ARTICLE |
Correspondence to: Graeme I. Murray, Dept. of Pathology, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
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Summary |
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Cytochrome P450 CYP1B1 is a recently identified member of the CYP1 P450 family. We have shown that this P450 displays increased expression in several types of human cancer, indicating that CYP1B1 is a potential tumor biomarker. In this study we developed monoclonal antibodies (MAbs) to CYP1B1 that are effective on formalin-fixed, paraffin-embedded tissue sections and investigated the presence of CYP1B1 in a series of primary breast cancers. The MAbs were generated using a synthetic peptide coupled to carrier protein as the immunogen. The MAbs specifically recognized CYP1B1 and did not recognize either CYP1A1 or CYP1A2, related CYP1 forms. The MAbs were tested by immunohistochemistry and were found to be effective on formalin-fixed, paraffin-embedded tissue sections. The majority of breast cancers showed positive immunoreactivity for CYP1B1, and in each case CYP1B1 was specifically localized to tumor cells. The presence of CYP1B1 in breast cancer cells is likely to contribute to their metabolism of estradiol because CYP1B1 is a specific estradiol hydroxylase. (J Histochem Cytochem 47:14571464, 1999)
Key Words: breast cancer, CYP1B1, cytochrome P450, immunohistochemistry
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Introduction |
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Cytochrome P450 (P450) CYP1B1 is the only known member of a recently identified subfamily of the CYP1 gene family. Human CYP1B1 was originally isolated from a dioxin-treated keratinocyte cell line (
Orthologous forms of this P450 have also recently been isolated from a benzanthracene-induced cell line derived from mouse embryo fibroblasts (
Breast cancer is the most common cancer to affect women and is usually an estrogen-dependent tumor. Human CYP1B1 expressed in yeast (S. cerevisiae) shows high specific activity towards the 4-hydroxylation of 17ß-estradiol (
Our initial immunohistochemical studies of CYP1B1 (
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Materials and Methods |
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P450 Sequence Alignment, Peptide Selection, and Immunization
Based on a combination of structural homology modeling and sequence alignment of the human CYP1B1 amino acid sequence with the human CYP1A1 and CYP1A2 amino acid sequences, a 148-amino-acid segment located in the C-terminal third of the CYP1B1 protein was predicted to contain regions of amino acids that would be located on the external aspect of the CYP1B1 protein. Peptides of either 14 or 15 amino acid residues corresponding to this segment of the CYP1B1 protein were synthesized in the University of Aberdeen Protein Facility. The individual peptide sequences and amino acid location on the CYP1B1 protein are listed in Table 1. Individual peptides were then conjugated to ovalbumin using glutaraldehyde, as previously described (
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Monoclonal Antibodies to CYP1B1
Four days after the final immunization with peptide conjugate, the mice were sacrificed, their spleens isolated, and splenic cells fused with mouse myeloma cells (Ag8.653). The resultant hybridoma clones were then screened for antibody production by enzyme-linked immunoassay (ELISA), using the relevant peptide conjugated with bovine serum albumin (BSA). The BSA conjugates were bound to an ELISA plate by incubation overnight at 4C in 50 mM sodium carbonate/bicarbonate buffer, pH 9.6, and the ELISA was performed as described previously (
Tissues
Samples of normal adult human tissues (liver, kidney, lung, pancreas, adrenal cortex, brain, stomach, jejunum, colon, breast, ovary, and endometrium) were obtained from fresh, unfixed tissue samples submitted to the Department of Pathology, University of Aberdeen for diagnosis. Samples of tissue were frozen in liquid nitrogen and stored at -75C before preparation of microsomes. Samples of primary breast cancer (n = 60) were obtained from samples of breast tissue submitted to the Department of Pathology, University of Aberdeen for diagnosis. All the samples of breast tissue were from needle core biopsies of palpable breast lumps performed as part of the diagnostic protocol before definitive treatment. The core biopsies were fixed in 10% neutral buffered formalin at room temperature (RT) for 1824 hr and then routinely embedded in paraffin.
The diagnosis of breast cancer was performed with hematoxylin and eosin-stained sections using standard histopathological criteria. All tumors were graded according to the Nottingham grading system (
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Expressed CYP1B1 and CYP1A1
Microsomes prepared from human lymphoblastoid cells containing expressed human CYP1B1, expressed human CYP1A1, or control lymphoblastoid cells that contained only vector were obtained from Gentest (Woburn, MA). The microsomes containing individual expressed P450s are supplied by Gentest with a specific P450 content per milligram of microsomal protein.
