Retinoblastoma protein monoclonal antibodies with novel characteristics

JOURNALOF IMMUNOLOGICAL METHODS ELSEVIER

Journal of ImmunologicalMethods 169 (1994) 231-240

Retinoblastoma protein monoclonal antibodies with novel characteristics Shu Fen Wen a, Margarita Nodelman a Karen Nared-Hood a John Duncan b, Joseph Geradts c, H. Michael Shepard a,, Department of Assay Development, Canji Inc., 3030 Science Park Road, Suite 302, San Diego, CA 92121, USA, b Scripps Lab., 8208 Torrel Way, San Diego, CA 92126, USA, c Department of Pathology, University of North Carolina School of Medicine CB 7525, Brinkhous-Bullitt Building, ChapelHill, NC 27599-7525, USA a

(Received 23 August 1993, revised received 27 October 1993, accepted 28 October 1993)

Abstract

We have developed a family of monoclonal antibodies directed against the retinoblastoma gene product (pll0RB). One of these monoclonal antibodies, 3C8, binds p l l 0 RB near the C-terminal end of the protein (aa886-aa905). It was characterized by immunobiotting, ELISA, fluorescence-activated flow cytometry and immunohistostaining. It was shown to be useful for the detection of p l l 0 RB in formalin-fixed and paraffin-embedded tissue sections. Because 3C8 binds outside of regions shown to be involved in p l l 0 RB interactions with other cellular proteins, it may be an especially useful reagent for the reliable detection of pll0 RB in tumor cells, and for the isolation by affinity chromatography of p l l 0 RB complexes with other cellular proteins. Key words: Retinoblastoma gene product; Tumor suppressor gene; (Diagnosis); (Prognosis)

1. Introduction

The retinoblastoma gene (RB) is one of the best-studied tumor suppressor genes (Lee, 1990;

* Corresponding author. Tel.: (619) 597-0177; Fax: (619) 5970237. Abbreviations: p110RB, wild type retinoblastoma gene product; RB, retinoblastoma gene; TAg, SV40 large T antigen; aa, amino acid; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IP, immunoprecipitation; IB, immunoblot; NBT, nitro blue tetrazolium; BCIP; 5-bromo-4-chloro-3-indolylphosphate; ABTS, 2.2-azino-bis(3-ethylhenz-thiazoline-6-sulfonic acid); DAB, diaminobenzidine tetra-hydrochloride; Kd, dissociation constant.

Weinberg, 1991). Mutations which prevent the normal expression of RB are associated with several h u m a n malignancies (Friend, 1986; Harbour, 1988; Lee, 1988; Stratton, 1989; Bookstein, 1990a,b; Cairns, 1991; Ishikawa, 1991; Strohmeyer, 1991; Xu, 1991). Recent data have correlated lack of normal RB expression with p o o r prognosis in acute myelogenous leukemia (Kornblau, 1992) and transitional cell bladder carcinoma (Cordon-Cardo, 1992; Logothetis, 1992). The RB gene product, p l l 0 RB, is also a target for inactivation by various viral oncoproteins, including adenovirus E 1 A (Whyte, 1988, 1989; Phelps, 1991), polyoma virus T A g (DeCaprio, 1988) and papillomavirus E7 (Phelps, 1991). It is believed

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that these oncoproteins bind to and inactivate an important function of p l l 0 RB, thereby mimicking the loss of retinoblastoma gene function. Studies have identified several cellular proteins which specifically interact with p l l 0 RB (Bagchi, 1991; Bandera, 1991; Chittenden, 1991; Kaelin, 1991, 1992; Hu, 1992; Shan 1992). However, understanding of the significance of p l l 0 RB association with other cytoplasmic or nuclear proteins is limited. Tools for such studies must include sensitive anti-p110 RB monoclonal antibodies which can detect p110 RB in patient specimens by simple methods, such as direct Western blotting, ELISA, immunohistochemistry or FACS analysis.

