Bcl-2 expression in breast cancer: a comparative study at the mRNA and protein level.

BACKGROUND: Bcl-2 is one of the most important antiapoptotic genes. Although it facilitates the survival of tumor cells, its expression has been consistently associated with a better prognosis for breast cancer. Virtually all studies on Bcl-2 conducted in breast cancer have been carried out by means of immunohistochemistry. The aim of this study was to examine for the first time the expression of Bcl-2 in a series of human breast cancer, both at the mRNA and protein level. MATERIALS AND METHODS: One hundred samples from previously untreated human breast cancers were used; Bcl-2 expression was determined both by means of immunohistochemistry using the bcl-2/100/D5 monoclonal antibody and differential RT-PCR. Additionally, the expression of hormone receptors (ER and PR), c-erb-B2, p53 and the proliferation-associated Ki-67 antigen were also studied by means of immunohistochemistry as part of the standard pathological workup. RESULTS: Any degree of immunohistochemical staining correlated significantly and inversely with c-erb-B2 expression (p = 0.0008), nuclear grade 3 (p = 0.0015), a Ki-67 labeling index > 10% (p = 0.02) and tumor size (p = 0.048), and in a direct fashion with estrogen (p = 0.0003) and progesterone receptor expression (p = 0.0002). mRNA expression of the Bcl-2 gene showed a significant inverse correlation with c-erb-B2 (p = 0.016) and p53 (p = 0.014) expression, as well as with a nuclear grade 3 (p = 0.006), and a direct correlation with estrogen (p = 0.0004) and progesterone (p = 0.001) receptor expression, as well as with nodal invasion (p = 0.04). CONCLUSION: The study of Bcl-2 expression in breast cancer by means of either immunohistochemistry or RT-PCR yields very similar results. In spite of its role opposing tumor cell death, Bcl-2 is associated with biological features of the tumors which define a better intrinsic prognosis, such as hormone receptor expression, low proliferation and absence of c-erb-B2 and mutant p53 expression. This may in great part explain why Bcl-2 expression has been invariably found to correlate with a better prognosis of breast cancer.

Developing chick embryos express a protein which shares homology with the nuclear pore complex protein Nup88 present in human tumors.

Nup88 is a nuclear pore complex protein which is overexpressed in a variety of human tumors of the stomach, colon, liver, pancreas, breast, lung, ovary, uterus, prostate and kidney. A monoclonal antibody crossreacting with the yeast Candida albicans and Nup88 was used to investigate the expression of cross-reactive antigens in chick embryos, in an attempt to identify an experimental model for studying the role played by Nup88 during cell development and differentiation. All cells in the trilaminar embryo were labeled with the antibody, but as development advanced and organogenesis was completed, expression of the corresponding antigen became more restricted. Thus, some structures continued to be intensely labeled (skin epithelium, oropharyngeal endothelium, perichondral mesenchymal tissue), whereas others ( muscular tissue, vascular endothelium, respiratory endothelium, digestive tract mucosa, peripheral nerves, medullary white matter and the retinal axons) were more moderately stained. No immunoreactivity was observed in the medullary grey matter or cartilage. A specific band of 53 kDa observed by Western blotting of chick embryo extracts suggested that the chicken antigen recognized by the monoclonal antibody is the homologue of human Nup88, which is associated with the high proliferation and low differentiation of tumor cells. The present results indicate that the role of Nup88 in cell differentiation and organ development could be fruitfully investigated using the developing chick embryo as an experimental model.

mRNA expression of the angiogenesis markers VEGF and CD105 (endoglin) in human breast cancer.

BACKGROUND: Both VEGF and CD105 (endoglin) have been identified as markers of tumor angiogenesis and prognosis in breast cancer. They have always been studied in this kind of tumor by means of immunological methods. OBJECTIVE: To study, by means of reverse-transcription polymerase chain reaction (RT-PCR), the expression of VEGF and CD105 (endoglin) at the mRNA level in a series of breast cancers and to correlate the results obtained with all available clinical and biological features of the tumors. MATERIALS AND METHODS: Fresh tumor tissue from 103 previously untreated breast cancer patients was studied for VEGF and CD105 (endoglin) expression. In addition, the following parameters were determined in all tumors: DNA ploidy by means of flow cytometry; hormone receptor (ER & PR), Ki67, c-erb-B2 and p53 expression by means of immunohistochemistry; and h-MAM (mammaglobin) expression by means of RT-PCR. Classical prognostic parameters of the tumors, such as histological and nuclear grade or axillary lymph node invasion, were also included into the statistical analysis. RESULTS: VEGF mRNA expression levels above the 25th percentile were significantly (p<0.05) associated with high proliferation (Ki67>10%) and aneuploidy of the tumors and inversely with estrogen receptor expression (p<0.01). CD105 (endoglin) mRNA expression levels above the 25th percentile only correlated significantly with nuclear grade 3 (p<0.05). The expression of both genes did not correlate with each other. CONCLUSION: VEGF mRNA expression levels seem to be directly associated with VEGF functions at the protein level, whereas this seems not to be the case for CD105 (endoglin) mRNA levels.

Nup88 mRNA overexpression is associated with high aggressiveness of breast cancer.

The nuclear pore complex protein Nup88 is overexpressed in tumor cells. Immunohistochemical studies have shown that this overexpression is linked to higher aggressiveness of colorectal carcinoma and to enhanced metastatic potential of melanoma cells. However, the antibodies so far developed against Nup88 have the drawback of recognizing a number of other, up to now unspecified antigens besides Nup88. For this reason, we devised the present study on Nup88 expression at the mRNA level. RNA was extracted from fresh tumor tissue corresponding to 122 breast cancer patients. Nup88 mRNA expression was measured by means of differential RT-PCR, standardizing against a constitutive internal control gene (beta-actin). The results were dichotomized into "high" and "low" expression levels, using the median value as cut-off. High Nup88 mRNA expression levels correlated significantly with ductal and tubular histology (p = 0.012), histologic and nuclear grade 3 of tumors (p < 0.001), absence of hormone receptor expression (p < 0.001), expression of the c-erb-B2 oncogene (p < 0.001), expression of mutant p53 protein (p < 0.001), high proliferation (defined by Ki67 labeling index >20%, p < 0.001), DNA aneuploidy (p < 0.001) as well as the most important ominous clinical prognostic factor, axillary node invasion (p < 0.001). We also found an inverse correlation (p < 0.001) with expression of the H-MAM (mammaglobin) gene, a marker of low biologic and clinical aggressiveness of breast cancer. All of these factors, without exception, define a highly aggressive tumor phenotype. These findings appear to be specific to Nup88 and not to nuclear pore proteins in general. Indeed, analysis of Nup107 (which is a limiting component of the nuclear pore complex) under the same conditions in the same tumors did not yield comparable results.