Fig. 1
MMP-8 expression in; primary normal and DCIS myoepithelial cells and a cell line model. a Normal and ductal carcinoma in situ (DCIS) tissue immunohistochemically stained for matrix metalloproteinase-8 (MMP-8) (Atlas, HPA02122, 1:1000). Clear MMP-8 staining can be observed in the normal myoepithelial cells (MECs), which is absent in the DCIS section. Magnification × 400. b Normal MECs and luminal epithelial cells (LECs) were isolated from reduction mammoplasty tissue and DCIS MEC and DCIS LEC were isolated from a patient with high-grade DCIS. Tissues were enzymatically digested and magnetic bead separation was used to isolated specific populations (MEC - ITGB4, LEC - EpCAM). RNA was extracted from the isolated cells and G361 cells (a positive control for MMP-8), converted to cDNA by reverse transcription and then subjected to nested PCR for MMP-8 or PCR of housekeeping gene 18S. The MMP-8 PCR produces a product of 150 bp in normal MEC and G361 lanes, and other lanes only give a weak band. 18S gives a size of 52 bp in all sample lanes. The water (H2O) control lane shows no band. c (i) RNA was extracted from cell lines; G361, N-1089 and β6-1089 and converted to cDNA as above. The cDNA was subjected to nested PCR for MMP-8 and bands were identified in G361 and N-1089 but only a weak band was observed in the β6-1089 lane. All samples demonstrated a band for 18S. (ii) RIPA protein lysates were produced from N-1089 and β6-1089 cells and 20 ug of protein was run on 8% PAGE then transferred to nitrocellulose and probed for MMP-8 (R&D Systems, mAb 908) αvβ6 (Santa Cruz Biotechnology, SC-6632) or HSC70 as a loading control. The gel indicates that N-1089 expresses higher levels of MMP-8 and much lower levels of αvβ6. While β6-1089 exhibit much higher levels of αvβ6 and lower levels of MMP-8. (iii) Densitometry of western blots indicates that MMP-8 expression in N-1089 is 2.5-fold higher than N-1089 and (iv) αvβ6 expression in β6-1089 is 10-fold higher than N-1089