immunohistochemical characterization of novel murine monoclonal antibodies against human...

Immunohistochemical characterization of

novel murine monoclonal antibodies against

human placenta-specific 1

Roya Ghods1,2

Mohammad Hossein

Ghahremani1,3∗

Maryam Darzi2

Ahmad-Reza Mahmoudi2

Omid Yeganeh2

Ali Ahmad Bayat2

Parvin Pasalar1

Mahmood Jeddi-Tehrani2

Amir-Hassan Zarnani4,5∗

1Department of Molecular Medicine, School of Advanced MedicalTechnologies, Tehran University of Medical Sciences, Tehran, Iran2Monoclonal Antibody Research Center, Avicenna Research Institute,ACECR, Tehran, Iran3Department of Pharmacology-Toxicology, Faculty of Medicine, TehranUniversity of Medical Sciences, Tehran, Iran4Nanobiotechnology Research Center, Avicenna Research Institute,ACECR, Tehran, Iran5Immunology Research Center, Iran University of Medical Sciences,Tehran, Iran

Abstract

Human PLAC1 (placenta-specific 1) is a new member ofcancer–testis antigens with 212 amino acids, and itsexpression is restricted to placenta and at much lower levelsto testis. Recently, ectopic expression of the PLAC1 transcripthas been demonstrated in a wide range of human tumors andcancer cell lines with a proposed function in tumor cell growth.No monoclonal anti-PLAC1 antibody applicable to immunohis-tochemical staining is available so far. To better understandthe PLAC1 expression and localization, we aimed to producemonoclonal antibodies (mAbs) against the extracellular regionof PLAC1. Mice were immunized with a synthetic peptidecorresponding to the C-terminal 11 amino acids of PLAC1conjugated with a carrier protein. Hybridomas were producedby standard protocol and screened for positive reactivity by

enzyme-linked immunosorbent assay. Reactivity of final twoclones was then assessed by Western blotting (WB),immunohistochemistry (IHC), and immunocytochemistry (ICC).Both clones showed a specific immunostaining pattern inhuman term placenta as the positive control. Reactivity wasmostly localized to the cytoplasm of syncytiotrophoblasts. Oneof the clones showed an excellent staining signal in breast,ovary, and prostate cancer cell lines. Importantly, no reactivitywas observed with human lymph node cells or prostate. Noneof the mAbs were able to detect PLAC1 in Western blot. Basedon the present results, these mAbs can be used for detectionof PLAC1 in IHC and ICC techniques. C© 2013 International Union ofBiochemistry and Molecular Biology, Inc. Volume 61, Number 3, Pages363–369, 2014

Keywords: Anti-PLAC1, immunohistochemistry, monoclonal antibody,placenta, PLAC1

Abbreviations: PLAC1, placenta-specific 1; WB, Western blotting; IHC,immunohistochemistry; ICC, immunocytochemistry; BSA, bovine serumalbumin; PBS, phosphate-buffered saline; FBS, fetal bovine serum; TBS,Tris-buffered saline; mAbs, monoclonal antibodies.∗Address for correspondence: Amir-Hassan Zarnani, Associate Professor,Nanobiotechnology Research Center, Avicenna Research Institute, ACECR,P.O. Box 19615-1177, Tehran, Iran. Tel: +982122432020; Fax:+982122432021; e-mail: [email protected]; or [email protected]; orMohammad Hossein Ghahremani, Associate Professor, Department ofMolecular Medicine, School of Advanced Medical Technologies, TehranUniversity of Medical Sciences, Tehran, Iran. Tel: +982166959102; Fax:+982188991117; e-mail: [email protected] 29 April 2013; accepted 8 November 2013DOI: 10.1002/bab.1177

1. IntroductionPLAC1 (placenta-specific 1) is a novel X-linked gene [1] and anew member of cancer–testis antigens [2,3]. Based on an openreading frame, human PLAC1 consists of 212 amino acids,whereas mouse Plac1 gene encodes a 173-amino-acid product.Human and murine PLAC1 proteins have 60% identity and 77%homology [1]. In silico analysis predicted that PLAC1 has atransmembrane region spanning from amino acids 23 to 40 ofthe N-terminus, suggesting that PLAC1 is localized to a mem-branous compartment [3,4]. In mouse placenta, the expression

