Vol. 39, No. 3, June 1996 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages553-561 IS-RT-PCR ASSAY DETECTION OF MT-MMP IN A HUMAN BREAST CANCER CELL LINE Larisa M. Haupt '~, Erik W. Thompson13,Lyn R. Griffiths=, and Michael G. Irving~ Griffith University Medical Research Institute, Griffith University Gold Coast, Queensland, Australia 4217 and ~Lombardi Cancer Centre and Departments of Cell Biology and Orthopedic Surgery, Georgetown University Medical Centre, Washington DC., USA. Received March 15, 1996 SUMMARY: The in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) is a method that allows the direct localisation ofgene expression. The method utilises the dual buffer mediated activity of the enzyme rTth DNA polymerase enabling both reverse transcription and DNA amplification. Labelled nucleoside triphosphates allow the site of expression to be labelled, rather than the PCR primers themselves, giving a more accurate localisation of transcript expression and decreased background than standard in situ hybridisation (ISH) assays. The MDA-MB-231 human breast carcinoma (HBC) cell line was assayed via the IS-RT-PCR technique, using primers encoding MT-MMP (membrane-type matrix metalloproteinase) and human 13-actin. Our results clearly indicate baseline expression of MT-MMP in the relatively invasive MDA-MB-231 cell line at a signal intensity similar to the housekeeping gene 13-actin, and results following induction with Concanavalin A (Con A) are consistent with our previous results obtained via Northern blotting. INTRODUCTION: We used IS-RT-PCR to investigate the expression of MT-MMP (membrane-type matrix metalloproteinase), the recently identified activator of pro-gelatinase-A, in human breast carcinoma cells (1). As tumour cells progress toward malignancy, the surrounding basement membrane is degraded (2). Although the biochemical mechanisms involved in basement membrane turnover under physiologic conditions remains unknown, it has been proposed that the basement membrane-degrading properties of tumour cells and surrounding stromal cells correlate with the metastatic potential of the tumour (3). Important turnout-host interactions during the onset and development of cancer include desmoplasia, angiogenesis, metastasis and immunosuppression (3). The central aim of investigations into molecular carcinogenesis, is the identification ofgene products involved in the progression of tumour cells toward invasion and metastasis (3). MMPs expressed at or around tumour sites are implicated in tumour invasion, and they can be produced by the stromal cells 553 1039-9712/96/030553~)9505.00/0 Copyright © 1996 by Academic Press Australia. All rights of reproduction in any form reserved.