Preparation of Microsomes
Frozen samples of tissues were thawed on ice in 0.01 M Tris-HCl, pH 7.4, containing 1.15% KCl. The thawed samples of tissue were dissected free of connective tissue and fat, finely chopped with a scalpel, and then homogenized in 0.01 M Tris-HCl containing 0.25 M sucrose and 15% glycerol using a Polytron PT3000 homogenizer (Kinematica; Lucerne, Switzerland). The homogenates were then centrifuged at 15,000 x g for 20 min at 4C using a Centrikon T-124 centrifuge (Kontron Instruments; Cumbernauld, UK). The resultant supernatants were then centrifuged at 180,000 x g (44,000 rpm) for 1 hr at 4C using a Centrikon T-1160 centrifuge (Kontron Instruments). The pellet obtained after centrifugation was resuspended in 0.1 M Tris-HCl containing 15% glycerol and 1 mM EDTA (SigmaAldrich; Poole, Dorset, UK) and centrifuged again at 180,000 x g (44,000 rpm) for 1 hr at 4C. The final microsomal pellet was resuspended in 0.1 M Tris-HCl containing 15% glycerol and 1 mM EDTA, and the microsomal samples were then stored at -75C before use. The protein concentration of each sample of microsomes was determined using Bradford's method (
Immunoblotting
Samples of microsomal proteins were electrophoretically separated at constant current in a 10% polyacrylamide gel using a Hoefer SE600 vertical gel electrophoresis apparatus (Amersham Pharmacia Biotech; Little Chalfont, Bucks, UK) and then transferred at constant current for 18 hr to nitrocellulose (Hybond ECL; Amersham Pharmacia Biotech) by electroblotting using a Hoefer TE42 blotting system (Amersham Pharmacia Biotech). After electrophoretic transfer, nonspecific protein binding sites were blocked by incubation of the nitrocellulose membrane for 60 min at RT in wash buffer consisting of 2% nonfat milk (Marvel; Premier Beverages, Stafford, UK) in 10 mM PBS containing 0.05% Tween-20 (Sigma). The nitrocellulose was then sequentially incubated with immune mouse serum (1:250) or CYP1B1 MAb (1:100) and goat anti-mouse immunoglobulin conjugated to horseradish peroxidase (1:2000; Bio-Rad, Hemel Hempstead, UK). After incubation with each antibody, the membrane was washed for five 10-min periods with wash buffer, and after removal of unbound secondary antibody the membrane was further washed in 10 mM PBS for five 10-min periods. Horseradish peroxidase was then demonstrated using an enhanced chemiluminescent technique (ECL plus; Amersham Pharmacia Biotech), which was performed as previously described (
Immunohistochemistry
Immunohistochemical detection of CYP1B1 was performed with a catalyzed signal amplification method (
The primary antibody was applied as a tissue culture supernatant at various dilutions (undiluted to 1:160) for 60 min at RT. After incubation in primary antibody, the sections were washed in TBS for three successive 5-min periods, and then peroxidase-conjugated rabbit anti-mouse immunoglobulin (1:100 in TBS containing 4% normal human serum; Dako, High Wycombe, UK) was applied for 30 min at RT. The sections were then washed in TBS and in TBS containing 0.05% Tween-20 (TNT buffer). The sections were then further washed in TNT buffer and fluorescein tyramide (NEN; Hounslow, Middlesex, UK) applied for 10 min at RT. The sections were then further washed in TNT buffer, followed by application of monoclonal mouse anti-fluorescein (1:20; Dako) for 30 min at RT. After further washing in TNT buffer, peroxidase-conjugated rabbit anti-mouse immunoglobulin (1:100 in TBS containing 4% normal human serum) was applied for 30 min at RT. After washing in TBS, sites of bound peroxidase were then demonstrated colorimetrically using a solution containing diaminobenzidine and hydrogen peroxide (Liquid DAB plus; Dako). After incubating the sections for 10 min at RT in the peroxidase substrate solution, the reaction was stopped by washing the slides in cold tapwater and the enzyme reaction product was intensified using 0.5% copper sulfate. The slides were then washed in cold tapwater, counterstained with hematoxylin, dehydrated in alcohol, cleared in xylene, and mounted in a synthetic mounting medium (DPX; BDH, Poole, Dorset, UK).
