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J Clin Pathol 1997;50:465-471

A new mdr-1 encoded P-170 specific monoclonalantibody: (6/1 C) on paraffin wax embedded tissuewithout pretreatment of sectionsElizabeth Moran, Annemarie Larkin, Gerard Doherty, Peter Kelehan, Susan Kennedy,Martin Clynes

AbstractAims-The generation and characterisa-tion ofa monoclonal antibody that specifi-cally recognises the mdr-I encodedprotein, P-glycoprotein (P-170), on rou-tinely processed formalin fixed, paraffinwax embedded tissue sections.Methods-The monoclonal antibody, des-ignated 6/1C, was produced following acombination of in vivo and in vitro immu-nisation regimens in Balb/c mice with asynthetic 12 amino acid peptide thatcorresponds to amino acids 21-32 (be-lieved to be intracellularly located) ofP-170 and has insignificant homology withthe mdr-3 encoded P-170. Antibody 6/1Cwas characterised by western blotting andimmunocytochemistry on cytospins ofpaired multidrug resistant or sensitive celllines, including mdr-1 and mdr-3 trans-fected cells, and by immunohistochemis-try on normal and malignant formalinfixed paraffin wax embedded tissue sec-tions.Results-Antibody 6/1C showed a singleband at 170 kDa on western blots ofmulti-drug resistant cell lysates and mdr-ltransfected cell lysates that was absent onsimilar preparations of drug sensitivecells and mdr-3 transfected cells. Immu-nocytochemical studies on cytospins ofmultidrug resistant cells and mdr-l trans-fected cells revealed strong inner plasmamembranelcytoplasmic staining. Stainingwas negligible on drug sensitive cells andcells transfected with the mdr-3 gene.Immunohistochemical studies on forma-lin fixed, paraffin wax embedded normaladult kidney, liver, and breast tissue and arange of fetal tissues exhibited stainingpatterns of a variety of secretory surfacesconsistant with documented mdr-l spe-cific staining. Specific staining of malig-nant cells in similarly treated sections ofbreast tumours was seen also with anti-body 6/1C. Staining on paraffin waxembedded tissue with this antibody didnot require any pretreatment of tissuesections.Conclusions-This new monoclonal anti-body, chosen for its specificity with themdr-l encoded P-170 and its reactivity onroutinely fixed paraffin wax embeddedtissue samples without pretreatment, ap-pears to be useful for the investigation ofP-170 in archival material. It is especiallyuseful for retrospective studies on pre-

treatment and post-treatment tissue sec-tions, and could help establish when andhow rapidly mdr-l associated drug resist-ance develops during chemotherapeuticregimens. Immunohistochemical assess-ment ofP-170 expression in many cancershas potential for diagnostic purposes andmay influence the choice of chemothera-peutic drugs used in the treatment ofrefractory tumours.(3 Clin Pathol 1997;50:465-471)

Keywords: monoclonal antibody; multidrug resistance;mdr-1; P-glycoprotein; P-170; paraffin wax embeddedsections

A major problem encountered during thechemotherapy of many tumour types is thedevelopment of resistance by the target tumourcells to a broad spectrum of drugs, a phenom-enon known as multidrug resistance. Multi-drug resistance is characterised by resistance toa broad range of structurally and functionallyunrelated chemotherapeutic drugs includingantracyclines, vinca alkaloids, epipodophyllo-toxins, and taxol. Several mechanisms of resist-ance have been identified including altered lev-els of multidrug resistance associated protein(an efflux pump),' 2 lung resistance relatedprotein (a major vault protein),3 topoisomer-ase II,s and glutathione S transferase.' How-ever, overexpression of the mdr-l gene prod-uct, P-glycoprotein (P-170), an integral plasmamembrane protein involved in the active effluxof cytotoxic materials from the cell, is consist-ently associated with multidrug resistance incultured cell lines selected for multidrug resist-ance and in certain tumours.7 P-170 isexpressed in a large number of normal tissuesincluding kidney, adrenal glands, large intes-tine, and liver indicating that it is involved innormal physiological functions including de-toxification and transport of lipophilic mol-ecules. Tumours arising from tissues thatnormally express P-170 may be intrinsicallyresistant to chemotherapeutic agents or, alter-natively, tumours that were initially responsiveto chemotherapy may develop multidrug resist-ance during the treatment regimen and subse-quently not respond to therapy."Owing to the cellular heterogeneity of most

