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Department of Genetics, Biology and Biochemistry (A.F., B.F., A.L.H., F.M.), Research Center for Experimental Medicine (A.F., B.F., A.L.H., F.M.), Department of Biomedical Science and Human Oncology (A.S., I.C.), University of Torino, Torino 10126, Italy; Department of Nuclear Medicine, University of Modena (B.B.), Modena 41100, Italy; and Serono International (G.G.), Geneva 1228, Switzerland
Address all correspondence and requests for reprints to: Fabio Malavasi, M.D., Department of Genetics, Biology and Biochemistry, via Santena 19, 10126 Torino, Italy. E-mail: fabio.malavasi{at}unito.it.
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Abstract |
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For this study, monoclonal antibodies (mAbs) were raised against the human LH receptor (LHR)/hCG receptor (hCGR), using Chinese hamster ovary LHR-transfected cells as the immunogen. Two reagents were then selected on the basis of their ability to recognize the full-length transmembrane re-ceptor expressed both by Chinese hamster ovary LHR-transfected cells and by a limited number of tumor cell lines.
One of these mAbs reacts with the LHR/hCGR in tissue sections of both frozen and paraffin-embedded specimens. This unique feature allowed us to map the cytological distribution of LHR/hCGR in human breast tissues at different stages of development in physiological and benign pathological conditions. The same mAb proved to be agonistic: receptor ligation elicits signals that modulate the growth of selected breast tumor cell lines. This observation suggests that the mAb recognizes an epitope that is included in the domain of the receptor involved in the interaction with the natural ligand.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LH and hCG (the ligands of LHR) are members of a single dimeric glycoprotein hormone family and share a common 
-subunit noncovalently bound to a hormone-specific ß-subunit that differs from hormone to hormone (1). The structural similarity of the ß-subunits mirrors the functional similarity between LH and hCG pituitary hormones that bind the same receptors and exert similar functions (7).
Until recently, LH and hCG were presumed only to regulate ovarian and testicular steroidogenesis (1). Current evidence reveals that the endocrine effects of LH and hCG are flanked by paracrine effects influencing the behavior of a number of tissues that express the specific receptors (e.g. the female and male reproductive tracts, the fetoplacental unit, brain, hippocampal cells, skin, breast, urinary bladder, and sperm) (8, 9, 10, 11, 12, 13).
Strategy for the prevention of breast cancer have been devised on the basis of observation that pregnancy at a young age offers protection against the development of breast cancer (14, 15). Russo et al. (16, 17, 18) have observed that the induction of mammary tumors in normal rats by dimethyl-benzanthracene can be delayed or inhibited by treatment with hCG. The in vivo results have been confirmed in vitro, where hCG exerts direct antiproliferative effects on human breast epithelial cells in culture (19). Contrasting results were reported over 20 yr ago, with hCG featuring growth-promoting activities on breast cancer cells in a different mouse model (20).
Dual effects have also been reported in other models. It is thought that hCG may also offer protection against prostate cancer (21), whereas it may enhance the development of epithelial ovarian carcinoma (22) and the growth of bladder (23) and lung cancer cell lines (24).
Like other hormone receptors, such as estrogen and progesterone receptors, LH/hCGR is characterized by heterogeneous expression by tumors of the same histotype. The stimulating or inhibitory effect of LH/hCG on the growth of tumors of different origins implies that the corresponding tissues express the appropriate receptors.
A means of rapidly identifying in vitro expression of the LH/hCGR would represent a clear asset to research and would also have tremendous diagnostic potential.
In the current study, mAbs specific for the LH/hCGR were raised and two specific reagents adopted. One of these mAbs reacts with the LH/hCGR even on formalin-fixed and paraffin-embedded tissues and displays agonistic activities in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female Balb/c mice (Charles River, Calco, Italy) were injected ip with 107 CHO cells transfected with the cDNA of human LHR (CHO-LHR) (kindly provided by T. Arulanandam, Serono Research Biotechnology Institute, Rockland, ME) and the injection was repeated four times every 15 d. Four days after the last injection, splenocytes were fused with the murine HGPRT- myeloma P3.X63.Ag8.653 (25). The selected primary cultures were cloned at least three times and expanded in vitro in conventional culture conditions and in vivo in Pristane-primed Balb/c mice.