Published online 1 May 2014 in Wiley Online Library(wileyonlinelibrary.com)

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of PLAC1 mRNA is detected at 7.5–14.5 days pc (postcoitus)in all of trophoblast linage [1, 5]. In healthy individuals, theexpression of PLAC1 is also relatively restricted to trophoblasts[6, 7] with constant levels of expression throughout the gesta-tion period [6]. In human placenta, PLAC1 protein is localizedto the membranous structure in syncytiotrophoblasts, includingthe microvillus plasma membrane surface [7, 8]. Much lowerlevels of PLAC1 transcript have been detected in the testis andcerebellum [2,3].

The function of PLAC1 is unknown. The Plac1 gene islocalized to a locus that is involved in placental growth [1]and has been shown to be important for the development anddifferentiation of placenta and embryo growth [9]. It has alsobeen hypothesized that spatiotemporal expression of Plac1 islinked to progressive and extensive vascularization. The roleof serving as a receptor for stabilization of the mother–fetusinterface has also been attributed to thismolecule [1]. Moreover,because of its high degree of homology between amino acids29 and 119 PLAC1 with the zona pellucida 3 domain [10], thisprotein may be involved in trophoblastic interactions with theuterus (maternal–placenta interface) [11].

Recently, ectopic expression of the PLAC1 gene has beendetected in a wide range of human malignancies such aslung [4], gastric [12], colorectal, hepatocellular [13], breast[4] and ovarian cancers [14], and cancer cell lines [2, 4, 13].In this context, PLAC1 is classified as a new member of thecancer–testis antigens. According to the published data, PLAC1is important for tumor cell growth and may impact overallpatient survival [3]. In vitro experiments in the MCF-7 breastcancer cell line have proposed that PLAC1 is involved inmigration, invasion, and proliferation [4].

The detection of PLAC1 in normal and cancerous tissues isimportant for cancer research and developing a better under-standing of the potential role that this molecule may serve. Inthis regard, monoclonal antibodies (mAbs) are considered asinvaluable tools. However, no monoclonal anti-PLAC1 antibodyapplicable to immunohistochemical staining is available so far.In this study, two mAbs were produced against PLAC1 that aresuitable for the detection of PLAC1 by immunohistochemistry(IHC) and immunocytochemistry (ICC) and might be used as aninvaluable tool for monitoring of PLAC1 expression in cancertissues.

2. Materials and Methods2.1. Epitope selectionThe prediction of continuous B-cell epitopes for PLAC1was performed by utilizing the following Web servers.Where appropriate, the specificity was set to 90%:Bcepred (http://www.imtech.res.in/raghava/bcepred/bcepredsub mission.html), BCPREDS (http://ailab.cs.iastate.edu/bcpred/predict.html) [15, 16], COBEpro (http://scratch.proteomics.ics.uci.edu/), BepiPred (http://www.cbs.dtu.dk/services/BepiPred/),and ABCpred (http://www.imtech.res.in/raghava/abcpred/ABC submission.html) [17]. All the proposed epitopes were

compared, and those with the highest scores were consid-ered. Among epitopes with highest scores, the sequencethat was offered by the majority of the aforesaid predic-tion tools (amino acid 166–177: CVFSEEEHTQVP) was se-lected. To ensure the unique nature of the selected epitope,BLAST analysis was performed by NCBI BLAST software(http://blast.ncbi.nlm.nih.gov/Blast.cgi).

2.2. Preparation of immunogenSynthetic peptide was conjugated separately with ImjectMaleimide-activated mcKeyhole Limpet Hemocyanin (ThermoScientific, Rockford, IL, USA) and Imject Maleimide-activatedbovine serum albumin (BSA) (Thermo Scientific). The efficacyof conjugation was evaluated by SDS-PAGE analysis of the BSAconjugate.