The sections were examined with brightfield light microscopy by two independent observers (MCEM, GIM) to establish the presence or absence of immunostaining, and its distribution, cellular localization, and intensity. The intensity of immunostaining in each tumor was assessed as strong (3), moderate (2), weak (1), or negative (0). The proportion of cells showing positive immunoreactivity was also assessed semiquantitatively using the following scale: less than 5% of cells (0), 525% (1), 2675% (2), and greater than 75% (3) of cells. A tumor was regarded as positive if more than 5% of tumor cells showed immunostaining. A tumor was classified as negative if there was complete absence of immunostaining in tumor cells or if less than 5% of tumor cells showed positive immunoreactivity. An overall CYP1B1 immunohistochemical score was obtained by assigning a numerical value from 03 for the results for the intensity of CYP1B1 immunoreactivity and the proportion of cells showing CYP1B1 immunoreactivity, respectively, and then combining these values. This resulted in an overall CYP1B1 immunohistochemical score ranging from 0 to 6. The overall CYP1B1 immunohistochemical score was grouped into four categories; 02 (score for weak immunoreactivity and low number of positive cells), 34 and 56 for comparison of CYP1B1 immunohistochemistry with histological grade, tumor type, presence of lymph node metastasis, and estrogen receptor status.
The positive control tissue used consisted of sections of a breast cancer that we had previously shown to contain CYP1B1 by both immunoblotting and immunohistochemistry with a polyclonal antibody to CYP1B1 (
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Results |
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Development of MAbs to CYP1B1
The immune response of sera from mice injected with each of the peptide conjugates was assessed by SDS-PAGE and immunoblotting, using expressed CYP1B1 as the antigen. The sera of mice injected with different peptides showed a variable immune response (Figure 1). Sera of mice that had been injected with Peptides D and E both showed recognition of CYP1B1 and were judged to have produced a positive immune response. Sera from mice injected with Peptide E gave a marginally stronger recognition of expressed human CYP1B1 than Peptide D. However, sera from mice injected with Peptides A and B and Peptides FJ showed no apparent recognition of CYP1B1 and were considered to have produced no significant immune response. Mice that had been immunized with Peptide E were therefore chosen to develop MAbs to CYP1B1. The peptide sequence used to develop the MAbs shows a high degree of similarity with corresponding rat CYP1B1 and mouse CYP1B1 sequences, with 13 of the 15 amino acid residues being identical (Figure 2).
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Five MAbs were developed from mice immunized with Peptide E. The individual antibodies were designated 5C4, 5D3, 5D9, 5E2, and 5G7, and isotyping showed that all the antibodies are IgG1 subtype. All the MAbs recognized a single immunoreactive band of molecular size 52 kD, corresponding to the expected molecular size of expressed human CYP1B1 by immunoblotting, and did not recognize expressed human CYP1A1 or any protein present in vector-only control microsomes or human liver microsomes (Figure 3). Serial dilutions of expressed CYP1B1 indicated that the minimal detectable amount of CYP1B1 by immunoblotting was 0.05 pmol of expressed CYP1B1 (Figure 4). CYP1B1 was not identified by immunoblotting of microsomes prepared from a range of normal adult human tissues, including kidney, stomach, small intestine, colon, lung, and endometrium (Figure 5).
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Immunohistochemistry on formalin-fixed, paraffin-embedded sections of breast cancer used as the positive control showed that three of the MAbs (5D3, 5E2 and 5G7) demonstrated strong staining, whereas two of the MAbs (5C4, 5D9) showed no immunoreactivity. All MAbs that showed positive immunoreactivity required an antigen-retrieval step for optimal immunohistochemical results, and all three MAbs showed an identical pattern of localization and distribution of immunohistochemical staining. Therefore, only one of the MAbs (5D3) was used for subsequent immunohistochemical studies of breast cancer. Liquid-phase preincubation of anti-CYP1B1 antibody with Peptide E before performance of immunohistochemistry almost completely abolished immunoreactivity.
