Role of the Cdc25A phosphatase in human breast cancer (original) (raw)
Patient population. This study was performed after approval by the Institutional Review Boards of the Dana-Farber Cancer Institute and of Brigham and Women’s Hospital.
Archival T1a,b breast carcinomas. A previously characterized series of breast carcinomas less than 1 cm in diameter (T1a,b) diagnosed between 1964 and 1994 was utilized (18). RNA preservation was adequate in 154 cases (18). In this study, a subset of 144 patients for whom tumor tissue was still available was analyzed by in situ hybridization with antisense riboprobes to Cdc25A. In this set of cases, p27 and Ki67 expression levels had been previously analyzed by immunohistochemistry (18). Population characteristics of this cohort have been previously published and are summarized in Table 1. The mean age of the patients was 60.7 years, and the median follow-up was 66.4 months. Follow-up data were obtained from patients’ charts and tumor registry records. Death from cancer was accepted when confirmed by autopsy or when there was convincing evidence of disease by clinical, pathologic, or radiographic parameters. Table 2 is a summary of the adjuvant therapy received in a subset of patients (n = 61).
Clinicopathological features of study population in relation to Cdc25A status
Methods of treatment versus p27 and Cdc25A levels in the tumors
Frozen breast carcinomas. Since no frozen tissue was available from any of the patients with T1a,b cancers, 21 frozen tissue samples of breast carcinoma were randomly selected from a series of over 100 patients who were operated upon between 1989 and 1994. Tumor specimens were characterized in terms of pathologic variables, including tumor size (13 T1c, 7 T2, and 1 T3) and histologic grade (9 grade II and 12 grade III). For each case a hematoxylin and eosin–stained frozen section was prepared to ensure the presence of tumor in the samples. Nontumor tissue surrounding the infiltrating cancer was dissected away so that at least 80% of each sample used for RNA and protein extraction was made up of carcinoma. Ten cases of normal breast tissue obtained from reduction mammoplasty specimens were utilized as control.
In situ hybridization. 1 μg of recombinant plasmid pBluescript II (Stratagene, La Jolla, California, USA) containing the 514-bp 5′ end of the human Cdc25A gene from the ATG start codon was linearized using BamHI and SalI restriction enzymes (Life Technologies Inc., Gaithersburg, Maryland, USA) to generate sense and antisense transcripts, respectively. Digoxigenin-labeled riboprobes were used. In situ hybridization was performed on the automated in situ hybridization instrument Gen II (Ventana Medical Systems, Tucson, Arizona, USA) using identical incubation and detection time as previously described, with minor modifications (hybridization at 60°C for 3 hours) (16, 19).
For the scoring of Cdc25A, a total of 1000 cells per case was counted independently by two investigators. Only tumor cells strongly positive for Cdc25A were counted. Categories were made on the basis of percent positive cells: 0 = negative cases, 1 = 0–25% positive cells, 2 = 25–50%, 3 > 50%. Cases were deemed to be overexpressers only when more than 50% of cells expressed high levels of Cdc25A (category 3). Patients were thus subdivided on the basis of expression as low expressers or nonexpressers (categories 0, 1, and 2) versus overexpressers (category 3) as previously described (16) (Table 1). All statistical analyses were performed comparing these two groups.
Immunoprecipitation, immunoblotting, and kinase assays. Frozen breast tumors were thawed and lysed in 200 μL of lysis buffer (10% sucrose, 20 mM Tris [pH 8.0], 137 mM NaCl, 10% glycerol, 2 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1% Nonidet P-40 substitute [Fluka, Ronkonkoma, New York, USA], and 1 μg/mL leupeptin). MCF-7 cells before and after transfection with antisense or sense Cdc25A oligonucleotides were lysed in RIPA buffer (1% Nonidet P-40 substitute [Fluka], 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate, 2 mM EDTA, 50 mM sodium fluoride, and 0.2 mM sodium vanadate) containing 100 mM PMSF, 100 mM DTT, and protease inhibitor cocktail (Boehringer Mannheim Biochemicals Inc., Indianapolis, Indiana, USA).
Conditions for immunoprecipitation and immunoblotting have been previously described (20). For immunoprecipitation, Cdk2 and Cdk4 rabbit polyclonal (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), and p27 mouse monoclonal (Transduction Laboratories, Lexington, Kentucky, USA) antibodies were used. For immunoblotting, cyclin E (2.5 μg/mL; Oncogene Research Products, Cambridge, Massachusetts, USA), Cdk2 (1 μg/mL; Santa Cruz Biotechnology Inc.), p27 (0.1 μg/mL; Transduction Laboratories), phosphotyrosine (2 μg/mL; Upstate Biotechnology Inc., Lake Placid, New York, USA), and Cdc25A (1 μg/mL; Santa Cruz Technology) antibodies were used. Densitometric analysis was performed on the developed Western blot for Cdc25A. To normalize the values, a ratio of the percent area of the tested samples to the one obtained with the control not treated by antisense oligonucleotides was used.