2.3. Immunization protocolAll research with animals was approved by the ethical commit-tees of the Avicenna Research Institute and Tehran Universityof Medical Sciences. Male Balb/c mice aged 8–10 weeks werepurchased from Pasteur Institute of Iran (Tehran, Iran). Threemice were immunized intraperitoneally with 60 μg conjugatemixed with complete Freund’s adjuvant, and four booster injec-tions of 40 μg antigen mixed with incomplete Freund’s adjuvantwere given at 3 week intervals between the first and secondinjection and 2 week intervals between subsequent injections.Ten days after the last immunization, blood was collected fromthe tail vein and antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) as described below. Basedon ELISA results, the mouse with the highest titer was selectedand injected intravenously with 20 μg of antigen. Three dayslater, cell fusion was performed as mentioned below.

2.4. Indirect ELISAAn ELISA was performed for titration of mouse sera, screeningof hybridomas, and reactivity assessment of purified mAbs.In brief, plates were coated with 5 μg/mL of the peptideprepared in phosphate-buffered saline (PBS, 0.15 M, pH 7.2)and incubated at 37 ◦C for 1 H followed by overnight incubationat 4 ◦C. Blocking was then performed with 3% skim milk at37 ◦C for 1.5 H. Hybridoma supernatants or serial dilutions ofmouse sera or purified antibodies were added subsequently for1.5 H. Signals were developed by successive addition of HRP-conjugated sheep anti-mouse immunoglobulin (Ig) (AvicennaResearch Institute, Tehran, Iran) and tetramethylbenzidine.Optical density (OD) was measured at 450 nm. After eachstep, plate washings were performed three times with PBScontaining 0.05% (v/v) Tween 20 (PBS/T). All incubations wereperformed at 37 ◦C for 1.5 H except coating as mentionedabove.

2.5. Cell cultureMurine myeloma cell line, SP2/0, breast (MCF-7, MDA-MB 231,and T47D), prostate (PC3, LNCaP), and ovarian cancer cell lines(SKOV-3 and Caov4) (all from National Cell Bank of Iran) werecultured in either Dulbecco’s modified Eagle medium (Gibco,

364 Anti-PLAC1 Monoclonal Antibodies

Invitrogen, CA, USA) (for MCF-7, MDA-MB 231) containing 5%fetal bovine serum (FBS) or RPMI 1640 (Gibco) (for other celltypes) containing 10% FBS (Gibco). Media contained 100 U/mLpenicillin and 100 μg/mL streptomycin (Gibco). Cultured cellswere incubated in a humidified atmosphere containing 5% CO2

at 37 ◦C.

2.6. Production of mAbsmAbs were generated as described elsewhere [18]. Spleencells of the immunized mouse were harvested and fused withSP2/0 at a 5:1 ratio using polyethylene glycol 1500 (Sigma,Milwaukee, WI, USA). Hybridoma cells were cultured in RPMIsupplemented with 20% FBS and hypoxanthine-aminopterin-thymidine selective medium (Sigma). Ten to 12 days afterfusion, supernatants of hybridomas were screened by indirectELISA as above. Specific antibody producing hybridomas werecloned four times by limiting dilution. The isotype of eachmAb in culture supernatant was determined by a mouse mAbisotyping kit (IsoStrip) (Roche, Indianapolis, IN, USA) accordingto the manufacturer’s protocol. Ascites fluid was preparedafter an intraperitoneal injection of anti-PLAC1 hybridomacells. mAbs were purified by a HiTrap protein G HP affinitychromatography column (GE Healthcare, Uppsala, Sweden)according to the manufacturer’s instruction.