Purification of endotoxin-free mAb
Ascitic fluids collected from the mice were purified by HPLC (Beckman Gold 125/166 and 126/166NM, Beckman, Milan, Italy). Briefly, samples were passed through a 100 x 25 mm Affi-Prep protein A MAPS column (Bio-Rad, Milano, Italy). Bound IgG were then eluted with citric acid (pH 4) and dialyzed against sodium phosphate buffer (pH 6.7). The eluted Ig were bound to the hydroxylapatite on a high performance hydroxylapatite preparative column and eluted with the appropriate gradient. The final purification step involved detoxi-gel chromatography on Polimyxin B-Sepharose (Pharmacia, Uppsala, Sweden). The presence of endotoxins in the final product was determined using the Limulus Amebocyte Lysate Pyrogent test (BioWhittaker, Inc., Walkersville, MD) (26).
Immunoprecipitation of the target antigen
Iodo-Gen (Pierce Chemical Co., Pero, Italy) was dissolved in chloroform at 0.25 mg/ml and the solvent evaporated. The CHO-LHR and CHO-mock cells (5 x 107) were added to 0.5 ml of PBS containing 1.85 MBq of carrier-free Na 125I (Amersham, Milano, Italy) for 15 min at 20 C. Samples were then transferred to saturated tyrosine-PBS solution and washed to remove unbound [125I]iodide. Immunoprecipitation was performed as described in (27). Briefly, 125I-surface-labeled cells were lysed and after preclearing postnuclear cell lysate with goat antimouse IgG (GaMIgG) bound to Sepharose, the target molecule was immunoprecipitated for 12 h at 4 C with 5 µg of purified mAb followed by the addition of GaMIgG bound to Sepharose for 2 h at 4 C. The immunocomplexes were then subjected to SDS-PAGE and the gels exposed for autoradiography.
Western blot analysis
It was performed using CHO-LHR and CHO-mock cells membranes isolated after cell lysis. Twenty micrograms of protein were electrophoresed on 10% SDS-PAGE under reducing conditions and electrotransferred onto polyvinylidene difluoride membranes. Membranes were incubated with 5% BLOTTO (Amersham) in Tris-HCl (pH 7.4), 0.15% Tween 20, for 2 h at room temperature. The selected mAbs (5 µg/ml) were applied onto the membrane overnight at 4 C. After rinsing with Tris-HCl, the membranes were incubated with peroxidase-conjugated GaMIgG antibody for 1 h at room temperature. Specific signals were detected using the enhanced chemiluminescence method. The molecular size of the proteins of interest was determined by running molecular size marker proteins in an adjacent line.
Characterization of the reactivity of the selected mAbs
The selected mAbs were analyzed on a panel of viable normal cells peripheral blood mononuclear cells and endothelial cells and tumor cell lines including MCF-7, MDA/MB-231, SKBR-3, and T47-D (human breast cancer), MCF-10 (nonmalignant breast), TSA and AG12 (murine breast cancer), NIH-OVCAR 3 (ovary cancer), HEC-1-B (endometrial cancer), Hep G2 (hepatocellular cancer), CF-PAC1 (pancreatic cancer), LoVo (colon cancer), Kato III (gastric cancer), PC-3 (prostatic cancer), U-251 and Lan-1 (neuroblastoma) and G4 (glioma). Briefly, cells (3 x 105/sample) resuspended in PBS were incubated for 30 min at 4 C with 100 µl of the supernatants from the hybridoma cultures. After washing, cells were incubated for 30 min at 4 C with fluorescein isothiocyanate-labeled GaMIg (LiStarFISH, Milano, Italy). Fluorescence was analyzed by means of a FACSort (Becton Dickinson, Milano, Italy). Background mAb binding was estimated by means of isotype-matched negative control mAbs.
