immunohistochemistry in normal tissues

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592 5J Clin Pthol 1994;47:592-596 Leukemi Reserch Fund Immunodignostic Unit, Deprtent of Cellulr Science, John Rdcliffe Hospitl, Oxford F Pezzell K Micklem K Pulford M Jones S Kocilkowski K C Gtter D

1 592 5J Clin Pthol 1994;47: Leukemi Reserch Fund Immunodignostic Unit, Deprtent of Cellulr Science, John Rdcliffe Hospitl, Oxford F Pezzell K Micklem K Pulford M Jones S Kocilkowski K C Gtter D Y Mson ICRF Moleculr Oncology Lbortory, John Rdcliffe Hospitl, Oxford H Turley R Bicknell K Smith A L Hrris Oncologi Sperimentle A, Istituto Nzionle dei Tumori, Miln, Itly D Deli A Aiello Correspondence to: Dr Frncesco Pezzell, Deprtment of Pthology, Europen Institute of Oncology, Vi Ripmonti 435, Milno, Itly Accepted for publiction 14 December 1993 Antibody for detecting p53 protein by immunohistochemistry in norml tissues F Pezzell, K Micklem, H Turley, K Pulford, M Jones, S Kocilkowski, D Deli, A Aiello, R Bicknell, K Smith, A L Hrris, K C Gtter, D Y Mson Abstrct Aims-To estblish whether PAb248 recognises humn p53 s weli s murine p53 nd if so, to determine its distribution in norml tissues. Methods-The bility of PAb248 to recognise humn p53 ws estblished by nlysis of the humn osteosrcom derived Sos-2 celil line, which lcks the p53 gene, before nd fter trnsfection with p53 cdna, using western blotting nd immunoprecipittion. Immunostining on norml tissues nd cell lines ws crried out using n immunoperoxidse technique. The two nti-p53 ntibodies PAb 240 nd DO-7 were used s controls. Results-The nti-p53 PAb248 monoclonl ntibody stined the Sos-2 cell line fter, but not before, trnsfection with p53 cdna. Both western blots nd immunoprecipittions performed with this ntibody reveled moleculr weight bnd. With immunostining, this ntibody detects p53 protein in most lymphoid nd humn epithelil cells in cytoplsmic-perinucler loclistion tht hs not been described before. In the sme tissues nucler stining could be seen in few scttered cells using the PAb240 ntibody. The topogrphicl distribution of wild type p53 ws not relted to proliferting res but, rther, to short-lived popultions of cells. Conclusions-Immunostining of wild type p53 is demonstrble not only in its nucler form using ntibody PAb240 but lso in its common cytoplsmic-perinucler loclistion in norml tissues using the PAb248 monoclonl ntibody. This opens up new possibilities for its study in both physiologicl nd pthologicl conditions. C( Clin Pthol 1994;47: ) It is widely believed tht only the ccumultion of bnorml p53 protein cn be demonstrted by immunohistochemistry.' However, severl reports, some more thn 10 yers old, suggest tht this is not entirely true. In 1981 Dippold et l reported p53 nucler stining in proliferting nd cytoplsmic positivity in quiescent cells in non-neoplstic humn cell lines.2 In 1983 Rotter et l showed tht p53 is expressed in the perinucler res of nontrnsformed murine cell lines but tht this becomes nucler fter chemiclly induced neoplstic trnsformtion.' In 1984 Milner described two different forms of murine p53: one in resting nd one in phytohemgglutinin (PHA) stimulted splenic lymphocytes4; PAb42 1 ntibody, which stins nuclei in tumours,5 precipitted the protein from PHA blsts but not from resting lymphocytes; PAb248 precipitted p53 only from the splenic smll lymphocytes.4 During the course of our investigtion of p53 expression in severl humn tumours we noticed tht the PAb248 ntibody ppered to give results discordnt with those obtined with other ntibodies on neoplstic tissues (Pezzell et l unpublished