title: Immunohistochemical detection of melphalan-DNA adducts in colon cancer cells in vitro and human colorectal liver tumours in vivo.
abstract: Melphalan is a chemotherapeutic drug that exerts its cytotoxic effect mainly through the formation of DNA adducts. We report the specific immunohistochemical detection and visualisation of melphalan-DNA adducts using the monoclonal antibody MP5/73 in cultured tumour cells and solid tumour tissue from colorectal liver metastases from patients treated with melphalan. The human colon cancer cell lines HT29, SW480 and SW1116, and the rat colon cancer cell line CC531 were exposed to different concentrations of melphalan. In addition, tumour samples from 17 patients with colorectal liver metastases treated by isolated hepatic perfusion with high dose melphalan (200mg) were collected. Cell lines and tumour samples were stained with the MP5/73 antibody against melphalan-DNA adducts and cell viability was determined by an MTT assay. Melphalan-DNA adducts could be visualised by immunohistochemistry in both cultured cells and solid tumour tissue. A correlation between melphalan exposure concentration, the subsequent melphalan-DNA adduct staining intensity, and melphalan cytotoxicity existed for each individual cell line, but the level of both parameters independently differed between cell lines. Specific staining for melphalan-DNA adducts also was feasible in the human solid tumour tissue. There was considerable variation in melphalan-DNA adduct staining, staining intensity, and distribution in the tumour stroma and the tumour epithelium among the different patients. Melphalan-DNA adducts appeared to be more intense in tumour cells at the border of the tumour nodules than in tumour cells in the centre. Thus, visualisation of melphalan-DNA adducts by immunohistochemistry allows the study of distribution of melphalan-DNA adducts in solid tumours.
authors.affiliation: Department of Surgery, Leiden University Medical Center, Albinusdreef 2, Leiden 2333, The Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: Rothbarth
authors.forename: Cindy
authors.initials: C
authors.lastname: Koevoets
authors.forename: Rob A E M
authors.initials: RA
authors.lastname: Tollenaar
authors.forename: Mike J
authors.initials: MJ
authors.lastname: Tilby
authors.forename: Cornelis J H
authors.initials: CJ
authors.lastname: van de Velde
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Mulder
authors.forename: Peter J K
authors.initials: PJ
authors.lastname: Kuppen
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D014408:Biomarkers, Tumor / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis; D003110:Colonic Neoplasms; D015179:Colorectal Neoplasms; D018736:DNA Adducts / Q000187:drug effects*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D007797:Laminin / Q000032:analysis; D017493:Leukocyte Common Antigens / Q000032:analysis; D008113:Liver Neoplasms; D008558:Melphalan / Q000494:pharmacology*; D051381:Rats; D011956:Receptors, Cell Surface; D014407:Tumor Cells, Cultured
chemical_list: D018952:Antigens, CD34; D018906:Antineoplastic Agents, Alkylating; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D018736:DNA Adducts; D007797:Laminin; D011956:Receptors, Cell Surface; C065686:carcinoembryonic antigen binding protein, human; D017493:Leukocyte Common Antigens; D008558:Melphalan