2.7. Reactivity assessment of mAbs by Westernblot analysisAs the positive control, human placenta lysate was prepared inRIPA buffer containing 50-mM Tris–HCl, pH 8, 150-mM NaCl,1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and1% protease inhibitor cocktail (Sigma). The lysate protein con-centration was measured by a BCA protein assay kit (ThermoScientific). Fifty micrograms of lysate was electrophoresedin 12% SDS-PAGE gel and transferred to the polyvinylidenedifluoride (PVDF) membrane (Roche, Mannheim, Germany).Membrane blocking was performed by washing buffer (PBS-T)containing 5% skim milk overnight at 4 ◦C. After washing,the membrane was incubated with purified antibodies at con-centrations ranging from 50 to 500 ng/mL for 1.5 H at 37 ◦C.Affinity-purified polyclonal antibody against a synthetic peptidenear the N-terminus of human PLAC1 (Avicenna ResearchInstitute) and preimmune rabbit Immunoglobulin G (IgG) andmouse sera were used as positive and negative primary anti-body controls, respectively. After washing, membranes wereincubated for 1 H with HRP-conjugated sheep anti-mouse Ig(Avicenna Research Institute). The bands were finally visualizedwith enhanced chemiluminescence substrate. β-Actin (Sigma)immunoreactivity was assessed as the internal loading controlafter reprobing of membranes.

2.8. ImmunohistochemistryIHC was performed on Formalin-fixed paraffin-embedded hu-man term placenta and normal human endometrium accordingthe protocol we published elsewhere with some modifications[19]. Paraffin blocks of human lymph node and normal prostatefrom Hasheminejad Hospital were prepared. Briefly, 3-μm

tissue sections were deparaffinized, rehydrated, and subjectedto heat-activated antigen retrieval in citrate buffer (10 mM,pH 6) at 95 ◦C for 30 Min in a water bath. Following threewashes with Tris-buffered saline (TBS), pH 7.4, endogenousperoxidase activity was quenched by addition of 1% H2O2 inTBS for 15 Min. After four times of washing, nonspecific bind-ing sites were blocked with 5% normal sheep serum in proteinblock solution (Dako, Carpinteria, CA, USA) for 30 Min andthen slides were tilted and incubated with 5 μg/mL anti-PLAC1mAbs for 60 Min at room temperature. Then the slides werewashed five times with TBS containing 1% BSA. Detectionwas carried out using the DAKO Envision detection kit (Dako,Glostrup, Denmark) for 15 Min. After washing as above, 3, 3′-diaminobenzidine substrate was added and slides were rinsedin H2O, counterstained with Harris hematoxylin, dehydrated,and mounted with Enthelan (Merck, Darmstadt, Germany).In negative reagent control slides, primary antibodies weresubstituted by preimmune mouse IgG. Digital images were cap-tured by a BX51 microscope and a DP70 CCD camera (Olympus,Tokyo, Japan). To assess the specificity of the produced an-tibodies, they were blocked with saturating concentration ofimmunizing peptide (1:100 molar ratio) prior to being appliedto the cells or sections as primary antibody.

2.9. ImmunocytochemistryCancer cell lines MCF-7 and MDA-MB 231 were harvested bycold PBS containing 0.03% ethylenediaminetetraacetic acid(EDTA). Other cell lines were harvested using trypsin–EDTA(Roche). Twenty-five thousand cells per well were either grownon multiwell slides overnight or cytospinned and then washedthree times with PBS. Cells were then dried and fixed with coldneutral-buffered Formalin for 15 Min. All subsequent stepswere performed as above.

3. Results3.1. Preparation of immunogenPeptide spanning amino acids 166 to 177 of the PLAC1 sequencewas selected based on overall high specificity and antigenicityscores for the generation of mAbs. Blast analysis togetherwith high antigenicity prediction of this peptide indicates thatthe selected peptide might be a suitable candidate for theproduction of highly specific and efficient antibodies againstPLAC1. Conjugation efficacy of immunogen was examined bythe electrophoresis pattern of a BSA conjugate. The smearpattern of electrophoresis movement of the conjugate and theabsence of free peptide clearly showed successful conjugation(Fig. 1).