Immunohistochemical analysis of the distribution of the LH/hCGR
Ovary and testis (positive controls) as well as breast biopsies were obtained from the file of the local Department of Pathology. Tissues were fixed in 10% formaldehyde buffer (pH 7.4) for 24 h, paraffin embedded, and processed for immunohistochemical analysis. Endogenous peroxidase and endogenous biotin were blocked by means of incubation with 6% H2O2 and with specific avidin- and biotin-blocking reagents (Biogenex, San Ramon, CA), respectively. Sections were incubated with normal goat serum (1 : 20) for 2 h at 37 C and successively with the primary 20C3 mAb (0.5 µg/ml) for 2 h at 37 C. After washing, sections were incubated for 13 min at 20 C with biotinilated GaMIgG (StrAviGen Multilink Kit, BioGenex), then washed and incubated with peroxidase-conjugated streptavidin for 13 min at 20 C. The reaction was developed in a solution containing 3,3'-diaminobenzidine (LiquidDAB Substrate Pack, BioGenex). Nuclei were counterstained with Mayer hemallum.
CHO-LHR and CHO-mock cells were centrifuged, and the pellets were processed either for cryostatic or paraffin sections which were used to test the mAb reactivity and specificity. Furthermore, the specificity of the selected mAb was assessed after adsorption with CHO-LHR cells and successively used on tissue sections.
Competition between mAbs and ligands to target hCGR
Purified mAbs and human recombinant LH (rLH) (Serono International, Geneva, Switzerland) were iodinated using the chloramine-T technique. The binding properties were characterized using a radioligand receptor assay with CHO-LHR cells as the target and CHO-mock cells as the negative control. Competition experiments were performed by coincubating cells (2 x 106/ml) with the relevant 125I-labeled ligand in the presence (or in the absence) of the selected mAb for 2 h at 4 C. After extensive washings, the bound radioactivity was evaluated in a 
-counter.
Analysis of signals mediated by LHR ligation
Intracellular cAMP assay. CHO-LHR cells were plated in 24-well plates (4 x 105/well) and incubated for 24 h in Iscove’s MEM (IMDM) (Sigma, Milano, Italy) supplemented with 5% FCS. Before treatment, cells were washed twice with serum-free IMDM and incubated in IMDM containing isobutylmethylxantine (0.1 mg/ml) for 15 min at 37 C. After adding increasing amounts of recombinant hCG (Serono International, Geneva, Switzerland) or purified mAbs, the incubation was increased to 90 min at 37 C. Then the medium was removed, the cells rinsed once with IMDM without isobutylmethylxantine and lysed in 0.1 M HCl. Intracellular cAMP concentrations were determined after acetylation of the samples using a direct cAMP enzyme immunoassay kit (sensitivity = 0.037 pmol/ml) (Sigma), according to the manufacturer’s protocol.
Cell proliferation assay. Cell proliferation was assessed by a 3[H]thymidine (Amersham) incorporation test. 1.2 x 104 cells/well were cultured in 96-well plates in the presence or in the absence of increasing amounts (range: 0.1 ng/ml–10 µg/ml) of soluble recombinant hCG or affinity purified pyrogen-free mAbs. Cultures were pulsed at predetermined times with 1 µCi/well 3[H]thymidine, incubated for another 12 h, harvested onto filter plates, and the counts analyzed in a ß-counter (Perkin-Elmer, Milano, Italy).
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Results |
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The hybridomas obtained were selected on the basis of their ability to react on CHO-LHR cells. The positive clones selected were unable to bind the CHO-mock cells. Two mAbs that showed the proper reactivity, namely 20C3 (IgG2a) and 4F4 (IgG1) (Fig. 1A
), underwent three cycles of cloning by limiting dilution. The hybridomas were expanded in vitro and the culture supernatant was used for all the staining in suspension cells. The hybridomas were also grown in vivo as tumor ascites, used for purification. The purified and depyrogenized mAbs were used for staining tissue sections and for signal transduction experiments.
