A comparative study of cytokine gene transcripts in normal and malignant breast tissue and primary cell cultures derived from the same tissue samples.
Using a differential centrifugation method followed by culture in selective medium, we have successfully isolated and maintained individual epithelial and stromal cells from normal (n = 10) and malignant (n = 6) human breast tissue and characterised their phenotype by immunocytochemistry. Further, we have studied expression of the cytokine genes IL-1beta, IL-6, IL-8 and TNF-beta in each cell fraction by RT-PCR and have compared these results with cytokine gene expression in tissue extracts from which primary cultures were derived. In breast tumours, there was near complete absence of IL-1beta in both whole tissue and cell fractions, and in normals it was present in only 3/10 tissue preparations, with increased expression in stromal (6/10) and epithelial (5/10) cell samples. IL-6 was constitutively expressed in all tumour-derived breast tissue samples but down-regulated in tumour cell cultures, with the opposite result in normal breast. Near identical levels of IL-8 expression were found throughout each preparation, irrespective of tissue origin. TNF-beta was expressed in all normal tissue samples, in 9/10 epithelial preparations but in only 6/10 stromal preparations. In tumours, TNF-beta was associated predominantly with whole tissue or stromal samples, with reduced expression in epithelial preparations. Our data confirm that primary cultures of normal and malignant human breast tissue can be successfully separated into epithelial and mesenchymal cell populations and their phenotype can be maintained in culture for up to 30 days. However, this cellular separation does alter the cytokine profiles; therefore, experimental findings with isolated cells should be treated with a caveat.
Speirs
V
V
Department of Medicine, University of Hull, UK.
Green
A R
AR
White
M C
MC
D001483:Base Sequence; D001940:Breast / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism*; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000378:metabolism
D016207:Cytokines; D017931:DNA Primers; D012333:RNA, Messenger; D014746:Vimentin; D015260:Neprilysin