tumours and the subsequent heterogeneity ofP-170 expression (resistant cells may be presentas clusters within a given tumour), it isimportant to use an investigational technique,such as immunohistochemistry, that can providedirect morphological confirmation of the pres-

National Cell andTissue Culture Centre,BioResearch Ireland,Dublin City University,Glasnevin, Dublin 9,IrelandE MoranA LarkinG DohertyM Clynes

Department ofPathology, NationalMaternity Hospital,Holles Street, Dublin2, IrelandP Kelehan

Department ofPathology, St Vincent'sHospital, Elm Park,Dublin 4, IrelandS Kennedy

Correspondence to:Dr Moran.

Accepted for publication8 April 1997

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Moran, Larkin, Doherty, Kelehan, Kennedy, Clynes

ence ofP- 170 rather than "bulk" methods suchas western blotting and some molecularbiological techniques.'0To date, several monoclonal antibodies have

been produced to different epitopes ofP-170"-" and subsequently used in studies ofvarious human tumour types.'0 1`1' However,many of these studies have been performed on

frozen tissue sections (in which cellular archi-tecture is not as well preserved as in paraffinwax embedded specimens) or paraffin wax

embedded tissue sections following the use ofaggressive antigen retrieval techniques (whichmay influence immunolocalisation of antigensor have adverse effects such as detachment oftissues),'9 20 long primary antibody incubationand/or the use of a high concentration ofexpensive primary antibody that may also influ-ence end results.2' On the other hand, there are

several reports on the advantages of antigenretrieval-for example, the use of antibodiesthat were previously unreactive on routinlyfixed paraffin wax embedded tissue.'o " Themajor disadvantage of having to use antigenretrieval methods appears to be the inter-laboratory variability of techniques and equip-ment (for example, microwave ovens).20 23 Anantibody that does not require antigen retrievalmethods has the advantage of potentiallyproducing more consistent results. A recentreport detailing a series of multi-institutionaltrials to assess sources of variability in assays todetect P-170 in tumour specimens recom-

mended standardisation of approaches to thedetection of P-170 in clinical specimens,including careful control of sample fixation andantigen preservation.24 This same report en-

couraged the development of new high affinityantibodies to P-170. We have successfully gen-

erated a monoclonal antibody, designated6/1C, which can be used to identify P-170positive cells specifically without any pretreat-ment (with a two hour incubation at room

temperature using 6-8 gg IgG/ml) in normaland malignant tissue that has been routinelyprocessed (formalin fixed, paraffin wax embed-ded) in the laboratory. This antibody is usefulin retrospective studies of archival tissuesections taken pre- and postchemotherapy andpossibly could determine when and to whatextent P-170 is expressed following chemo-therapy. It also has potential as a diagnostic toolfor determining the presence of P-170 positivecells in tumour samples that could influencechemotherapeutic regimens.