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). Moreover, immunoprecipitation obtained with either an irrelevant mAb or in the absence of the specific antibody failed to reveal the bands (data not shown), indicating that 20C3 and 4F4 specifically bind the LHR.
Immunoblotting analysis of CHO-LHR cells highlighted the presence of a major protein band of approximately 90 kDa identified by 20C3 and at a very low level by 4F4 mAb (Fig. 1C, lanes 3 and 4), whereas no reactivity was apparent on CHO-mock cells (Fig. 1C, lanes 1 and 2).
The binding of the selected mAbs was then analyzed on a panel of viable normal cells and tumor cell lines of different origins by means of the indirect immunofluorescence technique and FACS analysis. The results are summarized in Table 1. 20C3 and 4F4 mAbs displayed weak to moderate reactivity with all of the selected breast cancer cell lines so far analyzed and with the NIH-OVCAR-3 ovary carcinoma. The selected mAbs did not react with peripheral blood mononuclear cells (resting or activated), human keratinocytes, or tumor lines of the gastrointestinal tract. The characteristics of reactivity and fluorescence intensity on selected cell types were notably different for the selected mAbs. The range of 4F4 reactivity was much wider and included normal umbilical endothelial cells and selected brain tumors, i.e. LAN-1 (neuroblastoma) and G4 (glioma) cell lines.
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After noting that the 20C3 and 4F4 mAbs are reactive with breast cancer cell lines, the next step was to evaluate the presence of the LHR in normal and pathological human breast tissues. The analytical conditions were optimized in CHO-LHR cells processed as cryostatic samples and as paraffin-embedded tissues. On fixed CHO-LHR cells 4F4 showed a weak reactivity, whereas 20C3 showed a strong and reproducible reactivity at both the membrane and cytoplasmic levels (Fig. 2, A and C). No reactivity was observed in the CHO-mock cells (Fig. 2, B and D). 20C3 mAb reactivity on CHO-LHR cells was completely abolished by adsorption of the mAb with the same cells (data not shown).
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, E and F), whereas on testis it was restricted to the Leydig’s cells (Fig. 2, G and H).
The analysis of hCGR expression was performed in normal human mammary gland (tested in discrete differentiative steps) and benign breast lesions. The ductal structures diverging from the nipple were not stained in fetal breast tissues (two cases at 28 and 26 wk gestation, respectively) (Fig. 3A). In more developed mammary gland, the expression of LH/hCGR was mainly restricted to basal cells of lobular structures. In adolescent mammary tissues obtained from a 13-yr-old girl affected by juvenile fibroadenoma, hCGR was expressed on basal cells, on scattered epithelial luminal cells and on stromal cells of the fibroepithelial lesion (Fig. 3B).
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, C and D). The intensity of mAb staining varied among the lobules and the reactivity was completely abolished when sections were tested with adsorbed mAb (data not shown).
In normal lactating gland the reactivity was restricted to occasional basal cells (Fig. 3E). In postmenopausal women, basal cell expression of hCGR was barely visible around lobules (Fig. 3F).
Tissue samples of five fibroadenomas, two benign papillomas, and one sclerosing adenosis obtained from pre- and postmenopausal women displayed intense membrane immunostaining of basal cells, and of occasional luminal epithelial cells (Fig. 3, G and H).
Epitope analysis
The epitope analysis of the determinants recognized by 20C3 and 4F4 mAbs was performed by assaying the ability of 4F4 mAb to compete with the binding of 125I-labeled 20C3 mAb to CHO-LHR target cells, and, vice versa, the ability of 20C3 mAb to compete with 125I-labeled 4F4 mAb. The results demonstrated that 4F4 mAb was partially able to interfere with 20C3 binding in a dose-dependent manner when used at high concentrations. However, the competition never exceeded 35–40% of the total binding (Fig. 4A). On the contrary, 20C3 inhibited approximately 20% of the binding of 125I-labeled 4F4 mAb only when used at concentrations ranging from 10–20 µg/ml (data not shown).
