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A. Edorh et al.

tumors were associated with advanced clinical and histological markers. The estimation of the number of copies of c-erbB-2 is usually achieved by hybridization with a radio-labelled probe, by estimating directly the ąuantity of radio-labelled DNA fragments, or recently by competitive PCR (Orlando et al1994). Most of these methods give uncertain or approximate results.

For ascertaining the value of a non-radioactive method in the determination of the copy number of c-erbB-2 gene in human tumors, we have amplified this gene along with IFN-y. This latter one was chosen as the single copy reference gene. PCR of the DNA extracted from paraffin-embedded breast carcinomas was performed and its results were ąuantified by colorigenic and radioactive labelling of amplified DNA.

MATERIALS AND METHODS

Breast Tumors Sampling

Fourteen mammary Iesions, from archival materials of the Department of Pathology of the Centre Aiexis Vautrin (Vandoeuvre-lfcs-Nancy, France) were from either mastectomy or excision biopsy specimens (Edorh et al., 1995). Areas of tumor were identified macroscopically. Ali samples were fixed in 4% buffered formaldehyde for 12 hrs. and paraffin embedded and processed ovemight using a Tissue-Tek Vip Processor™ (Laboratoires Miles, Paris, France).

His to logie Evaluation

Hematoxylin-eosin-stained tissue sections were observed and the presence of an adequate volume of cancer tissue was assessed. The carcinomas were classified using the World Health Organization (WHO, 1981) Histological Classification of Breast tumors of 1981 and graded according to Bloom and Richardson (1957). Ali the selected Iesions were infiltrating ductal carcinomas: one of grade 1, seven of gradc II, six of grade III.

Reagents

Agarose NA was obtained from Pharmacia (St. Quentin-Yvelines, France) whereas Taq (Thermus aąuaticus) polymerase (E.C. 2.11.1) came from Eurobio (Paris, France), plastic bags from Gibco-BRL/Life Technologies (Cergy Pontoise, France), bromophenol blue and ethidium bromide from Sigma Chem. (St. Louis, MO, USA). Digoxigenin-l 1-dUTP (Digoxigenin-3-0-methylcarbonyl-e-aminocaproyl-[5-(3-aminoallyl)-2^,-deoxy-uridine-5'-triphosphate]tetralithium salt, dNTP (100 mM, stock solution), 5-bromo-4-chloro-3-indolyl-phosphate, 4-toluidine-salt (BCIP), 4-nitroblue tetrazolium chloride (NBT) were provided by Boehringer Mannheim (Meylan, France). Ali other Chemicals were purchased from Merck (Chelles, France). The nitrocellulose membranę used was from Stratagenc (La Jolla, CA, USA) and acrylamide/ bisacrylamide, ammonium persulfate, N, N, N\ N’-tetramethylethylenediamine (TEMED) from Bio-Rad Laboratories (Hercules, CA, USA).

DNA Extraction and Amplification Briefly, two sections (5 pm thick), placed in 1.5 ml microcentrifuge tubes with 1 mlofiso-octane, were peileted. The iso-octane wasdiscarded and this step was repeated once. Ethanol (100%, v/v) was added for in order to remove iso-octane and evaporated. Then, 100 pl of digestion buffer were added (50 mM Tris«HCl, 1 mM EDTA, 1 % w/v of proteinase K (EC 3.4.21.14, Boehringer Mannheim), pH 8.5). Samples were ineubated for 3 hrs. at 55°C, and proteinase K was inactivated by heating at 95°C, 10 min. PCR was carried out with 2 pi of supematant.

The primers were provided by Clontech Laboratories (Pało Alto, CA, USA): i) primers for c-erbB-2 (sense: ^yCCT-CTG-ACG-TCC-ATC-ATC-TC³', antisense: ⁵CGG-ATC-TTC-TGC-TGC-CGT-CG³'); ii) primers of IFN-y (sense: ⁵TCT-TTT-CTT-TCC-CGA-TAG-GT³', antisense: ⁵CTG-GGA-TGC-TCT-TCG-ACC-TC³).

The polymerase chain reaction was performed in a PCR⁷¹⁴ apparatus (Gene E Osi, Paris, France) with PCR buffer: 10 mM Tris*HCl, 1.5 mM MgCl₂, 50 mM KC1, 0.1 mg/ml gelatine, 0.2 mM dNTP, 100 ng of genomie DNA (2 pl), 20 pM of each primer set (c-erbB-2 and/F/V-y)and 1 unitof7fl<? DNA polymerase, pH 8.3. For radioactive labelling 0.1 pl radioactive ³²P-dCTP with a specific activity of 3000 Ci/ mmol (Institut de Chimie Nuclćaire, Orsay, France) was added to the PCR mixture (25 pl) as described by Sestini et al. (1994). For non-radioactive labelling, 0.2 mM of dTTP were replaced with a mixture of 0.7 mM digoxigenin-dUTP™ and 1.3 mM dTTP.

The amplification consisted of 30cyclcs of: i) denaturation at 94°C for 60 sec.; ii) primer annealing at 55°C for 60 sec., iii) primerextension at 72°C for 60 sec. The reaction was terminated by 10 min. of primer extension at 72°C. IFN-y and c-erbB-2 primers amplified 98 bp and 150 bp fragment, respectively.

Evaluation of the Number of Copies of c-erbB-2

- Non-radioactive method: Followingelectrophoresis the gels were ineubated in denaturing buffer for 30 min. (NaCl 1.5 M, NaOH 0.5 M) and equilibrated in neutral buffer for2x 15 min. (NaCl 1.5 M,Tris*HCI 0.5 M, EDTA 1 mM, pH 7.2). DNA samples were transferred to nitrocellulose filter paper (Nitrocellulose, Stratagene) by a dry transfer method for 4 hrs. according to Rihn et al. (1995b).

The DNA was fixed at 80°C and the membranę was washed inTPl buffer(Tris«HCl lOOmM.NaCI 150mM,pH 7.5) for 3 min., then ineubated in TP1 buffer containing 0.5% (w/v) Blocking Reagent™ (Boehringer Mannheim) for 30 min. and

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