Breast cancer proliferation measured on cytological samples: a study by flow cytometry of S-phase fractions and BrdU incorporation

Abstract

Summary Cell kinetics have been shown to be an important predictor of clinical evolution of operated breast cancer. We established a method for the estimation of the proliferative activity of tumour cells obtained by fine needle sampling without aspiration (FNS), using simultaneously S-phase fractions (SPF) measured on DNA histograms and 5-bromodeoxyuridine (BrdU) labelling index (BLI) Proliferative activity has consistently been found to be an important biological predictor of the clinical evolution of breast cancer (Tubiana et al., 1984;Silvestrini et al., 1985;Meyer, 1986;Hery et al., 1987). Traditionally, it has been measured by autoradiography of a radiolabelled precursor (3H-thymidine) incorporated in tumour-cell DNA.With the introduction of DNA flow cytometry (FCM), fast cell-cycle distribution analysis of large numbers of cells has become available (Barlogie et al., 1982). Retrospective studies have shown that S-phase fraction (SPF), computed from DNA histograms, is also a significant prognostic factor (Hedley et al., 1987;Kallioniemi et al., 1988) and a good predictor of response to neo-adjuvant chemotherapy (Remvikos et al., 1989). Nevertheless, the possibility of establishing SPF's correctly has been questioned, either from poor quality-or complex histograms presenting multiple aneuploid peaks (Meyer et al., 1984; Kallioniemi et al., 1985 In a prospective study of feasibility, we developed complementary methods for assaying SPF and BrdU labelling index (BLI) in vitro on the same tumour samples. Indeed, the substitution of 5-bromodeoxyuridine (BrdU) to 3H-thymidine has been proposed as a more convenient method of measuring DNA synthesis, due to the instant immunofluorimetric detection (Dolbeare et al., 1983;Schutte et al., 1987). Tumour cells were obtained directly from the patients by fine needle sampling without aspiration. The inherent difficulties of each methodology are discussed in order to define the best approach for an adequate estimation of the proliferative activity of each breast cancer before treatment decision. Materials and methodsOne hundred and eighty-nine consecutive patients were subjected to fine needle sampling without aspiration, using 23 or 25 gauge needles (Zajdela et al., 1987). The cytological samples were expelled in 1 ml of incubating medium: RPMI (Gibco), 10% foetal calf serum (Calbiochem), 30 pM BrdU (Sigma). The tubes were incubated at 37°C for 15 min, then 100 yl of DMSO were added and the tubes stored at -80C.FNS were processed for DNA-FCM according to a onestep protocol. The samples were thawed and rinsed with PBS at 1,500 r.p.m., 5 min. The pellets were resuspended in 600 tl of PBS, 0.2% Tween 80 (Sigma), 50 lg m -' propidium iodide (Sigma), 250 iLg ml-Ribonuclease A (B6ehringer), and left at room temperature for 20 min.For BrdU/DNA labelling, cell-rich samples were selected and fixed in 70% ethanol. In all cases, at least 50,000 cells were kept for DNA analysis as above. The fixed samples were centrifuged, digested with 3 ml of pepsin (Sigma

The Tumor Suppressor hSNF5/INI1 Modulates Cell Growth and Actin Cytoskeleton Organization

“…Where specified, cells were exposed to cycloheximide (Sigma) at 10 g/ml for 4 -12 h. Green fluorescent protein staining was observed using Fluorescent microscopy with Zeiss Axioplan 2 Microscope. Cells fixed in 70% ethanol and stained with anti-bromodeoxyuridine antibody (Harlan Sera-Lab, Hillcrest, United Kingdom) and propidium iodide (Sigma-Aldrich) were analyzed on a Becton Dickinson FACSscan as described previously (18,19). Detection of apoptotic cells by flow cytometry was carried out with annexin V-allophycocyanin according to the manufacturer's instructions (BD Biosciences PharMingen, Torreyana, CA).…”

Measurement of S-phase fraction and ploidy in sequential fine-needle aspirates from primary human breast tumours treated with tamoxifen

“…In four of these cases there was no clinical response to tamoxifen and all showed tumour progression of > 25% with a mean lag time of 7 months between the recorded post-tamoxifen sample and clinical progression. In two cases (patients 17 and 18 in Table II (Prey et al, 1985;Erhardt & Auer, 1986;Remvikos et al, 1991) which have all shown good correlation between sequential FNAs from the same tumour. Only one study (Mullen & Miller, 1989) has commented on significant variation in DNA analysis caused by intratumoral heterogeneity between two fine-needle aspirates taken from the same tumour.…”

Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2 and c-Myc expression

“…One day after transfection, cells were selected for 24 to 48 h in the presence of 1 mg/ml of puromycin then labelled with 30 mM of BrdU for 30 min at 378C. FACS analysis was performed as previously described (Remvikos et al, 1991). After washing in PBS 16 and ®xation in ethanol 70%, cells were digested with 0.5 mg/ml pepsine for 20 min at 378C and treated with 2 N HCl for 20 min at room temperature.…”