The role of bcl-2 expression in breast carcinomas (Review)

The proto-oncogene bcl-2 is demonstrated to block a final common pathway leading to apoptosis and is expressed in more than half of human breast cancers. Invasive breast cancer has reduced bcl-2 immunostaining compared with normal breast epithelia and preinvasive breast lesions. As an inhibitor of apoptosis, bcl-2 should correlate with highly aggressive tumor biology and resistance to hormonal/cytotoxic therapy. However, high bcl-2 expression has been shown to associate with a number of favorable prognostic factors including ER positivity, PgR positivity, low histological grade, well-differentiated tumor, absence of c-erbB-2 and p53. Unexpectedly, numerous studies have shown that tumors with high bcl-2 expression are more responsive to hormone therapy and have more favorable disease-free and overall survival. The clinical significance of bcl-2 in tumorigenesis and prognosis of breast carcinomas from the data recently published are reviewed. Its role in modulation of hormonal/cytotoxic therapy and future directions are also discussed.     

bcl-2 protein expression in the Barrett's metaplasia-dysplasia-carcinoma sequence

The bcl-2 proto-oncogene encodes a protein that blocks programmed cell death (apoptosis). Although bcl-2 has been shown to be involved in the development of follicular lymphoma via a chromosomal translocation t(14;18), little is known about its function in non-hematolymphoid neoplasms. The bcl-2 protein is normally expressed in the regenerative crypt compartment of the colon, small intestine, and stomach, and has been found to be abnormally overexpressed as an early event in the dysplasia-carcinoma sequences of both ulcerative colitis-related and gastric neoplasias. This study was undertaken to evaluate the role of bcl-2 in the Barrett's metaplasia-dysplasia-carcinoma sequence. Thirty-six esophageal resection specimens were studied, using a monoclonal antibody to the bcl-2 protein on fixed paraffin-embedded specimens. Barrett's mucosa was present in each specimen: low-grade dysplasia in 35, high-grade dysplasia in 34, intramucosal carcinoma (IMC) in 23, and submucosal carcinoma in 13. In addition, a section of the gastric resection margin was evaluated for bcl-2 immunoreactivity in each case. In all cases, the regenerative compartment of the gastric mucosa in the resection margins stained for bcl-2; however, no immunoreactivity was seen in any of the cases of Barrett's mucosa with or without dysplasia or carcinoma. We conclude that, in contrast to its role in gastric neoplasia, bcl-2 alterations are not an important molecular marker in the neoplastic progression of Barrett's mucosa.     

Clinicopathologic analysis of bcl-2 immunostaining in breast carcinoma

Tissue sections of 81 breast carcinomas and 19 benign breast tissues were immunostained with a monoclonal antibody to the bcl-2 gene product, a cytoplasmic protein that regulates apoptosis. The degree of immunoreactivity was then compared with clinicopathologic parameters and to immunostaining for mutated p53 gene product. Immunoreactivity for bcl-2 was present consistently in lymphocyte populations and in residual benign lobules. Apocrine metaplasia (n = 6) and lactating breast (n = 1) exhibited minimal bcl-2 expression, whereas duct hyperplasia (n = 10) showed staining of cells primarily at the periphery of the involved structure and adenosis (n = 7) displayed staining in a majority of cells. Neoplastic epithelial bcl-2 immunoreactivity was negative or minimally positive (staining in 1-5% of cells) in 42% of cases, heterogeneous (staining in 6-30% of cells) in 27% of cases, and diffuse (> 30% of cells) in 31% of cases. Immunostaining for bcl-2 correlated with the presence of estrogen receptor (bcl-2 negative, 16% estrogen receptor positive versus bcl-2 positive, 88% estrogen receptor-positive; P < 0.001), with differentiation (bcl-2 negative, 62% poorly differentiated versus bcl-2 positive, 8% poorly differentiated; P < 0.001) and with better disease-free survival (bcl-2 negative, 82% recurrence versus bcl-2 positive, 28% recurrence; P = 0.0001; 52-mo mean follow-up). Immunostaining for p53 in greater than 5% of tumor cells was observed in 39% of cases and was more frequent in bcl-2-negative tumors (18/35, 51%) as opposed to bcl-2-positive tumors (14/46, 30%); P = NS. Disease recurrence correlated with p53 staining, which was observed in 51% of tumors that relapsed versus only 22% of tumors that did not recur. We conclude that bcl-2 is expressed in benign breast tissues that retain proliferative capacity and partial differentiation. Moreover, in neoplastic breast tissue, it is better correlated with a differentiated, "hormonally responsive," prognostically favorable phenotype than with disabled p53 gene function.     

