Figure 1

Adrenomedullin expression by breast cancer cell lines and effects of adrenomedullin on tumor and bone cells. (A) Adrenomedullin (AM) RNA in human breast cancer cell lines that metastasize to bone. PCR detection of human AM mRNA in from left to right: normal tissues (kidney, prostate and breast), breast cancer cell lines (MCF-7, BT549, MDA-231, MCF-7, MDA-MB-436, ZR-75-1, T47D, MD-MB-361 and BT438) and three additional cancer cell lines that cause osteolytic bone metastases: MDA-MB-435S melanoma cells, PC-3 prostate cancer cells and A549 lung adenocarcinoma cells. cDNA (200 ng) was amplified for 30 cycles. The PCR product is 600 bp. It is unclear if kidney expresses only low amounts of AM mRNA or if the RNA was degraded. (B) Stable overexpression of human AM mRNA in MDA-MB-231 cells. Clones were tested for stable human AM mRNA expression using real-time PCR after 60 days of culture in the absence of antibiotic selection. G8-G31 are MDA-MB-231 control clones, and A15-A51 are MDA-MB-231 clones overexpressing AM. (C) Proliferation in vitro of breast cancer cells. Proliferation of two AM-overexpressing clones (A39, gray triangle; A51, black triangle) and two control clones (G8, gray square; G26, black square) were compared using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; tetrazolium blue dye) assay. Proliferation was compared after 1, 3 and 5 days of growth. OD, Optical density.