5.1. Transfecting well-differentiated prostatic cancer cell line LNCaP

A lipofection method has been set up for the transient transfection of LNCaP cells (I). The LNCaP cells were originated from a lymph node metastatic lesion of human prostatic adenocarcinoma. The relatively high degree of differentiation makes LNCaP cells difficult to maintain in cell culture conditions. Their low ability to attach to the growth support, the slow growth rate, the demand for a high percentage of serum in the culture medium, the tendency of making colonies, and the modest uptake of DNA are features typical of such well-differentiated cells. Lipofection was chosen as a method because it is the gentlest way for transfection. The disadvantage of the DEAE dextran method is the multiple wash steps, and the cytotoxicity of DEAE destroying most of the LNCaP cells. The high induction of positive control pMMTV with 10 nM androgen showed that LNCaP could be transfected after careful optimization of the conditions. Among three tested control plasmid, the cytomegalovirus promoter containing plasmid (pCMVβ -gal) was the most efficient one, while the pSVβ -gal and the pRSVβ -gal were only modestly active in LNCaP cells.

After the production of more efficient transfection reagents, DOTAP was replaced with FUGENE6. The method was modified by plating 5 x 105 cells/35mm plate for LNCaP, while 2 x 105 cells/35mm plate were grown for other cells before transfection. The total amount of DNA per plate was reduced to 2 µg. The longer culture time before and after transfection compared to other cells is still needed for LNCaP to obtain comparable protein levels for the consequent CAT assay and protein assay. Since LNCaP is the only commercially available cell line that expresses endogenous hPAP, transfecting LNCaP cells makes it possible to study the promoter activity of the hPAP gene, as well as the hormonal regulation of the hPAP promoter.