Androgens down-regulate bcl-2 protooncogene expression in ZR-75-1 human breast cancer cells

Although a large proportion of primary human breast cancers express the androgen receptor, and treatment with androgens exerts beneficial effects in women with breast cancer, the role and especially the mechanism of action of androgens in breast cancer development and growth are not well understood. The potential effect of androgens on bcl-2 protooncogene expression was investigated in a human breast cancer cell line whose proliferation is known to be inhibited by androgens. The estrogen-responsive ZR-75-1 cells were grown in the presence or absence of 5alpha-dihydrotestosterone (DHT), alone or in combination with 17beta-estradiol. DHT caused a marked down-regulation of Bcl-2 protein and messenger RNA levels in both the presence and absence of 17beta-estradiol. The inhibitory effect of DHT was completely prevented by coincubation with the pure antiandrogen hydroxyflutamide. The present data indicate that androgens can down-regulate bcl-2 protooncogene levels via an androgen receptor-mediated mechanism, thus providing a novel mechanism for their known inhibitory effect on breast cancer cell growth.     

Pathology of preinvasive and excellent prognosis breast cancer

BACKGROUND: Erythropoietin (EPO) therapy is a common and effective treatment for the correction of anemia in patients with end-stage renal disease. Simultaneous treatment with angiotensin-converting enzyme (ACE) inhibitors for the control of hypertension and/or heart failure is often necessary. Recent reports in the literature have raised concern about a potential interaction between these drugs, with a resultant decreased EPO efficacy. METHODS: To investigate whether this interaction occurs in chronic dialysis patients, we retrospectively reviewed the records of 175 patients receiving chronic dialysis. All study patients were treated with EPO for at least 3 months, and had normal iron indices. Patients were treated with ACE inhibitors for at least 3 months, at a constant daily dose for at least 1 month (group 1, n = 32), or did not receive ACE inhibitors (group 2, n = 143). Patients with infections or overt iron deficiency were excluded. Total weekly EPO doses and hematocrit (Hct)/hemoglobin (Hgb) values in the two groups were compared. Variables known to affect response to EPO were compared, including ferritin, transferrin saturation, dialysis dose and serum aluminum. RESULTS: Total weekly EPO dose was 17,358 +/- 6,871 units in group 1 and 17,612 +/- 7,744 units in group 2 (p = 0.854). The achieved Hct was 32.1 +/- 4.4% (group 1) and 30.5 +/- 4.0% (group 2) (p = 0.079). Similarly, Hgb, ferritin, transferrin saturation, Kt/V, and serum aluminum were not different. The dose or duration of ACE inhibitor therapy did not affect Hgb or Hct. Thus, ACE inhibitor therapy does not appear to affect response to EPO in chronic dialysis patients.     

bcl-2 but not p53 expression is associated with resistance to chemotherapy in advanced breast cancer

Programmed cell death is an important determinant of the response to chemotherapy. Among the factors controlling this process, a significant role is played by bcl-2 and p53, the expression of which, together with estrogen receptor content and tumor proliferative activity, was investigated by means of immunohistochemistry in 55 advanced breast cancer patients (median age, 60 years; range, 25-71 years). Analysis of bcl-2 expression identified two groups of patients with a significant difference in response rate. A total of 17 patients (31%) responded to chemotherapy (5 had a complete response and 12 had a partial response): 14 of 32 (44%) bcl-2-negative patients (< 40% stained cells) and only 3 of 23 (13%) bcl-2-positive patients (> or = 40% of stained cells; P = 0.019 by Fisher's exact test). The two groups were well balanced in terms of age, performance status, disease-free survival, menopausal status, and type of chemotherapy. bcl-2-negative tumors showed a tendency toward a higher p53 expression and proliferation rate, whereas an excess of bone as the dominant disease site was evident among the bcl-2-positive ones. However, the only variable to result significantly different between the two groups was estrogen receptor expression (P = 0.004). A multivariate logistic regression model showed that bcl-2 maintained its power of discriminating two groups with a different probability of responding to chemotherapy, although the greatest contribution was given by dominant disease site and type of chemotherapy. In conclusion, the results of this study suggest a possible role for bcl-2 in predicting resistance to chemotherapy.     