aprotinin/10 ~ g / m l leupeptin) on ice for 1 h. The p l l 0 RB was purified by sequential chromatography on CM Sepharose CL6B and DEAE Sepharose CL6B. Column fractions were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting (as described below). The fractions which contained pure p l l 0 RB were pooled and concentrated. TrpE-RB fusion proteins, RB cDNA fragments A v a i l / BgllI (exons 1-10; pRB 01-10), BgllI/BgllI (exons 10-19; pRB 10-19), HaelII/HindlII (exons 23-27; pRB 23-27) and BgllI/HindlII (exons, 19-27; pRB 19-27) were prepared as described (Wang, 1990a). 2.3. Hybridoma production

2. Materials and methods

2.1. Cells and cell culture All cell lines were obtained from ATCC. U2OS and SaOS2 (osteosarcoma), H128 (small cell lung carcinoma), HeLa (cervical carcinoma) and CEM (T cell leukemia) cell lines were routinely maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS) and 1% Fungi-Bact in a 7% CO z incubator (Vaugh, 1977) at 37°C. A549 (non-small cell lung carcinoma), H146, H69, and H209 (small cell lung carcinoma), and SK-OV-3 (ovarian adenocarcinoma), were maintained in Kaighn's Nutrient F - 1 2 / D M E high glucose medium with 10% heat-inactivated fetal calf serum, 1% Fungi-Bact and 1% L-glutamine. FO cell murine hybridoma fusion partners were maintained in DMEM. E. coli/BL21 and Sf-9 insect cells, a clonal isolate of Spodoptera frugiperda IPLB-Sf21-AE (Vaugh, 1977) were kindly provided by Dr. W.H. Lee. 2.2. Protein purification Recombinant p110 RB was expressed in Sf-9 insect cells infected with RB-containing recombinant baculovirus (AcNPV-Y4 RB) (Wang, 1990b). To recover p l l 0 RB, cells were first treated with lysis buffer (25 mM Tris-HCl, pH 8.0/150 mM NaCI/0.5% NP40/50 mM N a F / 1 0 / x g / m l /

Hybridomas were generated as described (Campbell, 1987). Briefly, purified p l l 0 RB, pRB 01-10 and pRB19-27 were used to immunize B A L B / c mice by intraperitoneal injection. After high titers of antibodies specific for p l l 0 RB, pRB 01-10 and pRB 19-27 were detected, representative mice were sacrificed and hybridomas were prepared by fusing mouse spleen cells to FO cells. All hybridomas were subcloned and propagated in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum. Monoclonal antibody isotype determination was done using a kit from Zymed Lab. 2.4. Immunoblotting Before analysis, tumor cells were solubilized in lysis buffer (as described above) on ice for 30-45 min and protein was quantitated (BioRad). For Western blotting, 20/zl of cell lysate was loaded into each gel slot and resolved on an 8-16% pre-cast SDS-polyacrylamide mini-gel (Novex). A multichannel immunoblot apparatus (BioRad) was employed for hybridoma screening. Protein was then electrotransferred to an Immobilon P membrane. After primary antibody incubation, membranes were washed three times, 10 min each, with 0.05% Tween 20 in PBS. Goat antimouse IgG conjugated to alkaline phosphatase diluted 1/7500 in 0.5% B S A / P B S was added to immunoblots for 1 h at 24°C. Blots were again

S.F. Wen et al. /Journal of Immunological Methods 169 (1994) 231-240

washed in 0.5% BSA/PBS three times for 10 min each. Alkaline phosphatase substrate, N B T / BCIP (Promega) was then added and incubated for 10-20 min to allow for detection of p l l 0 RB. 2.5. Immunoprecipitation

Epitopes of anti-p110 RB monoclonal antibodies were approximated by competitive immunoprecipitation in the presence of fusion protein fragments of p110 RB, or with synthetic peptides, utilizing a previously described protocol (Lee, 1981). Protein A-Sepharose was added to precipitate RB protein/antibody complexes. Immunoprecipitates were subsequently analyzed by immunoblotting. 2.6. TAg / p l l O Ra binding assay

