We are studying the effects of the cytokines IL-1, IL-6, and TNF alone and in combination on cell growth and estradiol stimulated metabolism of breast cancer cells. We found that all three cytokines inhibit cell growth in vitro with the following efficacy: TNF > IL-1 > IL-6. IL-1 acts additively with IL-6 to inhibit growth and antagonize E2 stimulation of growth of ER+ cells MCF-7 and T47D. TNF blocks E2 stimulated growth and down-regulates the ER. This also indicates an important interaction between the immune and endocrine systems. The effects of the cytokines on steroid receptors are at the posttranscriptional level. TNF induces sustained expression of TNF mRNA and secretion of TNF protein and arrests growth at G(0)G(1). TNF, however, does not act additively with IL-1. TNF and IL-1 both stimulate secretion of TGFbeta by MCF-7 cells in a time and dose- dependent manner; stimulation is antagonized by estradiol. TNF increases secretion of both the biologically active and inactive forms of TGFbeta. This occurs without a change in the structure of the subunit or glycosylated forms as determined by immunoblotting. Studies are in progress to determine the particular isoform(s) of TGF which is stimulated by TNF and IL-1, and whether these cytokines are acting at the transcriptional or posttranscriptional level to increase secretion. A second important study is the establishment of long-term human breast carcinoma cell lines from primary solid tumors. We have developed a method involving enzymatic digestion of fresh tumor, maintenance of cell monolyers in defined condition media and the cloning of subcultures. Cellular characterization includes cell morphology, keratin staining and FACS analysis with antibodies specific for epithelial and breast carcinoma. Growth curves are constructed in vitro and in vivo in nude mice. Karyotyping studies are being performed to identify chromosomal aberrations, and in situ hybridization to identify specific genetic changes, including the evaluation of multi drug resistance and oncogene expression. These cell lines will allow more detailed characterization of chromosomal, genetic and transcriptional abnormalities, and the role of cellular and human cytotoxicity in the treatment of this malignancy.