Apoptosis loss and bcl-2 expression: key determinants of lymph node metastases in T1 breast cancer

The Bcl-2 proto-oncogene extends cell survival but does not confer any proliferative advantage to cells that express it. Thus, the loss of apoptosis may have a role in progression allowing the acquisition of additional mutations. To determine whether apoptosis loss at diagnosis is associated with the metastatic advantage of ductal breast carcinomas and to examine the relationship between Bcl-2 expression, p53, and tumor cell death status, we examined tumor samples from 116 patients diagnosed with T1 (2 cm or less) breast cancer with (n = 49) or without (n = 67) lymph node metastases. Apoptosis loss in histological sections was considered when <1% of tumor nuclei were stained with terminal deoxynucleotidyl transferase labeled with biotin. We studied the expression of Bcl-2 and p53 by immunohistochemistry and in 37 p53 mutations by single-strand conformational polymorphism analysis and cycle sequencing. Multivariate logistic regression modeling was used to estimate prevalence odds ratios (pORs) for apoptosis loss and presence of lymph node metastases. Patients with marked apoptosis loss in their tumor cells were about 5 times more likely to present lymph node metastases than those with no apoptosis loss in their tumor cells (adjusted pOR, 4.7; 95% confidence interval, 1.4-15.6; trend test, P = 0.008). Bcl-2 expression was strongly associated with both apoptosis loss (pOR, 6.9; trend test, P < 0.0001) and presence of lymph node metastases (pOR, 5.7; trend test, P = 0.002). These associations were more evident in histological grade I and II tumors than in poorly differentiated histological grade III tumors and in p53-negative tumors than in p53-positive tumors. This study demonstrates for the first time that the lymphatic progression of T1 human breast cancer is strongly related to apoptosis loss.     

Enhanced expression of bcl-2 inhibits apoptosis in cultured human keratinocytes

Intravenous heparin followed by oral warfarin sodium is effective for preventing recurrent thromboembolism in patients who have pulmonary embolism or proximal vein thrombosis. The effectiveness of intravenous heparin depends on obtaining an adequate anticoagulant response early during therapy. A validated heparin protocol should be used to ensure that an adequate anticoagulant response is obtained as soon as possible. Low molecular weight heparin has the practical advantage that it does not require monitoring and dose finding. If thrombolytic therapy is indicated, it is safer for many patients to base management on the noninvasive diagnosis rather than performing pulmonary angiography. In patients suspected to have pulmonary embolism who have nondiagnostic lung scan and adequate cardiorespiratory reserve, serial noninvasive leg testing is a practical approach that avoids pulmonary angiography, identifies patients who have proximal vein thrombosis requiring treatment, and avoids the risks of anticoagulant treatment in the majority of patients.     

p53 protein expression in benign and malignant breast lesions

OBJECTIVE: To investigate p53 protein expression in imprints from benign and ductal breast carcinoma cases in relation to the histologic grade of malignancy and clinical stage. STUDY DESIGN: The study group consisted of 60 cases of primary ductal breast carcinomas and 20 benign lesions. For the demonstration of p53 protein expression, an immunocytochemical avidin-extravidin complex technique was applied. Monoclonal antibody p53 was used as the primary antibody, diaminobenzidine as the chromogen and hematoxylin as the counterstain. RESULTS: Forty-five percent of breast cancer cases showed positive expression of p53. A statistically significant difference in p53 protein expression was observed between grade 1, 2 and 3 carcinomas and stage I, II and III cases. All benign lesions were negative for p53 protein expression. CONCLUSION: Immunocytochemical p53 protein expression in cytologic material is a simple method that can be applied in routine cytologic laboratories for the identification of genetic alterations in primary ductal breast cancer.     

Analysis of the expression of the hybrid gene bcl-2/IgH in follicular lymphomas

To investigate the clinical and biologic significance of the circulating t(14;18)-carrying cells in follicular lymphoma (FL) patients, we analyzed the mbr/JH junction of the hybrid bcl-2/IgH gene simultaneously at the DNA and RNA levels by polymerase chain reaction (PCR) in 37 peripheral blood samples from 37 patients in different remission status: 4 before treatment, 8 during treatment, and 25 in complete remission (CR). Of these 37 patients, 22 were positive either at the DNA or RNA level (8 with active disease and 14 in CR). Among these positive patients, RNA was more often negative for patients in CR (9 of 14 [64%]) than for patients with active disease (2 of 8 [25%]; Fisher's exact test, P = .09). Among the 14 patients in CR with residual disease, 2 of 5 with RNA positivity relapsed, whereas 1 of 9 with RNA negativity and DNA positivity relapsed with a median follow-up after sample collection of 8 months (range, 4 to 18 months). Simultaneous analysis of the bcl-2/IgH gene at the DNA and RNA level showed heterogeneous patterns of PCR positivity in regards to the evaluation of the biologic activity of the t(14;18)-carrying cells. A larger study and long-term follow-up will help in determining whether the expression patterns in turn reflect the functional status of disease activity in FL patients.     