A modified ELISA protocol (Campbell, 1987) was employed. 50/xl of 1 / z g / m l purified TAg in coating buffer (40 mM borate decahydrate/0.15 M NaCl, pH 8.0) was bound to wells in microtiter plates at 4°C overnight. After blocking with 1% BSA/PBS, various concentrations (2 /~g/ml to 0.016 /xg/ml) of purified p l l 0 RB were added into each well. The plate was incubated for 1 h at room temperature. After washing four times with PBS/Tween 20, biotinylated antibody was added to the appropriate wells and the plate was incubated for 1 h at room temperature. The plate was then washed four times as described above. 1/2500 dilution of Streptavidin-peroxidase (POD; Boehringer) was added to the wells and incubated for 1 h at room temperature. ABTS (Sigma) was used as chromogen. Color development was measured after 20-30 min incubation and the absorbance (405 nm) was determined by Coulter microtiter plate autoreader. 2. 7. Flow cytometry analysis

Fluorescence-activated flow cytometry was performed using a Becton Dickinson FacScan flow cytometer. Tumor cells were fixed in 50% ETOH for 2 h. The cells were washed and resuspended in PBS containing 5% newborn calf serum (NCS/PBS) and were resuspended at 4 × 107

233

cells/ml. 25 ~1 aliquots were reacted with 50/.d of either non-specific MulgG or 3C8 at 0.5 ~ g / m l for 30 min at 4°C, then washed 2 times with NCS/PBS. Subsequently, cells were resuspended in 50 /xl goat anti-mouse FITC (Cal TAg) and incubated 30 min at 4°C. After incubation the cells were washed 2 times in 5% NCS/PBS and resuspended for flow cytometry analysis. 2.8. Immunohistochemical staining

For immunohistochemical staining of paraffinembedded sections with antibody 3C8, we utilized a recently developed protocol including an antigen retrieval step. Briefly, deparafinized sections were incubated in 0.1 M sodium citrate buffer (pH 6.0) at 95°C for 20 min. Subsequently, the slides were placed in a humidity chamber and all subsequent steps followed the vector ABC Elite Kit protocol (Vector Laboratories), with the exception that primary antibody and secondary reagents were diluted in 10% horse serum in PBS/0.1% BSA. Monoclonal antibody 3C8 was routinely used at a concentration of 1.0 ~ g / m l .

3. Results 3.1. Monoclonal antibody generation

Purified p l l 0 RB or TrpE-fusion proteins with portions of p l l 0 RB were used for immunization. After the third boost, the most responsive mice as determined by ELISA were chosen for the preparation of hybridomas. In these fusions all of the monoclonal antibodies were found to recognize only sequences between aa330-612 of p l l 0 RB. In order to obtain antibodies that target different regions of p l l 0 RB, we used E. coli trpE-Rb fusion proteins encompassing aa41-330 and 612928, respectively, as immunogens. Positive hybridomas were identified by single cell cloning and analysis of culture supernatants by ELISA and immunoblotting. After single cell cloning of ELISA and immunoblotting positive hybridomas, the locations of their epitopes on p l l 0 RB were determined by competition with fusion proteins a n d / o r with synthetic polypeptides in the ELISA

234

S.F.

Wen et aL /Journal of Immunological Methods 169 (1994) 231-240 Table 1 Characterization of a n t i - p l l 0 RB monoclonal antibodies

116~

m

I

2

3

..

4

5

6

7

Fig. 1. Immunoblot analysis o f p110 RB expression in h u m a n tumor cell lines. Lane 1: purified pllORB; lanes 2-7: lysates of SK-OV-3, A549, H596, H209, H146 and HeLa. 30/~g of total protein was loaded for each cell line. The p l l 0 RB was detected with 3C8 ( 1 / ~ g / m l ) The p110 RB phenotype of the cells are SK-OV3 (positive, unpublished data); A549 (positive) (Yokota, 1987); H596 (negative) (Harbour, 1988); H209 (positive, codon 706 RB mutant) (Hensel, 1990); H146 (negative) (Hensel, 1990); HeLa (positive) (Scheffner, 1991).

and during immunoprecipitation/immunoblotting (IP/IB). A negative result, loss of detectable p110 RB by immunoprecipitation/immunoblotting (IP/IB), indicated effective competition by the synthetic peptide or fusion protein. The epitope assignments and antibody isotypes are given in Table 1.

