[Expression and intranuclear distribution of nucleolin in estrogen receptor-negative and estrogen receptor-positive breast cancers in women measured by laser scanning cytometry]

Abstract

Introduction: Nucleolin (NU) is one of the most abundant nucleolar proteins. Nucleolin is mainly involved in ribosome biogenesis that is supported by the ability of NU to bind rDNA and modify the structure of chromatin by binding to histon H1. Estrogen receptor alpha (ERalpha) is a DNA-binding transcriptional factor. It is estimated that 69-85% of breast cancers in women are ERalpha-positive. The aim of the study was to assess the expression and intranuclear distribution of NU in invasive ductal and lobular breast cancers in women and their relationship to ERalpha-status, histologic type and grade of breast cancer, and lymph node status. For this purpose, laser scanning cytometry (LSC) was used.

Material and methods: Measurements were done in cytospins of cancer cells of 87 ductal and 11 lobular invasive breast cancers. The cells were labeled with mouse anti-human NU antibody followed by F(ab')2 fragments of FITC-conjugated goat anti-mouse antibody. Nuclei were counterstained with 5 microg/mL of propidium iodide in the presence of 100 microg/mL of RNase A. All measurements were performed using LSC. The following parameters of individual cancer cells were calculated: NU fluorescence within the nucleus, within nucleolin aggregates (NUA) and in the remaining karyoplasm, number of NUA, area of the nucleus and NUA. The percentage of ER-positive breast cancer cells was calculated in parallel by the automated image analysis in formalin-fixed sections using immunohistochemistry with anti-ERalpha antibody. The cut-off value for ER-negative tumors was set at 10% of positively stained nuclei. Statistical analysis was done using the Statistica 5.0 software. P values less than 0.05 were considered statistically significant.

Results: The mean area of the nucleus of ductal cancer cells was significantly higher and NU expression lower in ERalpha-negative cancers than in ERalpha-positive ones (p = 0.007 and p = 0.04, respectively). The mean area of NUA and NU expression in ductal cancers were higher than in lobular cancers (p = 0.03 and p = 0.02, respectively). The expression of NU within the nucleus and within the karyoplasm besides NUA was significantly higher in ductal than in lobular cancers (p = 0.02 and p = 0.04, respectively). The expression of NU in the remaining karioplasm of tumor cells of lymph node-positive cancers was lower than in node-negative ones (p = 0.04). The same relation was found for ductal cancers (p = 0.02).

Conclusions: The differences in nucleolin expression and its intranuclear distribution in ERalpha-negative and ERalpha-positive breast cancers, as well as ductal and lobular cancers point to biologic differences between these carcinomas. The method used in the study may be applied to measurements of expression and intranuclear distribution of other nuclear proteins or to simultaneous measurement of expression and distribution of nuclear and cytoplasmic proteins.