3.2. Immunization of mice and screening and selectionof anti-PLAC1 hybridomasFollowing immunization of mice, the presence of a specificantibody against peptide was detected in mice sera by indirectELISA. The mouse with the highest titer of specific antibodywas selected for mAb production (Fig. 2). After cell fusion,supernatants of all growing hybridomas were screened for

Biotechnology and Applied Biochemistry 365


FIG. 1Assessment of peptide conjugation. BSA (lane 2),BSA-linker (lane 3), and BSA-conjugated peptide(lane 4) were electrophoresed on SDS-PAGE gel toassess conjugation efficacy. The smear pattern ofelectrophoresis movement of conjugate showssuccessful conjugation. Lane 1: molecular weightmarkers.

FIG. 2Titration of anti-PLAC1 antibody in immunizedmouse serum by indirect ELISA. Hyperimmunizedserum was serially titrated on a peptide-coatedplate.

specific antibody against the synthetic peptide as above. Afterfour successive clonings, two final clones named 1A4A8 and4D7D12 were achieved. The isotypes of 1A4A8 and 4D7D12were found to be IgG3/κ and IgG1/κ, respectively. Ascitesfluids from both clones were purified by the protein G column,yielding 1.29 and 2.35 mg/mL purified antibodies.

FIG. 3Reactivity assessment of purified anti-PLAC1mAbs by indirect ELISA. Purified antibodies wereserially diluted on a peptide-coated plate.

3.3. Reactivity assessment of purified mAbsReactivity of purified mAbs was tested by indirect ELISA.The results (Fig. 3) showed excellent reactivity of both clonesagainst immunizing peptide. The two mAbs demonstratedsimilar trends of reactivity reaching to plateau at as low anantibody concentration as 500 ng/mL. This implies reasonableaffinity of produced antibodies.

3.4. Western blot analysisHuman placenta lysate was separated on SDS-PAGE. Aftertransfer of protein to PVDF, the membrane was incubatedwith protein G-purified mAbs in conjunction with polyclonalanti-PLAC1 antibody as the positive reagent control. None ofthe mAbs was able to detect PLAC1 in human term placenta.A sharp band of approximately 24 KD corresponding to theestimated molecular weight of PLAC1 was detected withpolyclonal antibody (Fig. 4). Although some nonspecific bandswere seen, they were attributed to the nonspecific reaction assuch bands were also present in the negative control lane. Thisexperiment clearly showed no cross-reactivity of the antibodywith unidentified proteins.

3.5. IHC and ICCThe reactivity of anti-PLAC1 mAbs was checked by IHC and ICC.The results of PLAC1 immunostaining in human term placenta,normal human endometrium, prostate and lymph node, breast,ovary, and prostate cancer cell lines are shown in Figs. 5 and6. Immunostaining of human term placenta showed that bothproduced mAbs against PLAC1 have a specific pattern of re-activity, with higher signals obtained using the 4D7D12 clone.4D7D12-recognized PLAC1 in differentiated trophoblasts andlocalization was restricted mostly to a cytoplasmic compart-ment and to some extent to themicrovillus plasmamembrane ofsyncytiotrophoblasts. A very small number of cytotrophoblastsexhibited specific immunoreactivity. As a tissue adjacent toplacenta, the endometrium was assessed for its reactivity withmAbs with completely negative results. The human lymph nodeas a source of lymphocytes and normal prostate as the repre-sentative of epithelial tissues did not show reactivity with our


FIG. 4Detection of PLAC1 protein in human termplacenta lysate using anti-PLAC1 mAbs byWestern blot analysis. Human term placenta wasseparated on 12% SDS-PAGE and transferred toPVDF membrane. Each lane was incubated withantibodies as follows: Lane 1: affinity-purifiedpolyclonal anti-PLAC1 antibody. Lane 2:preimmune rabbit IgG as negative control. Lane 3:mAb anti-PLAC1, clone 4D7D12. Lane 4: mAbanti-PLAC1, clone 1A4A8. Lane 5: preimmunemouse serum. β-Actin was used as internalloading control.

mAbs. Control sections showed an absence of specific stainingin tissues exposed to preimmune serum from the same animalor immunizing peptide-adsorbed antibody.