M O A B Epitope a E L I S A b IB e amino acid (EXON)

IP/ IB d

IS e

Isotype

3C8

886-905 (25-27)

+++

+++

+++

+++

IgC2a

4G2

41-330 (1-10)

+

+ +

+

+ +

IgG1

7F12

280-300 (1-10)

+

+

+

-

IgG1

4H6

121-140 (1-10)

+ +

+ +

+

+ +

IgG1

1D1

571-590 (18)

+ +

+

+

-

IgG1

1E5

511-530 (17)

+

+ +

+ +

-

IgG1

1G5

371-390 (11-12)

+ +

+ +

+ +

+ +

IgG1

a Summary of the epitopes location on p l l 0 RB. b ELISA was read at 405 nm. + : 0.5-1.0; + + : 1.0-1.5; +++: >1.5. c 1B: results of immunoblotting with p l l 0 RB. + : moderate immunoreaction; + + : strong immunoreaction; + + + : very strong immunoreaction. d I P / I B : results of immunoprecipitation (IP) and immunoblotting (IB) of p l l 0 RB. e IS: results of immunocytochemical staining with U20S cell line. - : staining patterns equal to negative control; + + : strong staining patterns: + + + : very strong staining pattern.

3.2. p l l O RB Expression analysis by direct immunoblotting

Lysates were prepared as described in the materials and methods section, and subjected to electrophoresis and immunoblotting with 3C8 (Fig. 1). A tight cluster of bands consistent with the presence of phosphorylated and unphosphorylated p l l 0 RB proteins with apparent molecular weights between 110 kDa and 116 kDa were detected in SK-OV-3, HeLa and A549 cell lysates. In contrast, these bands were undetectable in H146 and H596 cells. H209 showed an unphosphorylated p l l 0 Ra pattern. To test the sensitivity of 3C8, we serially diluted A549 cell lysate. As shown in Fig. 2, p l l 0 RB could be detected in as little as 1.25 /zg of total protein of A549 cell lysate.

116 84 58 48 36 26 1

2

3

4

5

6

7

8

Fig. 2. Immunoblotting with 3C8 monoclonal antibody is a sensitive method for detecting p l l 0 RB. Two-fold serial dilutions (lanes 1-8) of an A549 cell lysate (initial protein concentration 1 m g / m l ; lane 1) were immunoblotted and detected with antibody 3C8 ( l ~ g / m l )

S.F. Wen et al. /Journal of Immunological Methods 169 (1994) 231-240 1.5-

3.3. A quantitative ELISA may be a valuable aid to clinical evaluation of pl lO nB in tumor samples Expression of p l l 0 RB in tumor cells was also evaluated using analysis by ELISA. 4G2 (which binds an N-terminal epitope in p110 RB, Table 1) was used as a coating antibody in combination with the 3C8 monoclonal antibody. Cell lysates were prepared from A549, H146, H69, H209, HeLa and SK-OV-3. Purified p l l 0 RB was used as standard to quantitate the expression level of p l l 0 RB in these tumor cell lines (Fig. 3). Western blotting (Fig. 1) of cell lysates prepared from cell lines were in general agreement with ELISA resuits (Fig. 3). One difference between these two methods is evident in the comparison of SK-OV-3 and A549 cell lines. SK-OV-3 cells appear to have relatively more p l l 0 Ra as compared to A549 by immunoblotting (Fig. 1) as compared with ELISA (Fig. 3). The reason for this discrepancy is unclear, but current data (not shown) indicate that ELISA is more quantitative and reproducible than immunoblotting as a method to measure p l l 0 RB levels in cell lysates.

235

(.)

E

d 6

1.0

0.5-

0,0

|

-3

-1 Iog[biotlnylated

antibody

(uglml)]

1.5 (b)

E_

1.0,

~"

0.5-

3.4. Antibody /epitope selection is important for the analysis of pl lO RB expression In some cell types the retinoblastoma protein 30

0.0

2

o.

