcinoms nd squmous cell crcinoms, using the Mnn Whitney U-test nd the Spermn rnk correltion method. When ny prmeters significntly correlted with reltive p73 expression, we used the prtil correltion nlysis to exclude the interction mong these prmeters. Liner regression ws used to test the ssocition between p73:β-ctin expression rtio nd clinicl chrcteristics; the optiml regression model ws chosen using the bckwrd step-wise selection of vribles. Moreover, reltive p73:β-ctin expression rtio ws compred with p53 gene ltertion in 49 of the bove 53 denocrcinoms nd squmous cell crcinoms using the Mnn Whitney U-test. All P-vlues < 0.05 were considered significnt. RESULTS To check the liner reltionship between mount of RNA nd rdiolbelled PCR products in the semi-quntittive RT-PCR method, vrious mounts of totl RNA were processed by RT-PCR mplifiction. The men vlue of the duplicte p73 PCR products mplified by using P1 nd P2 incresed dose-dependently t 35 cycles of mplifiction (Figure 1A), nd similr reltionship between β-ctin PCR products nd mounts of RNA ws lso observed (Figure 1B). Figure 2 shows the representtive p73 RT-PCR products from pired norml lungs nd tumours, in which incresed p73 expression ws observed in tumours compred with norml lung tissues. Use of nother set of primers, P3 nd P4, confirmed the results of PCR using P1 nd P2 (dt not shown). In 87% of the cses (52/60), the reltive p73:β-ctin rtio in tumours is more thn twice s high s tht in norml tissues, but the reverse is found in only 5% of the cses (3/60, No 85, 130 nd 147 in Tble 1). Distribution of the reltive p53:β-ctin rtio both in norml lung tissues nd in tumours in shown in Figure 3. The expression of p73 mrna in lung tumours is distributed significntly higher thn tht in norml lungs (P < 0.0001, Mnn Whitney U-test); the mens mounts of p73 mrna in lung tumours nd norml tissues were 6.76 nd 1.10 respectively. In order to exmine whether the overexpression of p73 exclusively observed in lung tumours ws due to the gene mplifiction, Southern blot nlysis ws performed using genomic DNAs.
p73 overexpression in lung cncer 1627 A Cse No. p73 (P1 nd P2) 196 200 201 202 203 205 206 208 L T L T L T L T L T L T L T L T A Cse No. 69 94 99 L T L T L T B β-ctin Figure 2 Representtive utordiogrm of RT-PCR products of p73 (A) nd β-ctin (B) mrna in lung cncer nd norml tissue. T, lung cncer tissue; L, pired norml lung tissue. (A) p73 expression mplified with P1 nd P2 s described in Mterils nd Methods; (B) mplified with P5 nd P6 9.42 kb 2.02 kb P < 0.0001 60 30 B Cse No. 69 94 99 L T L T L T 25 p73:β-ctin rtio 20 15 10 5 Figure 4 Southern blot nlysis of p73 gene using genomic DNA extrcted from three representtive cses in which p73 mrna in lung cncer tissues ws expressed stronger thn in norml lung tissues. (A) Ethidium bromide stined gel electrophoresis fter digestion of 10-µg genomic DNA with HindIII; (B) Southern blot hybridiztion with the lbelled p73 cdna probe (contining exons 5, 6 nd prt of exon 7 of p73), which ws mplified by RT-PCR with P1 nd P2 s described in Mterils nd Methods. T, lung cncer tissue; L, pired norml lung tissue 0 Norml lung Genomic DNAs were extrcted from three representtive cses in which p73 mrna expression ws mrkedly higher in tumours thn in norml tissues: none of these cses showed the mplifiction of p73 gene (Figure 4), thus eliminting mplifiction s the cuse of p73 overexpression in these three cses. Next, we nlysed the reltionship between the reltive p73:β-ctin rtio nd the clinicoptholigicl fetures in 53 lung denocrcinoms nd squmous