Cdk2 histone H1 kinase assay was performed as previously described (21). Cdk4 kinase assay was performed essentially with the same methodology, substituting histone H1 with 5 mg/mL of Rb peptide (Santa Cruz Biotechnology Inc.) as substrate. HeLa cell lysate was used as a positive control. Densitometric analysis was performed on the developed autoradiography. To normalize the values, a ratio of the percent area of the tested samples to the one obtained with the HeLa control in each gel was used. To account for variability from gel to gel, this ratio was, in turn, related (as a ratio) to the total area under all curves in a given gel. Normalized values were used in the statistical analysis.
Semiquantitative RT-PCR for Cdc25A levels. Frozen tissue samples were ground by a tissue homogenizer, and both frozen tissue and cell line RNA were extracted using a guanidine isothiocyanate protocol (TRI Reagent; Molecular Research Center Inc., Cincinnati, Ohio, USA). Cdc25A cDNA was synthesized from equal amounts of RNA (1 μg) with the downstream primer, 5′-CCGGTAGCTAGGGGGCTCACA-3′, encompassing the catalytic domain, using murine leukemia virus reverse transcriptase (2.5 U/μL) (Perkin-Elmer Corp., Norwalk, Connecticut, USA). PCR was performed in 25-μL reaction mixture containing DNA template, PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 1.5 mM MgCl2, 200 μM of each dNTP, 0.4 μM of each primer (upstream primer 5′-AGCCCCAAAGAGTCAACTAATCCAGA-3′), and 0.75 units of AmpliTaq DNA polymerase (Perkin-Elmer Corp.) to amplify a 570-bp product. PCR was performed for 35 cycles consisting of 95°C for 1 minute, 63°C for 2 minutes, and 72°C for 1 minute. Levels of Cdc25A were compared with those of the small trimeric G protein Gsα (upstream primer 5′-GTGATCAAGCAGGCTGACTAT-3′ downstream primer 5′-GCTGCTGGCCACCACGAAGATGAT-3′), which was reverse-transcribed and amplified (200-bp product) using the same conditions described above for Cdc25A. The resulting amplification products were then analyzed by agarose gel electrophoresis using standard methods, and densitometry was performed.
For statistical analysis, the ratio of the densitometric values of Cdc25A to those of Gsα was calculated and the median value (median = 1) was utilized as a cutoff for the categorization of cases into high and non-/low expressers. A sample was considered a high expresser for Cdc25A when this ratio was ≥ 1; non-/low expressers showed a ratio < 1.
Cdc25A antisense oligonucleotide transfection. The breast carcinoma cell line MCF-7 (American Type Culture Collection, Rockville, Maryland, USA) was maintained in DMEM supplemented with 10% FBS (Life Technologies Inc.). MCF-7 cells, 60% confluent, were transfected with antisense (TCAAACACAAACACGACT) or scrambled (ATCCGAACTCAACAAACA) oligonucleotides (New Second Generation Chimeras, Oligos Etc. Inc., Bethel, Maine, USA) for Cdc25A according to Life Technologies Inc. directions. MCF-7 cells were incubated with a DNA-lipid solution consisting of 400-nM oligonucleotides, 12 μL of lipofectin reagent (Life Technologies Inc.) and Opti-MEM I reduced serum media (Life Technologies Inc.) for 6 hours at 37°C. Cells were harvested 24 hours and 48 hours after transfection.
Antisense experiments were also performed cotransfecting (a) the Cdk2AF mutant (7.8 μg), in which the phosphorylatable sites Thr14 and Tyr15 were replaced with nonphosphorylatable residues (Ala14 and Phe15); and (b) wild-type Cdk2 (10 μg). Cdk2AF cannot be phosphorylated in the critical residues normally dephosphorylated by Cdc25A phosphatase (22).
Proliferation and cell cycle analysis. Cells were pulse-labeled with 10 mM bromodeoxyuridine (BrdU) at 37°C for 30 minutes, harvested, fixed in 70% ethanol, and treated with 0.2 N HCl for 30 minutes. Cells were then stained with anti-BrdU FITC-conjugated (Boehringer Mannheim Biochemicals Inc.) and counterstained with propidium iodide (5 μg/mL). Flow cytometric analysis was performed using a Becton Dickinson FACScan flow cytometer (Becton Dickinson and Co., Franklin Lakes, New Jersey, USA).
Statistical analysis. In the archival T1a,b breast carcinomas, the primary study outcome was disease-free survival, which was measured from the date of surgery to the date of last follow-up or death. Survival was censored if the patient was still alive or died from other causes. Survival curves for Cdc25A and p27 were constructed using the Kaplan-Meier method. Univariate survival curves were compared using a Wilcoxon procedure, and differences between prognostic factors were tested for statistical significance with the Logrank analysis. A P value of less than 0.05 was required for significance. Fisher’s exact test was used to test for an association between Cdc25A and Ki67 expression levels and to compare patients who received adjuvant treatment with those who did not in the four categories stratified according to Cdc25A and p27 status.
All data obtained from the frozen breast cancer tissues were analyzed by a linear regression model. This was used to explain Cdk2 enzymatic activity in terms of p27 (before or after immunoprecipitation), Cdk2, cyclin E, and Cdc25A levels.