Prognostic value of bcl-2 oncoprotein expression in stage II colon carcinoma

The bcl-2 proto-oncogene encodes a Mr 25,000 protein that has been shown to prevent apoptosis or programmed cell death. The bcl-2 protein is detectable in basal cells of normal colonic epithelium, and an altered topographic distribution of this protein is found in colonic neoplasms. However, the clinical significance of abnormal bcl-2 expression in colon carcinomas remains unknown. We examined the prognostic value of the bcl-2 protein in TNM stage II colon carcinomas and its relationship to DNA ploidy, cell proliferation indices, p53 expression, and clinicopathological features. We analyzed 119 resected and otherwise untreated, paraffin-embedded stage II colon carcinomas for bcl-2 and p53 protein expression using immunohistochemistry. DNA ploidy and proliferative index (% S-phase + % G2-M) were determined by flow cytometry, and tumor grade and vascular microinvasion were assessed on histological sections. Cytoplasmic expression of the bcl-2 protein was detected in 72 (66%) of 110 carcinomas, and a high level of expression was significantly correlated with diploid DNA content (P = 0.02) and low proliferative activity (P = 0.005). bcl-2 was not associated with nuclear p53 expression. In a univariate analysis, a higher fraction of bcl-2-positive tumor cells was associated with better relapse-free survival (P = 0.02) and overall survival (P = 0.05) rates. Moreover, a high level of bcl-2 expression was an independent predictor of better relapse-free survival (P = 0.04), but not overall survival (P = 0.14), after adjustment for other variables, including proliferative index, DNA ploidy, and race. In conclusion, bcl-2 overexpression is associated with favorable prognostic features and may predict clinical outcome in stage II colon carcinomas.     

bcl-2 and p53 oncoprotein expression during colorectal tumorigenesis

AMS is a preventable disease about which travellers are frequently uninformed and one which physicians may wrongfully assume is limited to the population of ultra high altitude adventurers. These studies on incidence, while not without flaws, point out the frequency of AMS, as well as its significant incidence at moderate and commonly frequented altitudes. The current literature does not fully answer questions about incidence at moderate altitudes, nor about the full effects of altitude on children. Certainly AMS is not a rare complication of travel to altitudes and may indeed be under-recognized and under-treated. Both acetazolamide and dexamethasone provide adequate prophylaxis, and the choice of medications can be to some extent based on experience and patient profile. The best prophylaxis is a slow stepwise ascent, and the best treatment descent. The availability of medications for the amelioration or prevention of symptoms, and succinct advice on prevention by travel planning will make many of our patients' holidays more enjoyable and business trips more productive.     

Prognostic value of bcl-2 expression in patients with operable carcinoma of the uterine cervix

The nucleus retroambiguus (NRA) is a group of premotor neurons at the transition between brainstem and spinal cord. It projects to certain motoneuronal cell groups, among which is a distinct set of motoneurons in the lumbar enlargement innervating muscles including iliopsoas, adductor longus, and hamstrings. To find out whether these NRA-motoneuronal projections are monosynaptic, injections of wheat germ-agglutinin horseradish peroxidase (WGA-HRP) into the NRA were combined with injections of cholera toxin subunit b (CTb) into the hamstring muscles. Electron microscopical examination revealed that the NRA terminal profiles make monosynaptic contacts with dendrites of motoneurons innervating these muscles. The NRA terminal profiles formed asymmetrical synapses, and contained spherical and a few dense core vesicles. These findings provide evidence of monosynaptic NRA-hindlimb motoneuronal projections which are likely to be excitatory.     