MethodsCHOICE OF IMMUNOGEN FOR MONCLONALANTIBODY PRODUCTIONThe peptide used for the production ofantibody 6/1C was selected after alignmentsearches of the EMBL Swiss-Prot proteinsequence database using the Mail-FASTA pro-gram. The entire amino acid sequence of the

mdr-1 encoded P-170 (deduced from theknown cDNA sequence)2" was obtained fromthe data bank above using the NETSERV pro-gram. From this deduced sequence, a 12 aminoacid peptide was chosen corresponding to theintracellularly located amino acids 21-32 of

P-170. The peptide was synthesised by BioSynLtd (Belfast, Northern Ireland) and waspurchased both in the free form and conju-gated to bovine serum albumin (BSA) forimmunisation and screening purposes. Thechosen peptide had insignificant homologywith the mdr-3 encoded P-170 that does notappear to be involved in drug resistance.26

IMMUNISATION REGIMENSA Balb/c mouse was injected twice with thechosen peptide conjugated to BSA. Primaryimmunisation was with 100,g conjugatedpeptide in complete Freund's adjuvant, andsecondary immunisation (following a threeweek interval) was with 100,g conjugatedpeptide in incomplete Freund's adjuvant. Fourweeks after immunisation the spleen wasremoved and the spleen cells immunised with60 ,ug unconjugated peptide following instruc-tions provided with the in vitro immunisationkit (Cel-Prime; Immune Systems Ltd, Paign-ton, UK). Three days after in vitro immunisa-tion, the spleen cells were fused with SP2myeloma cells according to a modification ofthe method of Kohler and Milstein.27 Initially,all clones produced were screened by enzymelinked immunosorbent assay (ELISA) againstthe peptide/BSA complex and against BSAalone. Only clones positive for the peptide/BSAcomplex and negative for BSA alone were cho-sen for further expansion. Clone 6/1 C wascloned twice by limiting dilution and finallypropagated as ascites in Balb/c mice. Thisascites was used for characterisation of theantibody by western blotting and immunocyto-chemical methods.

CONTROL CELL LINESWestern blotting studies and initial immunocy-tochemical studies were performed on a multi-drug resistant non-small cell lung carcinomacell line, DLKP-A (made resistant by exposureto increasing concentrations of adriamycin)and its drug sensitive counterpart, DLKP.28Overexpression of P-170 in DLKP-A cellscompared with the parental DLKP cells hadbeen confirmed in previous studies using othercommercially available antibodies raisedagainst P-170 (C219, Centocor, France; andJSB-1, Serotec, UK). Control cells were usedto test antibody 6/1C for mdr-1 specificity. Themdr-3 transgenic fibroblast cell line, VOl VO 1was kindly provided by Professor Piet Borst(department ofmolecular biology, NetherlandsCancer Institute, Amsterdam, Netherland) andmdr-1 transfected DLKP cells were obtainedfrom Dr Seamus Coyle in our laboratory. Cyto-spin preparations were made on poly-L-lysinecoated slides, air dried overnight, wrapped infoil, and stored at -20°C until required.

PEPTIDE NEUTRALISATION STUDIESIn addition to the control cells used todetermine the specificity of antibody 6/1C forthe mdr-1 encoded P-170, competitive inhibi-tion studies with the peptide used as theimmunogen were performed. Both purified6/1C antibody (ascites purified using anImmunoPure (A/G) IgG purification kit from

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New mdr-1 antibody reactive on paraffin wax embedded tissue

Pierce and Warriner, UK) and unpurifiedsupernatant from the hybridoma secreting6/1C antibody were preincubated with a10-fold (by weight) excess of the peptide(unconjugated form) in phosphate bufferedsaline (PBS, Dulbecco A, 10 mM, pH 7A)overnight at 4°C. To reduce background(caused by "sticky" aggregates) the antibody-peptide complex was centrifuged at full speed(15 000 xg) in a microcentrifuge (HeraeusInstruments, Germany) and the supernatantremoved carefully before use in immunocyto-chemical studies on cytospins of control cells.

TISSUE SECTIONSFormalin fixed, paraffin wax embedded tissuewas donated by the histopathology depart-ments of St Vincent's Hospital and TheNational Maternity Hospital, Holles St, Dub-lin, Ireland. Sections (5 gm) of tissue blockswere cut, mounted on to poly-L-lysine coatedslides, and dried overnight at 37°C. Slides werestored at room temperature until required.