CYP1B1 immunoreactivity was identified in 46 (76.7%) of cases of breast cancer, whereas there was no detectable CYP1B1 immunoreactivity in 14 cases (23.3%). In each case in which CYP1B1 immunoreactivity was observed, the immunoreactivity was localized to the cytoplasm of tumor cells (Figure 6). The intensity of the CYP1B1 immunoreactivity ranged from strong in 10 (16.7%) cases to moderate in 12 (20%) cases, and weak immunoreactivity was observed in 24 (40%) cases. The proportion of cells showing CYP1B1 immunoreactivity ranged from high in 10 (16.7%) cases to moderate in 27 (45%) cases, to low in nine (15%) cases. The relationship between the intensity of CYP1B1 immunoreactivity and the proportion of cells showing CYP1B1 immunoreactivity is summarized in Table 3. Twelve (20%) cases had an overall CYP1B1 immunohistochemical score of 5 or 6, 26 (43.4%) cases had a score of 3 or 4, 8 (13.3%) cases had a score of 2, and 14 (23.3.%) cases had a score of 0. The presence of CYP1B1 in different grades, histological types of breast cancer, lymph node status, and estrogen receptor status is summarized in Table 4 Table 5 Table 6 Table 7. There was no statistical relationship between the presence of CYP1B1 and the histological type of the tumor, tumor grade, the presence or absence of lymph node metastasis, or estrogen receptor status. There was no CYP1B1 immunoreactivity in stromal cells or connective tissue in the following cell types, including lymphocytes and plasma cells, when they were present in individual biopsy specimens.
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Discussion |
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CYP1B1 shows increased expression in a variety of tumors, including breast cancer (
The strategy we used to develop MAbs to CYP1B1 was a combination of structural molecular modeling and sequence alignment to identify regions of CYP1B1 that were likely to be located on the external aspect of the CYP1B1 protein and therefore were likely to be immunogenic. We have recently used a similar approach to develop MAbs to matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases (
In this study, we were not able to detect CYP1B1 by immunoblotting in a range of normal human tissues. The absence of CYP1B1 protein in both normal human liver and a range of extrahepatic tissues is consistent with our previous immunohistochemical studies (
A further major aim of this study was to develop antibodies to CYP1B1 that could be used in immunohistochemistry and that were effective on formalin-fixed, paraffin-embedded sections of tissue. All the MAbs were evaluated by immunohistochemistry on formalin-fixed, paraffin-embedded sections of breast cancer that we have previously shown by immunoblotting to contain a relatively high level CYP1B1 (
In this study, we found that 77% of breast cancers contained CYP1B1, and in each tumor CYP1B1 was specifically localized to tumor cells. The high frequency of expression of CYP1B1 in breast cancer is very similar to the findings of our previous studies of a small number of breast cancers and support the concept that CYP1B1 is a major form of cytochrome P450 present in breast cancer (leucine) at codon 432 and estrogen receptor status in breast cancer has been described (
Because CYP1B1 is involved in estrogen metabolism, acting as a specific C4 hydroxylase of 17ß-estradiol (
The development of MAbs to CYP1B1 that are effective in formalin-fixed, paraffin-embedded sections should make them particularly useful for investigating CYP1B1 expression in different tumor types and related preneoplastic lesions.
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Acknowledgments |
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Supported by a grant from the UK Medical Research Council.
We thank Mr Ian Davidson of the University of Aberdeen Protein Facility for synthesis of the peptides.
Received for publication February 8, 1999; accepted June 8, 1999.
