Bcl-2 Expression and DNA Fragmentation in Breast Carcinoma, Pathologic and Steroid Hormone Receptors Correlates

Abstract

B-cell leukemia/lymphoma (bcl-2) expression can override the apoptosis development in lymphoid and hormonally regulated tissue-like breast. The presence of estrogen receptor (ER), progesterone receptor (PR), and androgen receptor (AR) have revealed in breast carcinomas, but they have not been correlated to the bcl-2 protein expression and DNA fragmentation markers. We evaluated the immunohistochemical expression of bcl-2 protein and hormonal receptors (ER, PR, AR) and differentiation grade in 37 infiltrating ductal carcinomas of the breast for which frozen tissues were available for DNA extraction. The immunohistochemical reaction for bcl-2 was considered positive if more than 50% of neoplastic cells had intense cytoplasmic staining, whereas for steroid receptor evaluation Battifora's criteria were used. The DNA was extracted according to the phenol-chloroform procedure and used for bcl-2 gene rearrangement study of the major breakpoint region (Southern blot) and for membrane-based end-labeling using digoxigenin-labeled nucleotides and E. coli DNA polymerase I (Klenow fragment). The results were quantified by three different observers. Low-grade carcinomas were positive for bcl-2 protein (27/28, 96.4%) and ER (15/28, 53.6%), whereas the remaining neoplasms were negative for bcl-2 (9/9, 100.0%) and ER (8/9, 53.6%) (p < 0.001). No statistically significant differences were revealed at the bcl-2, PR and AR comparisons. The Southern blot analysis for bcl-2 major breakpoint region showed neither rearrangement nor genetic amplification (densitometric study). Only the membrane-based end-labeling of DNA fragments showed correlation with bcl-2 protein and ER expressions: all except one bcl-2-negative tumor and two bcl-2-positive tumors had positive labeling using 7 pg of DNA at dot blot analysis (p < 0.002). The bcl-2 protein expression would allow both proliferation and cell progression by blocking apoptosis in well-differentiated, ER-positive breast carcinomas. In these neoplasms, DNA fragmentation as a molecular marker of apoptosis was prevented by bcl-2 expression.

Kinetic profiles of intraepithelial and invasive prostatic neoplasias: the key role of down-regulated apoptosis in tumor progression

“…A continuous increase in proliferation markers normally defines tumor progression, with a parallel increase in apoptosis [43]. Prior to any significant difference in proliferation markers, intraepithelial lesions seem to down-regulate apoptosis, resulting in a kinetic advantage and monoclonal expansions specifically of these lesions, as has been shown in breast DCIS [12], benign adrenal cortical proliferative lesions [8,13], and adrenal medullary hyperplasia [10,16] or C-cell hyperplasias in MEN-2A [9,14,51]. Decreased apoptotic cell loss would increase the cellular pool and allow the accumulation of genetically damaged cells, ending in a convergent cellular selection.…”

“…After incubation in 2×SSC buffer (80°C, 20 min) and protein digestion (500 µg/ml pronase in 10 mM Tris-HCI pH 7.5, 10 mM EDTA, 0.5% SDS, at room temperature for 25 min), the sections were incubated with the Klenow fragment of E. coli DNA polymerase I under appropriate conditions (20 U/ml in 50 mM Tris HCI pH 7.5, 10 mM Mg 2 Cl, 1 mM dithiothreitol, 250 µg/ml BSA with 100 µM of each dNTP, maintaining a proportion of 11-digoxigenindUTP/dTTP of 0.35/0.65; 2 h at 37°C). The dig-labeled DNA fragments were immunoenzymatically detected using an antidigoxigenin polyclonal Fab fragment labeled with alkaline phosphatase (1/100 dilution, Boehringer-Mannheim, Germany); the enzymatic reaction was developed under microscopic control with nitroblue-tetrazolium and X-phosphate [12]. The sections were counterstained with diluted hematoxylin (25%), dehydrated, and mounted.…”

Heterogeneous topographic profiles of kinetic and cell cycle regulator microsatellites in atypical (dysplastic) melanocytic nevi

“…31,32 After routine dewaxing and hydration, the sections were incubated in 2 Â standard saline citrate (20 min at 801C) and digested with pronase (500 mg/ml, 25 min, room temperature) in a moist chamber.…”

Clonality as Expression of Distinctive Cell Kinetics Patterns in Nodular Hyperplasias and Adenomas of the Adrenal Cortex

“…22,23 After routine dewaxing and hydration, the sections were incubated in 2ϫ standard saline citrate (20 minutes at 80°C) and digested with proteinase K (100 g/ml in Tris-HCl, pH 7.6, for 30 minutes at room temperature) in a moist chamber.…”