IMMUNOFLUORESCENCE STUDIES ON LIVE CELLSPaired resistant and sensitive viable cells weretested for reactivity with antibody 6/1C byindirect immunfluorescence studies. Whenimmunofluorescence studies are performed onviable cells only cell surface components arerecognised.29 Briefly, test cells were adjusted toa concentration of 1 x 106 cells/ml in PBS and100 pl of the cell suspension was aliquoted intoeach of two Eppendorf tubes. A volume of100 pl antibody 6/1C (1 in 100 dilution ofascitic fluid in PBS) was added to one tube and100 pl of control irrelevant mouse ascites(diluted 1 in 100 in PBS) was added to theother. The tubes were mixed and incubated for30 minutes at 4°C. The primary antibody wasremoved by centrifugation of cells at 1000 rpmfor five minutes. The cells were washed threetimes with PBS using the same procedure and100 pl of fluorescein isothicyanate linked(FITC) sheep antimouse IgG (BoehringerMannheim, Germany) diluted 1 in 50 in PBSwas added. The tubes were mixed andincubated for 30 minutes at 4°C after whichthe secondary antibody was removed and thecells were washed as above. Each cell pellet wasresuspended in a minimum amount of Vecta-shield (Vector Laboratories, UK) mountingmedium and the cells were viewed using aNikon phase contrast microscope fitted with anFITC filter.

IMMUNOHISTOCHEMISTRYAll immunocytochemical studies on cytospinsof cell lines or on formalin fixed paraffin waxembedded tissue sections were performedfollowing the method of Hsu et al, 0 using anavidin-biotin horseradish peroxidase (HRP)conjugated kit (ABC) plus an appropriate sec-ondary antibody from Dako, UK. Briefly,cytospin preparations were fixed for oneminute in ice cold acetone and allowed to airdry for at least 15 minutes before immuno-staining. Paraffin wax embedded tissue sectionswere dewaxed in xylene (2 x 5 minutes), rehy-drated in graded alcohols, and placed in Tris

buffered saline (TBS)/0.05% (vol/vol) Tween20. Endogenous peroxidase activity was quen-ched by placing cytospins in 0.6% (vol/vol)H202/methanol and tissue sections in 3% (vol/vol) H202/distilled H2O for five minutes atroom temperature. All slides were blocked fornon-specific staining with 20% (vol/vol) nor-mal rabbit serum/TBS for 20 minutes at roomtemperature. Antibody 6/1C was applied toeach sample (1 in 100 dilution in TBS/0.05%(vol/vol) Tween 20 for tissue sections and 1 in40 dilution in the same diluent for cytospinpreparations) for two hours at room tempera-ture. This was followed by a 30 minute incuba-tion with biotinylated rabbit antimouse IgG (1in 300 dilution in TBS/0.05% (vol/vol) Tween20) at room temperature. Finally, ABC (HRPconjugated) was applied for 25 minutes at roomtemperature. The peroxidase substrate, 3',3-daminobenzidine tetrahydrochloride (DAB;Dako) containing 0.02% (vol/vol) H202, wasapplied for 10-15 minutes at room tempera-ture. All slides were washed after each incuba-tion in three changes of TBS/0.05% (vol/vol)Tween 20 over 15 minutes. Tissue sections andcells were lightly counterstained with Cole'shaematoxylin, differentiated in 1% (vol/vol)acid alcohol, and blued in Scott's tap water.Following dehydration in graded alcohols,slides were cleared in xylene and mounted inDPX (BDH, UK). Negative control slides inwhich antibody 6/1C was replaced by controlmouse ascites (Sigma, UK) at the samedilution were included in all experiments.Antibody 6/1C has also been used in a separatestudy of ocular melanoma using the alkalinephosphatase anti-alkaline phosphatase tech-nique on cryostat sections.3'