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). Expression of the 20C3 and 4F4 epitopes was increased by the addition of recombinant LH, as witnessed by the binding of the 125I-mAbs in the presence of LH. The effect was weak (ranging from 10–25%) but dose-dependent and reproducible in several experiments (Fig. 4C). Analysis of signals mediated by hCGR
Intracellular cAMP assay.
Treatment of CHO-LHR cells with hCG resulted in a significant dose-dependent production of cAMP compared with CHO-mock cells. Ligation of LH/hCGR by the 20C3 mAb was followed by the induction of intracellular cAMP, although at lower levels than after hormone binding (Fig. 5). The addition of the 4F4 mAb in the same experimental conditions was unable to induce a detectable cAMP production.
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, A and B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The focus of the present work was initially on the hCGR, which has been studied until now by exploiting hCG itself. The only mAbs reported to be specific for the receptor were raised against a chimeric protein (33) and are difficult to procure. The difficulties inherent in the direct use of hCG, together with the lack of a simple means of studying the distribution of the receptor or the signals channeled in normal and pathological samples prompted us to raise a panel of conventional mAbs specific for the human LHR/hCGR.
The strategy adopted allowed us to produce reagents recognizing the actual molecule expressed by various tissues, and was proven successful given that 1) one of the mAbs selected reacts with the hCGR in tissue sections, and, more importantly, 2) this occurs even when the tissues are fixed with conventional techniques. This makes it now possible to evaluate the samples previously stored in the Pathology Department files. The second major result of this work is that the epitope of one of the mAbs produced (namely 20C3) is involved in (or includes) the domain of the receptor specific for the interaction with the ligand. Indeed, the binding between 20C3 mAb and the receptor is followed by the delivery of signals, which are identical in some aspect to those channeled through the physiological interactions between receptor and ligand.
The target antigen immunoprecipitated from 125I-labeled CHO-LHR cells demonstrates that 20C3 and 4F4 mAbs recognize a dominant surface protein with an apparent molecular mass of approximately 90 kDa, close to that predicted from the amino acid sequence of the mature transmembrane receptor (34). The protein of lower molecular mass (
50 kDa) may reflect a partial degradation of the larger protein.
LH/hCGRs have been found in man, monkey, mouse, rat, pig, cow, sheep, turkey, and fish (35, 36, 37), confirming that their expression is not species specific. The expression of LH/hCGR has been investigated by means of a wide range of technical approaches, including gene transcription, radio binding, signaling pathways, and biological responses (38, 39, 40). The availability of specific mAbs allowed the immunohistochemical localization of the LH/hCGR in human tissues even after fixation, which is the routine approach used in pathology. The tissue fixation procedure appears to be a crucial step in regulating successive immunostaining: indeed, the hCGR proves to be labile and easily degraded. Based on these findings, 20C3 mAb was selected as the analytical probe for the study of the tissue distribution of hCGR. The reactivity of 20C3 mAb was initially evaluated on ovary and testis to compare the results obtained in this work with the localization of the receptor determined with different techniques by other groups (41, 42). The results indicate that the hCGR was detected on theca interna cells of the antral follicles and on the Leydig cells of testis. The present results fit perfectly with those reported so far following different technical approaches (43), indirectly confirming the specificity of 20C3 mAb for the LH/hCGR.
The reactivity of 20C3 mAb on formalin-fixed tissues allowed us to map the cytological distribution of hCGR in human breast tissue at different stages of development. hCGR could not be detected in the mammary gland of female fetus. This is in line with the observation that women with hCG resistance have spontaneous breast development, whereas hCG stimulation is necessary for normal ovarian steroidogenesis and ovulation (44). In physiological and pathological conditions of the adult life, hCGR expression is detectable in breast tissue, as previously reported (33). The difference from previous reports is that 20C3 mAb detects the target molecule on the membrane of the basal cell layer, which is constituted of non differentiated cells and myoepithelial cells. Only occasional luminal epithelial cells are stained. The expression of the hCGR by basal cells is lower in postmenopausal samples. This result is in line with the notion that the hCGR is generally not detectable in the postmenopausal ovary, and down-modulated in other target organs during the same phase of life (45). Analysis of the distribution of hCGR on breast cancers using the 20C3 mAb is currently underway.