Based on its higher immunoreactivity, we used only the4D7D12 clone in cancer cell lines ICC. We selected some celllines with a reported expression of PLAC1 transcript, includ-ing SKOV-3, LNCaP, and MCF-7. Some other cell lines with noprevious report on PLAC1 expression were also tested. Interest-ingly, the immunostaining pattern was neither identical amongcancer cell lines nor homogeneous in cells of a given cell line.Notably, in such cell lines, LNCaP and Caov4 immunoreactivitywas mainly restricted to the cell membrane, whereas bothcytoplasmic and membrane staining patterns were observedin other cancer cell lines. The percentage of cells expressingmembrane PLAC1 differed among cell lines examined with thehighest percentage in LNCaP cells. Cell lines with the samehistologic origin also showed various PLAC1 expressions. Stain-ing intensity was also shown to have a heterogenic patternwithin cells of the same origin with some cells showing a strongsignal and others showing comparatively weaker staining. Ofnote, cells with a higher signal were smaller with a lowercytoplasm/nuclear ratio, whereas proliferating cells and thosewith looser chromatin exhibited lower PLAC1 expression.

4. DiscussionDetection of tumor-specific antigens in cancer tissues is aprerequisite for a better understanding of cancer biology. Theexpression of the PLAC1 transcript has been reported in avariety of cancer tissues and a wide range of cancer cell lines,but not in normal tissues. In this context, mAbs with a specificpattern of reactivity would be invaluable tools for research and

FIG. 5Assessment of anti-PLAC1 mAbsimmunoreactivity in IHC. Sections of normalhuman term placenta (a–e), endometrium (f),prostate (g), and lymph node (h) were preparedand stained with anti-PLAC1 mAbs. (a and b)Placenta: clone 4D7D12. (c and d) Placenta: clone1A4A8. (e) Placenta: negative reagent control.(f) Endometrium: clone 4D7D12. (g) Prostate: clone4D7D12. (h) Lymph node: clone 4D7D12.



FIG. 6Immunoreactivity of anti-PLAC1 mAb (clone4D7D12) in human cancer cell lines: (a) MCF-7,(b) MDA-MB 231, (c) T47D, (d) PC3, (e) LNCaP,(f) Caov4, (g) SKOV-3, and (h–n) correspondingnegative controls. In negative controls,peptide-adsorbed anti-PLAC1 mAb was used asprimary antibody.

diagnostic approaches. In this study, we produced mAbs againstPLAC1 and characterized their reactivities by IHC/ICC and WB.

Based on the predicted topology and the structure of PLAC1protein, a PLAC1-specific peptide in the extracellular regionof the molecule was designed and used for the productionof mAbs. Produced clones worked well in ELISA, even atlow antibody concentrations, implying excellent affinity. InWestern blot, both mAbs failed to detect PLAC1. This findingimplies that these antibodies may react with conformationalepitopes that are related to proper folding. Both mAbs showed aspecific pattern of immunostaining with human placental tissuesections without a nonspecific background. The 4D7D12 clone,however, showed a stronger reaction compared with the 1A4A8clone. This may reflect a difference in epitopes that each mAbrecognizes. In addition, as demonstrated by ELISA, the higherreactivity of 4D7D12 in IHC may stem from its relatively higheraffinity. Specificity of these mAbs was confirmed using IHCstaining of the normal human endometrium as most adjacenttissue to placenta. According to published data, normal humantissues except testis do not express PLAC1 [3]. As expected, nopositive staining was observed in human endometrium IHC.Regarding the expression of PLAC1 in trophoblasts as epithelialcells, normal human prostate with an enriched populationof epithelial cells was examined for the expression of thismolecule. As with other normal human tissues, the prostatedid not express PLAC1 at the protein level. Such data were alsoobserved in a lymph node, implying that PLAC1 expression ismostly restricted to placenta and cancerous tissues.