dt) nd, furthermore, to produce stining on norml tissue present in the sections. The PAb248 monoclonl ntibody ws believed to be specific for murine p536 but since it recognises n epitope highly preserved between mouse nd mn6 we believed tht it might lso identify humn p53, perhps in previously unrecognised form. This study ws designed to estblish whether PAb248 recognises humn p53 nd if so, its distribution in norml humn tissues. Methods Fresh frozen humn tissues were obtined from the histopthology deprtment t the John Rdcliffe Hospitl, Oxford. Frozen smples were stored t - 70 C until use. The following nti-p53 monoclonl ntibodies were used: PAb2486; PAb2407 8; nd DO-7.9 The nti-vimentin V9 (Dko) ntibody ws used s positive control for immunostining of the Sos-2 cell line nd s negtive control in the immunoprecipittion-immunoblot experiments. Five cell lines estblished from humn tumours were used. The two lines in which p53 protein is not expressed, becuse of lrge deletions in the gene, were the osteosrcom derived Sos-2 cell line,'0 obtined from the Americn Tissue Culture Collection (ATCC) (Rockville, Mrylnd, USA) nd the promyelocytic leukemi cell line HL The other three were cell lines expressing p53 protein: the brest crcinom cell line MCF-7, in which the protein is norml'2; the colorectl crcinom cell line HT-29 expressing only mutted p53 protein"3; nd the cell line K 422, for which the gene sequence is s yet unpublished.'4 Immunoblotting ws performed under reducing conditions ccording to the method

Antibodyfor detecting p53 protein by immunohistochemistry in norml tissues 593 6i7kD -o-i. K422 Immunoblot HL60 D07 248 D07 248 p ~ ~. 45kD - 36kD b Immunoblot HT29 D07 D07 421 248 V9."IRM to.

2 Antibodyfor detecting p53 protein by immunohistochemistry in norml tissues 593 6i7kD -o-i. K422 Immunoblot HL60 D D p ~ ~. 45kD - 36kD b Immunoblot HT29 D07 D V9."IRM to..s m munoprecipittion/ D07 immunoblot SAOS-2 D V9 Figure 1 Chrcteristion ofpab248 by immunoblotting nd combined immunoprecipittion nd immunoblotting. (A) Immunoblot: lne 1, DO-7 used s positive control; lne 2, PAb248 shows unique bnd of moleculr weight from p53 positive line (K 422); but notfrom the HL-60 cell line (lnes 3 nd 4); in which the protein is not expressed becuse of lrge deletions in the gene. (B) Immunoprecipittion nd immunoblot nlysis ofht-29 cell line expressing mutted p53 nd SAOS-2 cell line not expressing the protein, becuse of biulelic gene deletion, with nti-p53 ntibodies. Lne 1, immunoblot ofht-29 cells using DO-7 ntibody; lnes 2 nd 6, immunoprecipittion using DO-7 ntibody from HT-29 (lne 2) nd SAOS-2 (lne 6) cells; lnes 3 nd 7, immunoprecipittion using ntibody PAb421 from HT-29 (lne 3) nd SAOS-2 (lne 7) cells; lnes 4 nd 8, immunoprecipittion using PAb248 ntibody from HT-29 (lne 4) nd SAOS-2 (lne 8) cells; lnes 5 nd 9, negtive control immunoprecipittion using nti-vimentin ntibody V9 (Dko) from HT-29 (lne 5) nd SAOS-2 (lne 9) cells. p53 in the unlbelled immunoprecipittes ws detected by immunoblotting with DO-7. The positions of moleculr weight mrkers nd the p53 bnd re indicted. described by Deli et l,'5 nd modified by C Schneider et l (Ntionl Lbortory CIB, Pdricino, Trieste, Itly, mnuscript in preprtion) to enhnce the ntibody binding. Cells (107/trck) were solubilised in nonionic detergent nd buffer contining protese inhibitor. Immunoprecipittion ws performed using the ntibodies dsorbed to Protein G-Sephrose. Immunoprecipittes were nlysed by sodium dodecyl sulphte polycrylmide gel electrophoresis (SDS- PAGE) under reducing conditions nd trnsferred to Immobilon membrnes. For the immunoblot, whole