0.0

015

110

115

210

2.5

20' p110RB

o*

¢oneentratlon(uglml)

Fig. 4. Association o f viral oncoproteins with p l ] 0 RB can E

10' t, ¢¢ Y,/,'~,~,//,/,/,I, H146

SKOV-3

H596

A549

H209

Fig. 3. Quantitation of p l l 0 RB expression in various tumor cell lines. 4G2 was coated on microplates. Cell lysates were then added into each well. Biotinylated 3C8 was used to detect bound p110 RB. Total cell lysate protein was determined by BioRad protein assay. * Undetectable level ( < 7.8 ng pll0RB/mg of cell lysate)

inhibit antibody detection. (a) Purified p l l 0 RB was coated on a microwell plate, serial dilutions of biotinylated 3C8 (©) or 1E5 (A) was added to detect p l l 0 RB as described in the materials and methods section. (b) Purified TAg was coated on plates, serial dilutions of p l l 0 RB was added to allow complex formation with TAg, then biotinylated 3C8 (C-terminal epitope; 0.2 /.Lg/ml (©) or biotinylated 1E5 (Pocket domain; 1 /zg/ml (A) was used to detect bound p l l 0 RB.

is complexed with viral oncoproteins. The viral oncoproteins bind specifically to the p l l 0 RB pocket domains. One such viral oncoprotein which

236

S.F. Wen et al. /Journal

of Immunological Methods 169 (1994) 231-240

S.F, Wen et aL /Journal of Immunological Methods 169 (1994) 231-240

binds p l l 0 RB is SV40 large TAg (DeCaprio, 1988). When TAg/p110 RB complexes were prepared in vitro, p110 RB detection by ELISA was reduced when antibodies recognizing epitopes in the pocket binding domain were employed (Fig. 4a,b). However, the 3C8 monoclonal antibody, which binds C-terminal to the pocket domains, maintained its ability to recognize the complexed p110 RB (Fig. 4b). Thus the choice of antibodies is critical for detecting p110 Ra in different cellular backgrounds.

>-

0

(a)

loo

lo,

°j

10z Fluorescence Units

10 3

104

10z

1~

10¢

z

3.5. Immunohistochemical evaluation of pllO RB protein expression Monoclonal antibody 3C8 was evaluated for detection of p110 RB expression in formalin-fixed and paraffin-embedded tissues. Staining of processed tissues was greatly enhanced by immersion of the tissue sections in hot citrate buffer. Reactivity was nuclear, with little cytoplasmic background staining (Fig. 5). Varying degrees of staining intensity may reflect cell cycle a n d / o r differentiation-dependent variation in p110 Ra expression within a single sample. We tested a small number of prostatic adenocarcinomas, all of which displayed normal levels of p110 RB (Fig. 5a). The majority of bladder carcinomas also showed normal p110 RB expression (Fig. 5b). In agreement with previous reports by other investigators (Cordon-Cardo, 1992; Logothetis 1992), we found that a subset 25% of bladder cancers failed to show immunohistochemical reactivity for p110 Ra (Fig. 5c). Normal staining of vascular endothelial cells provides a positive control for staining in these latter samples.

237

i

0

(b)

-

10o

lol

Fluorescence Units

Fig. 6. Flow Cytometry of H128 and CEM cells. Histograms of the green fluorescence (x axis) plotted against cell number (y axis). 1 × 106 cells were fixed by ethanol and processed for indirect immunofluorescence detection using 3C8 (filled peak) or MuIgG (open peak). H128 cells (a) are negative and CEM cells (b) are positive for p110 Rn stained with antibody 3C8 in immunocytostaining assays.

monitor for background fluorescence, identically processed fixed cells that had been incubated with a murine IgG control were analyzed in parallel. The results (Fig. 6), showed that the pll0Ra-dependent fluorescence signal of the CEM (pll0 mB positive) cell line was found to have a much greater intensity than the fluorescence signal obtained from H128 (pll0 RB negative) tumor cells.

