Apoptosis (programmed cell death) can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. We describe a new staining method for formalin-fmed, paraffii-embedded tissue sections that involves an in situ end-labeling (ISEL) procedure.
Summary The aim of this study was to assess relationships between Bcl-2 expression, response to chemotherapy and a number of pathological and biological tumour parameters in premenopausal, lymph node-negative breast cancer patients. Expression of Bcl-2 was determined using immunohistochemistry on paraffin-embedded sections in a series of 441 premenopausal, lymph node-negative breast cancers of patients randomised to receive perioperative chemotherapy (5-fluorouracil, doxorubicin, cyclophosphamide) or no perioperative chemotherapy. Immunohistochemistry of Bcl-2 was evaluated by scoring both staining intensity (0-3) and number of positive cells (0-2). Using these scores tumours were grouped into categories 0-6. It was found that 9.2% of the tumours were completely negative (0), 17.2% weakly (1 + 2), 41.6% moderately (3 + 4) and 31.9% strongly positive (5+6) for Bcl-2. A positive correlation was found between high Bcl-2 expression and oestrogen (P<0.001) and progesterone receptor positivity (P<0.001) and low tumour grade (P<0.001), whereas high Bcl-2 expression was negatively correlated with p53 (P<0.001) and c-erb-B-2 positivity (P<0.001), high Ki-67 index (P<0.001), mitotic index (P<0.001) and large tumour size (P=0.006). Patients with tumours expressing high levels of Bcl-2 (overall score 3-6) had a significantly better disease-free (P=0.004) and overall (P=0.009) survival. However, in a multivariate model this association no longer remained significant. There was a trend for an effect of adjuvant chemotherapy on disease-free survival both for patients with Bcl-2-positive (HR-0.61, 95% CI 0.35-1.06, P=0.07) and negative (HR=0.55, 95% CI 0.27-1.12, P=0.09) breast tumours at a median follow-up of 49 months. The level of Bcl-2 expression does not seem to predict response to perioperative chemotherapy in premenopausal, lymph node-negative breast cancer patients. High levels of Bcl-2 are preferentially expressed in well-differentiated tumours and are associated with favourable prognosis. However, Bcl-2 expression is not an independent prognostic factor in this patient series.
After pulse labeling of mammalian cells in vivo or in vitro with 5-bromodeoxyuridine (BrdUrd), followed by immunostaining with a monoclonal antibody to DNA-incorporated BrdUrd, various intranuclear staining patterns are observed. These results are obtained in various labeling, fixation, denaturation, and staining conditions. We defined five different patterns in immunoperoxidasestained monolayers of human and rodent cancer cells and compared mean nuclear areas as measured by computerized planimetry. Furthermore, we evaluated frequency distributions of these patterns in partly synchronized cell populations and correlated these results with flow cytometric DNA histograms.The results indicate that the observed patterns reflect the spatial and temporal organization of DNA synthesis, which seems to be characterized by at least three successive stages of replication. Evaluation of these patterns may have various applications in studies on cell cycle kine tics.Key terms: DNA replication, B-bromodeoxyuridine, monoclonal anti-BrdUrd antibodies, interphase nucleus There is much evidence from (3H)-thymidine (3HTdR) labeling studies that DNA synthesis in an eukaryotic nucleus consists of an ordered cascade of initiations of replicons (4), controlled by an intranuclear mechanism (36). This is particularly clear from studies on polytene chromosomes of dipteran larvae, describing various patterns of DNA synthesis, tentatively assigned to specific periods of the S-phase (1,2,28). In general, GC-rich euchromatin replicates early, whereas AT-rich heterochromatic DNA replicates late (13); these early and late DNA synthesizing sites roughly correspond to R-and G-bands on trypsinGiemsa-stained chromosomes, respectively (6). However, also in euchromatin, distinct early-and late-replicating DNA fractions have been decribed (24); Goldman et al. (1 1) have presented evidence that genes that are obligatory or potentially active in a given cell type replicate early in that cell type, whereas genes that are permanently inactive replicate late.The characteristic grain distributions observed in eukaryotic nuclei after in situ autoradiography of incorporated 3HTdR (35) may reflect both the structural organization of replicon domains and a nonrandom localization of chromosomes in the interphase nucleus (12,161. In particular, centromeric and other highly repeated nontranscribed sequence domains may be positioned on the nucleolus or nuclear membrane and act as general organizing centers for cell type-specific interphase patterns (21). Multiple adjacent replicons appear to be clustered on fixed sites of the nuclear matrix (17,271, and it has been suggested that clustered replication origins are activated simultaneously (25). Early DNA synthesis then seems to occur in replicon clusters evenly distributed over the nucleus, whereas late DNA synthesis, being primarily heterochromatic, is preferentially restricted to domains on the periphery of the nucleus and in nucleoli (35,361. However, the assignment of autoradiographic labeling patterns to spe...
Summary Bcl-2 has been demonstrated to inhibit apoptosis in breast cancer cells in vitro, and the ratio between Bcl-2 and its proapoptotic homologue Bax seems to be an important determinant of cellular sensitivity to induction of apoptosis. However, little information is available on the relationship between Bcl-2 and the rate of apoptotic and necrotic cell death in breast tumours. From a series of 441 premenopausal, lymphnode-negative breast cancer patients, a subset of 49 tumours was selected in which immunostaining for the 26-kDa isoform of Bcl-2 was either absent (n = 23) or very high (n = 26). High expression of Bcl-2 was found to be strongly associated with low rates of apoptotic (P < 0.001) and necrotic cell death (P < 0.001). The mean value of the apoptotic index was 2.69% ± 1.40% in Bcl-2-negative tumours and 0.68% ± 1.00% in Bcl-2-positive tumours. Expression of the proapoptotic protein Bax correlated neither with Bcl-2 nor with the frequency of apoptotic cells. Immunostaining for the antiapoptotic Bcl-2 homologue Bcl-XL correlated with Bcl-2 expression (P < 0.001) but not with apoptosis. High proliferation rate and high tumour grade (Bloom-Richardson) were strongly associated with absence of Bcl-2 expression (P < 0.001). p53 accumulation was associated with absence of Bcl-2 expression and increased apoptotic activity. Loss of Bcl-2 expression was strongly correlated with increased apoptotic and necrotic cell death, high proliferation rate and high tumour grade, supporting a model in which Bcl-2 not only mediates cell death, but also cell division in breast cancer tissue, and in which regulation of cell division and cell death are tightly linked.
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