cells (107) were solubilised nd pplied to the gel. The membrne ws incubted with DO-7 ntibody nd bound ntibody visulised using enhnced chemiluminescence regents (Amershm UK). Sos-2 cells obtined from the ATCC (Rockville, Mrylnd) were trnsfected with pcmv-neo-bm plsmid crrying either the wild type pc53-sn3 or the mutted pc53- SCX3 p53 cdna16 by electroportion, using Bio-Rd instrument under the following conditions. Cells (106) in 400 pl RPMI culture medium contining 5% fetl clf serum were incubted directly in the electroportion cell (10 minutes on ice) with 10 ug of plsmid previously electroported. Cells were trnsferred in Petri dishes nd cultured with 400 ng/ml G418 (Gibco). Colonies were tested for p53 protein expression. Immunostining ws performed by the immunoperoxidse technique, s described before. 7 PAb240 nd PAb248 ntibodies were tested on prffin wx embedded sections fter microwve pretretment ccording to the method of Cttoretti et l.'8 Briefly, sections were dewxed in the usul wy, then plced in glss continer contining 001M solution of sodium citrte. The slide continer ws plced in microwve oven nd heted t 700W (equivlent) twice for five minutes before being trnsferred to TRISbuffered sline (TBS) nd continuing the immunocytochemistry s usul. Results PAb248 recognises humn p53 by immunoblotting (fig 1A) nd by immunoprecipittion-immunoblotting (fig 1B). Its bility to recognise specificlly humn p53 is further demonstrted by its filure to stin the humn osteosrcom Sos-2 cell line (which lcks p53 coding exons'0) (figs 2A nd B). However, fter trnsfection with either the wild type or the mutnt gene (figs 2C nd D)

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3 =..... = '- SI '- '' ''-'-'-'- --' '' f.w.,. ".,. r., s.: ks.t. tt. * *.f.s:.. _ }+*,, I t e & f g Figure 2 Chrcten'stion o IAb 248 by immunostining nd demonstrtion of two different forms ofp53. Humn osteosrcom Sos-2 cell line, in which on both lleles of the p53 gene the coding exons re deleted (Msud, et l, 1987), is not stined with (A) PAb248 nd (B) PAb240. After trnsfection with mutted p53 gene, PAb 248 produces pronounced cytoplsmic perinucler stining (C), chrcteristic of this ntibody; PAb240 stins nuclei nd, diffusely, the cytoplsm (D). Immunostining of the cell lines MCF-7, expressing wild type p53 (Csey et l 1991) (E-F) nd HT-29, expressing mutted p53 (G-H) produces the sme pttern seen in the trnsfected cells with PAb248, reveling perinucler ps3 (E-G) nd PAb240, producing nucler stining (F-H). h Wild type p53 expression in norml humn tissues PAb248: Perinucler stining PAb240: Nucler stining Pentherl blood nd lymphoid tissues Peripherl blood Negtive Negtive lymphocytes Neutrophils Positive (ll) Positive ( few) Lymph node, tonsil, nd spleen: Prcorticl re Positive Positive ( few) Mntle zone Positive Negtive Germinl centres Positive Positive ( few) Endotheli Negtive Negtive Thymus Cortex Positive Positive ( few) Medull Positive Negtive Other tissues Liver (heptocytes) Negtive Negtive Stomch Positive ( few) Negtive Duodenum Positive Negtive Colon Positive Negtive Respirtory epithelium Positive Negtive Kidney Negtive Negtive Prostte Positive Negtive Pezzell, Micklem, Turley, Pulford, Jones, Kocilkowski, et l into Sos-2 chrcteristic perinucler stining cn be seen. Stining with PAb240, which is lso negtive on Sos-2 cells (fig 2B), produces nucler nd diffuse cytoplsmic stining fter trnsfection (fig 2D). Both PAb240 nd PAb248 ntibodies produce stining on frozen tissue but, in our hnds, no stining ws obtined on prffin wx-embedded mteril either before or fter pretretment with microwves. PAb248 