WESTERN BLOTTINGDenaturing polyacrylamide gel electophoresis(SDS-PAGE) followed by western blotting forthe detection of P-170 were performed by themethods of Laemmli"2 and Towbin et al,33respectively. Briefly, whole cell lysates ofcontrol cells were prepared by a modification ofthe method of Gerlach et ar4 and separated on7.5% polyacrylamide gels followed by electro-blotting on to polyvinylidene difluoride sup-port material. Following incubation of blotswith antibody 6/1C, diluted 1 in 200 to 1 in300 in TBS overnight at 4°C, blots were incu-bated with biotinylated rabbit antimouse IgG,diluted 1 in 2000 in TBS for one hour at roomtemperature. Blots were then incubated withABC, HRP conjugated, diluted according tothe manufacturer's instructions, for 30 minutesat room temperature before being developedusing the enhanced chemiluminesence method(Amersham, UK). In all cases negative controlblots were included in which antibody 6/1Cwas substituted with diluent or control mouseascites.

ResultsWESTERN BLOTTrINGRecognition by antibody 6/1C (IgG, isotype) ofa single band at 170 kDa (associated with thepresence of P-170) on preparations from drugresistant cells and cells transfected with the

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transfected cells (fig 2). Other paired sets ofdrug resistant/drug sensitive cell lines wereexamined with antibody 6/1C and similarresults were obtained.

PEPTIDE NEUTRALISATION STUDIESImmunocytochemical studies on fixed controlcell lines showed that the reactivity of antibody6/1C (both in its purified form and inhybridoma supernatants) was obliterated fol-lowing incubation with the peptide used as theimmunogen for production of the antibody.

Figure 1 Recognition by antibody 611C (IgG, isotype) ofa single band at 170 kDa (associated with the presence ofP-1 70) on preparations from drug resistant cells(DLKP-A) plus the lack of reactivity on preparations ofdrug sensitive cells (DLKP) and cells transfected with themdr-3 gene (VOl VO1) confirming the specificity of 6/1Cfor mdr-1 encoded P-170.

mdr- 1 gene plus the lack of reactivity onpreparations of drug sensitive cells and cellstransfected with the mdr-3 gene (VO 1 VO1cells) confirmed the specificity of this antibodyfor mdr-l encoded P-170 (fig 1).

IMMUNOFLUORESCENCE STUDIES ON LIVE CELLSThere was no detectable staining of live resist-ant cells, indicating that the antigen recognisedby antibody 6/1C is not expressed on the cellsurface of these cells. In addition, staining ofsensitive cells was not seen with this antibody.

IMMUNOCYTOCHEMICAL STUDIES ON FIXEDCELLSStrong staining of the plasma membrane/cytoplasm was seen with antibody 6/1C onresistant DLKP-A cells and cells transfectedwith the mdr-1 gene, whereas negligible stain-ing was detected on drug sensitive and mdr-3

IMMUNOHISTOCHEMICAL STUDIES ON FORMALIN

FIXED, PARAFFIN WAX EMBEDDED TISSUEThe suitability of antibody 6/1C for use onroutinely fixed, paraffin wax embedded tissuewithout pretreatment of the tissue was demon-strated initally on normal adult tissue. Underthe aforementioned conditions antibody 6/1Cgave more intense staining of cells than eitherantibodies C2 19 or JSB-1 although the overallpercentage of staining was similar. Normaladult kidney showed specific mdr-1 staining incollecting and proximal tubules (fig 3A). Innormal adult liver the overall mdr- 1 specificstaining was low compared with the kidney,and low levels of cytoplasmic staining wereseen in hepatocytes. In normal breast tissue,some ducts showed low mdr- 1 positivity andsome non-specific staining of adipose tissuewas seen (similar staining was seen with theantibody JSB-1). A number of normal fetal tis-sues were also investigated with antibody 6/1Cincluding lung, small intestine, adrenal gland,liver, and placenta.Lung-Epithelial cells lining the alveoli, epithe-lial cells, and cilia of the bronchus, macro-phages, and some interstitial cells (probablyerythroid precursor cells) showed specificmdr-1 staining (fig 3B).Small intestine-Enterocytes, the absorptivecells of the small intestine, showed intensemdr-1 staining. In general, cells at the bottomof crypts stained much less than those in thevilli. The goblet cells were negative. The mus-cularis propria also stained intensely (fig 3C).Adrenal gland-A low level of staining waspresent in the medulla (but neuroblast cells