The results obtained thus far using the two mAbs suggest the existence of two discrete epitopes on the receptor. Binding competition experiments showed that 4F4 mAb can partially interfere with the binding of 20C3 mAb on the target cells. This suggests that the reagents likely recognize two different epitopes of the receptor and that steric hindrance is likely responsible for the partial displacement observed. The hypothesis of steric hindrance might also explain the interference observed in mAb and LH binding. An alternative explanation is that 20C3 mAb ligation induces conformational changes that affect the interaction of LH with its binding site. However, a difference in affinity of the mAbs compared with that of the natural ligand may be responsible for the effects observed.
Several breast cancer cell lines reportedly show a diminished growth when cultured in the presence of hCG (46). In the present work, the growth inhibitory effects mediated by the addition of hCG in culture are reproduced by the addition of the 20C3 mAb to the SKBR-3 and MCF-7 cell lines. It is reasonable to think that a mAb/receptor engagement is related to the hCGR-mediated signaling. This is consistent with the premise that hCG exerts a protective role against breast cancer in humans. It has been proposed that hCG induces the proliferative terminal end buds of mammary glands to differentiate into secretory type lobules in vivo, rendering the cells less susceptible to neoplastic transformation (47, 48). This hypothesis is supported by epidemiological studies indicating that early parity, late menarche and early menopause have protective effects against breast cancer (15). However, contradictory results were obtained by another group in a different mouse mammary adenocarcinoma model (20). The reasons for these experimental discrepancies are still unknown.
hCG binding to the LH/hCGR is coupled to the adenylyl cyclase and PLC signal pathways, thus leading to the production of cAMP and IP3 as intracellular signal molecules (34, 49). Engagement of the hCGR by the 20C3 mAb is followed by increased intracellular concentrations of cAMP, although significantly lower than those induced by ligand binding. This observation likely reflects the lower efficiency of mAb/receptor binding compared with the physiological hormone/receptor interactions. This finding is not surprising in light of existing evidence that hCG and its receptor undergo multistep interactions with alteration of the receptor geometry, physical shape, and flexibility, leading eventually to total activation of the receptor (50, 51). It is likely that mAb ligation of the receptor is unable per se to induce the structural modifications necessary for eliciting efficient production of cAMP. Alternatively, the affinity of the mAb might be too low to elicit efficacious intracellular signals. The resolution of the crystal structure of hCG has provided clues about the early steps of hormone/receptor interactions and has made it possible to localize the binding domains of the receptor (52). Nonetheless, detailed information about gonadotropin pharmacology is limited, hampered by the difficulties in crystallizing the gonadotropin receptors, which are seven-transmembrane amphipathic proteins. However, large-scale production of the receptor ectodomain-hormone soluble complexes was recently proposed as a realistic strategy for obtaining soluble and secreted molecules stable enough to allow structural analysis and crystallization (53). This approach will offer new insights into the physiological mechanisms regulating the cross-talk of hormone and receptor.
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Footnotes |
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Abbreviations: CHO, Chinese hamster ovary; CHO-LHR, CHO cells transfected with the cDNA of human LHR; GaMIgG, goat antimouse IgG; hCG, human chorionic gonadotropin; hCGR, hCG receptor; IMDM, Iscove’s MEM; LHR, LH receptor; mAb, monoclonal antibody; rLH, human recombinant LH.
Received June 5, 2003.
Accepted August 11, 2003.
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References |
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-reductase and androgen receptor protein levels in women’s skin. J Clin Endocrinol Metab 83:1776–1782-human chorionic gonadotropin antibody. J Natl Cancer Inst 84:42–47This article has been cited by other articles:
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