produces perinucler stining of both the MCF-7 cell line (fig 2E) expressing norml p53 protein nd the HT-29 cell line (fig 2G) producing only mutted protein. PAb240 stins nuclei of only few cells in the MCF-7 cell line (fig 2F) nd of lmost ll the HT-29 cell lines (fig 3). PAb248 stins wild type p53 in numerous cells from norml tissues (tble, figs 3 nd 4). In the presence of high protein concentrtions (for exmple, trnsfected Sos-2 or HT-2913 cell lines) both ntibodies produce, in ddition to their specific ptterns, diffuse cytoplsmic stining of vrible intensity (figs 2C nd D). Omission of the first ntibody lwys resulted in bsence of stining. Discussion In this study we hve shown tht p53 is detectble in mny cells from norml humn tissues by immunostining, nd tht the protein in these cells is present in two forms: cytoplsmic-perinucler, s stined by PAb248; nd nucler, this pttern being only occsionlly detectble, with PAb240. This cytoplsmic-perinucler loclistion hs not been described before in norml humn tissues. However, it is not totl surprise becuse of its description in n embryonic murine cell line3 nd the evidence for puttive cytoplsmic protein ble to link with p53.'9 Furthermore, perinucler stining ws described in the humn brest cncer cell line MDA 157 by Brtek et P0 by immunostining with PAb240. The uthors comment tht lthough this pttern of stining could hve been due to the presence of protein crossrecting with PAb240, this is unlikely, nd tht it might insted be cused by the presence of perinucler p53. They support this conclusion on the grounds tht perinucler loction is chrcteristic of centrosomes nd tht it hs been reported tht, "p53 cn bind to murine p34cdc2 nd tht p34 cdc2 is present in the centrosome."" They lso point out tht on the sme cell line stining with two other nti-p53 ntibodies (PAb421 nd PAb180I) ws negtive nd consequently if the protein in question is p53, it is " p53 molecule which hs lost both the PAb421 nd PAb1801 epitopes".'' Our findings strongly support their view tht the p53 protein molecule exists in different conformtions, nd show tht one of these hs high ffinity for PAb248. Nucler stining for p53 in occsionl norml cells hs been reported both in proliferting germinl centre cells2' nd quiescent cells, such s epithelil prbsl cells of the

Antibody for detecting p53 protein by immunohistochemistry in norml tissues 595 d-.4 Figure 3 P53 expression in norml humn peripherl blood nd rective tonsil.

4 Antibody for detecting p53 protein by immunohistochemistry in norml tissues 595 d-.4 Figure 3 P53 expression in norml humn peripherl blood nd rective tonsil. (A) PAb248 does not stin peripherl blood smll lymphocytes, but neutrophils show the chrcteristic perinucler positivity (rrows). Stining of rective tonsil shows diffuse positivity with PAb248 (B) in gertninl centre (GC), mntle zone (AMIZ), nd interfolliculr res (IF), nd few cells positive with PAb240 (C) (rrows). Higher mgnifiction shows tht PAb248 gives strong cytoplsmic perinucler stining (D) wheres PAb240 stins nuclei (E) (rrows). oesophgel mucos.22 Our report supports the evidence by Ssno et 1 22 tht p53 expression is not relted to prolifertion in norml tissues. Our dt show tht p53 expression, s detected by immunostining with the PAb248 nd PAb240 ntibodies, is minly present in tissues chrcterised by occurrence of cell deth by poptosis. Indeed, its expression in lymphoid, myeloid, nd epithelil cells is complementry to tht of bcl-2 protein, which protects cells from deth by poptosis.2' Peripherl blood lymphocytes re bcl-2 positive24 but p53 negtive (fig 3A). Smll lymphocytes in tissues