cell crcinoms, using the Mnn Whitney U-test nd the Spermn rnk correltion. The Lung cncer Figure 3 Distribution of the reltive p73; β-ctin expression rtio in 60 pired norml lung tissues nd lung tumours. Circles with brs indicte the men vlue nd the stndrd devition reltive p73:β-ctin rtios in tumours were found to significntly correlte with differences of histology (denocrcinom or squmous cell crcinom), ge, sex nd smoking index (Tbles 2 nd 3). However, we could not ignore inter-reltionships within these prmeters, so we pplied the prtil correltion nlysis. We found tht only differences in histology nd ge significntly correlted with the reltive p73:β-ctin rtio independently of other fctors (Tble 4). This result ws reconfirmed by the optiml multivrite liner regression model with the bckwrd step-wise selection of vribles: the finl model obtined fter the steps included ge nd histologicl types only s significnt nd independent vribles. Concerning the comprison of p73 expression in norml lung tissues nd clinicopthologicl prmeters, the reltive p73:β-ctin expression rtio in norml tissues is significntly ssocited with the ge of the ptients nd p73 expression in pired tumours (P<0.05 respectively, the Spermn rnk correltion). Accordingly, the reltive p73:β-ctin expression rtio in norml tissues lso increses with ge, lthough it is fr lower thn in tumours. We lso exmined muttionl sttus of p53 gene in 92% of the cses (49/53) with denocrcinoms or squmous cell crcinoms
1628 Y Tokuchi et l Tble 2 The reltionship between the mount of p73 expression nd clinicopthologicl fetures in 53 denocrcinoms nd squmous cell crcinoms Clinicl fetures No. of cses p73 : β-ctin rtio P-vlue b Age <61 26 3.74 ± 3.47 0.018 61 27 7.91 ± 7.69 Sex Men 38 6.95 ± 6.93 0.023 Women 15 3.11 ± 3.08 Histology Adenocrcinom 34 3.73 ± 3.60 0.001 Squmous cell crcinom 19 9.68 ± 8.20 p-stge I 18 5.01 ± 6.97 0.15 II 35 6.30 ± 6.00 Smoking index <400 20 2.80 ± 2.79 0.001 400 33 7.72 ± 7.12 p53 gene sttus Wild 28 5.17 ± 6.49 0.11 Mutnt 21 6.19 ± 4.34 Men ± s.d. b Mnn Whitney U-test. by PCR-SSCP, in exons 4 8 nd exon 10, nd subsequent direct sequencing: we found tht 43% (21/49) hd mutnt p53 (Tble 1), much the sme frequency s in previous reports (Kishimoto et l, 1992). The men levels of p73 expression in 21 mutnt p53 cses nd 28 wild p53 cses re 6.19 nd 5.17 respectively, nd there ws no sttisticlly significnt correltion between p53 gene ltertion nd p73 expression (P = 0.11, Tble 2). Moreover, even when we divided the subjects into two groups by histologicl difference, no ssocition ws found between p53 gene ltertion nd p73 expression. DISCUSSION The p53 gene nd its protein product hve been the centre of intensive cncer studies since more thn 50% of humn cncers contin this gene bnormlity (Levine, 1997). Becuse p73 closely resembles p53 in trnsctivtion (29% identity with p53 mino cids), DNA binding (63% identity with p53), nd p53 oligomeriztion (38% identity with p53) domins, p73 is thought to ply n importnt role in the development nd/or progression of vrious types of humn cncers (Dickmn, 1997; Kghrd et l, 1997; Oren, 1997). In fct, p73 cn enhnce levels of endogenous p21/wf1 protein, the representtive trget of p53, nd p73 cn lso inhibit cell growth by inducing poptosis in p53-like mnner (Jost et l, 1997; Kghrd et l, 1997). In the present study we clerly showed tht levels of p73 mrna expression is higher in tumours thn in norml tissues, s two previous ppers deling with only few smples hd reported (Mi et l, 1998; Tkhshi et l, 1998). In four reports the uthors filed to