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Figure 2 (A) Strong staining of the plasma membranelcytoplasm with 6/1C on multidrug resistant cells (DLKP-A). (B) Negligible staining detected ondrug sensitive (DLKP) cells. (Original magnification x40).

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Figure 3 (A) Normal adult kidney showing specific mdr-1 staining in collecting and proximal tubules (originalmagnification x40). (B) Epithelial cells lining the alveoli, epithelial cells, and cilia of the bronchus, macrophages, and someinterstitial cells (probably erythroid precursor cells) showing specific mdr-1 staining (original magnification x40).(C) Enterocytes of small intestine showing intense mdr-1 staining. In general, cells at the bottom of crypts stained much lessthan those in the villi; the goblet cells were negative. The muscularis propria also stained intensely (original magnificationx60). (D) Tropoblasts in placental villi showing mdr-1 positive staining. This was localised to the inner layer ofcytotrophoblastic cells. The syncytiotrophoblastic cells were negative, but the brush border region was positive. Hoffbaur cells(macrophage type cells) showed positive cytoplasmic staining (original magnification x40). (E) Ductal breast carcinoma insitu showing strong specific 611C staining. The centre of the ductal carcinoma had undergone necrosis and is mdr-1 negative(original magnification x10). (F) Infiltrating tumour cells in a ductal breast carcinoma staining intensely positive formdr-1, this staining was located in the inner plasma membranelcytoplasm (original magnification x40).

within the medulla were negative) but the cor- myeloid precursor cells. Bile duct epithelialtex region was mdr-l negative. cells in portal tracts appeared to be negative forLiver-The liver had low levels of overall mdr- 1 mdr- 1.positivity; hepatocytes exhibited faint cytoplas- Placenta-Trophoblasts in placental villi weremic staining. Erythroid precursor cells showed mdr-1 positive. This positivity was localised tovery intense cytoplasmic staining as did the inner layer of cytotrophoblastic cells. The

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syncytiotrophoblastic cells were negative, butthe brush border region was positive (plasmamembraneous staining). Hoffbaur cells (mac-rophage type cells) showed positive cytoplas-mic staining (fig 3D).

Six malignant breast tumours were exam-ined for mdr-1 staining. Representative exam-ples of the type of staining seen in thesetumours are shown in figures 3E and F. Figure3E shows a ductal carcinoma in situ whichexhibited strong specific staining with antibody6/1C. The centre of the ductal carcinoma hadundergone necrosis and is mdr-1 negative. Fig-ure 3F shows infiltrating tumour cells in a duc-tal breast carcinoma staining intensely positivefor mdr- 1, this staining was located in the innerplasma membrane/cytoplasm. Overall, the insitu component did not stain as intensely asinfiltrating cells. Normal breast ducts adjacantto tumour cells showed low level staining andthere was some non-specific staining of adiposetissue.

DiscussionA monoclonal antibody specific for mdr-1encoded P-170 that was reactive on routinelyprocessed, paraffin wax embedded tissue sec-tions without pretreatment has been generatedand characterised. To date, immunohisto-chemical studies of P-170 on archival materialhas been less than satisfactory due mainly tothe lack of reproducibility of results with theantibodies available on paraffin embeddedtissue. Differences in antigen retrieval methodsused on tissue sections may be responsible formuch of this lack of reproducibility. Many ofthe studies on the distribution of P-170 in nor-mal tissues and tumours have been performedon frozen sections.35"- During characterisationstudies in which the localisation of P-170 withantibody 6/1C was examined on paraffin waxembedded tissue we compared our results tothose documented for both frozen tissue andthe variable results obtained on paraffin waxembedded material to date.