IHC is a common detection method in pathology andclinical diagnosis. In biology, IHC staining is also widely used tounderstand the localization and distribution of molecules. It isalso notable that in spite of several papers reporting expressionof PLAC1 transcript in various tissues or cells, the absence of aspecific monoclonal anti-PLAC1 antibody was the main obstaclefor assessing this molecule at the protein level [20]. Based onour results, mAbs presented here are suitable candidatesfor in situ detection of PLAC1. Immunostaining of PLAC1 inMCF-7 cell line and also placental syncytiotrophoblasts showedlocalization of this molecule mainly restricted to cytoplasmwith some degree of membrane distribution. There is onlyone paper on PLAC1 localization in the MCF-7 cell line [4].Interestingly, and according to this paper, PLAC1 is restrictedto the plasma membrane. This result is not in accordance withwhat is presented here. Notably, Koslowski et al. [4] did notreport whether all or a part of the MCF-7 cells express PLAC1on their plasma membrane, and the presented figure is notconclusive. Different localization of PLAC1 was observed indifferent cancer lines examined with predominant membraneexpression in such cell lines as LNCaP and Caov4, whereasothers primarily expressed this molecule in their cytoplasm.The reason behind these various localizations of PLAC1 isnot clear at the moment but may conceivably imply differentmolecular functions. Profiling of protein localization in differentcellular compartments is as important as profiling proteinexpression and posttranslational modification patterns. More


importantly, the activation state, interaction network, andbiological function are among the information that is mainlydependent on the precise localization of proteins. Nonetheless,surface expression of a tumor marker is among the idealcriteria for cancer antigens [21]. As with MCF-7, the PLAC1staining pattern of other cell lines was not homogeneous,with some cells expressing higher amounts of PLAC1. Basedon the cell phenotype and expression pattern, it is possiblethat the expression of PLAC1 in cancer cells correlates withthe cell cycle. Interestingly, siRNA-mediated PLAC1 knockingdown in MCF-7 induces the G1-S cell cycle block with completeabrogation of proliferation [4]. Its expression may therefore riseduring the mitosis phase and downregulate after cell division.In line with this assumption, PLAC1 knockdown MCF-7 cellshave impaired proliferation capacity. Moreover, the MCF-7 cellline has reportedly six subpopulations with different growthrates [22]. Therefore, the expression levels of PLAC1 may differin each subpopulation.

5. ConclusionThis is the first report on the localization of PLAC1 protein in aseries of cancer cells using mAb. We showed that anti-PLAC1mAbs presented here are excellent biological tools for in situmonitoring of PLAC1 expression. IHC results using such mAbsrevealed that the localization and expression level of PLAC1differ considerably in a cell-type-dependent manner. Suchvariability was also evident in different subpopulations withina particular tumor cell. Based on the differential expressionof PLAC1 protein in normal and cancer cells, this moleculemay be considered as an apt and invaluable target of cancerimmunotherapy. Nonetheless, functional assays must be donebefore such a conclusion could be drawn.

6. AcknowledgementsThis study was funded by grants from the Iran University ofMedical Sciences (Grant No.: 90-03-13-13530) and the AvicennaResearch Institute (Grant No.: 910108-027). The authorswould like to thank Mrs. J. Ghasemi and Mr. E. Mirzadeganfor technical assistance. The authors declare no conflict ofinterest.

7. References[1] Cocchia, M., Huber, R., Pantano, S., Chen, E. Y., Ma, P., Forabosco, A., Ko, M.

S., and Schlessinger, D. (2000) Genomics 68(3), 305–312.[2] Silva, W. A., Jr., Gnjatic, S., Ritter, E., Chua, R., Cohen, T., Hsu, M., Jungbluth,

A. A., Altorki, N. K., Chen, Y. T., Old, L. J., Simpson, A. J., and Caballero, O. L.(2007) Cancer Immun. 7, 18.

[3] Fant, M., Farina, A., Nagaraja, R., and Schlessinger, D. (2010) Prenat. Diagn.30(6), 497–502.

[4] Koslowski, M., Sahin, U., Mitnacht-Kraus, R., Seitz, G., Huber, C., and Tureci,O. (2007) Cancer Res. 67(19), 9528–9534.