find p73 genomic muttion despite intensive serch (Kghrd et l, 1997; Mi et l, 1998; Nomoto et l, 1998; Tkhshi et l, 1998). Judging from these results, it ws possible tht wild-type p73, not mutnt p73, might be overexpressed exclusively in tumours. Furthermore, our results reveled tht p73 overexpression ws not due to gene mplifiction but, in ll probbility, to induction of trnscription or to stbiliztion of p73 mrna. Moreover, we showed tht p73 overexpression is independent of p53 gene ltertion. There re two possible explntions of the reltionship between p73 overexpression nd lung crcinogenesis. The first is tht p73 my work s security gurd (Dickmn, 1997): we found no direct reltionship between p53 muttion nd p73 overexpression in the present study, but the cell cycle in cncer cells is generlly fster thn in norml cells regrdless of p53 gene sttus, so p73 might work to rrest the cell cycle s tumour suppressor gene. The Tble 3 Spermn rnk correltion coefficients for p73 : β-ctin rtio in tumours nd clinicopthologicl chrcteristics in 53 denocrcinom nd squmous cell crcinoms Sex Age Histology Smoking index p-stge p73/β-ctin rtio Sex 1.0 Age 0.10 1.0 Histology 0.47 0.21 1.0 Smoking index 0.75 0.09 0.38 1.0 p-stge 0.02 0.02 0.09 0.08 1.0 p73:β-ctin rtio 0.32 b 0.40 0.46 b 0.30 0.10 1.0 P < 0.01, b P < 0.05. Tble 4 Prtil correltion nlysis for p73 : β-ctin rtio in tumours nd clinicopthologicl chrcteristics in 53 denocrcinom nd squmous cell crcinoms Prtil correltion coefficients Vribles under control Sex 0.05 Age, Histology, Smoking index, p-stge Age 0.39 Sex, Histology, Smoking index, p-stge Histology 0.34 b Sex, Age, Smoking index, p-stge Smoking Index 0.04 Sex, Age, Histology, p-stge p-stge 0.05 Sex, Age, Histology, Smoking index P < 0.01; b P < 0.05
p73 overexpression in lung cncer 1629 second possibility is tht p73 might ct s n oncogene in up-regultion of cell growth, s Mi et l (1998) pointed out. Kghd et l (1997) reported tht p73 protein is neither stbilized nor ctivted by DNA dmge, including dmge from UV rdition or ctinomycin D, so it is clerly different from p53. Becuse the expressions of p73 nd p53 re induced in different mnners, it is possible tht p73 my hve role different from tht of p53 in lung cncer crcinogenesis. And since, in the present study, p73 overexpression ws observed exclusively in lung tumour, p73 my possibly work s n oncogene in lung crcinogenesis. In the present study, our results suggest tht p73 overexpression in tumours correltes with histologicl type nd ge of ptients. Furthermore, p73 expression in norml lung tissues lso correltes with ge of ptients, lthough p73 expression in norml lung tissues is much lower thn tht in tumours. Accordingly, p73 expression might be ssocited with the chnges ccompnying ging of the host or differences of histologicl construction in tumors. These observtion will provide some clue s to why p73 is overexpressed in lung tumour. In conclusion, p73 expression in lung tumours is obviously greter thn its expression in norml tissues, independent of p53 gene ltertion, indicting tht the role of p73 in lung crcinogenesis might be different from its role in neuroblstom. Further study is needed of this possible new function of p73 protein. ACKNOWLEDGEMENTS We thnk Drs H Sugno (Cncer Institute) nd T Kozu (Sitm Cncer Center) for their helpful dvice. We lso thnk Drs S Tsuchiy nd S Okumur t Cncer Institute Hospitl, for kindly providing lung cncer smples. The technicl ssistnce of T Yoshikw nd Y Ymok is grtefully cknowledged. 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