4. Discussion

3.6. Flow cytometry Analysis of p110 RB expression in H128 and CEM was also performed by flow cytometry. To

Previous studies have indicated that the region of p l l 0 RB needed for binding to DNA tumor virus oncoproteins E1A, E7 and large TAg maps

Fig. 5. Immunohistochemical staining of formalin fixed and paraffin embedded tumors. 5/~m paraffin sections were reacted with monoclonal antibody 3C8 (1 p,g/ml). (a) Prostatic adenocarcinoma. Virtually all tumor cells and many stromal cells show p l l 0 Re reactivity (straight arrows). A subset of nuclei do not stain (hooked arrows).(b) High grade transitional cell carcinoma of the bladder. Tumor and stromal cell nuclei display variable degrees of staining intensity (straight arrows). A subset of nuclei do not stain at all (hooked arrows).(c) Transitional cell carcinoma of bladder. The tumor cells are completely negative for p l l 0 RB (hooked arrows). Stromal cells serve as a positive internal control in that most of them show distinct nuclear p l l 0 Ra reactivity (straight arrows). Magnification 400 × .

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S.F. Wen et al. /Journal of lmmunological Methods 169 (1994) 231-240

in the region from aa393-571 and 649-793 (Hu, 1990; Huang, 1990). There is also evidence that cells produce one or more proteins that interact with the pocket domains of p l l 0 RB (DeCaprio, 1988; Whyte, 1988, 1989; Kaelin, 1991; Phelps, 1991; Shan, 1992). If so, an antibody that interacts with this region of p l l 0 Ra might only recognize the uncomplexed form of p l l 0 Ra, thus resuiting in a false negative conclusion regarding p l l 0 RB expression. This assumption was confirmed by using 1E5 in a T A g / p l l 0 RB binding assay. 1E5, which recognizes an epitope located within the TAg binding site (aa393-aa572), failed to recognize p l l 0 RB in the complex with TAg. The epitope recognized by 3C8, in addition to its high affinity for p l l 0 RB ( K d 2.9 × 10 -9 M, unpublished results), makes it very attractive for affinity chromatography or immunoprecipitation studies to characterize other cellular proteins associated with p l l 0 RB (Dyson, 1989; Hu, 1991; Shan 1992) (Eva Lee, personal communication). Previous studies have shown that functional loss of p l l 0 Ra may contribute significantly to tumor progression (Cance, 1990; Cordon-Cardo, 1992; Logothetis, 1992; Reissmann, 1993), In this regard, loss of p l l 0 RB tumor suppressor protein expression may be similar to the prognostic value of estrogen receptor loss in breast cancer (EORTC, 1973; McGuire, 1988). To resolve whether RB status plays a key role in tumor progression, an accurate and fast screening method should be established to reflect the frequency of loss of p l l 0 RB in tumor biopsies. Molecular genetic approaches (e.g., Southern analysis) may not detect small molecular alterations (Horowitz, 1989; Cairns, 1991; Figge, 1991; Reissmann, 1993). Also, contamination of tumor tissue with normal cells may confound Southern or Northern analysis, or PCR-based analyses (Cairns, 1991; Murakami, 1991; Reissmann, 1993). Immunohistochemical staining may represent the most reliable method for analyzing tumor samples for p l l 0 RB expression because this method allows direct visualization of tumor cells within biopsy specimens. Antibody 3C8 is a sensitive reagent to detect p l l 0 RB in neoplastic and non-neoplastic cells. Nuclear staining tends to be strong, and cytoplasmic background low. Thus,

this reagent may be a useful tool to investigate patterns of p l l 0 RB expression in archival tissues. In addition to immunohistochemical staining, quantitative ELISA testing or simple direct immunoblotting procedures for detection of p l l 0 RB may be a helpful aid to clinical evaluation. An antibody with high sensitivity will be necessary for this purpose. The sensitivity of the 3C8 monoclonal antibody in flow cytometric analyses may be useful for determining p l l 0 RB expression in hematologic malignancies, or for urinary cytological analysis of bladder cancers. The availability of the family of monoclonal antibodies described here will provide an improved means to screen for p l l 0 RB expression in cells and histological specimens. Such work will therefore allow for a more critical analysis of the prognostic value of p l l 0 RB expression in human cancers.

4. Acknowledgements We wish to thank Dr. Wen Hwa Lee and Dr. Eva Lee for their help and encouragement during the course of this work.

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