[5] Rawn, S. M., and Cross, J. C. (2008) Annu. Rev. Cell Dev. Biol. 24, 159–181.[6] Fant, M., Weisoly, D. L., Cocchia, M., Huber, R., Khan, S., Lunt, T., and

Schlessinger, D. (2002) Mol. Reprod. Dev. 63(4), 430–436.[7] Massabbal, E., Parveen, S., Weisoly, D. L., Nelson, D. M., Smith, S. D., and

Fant, M. (2005) Mol. Reprod. Dev. 71(3), 299–304.[8] Fant, M., Barerra-Saldana, H., Dubinsky, W., Poindexter, B., and Bick, R.

(2007) Mol. Reprod. Dev. 74(7), 922–929.[9] Jackman, S. M., Kong, X., and Fant, M. E. (2012) Mol. Reprod. Dev. 79(8),

564–572.[10] Jovine, L., Janssen, W. G., Litscher, E. S., and Wassarman, P. M. (2006) BMC

Biochem. 7, 11.[11] Concu, M., Banzola, I., Farina, A., Sekizawa, A., Rizzo, N., Marini, M., Caramelli,

E., and Carinci, P. (2005) Fetal Diagn. Ther. 20(1), 27–30.[12] Chen, J., Pang, X. W., Liu, F. F., Dong, X. Y., Wang, H. C., Wang, S., Zhang, Y.,

and Chen, W. F. (2006) Beijing Da Xue Xue Bao 38(2), 124–127.[13] Dong, X. Y., Peng, J. R., Ye, Y. J., Chen, H. S., Zhang, L. J., Pang, X. W., Li,

Y., Zhang, Y., Wang, S., Fant, M. E., Yin, Y. H., and Chen, W. F. (2008) Int. J.Cancer 122(9), 2038–2043.

[14] Tchabo, N. E., Mhawech-Fauceglia, P., Caballero, O. L., Villella, J., Beck, A. F.,Miliotto, A. J., Liao, J., Andrews, C., Lele, S., Old, L. J., and Odunsi, K. (2009)Cancer Immun. 9, 6.

[15] El-Manzalawy, Y., Dobbs, D., and Honavar, V. (2008) Comput. Syst. Bioinfor-matics Conf. 7, 121–132.

[16] El-Manzalawy, Y., Dobbs, D., and Honavar, V. (2008) J. Mol. Recognit. 21(4),243–255.

[17] Saha, S., and Raghava, G. P. (2006) Proteins 65(1), 40–48.[18] Shojaeian, S., Allameh, A., Zarnani, A. H., Chamankhah, M., Ghods, R., Bayat,

A. A., and Jeddi-Tehrani, M. (2010) Immunol. Invest. 39(2), 114–131.[19] Zarnani, A. H., Shahbazi, M., Salek-Moghaddam, A., Zareie, M., Tavakoli,

M., Ghasemi, J., Rezania, S., Moravej, A., Torkabadi, E., Rabbani, H., andJeddi-Tehrani, M. (2010) Fertil. Steril. 93(8), 2738–2743.

[20] Shantha Kumara, H., Grieco, M. J., Caballero, O. L., Su, T., Ahmed, A., Ritter,E., Gnjatic, S., Cekic, V., Old, L. J., Simpson, A. J., Cordon-Cardo, C., andWhelan, R. L. (2012) Cancer Immun. 12, 16.

[21] Cheever, M. A., Allison, J. P., Ferris, A. S., Finn, O. J., Hastings, B. M., Hecht,T. T., Mellman, I., Prindiville, S. A., Viner, J. L., Weiner, L. M., and Matrisian,L. M. (2009) Clin. Cancer Res. 15(17), 5323–5337.

[22] Resnicoff, M., Medrano, E. E., Podhajcer, O. L., Bravo, A. I., Bover, L., andMordoh, J. (1987) Proc. Natl. Acad. Sci. USA 84(20), 7295–7299.


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