Haematopoietic response and bcl-2 expression in patients with acute myeloid leukaemia

In the Bethesda System for reporting cervicovaginal cytology results, 1 criterion for smear adequacy is an adequate squamous component. The accuracy of a cytologist's estimate that 10% of the slide is covered by squamous cells, the adequacy threshold, has not been determined. The percentage of the surface of a glass slide covered by squamous cells was independently estimated by 4 cytologists on 2 occasions by microscopic examination of 83 buccal smears prepared to display estimated coverage of 1% to 20% of the slide surface. The accuracy of visual estimates was compared with measurements by the TracCell System. Each observer made a third set of estimates after receiving 5 slides with known coverage. Median coverage by visual estimation ranged from 4% to 25%, but as measured by the TracCell system was 2%. Median estimated coverage was significantly different for 2 of 4 observers between first and second viewings and between all but 1 pair of observers. For all observers, it was significantly higher than the true coverage. A visual estimate of 10% coverage corresponded to a true median coverage of 3%. When provided with a physical standard, the median estimated coverage by 3 of 4 observers was not statistically different from the true coverage, and interobserver kappa values improved. Unaided visual estimation of the adequacy of squamous cell coverage is neither reproducible nor accurate. What most cytologists consider "adequate" coverage represents only 3% coverage. The availability of a physical standard dramatically increases reproducibility and accuracy.     

Mineralocorticoid receptors regulate bcl-2 and p53 mRNA expression in hippocampus

The present study addressed the hypothesis that the neuronal mineralocorticoid receptor (MR) regulates genes associated with cell death, such as bax and p53, and cell viability, including bcl-2, BDNF, and NT-3. Rats were pretreated with either oil vehicle or the MR antagonist spironolactone (SPIRO) and subsequently injected with saline or kainic acid (KA). MR blockade significantly decreased basal mRNA expression of bcl-2 in CA1 of saline-treated animals and attenuated KA-induced increases in p53 mRNA levels in CA3. SPIRO pretreatment had no significant effect on expression of bax, NT-3, or BDNF mRNAs. The data suggest that the neuronal MR contributes to regulation of select cell survival and cell death-related genes in hippocampal pyramidal neurons.     

Differential expression of rat brain bcl-2 family proteins in development and aging

1. The effects of YM-60828, a newly synthesized factor Xa inhibitor, were investigated to analyse the relationship between its antithrombotic effects and its prolongation of template bleeding time in rats. YM-60828 was compared with argatroban, heparin and dalteparin. All agents were intravenously administered as a bolus. 2. In ex vivo studies, YM-60828 and argatroban prolonged both prothrombin time and activated partial thromboplastin time in a dose-dependent manner, while heparin and dalteparin prolonged only activated partial thromboplastin time. 3. In a venous thrombosis model, all agents exerted antithrombotic effects in a dose-dependent manner. The ID50 values of YM-60828, argatroban, heparin and dalteparin were 0.0081 mg kg(-1), 0.011 mg kg(-1), 6.3 iu kg(-1) and 4.7 iu kg(-1), respectively. 4. In an arterio-venous shunt model, all agents exerted antithrombotic effects in a dose-dependent manner. The ID50 values of YM-60828, argatroban, heparin and dalteparin were 0.010 mg kg(-1), 0.011 mg kg(-1), 10 iu kg(-1) and 4.2 iu kg(-1), respectively. 5. In bleeding time studies, all agents prolonged template bleeding time in a dose-dependent manner. ED2 values, the doses causing a 2 fold prolongation of bleeding time in the saline group, of YM-60828, argatroban, heparin and dalteparin were 0.76 mg kg(-1), 0.081 mg kg(-1), 18 iu kg(-1) and 25 iu kg(-1), respectively. 6. The ratio (ED2/ID50) of YM-60828 was more than 30 fold greater than that of heparin and more than 10 fold greater than those of argatroban and dalteparin. 7. These data show that YM-60828 can exert its antithrombotic effects with little prolongation of bleeding time compared with the other currently used anticoagulant agents.     

Loss of heterozygosity at chromosome 6q in preinvasive and early invasive breast carcinomas

We have used polymerase chain reaction (PCR) analysis to study the incidence of allelic imbalance at four polymorphic microsatellite markers on chromosome 6q25.1-27, three dinucleotide repeats and one trinucleotide repeat, for microdissected tumour foci from a group of 75 'early' breast carcinomas. The tumours comprised 16 preinvasive cases of ductal carcinoma in situ (DCIS) and 59 mammographically detected early invasive carcinomas. Loss of heterozygosity (LOH) was detected at all four loci and in all types and grade of disease. The frequency of LOH ranged from 23% to 50% depending on the marker studied. The highest frequency of LOH was observed at the D6S186 locus for the cases of DCIS and at the oestrogen receptor locus for the invasive carcinomas. These data suggest that the inactivation of tumour-suppressor genes within this region on chromosome 6q is important for the development of these early lesions.