re bcl-224 nd perinucler p53 positive (fig 3D) but bcl-2 negtive germinl centre cells,24 most of which re going to die by poptosis, retin cytoplsmic p53, nd occsionlly express nucler p53 (figs 3B- E). Circulting grnulocytes re bcl-2 negtive,2' but p53 positive (fig 3A). In orgns such s prostte nd lung, bsl proliferting cells re p53 negtive (figs 4A nd B) nd bcl- 2 positive.2526 The upper lyers of well differentited cells show cytoplsmic-perinucler p53 (figs 3A nd B) but bcl-2 is bsent.2526 The dt re consistent with the hypothesis tht these two proteins belong to two different pthwys: one, which includes bcl-2, prevents cells from poptosis23 while the other, to which p53 belongs, is likely to rrest cellulr growth nd perhps to induce cell deth e A P53 is tumour suppressor gene nd its inctivtion by deletion of both lleles, point muttions in the gene, or binding to nother protein such s MDM2," is considered to be n importnt step in oncogenesis. Antibodies such s PAb240 hve demonstrted p53 protein expression in the nuclei of neoplstic cells, which in some cses, is due to n underlying point muttion of the gene. PAb248 ntibody will permit full evlution of p53 expression by immunohistochemistry, mking it possible to identify tumours truly lcking detectble protein, or those with norml pttern of p53 expression. Furthermore, the use of PAb248 now gives reserchers the possibility of investigting the expression of humn p53 "in vivo" nd "in vitro" to rech n understnding of its norml function. We thnk Professor Dvid Lne for kindly providing ll the nti-p53 ntibodies used in this study nd Mr Enrico Fontnell for his excellent technicl ssistnce. 1 Iggo R, Gtter K, Brtek J, Lne D, Hrris AL. Incresed expression of mutnt forms of p53 oncogene in primry lung cncer. Lncet 1990;335: Dippold WG, Jy G, DeLeo AB, Khoury G, Old U. p53 trnsformtion-relted protein: detection by monoclonl ntibody in mouse nd humn cells. Proc Nd Acd Sci USA 1981;78: Rotter V, Abutbul H, Ben-Ze'ev AA. P53 trnsformtionrelted protein ccumultes in the nucleus of trnsformed fibroblsts in ssocition with the chromtin nd is found in the cytoplsm of non-trnsformed fibroblsts. EMBOJ 1983;2:

5 596 i2;.6.< *5.X.*.Y*<KX C e X :%f :s *.t.ȧj^ Xe.1...h. of e.-s v Figure 4 P53 expression in norml humn tissues. With PAb248 prosttic (A) nd respirtory (B) epithelium show positive cytoplsmic-perinucler stining in the upper lyer of non-proliferting well differentited cells (rrows). Bsl cells (*) re mostly negtive. Colonic mucos (C) is mostly positive with PAb248. In liver (D) heptocytes re PAb248 negtive wheres spordic lymphoid cells re positive (rrows) showing tht p53 is not expressed in ll tissues. * f, 1. '-P' L...t'.'..j A.: 4 Milner J. Different forms of p53 detected by monoclonl ntibodies in non-dividing lymphocytes. Nture 1984; 310: Vn Den Berg FM, Tigges AJ, Scipper MEI, Den Hrtog- Jger, Kroes WGM, Wlboomers JMM. Expression of the nucler oncogene p53 in colon tumours. I Pthol 1989;157: Yewdell JW, Gnnon JV, Lne DP. Monoclonl ntibody nlysis of p53 expression in norml nd trnsformed cells. 