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Literature Cited |
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Bailey LR, Roodi N, Dupont WD, Parl FF (1998) Association between cytochrome P450 1B1 (CYP1B1) polymorphism with steroid receptor status in breast cancer. Cancer Res 58:5038-5041[Abstract]
Bhattacharyya KK, Brake PB, Eltom SE, Otto SA, Jefcoate CR (1995) Identification of a rat adrenal cytochrome P450 active in polycyclic hydrocarbon metabolism as rat CYP1B1. J Biol Chem 270:11595-11602
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye-binding. Anal Biochem 72:248-254[Medline]
Crespi CL, Penman BW, Steimer DT, Smith T, Yang CS, Sutter TR (1997) Development of a human lymphoblastoid cell line constitutively expressing human CYP1B1 cDNA: substrate specificity with model substrates and promutagens. Carcinogenesis 12:83-89[Abstract]
Duncan ME, McAleese SM, Booth NA, Melvin WT, Fothergill JE (1992) A simple enzyme-linked immunosorbent assay (ELISA) for the neuron-specific isozyme of human enolase (NSE) using monoclonal antibodies raised against synthetic peptides corresponding to isozyme sequence differences. J Immunol Methods 151:227-236[Medline]
Elston CW, Ellis IO (1991) Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology 19:403-410[Medline]
Hakkola J, Pasanen M, Pelkonen O, Hukkanen J, Evisalmi S, Anttila S, Rane A, Mäntylä M, Purkunen R, Saarikoski S, Tooming M, Raunio H (1997) Expression of CYP1B1 in human adult and fetal tissues and differential inducibility of CYP1B1 and CYP1A1 by Ah receptor ligands in human placenta and cultured cells. Carcinogenesis 18:391-397[Abstract]
Hayes CL, Spink DC, Spink BC, Cao JQ, Walker NJ, Sutter TR (1996) 17ß-estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc Natl Acad Sci USA 93:9776-9781
King G, Payne S, Walker F, Murray GI (1997) A highly sensitive detection method for immunohistochemistry using biotinylated tyramine. J Pathol 183:237-241[Medline]
Liehr JG, Ricci MJ (1996) 4-Hydroxylation of estrogens as marker of human mammary tumors. Proc Natl Acad Sci USA 93:3294-3296
McKay JA, Melvin WT, Ah-See AK, Ewen SWB, Greenlee WF, Marcus CB, Burke MD, Murray GI (1995) Expression of cytochrome P450 CYP1B1 in breast cancer. FEBS Lett 374:270-272[Medline]
Murray GI, Duncan ME, Arbuckle E, Melvin WT, Fothergill JE (1998a) Matrix metalloproteinases and their inhibitors in gastric cancer. Gut 43:791-797
Murray GI, Duncan ME, O'Neil P, McKay JA, Melvin WT, Fothergill JE (1998b) Matrix metalloproteinase-1 is associated with poor prognosis in oesophageal cancer. J Pathol 185:256-261[Medline]
Murray GI, Taylor MC, McFadyen MC, McKay JA, Greenlee WF, Burke MD, Melvin WT (1997) Tumor specific expression of cytochrome P450 CYP1B1. Cancer Res 57:3026-3031[Abstract]
Savas Ü, Bhattacharyya KK, Christou M, Alexander DL, Jefcoate CR (1994) Mouse cytochrome P-450EF, representative of a new 1B subfamily of cytochrome P-450s. J Biol Chem 269:14905-14911
Savas Ü, Carstens C-P, Jefcoate CR (1997) Recombinant mouse CYP1B1 expressed in Escherichia coli exhibits selective binding by polycyclic hydrocarbons and metabolism which parallels C3H10T1/2 cell microsomes but differs from human recombinant CYP1B1. Arch Biochem Biophys 347:181-192[Medline]
Schmidt JV, Bradfield CA (1996) Ah receptor signaling pathways. Annu Rev Cell Dev Biol 12:55-89[Medline]
Shen Z, Liu J, Wells RL, Elkind MM (1994) cDNA cloning, sequence analysis, and induction by aryl hydrocarbons of a murine cytochrome P450 gene, Cyp1B1.. DNA Cell Biol 13:763-769[Medline]
Shimada T, Hayes CL, Yamazaki H, Amin S, Hecht SS, Guengerich FP, Sutter TR (1996) Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res 56:2979-2984[Abstract]
Sutter TR, Tang YM, Hayes CL, Wo Y-YP, Jabs EW, Li X, Yin H, Cody CW, Greenlee WF (1994) Complete cDNA sequence of a human dioxin-inducible mRNA identifies a new gene subfamily of cytochrome P450 that maps to chromosome 2. J Biol Chem 269:13092-13099
Tang YM, Wo Y-Y, Stewart J, Hawkins AL, Griffins CA, Sutter TR, Greenlee WF (1996) Isolation and characterization of the human CYP1B1 gene. J Biol Chem 271:28324-28330
Walker NJ, Gastel JA, Costa LT, Clark GC, Lucier GW, Sutter TR (1995) Rat CYP1B1: an adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals. Carcinogenesis 16:1319-1327[Abstract]
Wang F, Hoivik D, Pollenz R, Safe S (1998) Functional and physical interactions between the estrogen receptor Sp1 and nuclear aryl hydrocarbon receptor complexes. Nucleic Acids Res 26:3044-3052