Initial specificity and localisation studieswere performed on normal adult and fetaltissue. The positivity in the kidney of the proxi-mal and collecting tubules for mdr-1 has beendescribed using both paraffin embedded andfrozen tissue samples. Bittl et al" describedstaining of collecting ducts in paraffin embed-ded kidney tissue with antibodies C494 and4E3 but reported that C219 did not give anyconclusive staining pattern. Sugawara et al16reported staining of proximal tubules withantibody MRK1 6 on frozen sections of kidney.Staining patterns on normal liver using variousantibodies against P-170 are inconsistent. Thelow level mdr-1 positivity that we found local-ised to the cytoplasm of hepatocyes was inagreement with a study performed by Bittl etal" on frozen tissue using antibody JSB-1. Inthis same study, positive staining on bile ductsand bile canaliculi was seen with antibodyC219. These differences in staining patternsmay be accounted for by the reported differ-ences in mdr- 1 and mdr-3 distribution in sometissues. The P- 170 encoded by mdr-3 isreported to be more abundantly expressed in

liver than the mdr-1 encoded protein,42 whichmay account for the low level staining seen withthe mdr-1 specific 6/1C antibody; C219 recog-nises both mdr-1 and mdr-3 encoded P-170.2'In normal breast tissue the low level of mdr-1staining observed with antibody 6/1C isconsistent with documented data,43 althoughthere are also reports ofmdr- 1 negativity foundin normal breast tissue.40The expression of mdr-1 in the fetal tissues

surveyed was similar to the patterns seen in thecorresponding adult tissue with the exceptionof the adrenal gland. Fetal liver showed lowlevel mdr-1 positivity, however, intense mdr-1positivity was observed in erythroid precursorand myeloid precursor cells. Reactivity of stro-mal macrophages and endothelial cells withantibodies C2 19 and MRK16 has beenreported44; with antibody C219, positivity waslocalised to monocytes, histocytes, andfibroblasts.45 Using antibody 6/1C, we ob-served positive staining of macrophages in fetallung and placenta (Hoffbaur cells). Positivemdr-1 staining of placental trophoblasts hasbeen described previously using antibodyMRK16."6 Generally, the pattern of mdr-1staining in the small intestine reflected thatseen in the adult tissue with greater positivityseen towards the villi compared to the bottomof the crypt; this differentiation dependentpathway of P-170 expression (as normalmucosal cells move up the crypt towards thesurface) has been described in the colon by DeAngelis et al46 In the lung, the positive stainingof alveoli, cilia, and epithelial cells of the bron-chus with antibody 6/1 C corresponds tosimilar results observed in the epithelium of thebronchus and digestive tract.47 48 It has beenshown that mdr-1 encoded P-170 is expressedin the medulla and cortex of the adult adrenalgland,47 and it has been suggested that P-170plays a particularly important physiologicalrole in the adult adrenal and that this role mayincrease with age as P-170 was not found in thefetal adrenal gland.'6 Binding studies with anti-body 6/1C on fetal adrenal tissue showed nomdr-I positivity in the cortex and a very lowlevel of staining in the medulla region. Initialstudies on malignant breast tumours show thatantibody 6/1C recognised mdr-i-positive cellsspecifically within these tumours, indicatingthat this novel antibody may prove clinicallyuseful for the detection of P-170 positive cellswithin a tumour mass. The reactivity of thisantibody on routinely fixed tissue could enablelarger studies on archival material to be carriedout in an attempt to establish a prognostic rolefor P-170.

The authors acknowledge the expert advice and technicalassistance with animal handling ofMr Nick O'Connor and staffat the Biomedical Research Facility, Beaumont Hospital, Dub-lin, Ireland.

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