7 Virol 1986;59: Pezzell, Micklem, Turley, Pulford, Jones, Kocilkowski, et l 7 Gnnon JV, Greves R, Iggo R, Lne DP. Activting muttions in p53 produce common conformtionl effect. A monoclonl ntibody specific for the mutnt form. EMBOJ 1991;9: Rivs CI, Wisniewski D, Strife AB, et l. Constitutive expression of p53 protein in enriched norml humn mrrow blst cell popultions. Blood 1992;78: Vojtesek B, Brtek J, Midgley CA, Lne DP. An immunochemicl nlysis of the humn nucler phosphoprotein p53. YImmunol Methods 1992;151: Msud H, Miller C, Koeffier HP, Bttifor H, Cline MJ. Rerrngement of the p53 gene in humn osteogenic srcom. Proc NtlAcd Sci USA 1987;84: Wolf D, Rotter V. Mjor deletions in the gene encoding the p53 tumor ntigen cuse lck of p53 expression in HL-60 cells. Proc NtlAcd Sci USA 1985;82: Csey G, Lo-Hsueh M, Lopez ME, Vogelstein B, Stnbridge EJ. Growth suppression of humn brest cncer cells by the introduction of wild-type p53 gene. Oncogene 1991;6: Rodriguez NR, Rown A, Smith MFE, et l. P53 muttions in colorectl cncer. Proc Ntl Acd Sci USA 1990; 87: Dyer MJS, Fisher P, Nchev E, Lbstide W, Krps A. A new humn B-cell non-hodgkin's lymphom cell line (Krps 422) exhibiting both t(i4;,18) nd t(4;- 1) chromosoml trnsloctions. Blood 1990;75: Deli D, Aiello A, Soligo D, et l. bcl-2 proto-oncogene expression in norml nd neoplstic humn myeloid cells. Blood 1992;79: Bcker SJ, Mrkowitz S, Feron ER, Willson JK, Vogelstein B: Suppression of humn colorectl crcinom cell growth by wild type p53. Science 1990;249: Mson DY, Abdulziz ZA, Flini B, Stein H. Single nd double immunoenzymtique techniques for lbelling tissue sections with monoclonl ntibodies. Ann N YAcd Sci 1984;420: Cttoretti G, Pileri S, Prrvicini C, Becker MHG, Poggi S, Bifulco C, et l. Antigen unmsking on formlin-fixed, prffin-embedded tissue sections. Y Pthol 1993;171: Gnnon JV, Lne DP. Protein synthesis required to nchor mutnt p53 protein which is temperture-sensitive for nucler trnsport. Nture 199 1;349: Brtek J, Iggo R, Gnnon J, Lne DP. Genetic nd immunochemicl nlysis of mutnt p53 in humn brest cncer cell lines. Oncogene 1990;5: Villuends R, Piris MA, Orrdre JL, et l. P53 protein expression in lymphoms nd rective lymphoid tissue.. Pthol 1992;166: Ssno H, Miyzki S, Gooukon Y, Nishihir T, Swi T, Ngur H. Expression of p53 in humn esophgel crcinom. An immunohistochemicl study with correltion to proliferting cell nucler ntigen expression. Hum Pthol 1992;23: Hockenberry D, Nunez G, Millimn C, Screiber RD, Korsmeyer SJ. Bcl-2 is n inner mitochondril membrne protein tht blocks progrmmed cell deth. Nture 1990;348: Pezzell F, Tse A, Cordell JL, Pulford KAF, Gtter KC, Mson DY. Expression of the bcl-2 protein is not specific for the 14;18 chromosoml trnsloction. Am Y7 Pthol 1990;137: Hockenbery DM, Zutter M, Hickey W, Nhm M, Korsmeyer SJ. BCL2 protein is topogrphiclly restricted in tissues chrcterized by poptotic cell deth. Proc NtlAcd Sci USA 199 1;88: Pezzell F, Turley H, Kuzu I, et l. Bcl-2 protein in nonsmll-cell lung crcinom. Immunohistochemicl evidence for bnorml expression nd correltion with survivl. NEnglJ Med 1993;329: Yonis-Rouch E, Resnitzky D, Lotem J, Schs L, Kimchi A, Oren M. Wild type p53 induces poptosis of myeloid leukemic cells tht is inhibited by interleukin-6. Nture 199 1;352: Shw P, Bovey R, Trdy S, Shli R, Sordt B, Cost J. Induction of poptosis by wild-type p53 in humn colon tumor-derived cell line. Proc Ntl Acd Sci USA 1992;89: Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jcks T. p53 is required for rdition-induced poptosis in mouse thymocytes. Nture 1993;362: Clrke AR, Purdie CA, Hrrison DJ, et l. Thymocytes poptosis induced by p53-dependent nd independent pthwys. Nture 1993;362: Oliner JD, Kinzler KW, Meltzer PS, George DL, Vogelstein B. Amplifiction of gene encoding p53- ssocited protein in humn srcoms. Nture : t 0,.fl-

Plaque Assay of Avian Sarcoma Viruses Using Casein

Plaque Assay of Avian Sarcoma Viruses Using Casein JOURNAL OF VIROLOGY, Sept. 1975, p. 707-711 Copyright 0 1975 Americn Society for Microbiology Vol. 16, No. 3 Printed in U.S.A. Plque Assy of Avin Srcom Viruses Using Csein PIERO C. BALDUZZI*l AND HELEN