Phenotypic and genotypic characterization of commonly used human prostaticcell lines

Objective To investigate and catalogue systematically the phenotypic and ge notypic characteristics of the commonly used prostatic cell lines using imm unocytochemistry and polymerase chain reaction (PCR) of hypervariable seque nces within the genome to provide a 'fingerprint' characteristic of each ce ll line. Materials and methods Malignant (LNCaP, LNCaP-r, PC-3, DU-145) and benign i mmortalized prostatic cell lines (PNT-1A, PNT-1B, BPH-1) were grown on four -well slides, fixed and subjected to indirect streptavidin-biotin immunocyt ochemistry. Twenty-three antibodies were used in the following groups: cyto skeletal elements: cytokeratins (CK)-5, -7, -8, -14 (two), -16, -18, -19 (t hree), -20, vimentin and desmin; MUC1 (three); cell adhesion molecules (E-c adherin, alpha-beta-and gamma-catenin); and prostatic associated proteins: prostate specific antigen (PSA), prostatic acid phosphatase (PAP) and andro gen receptor (AR). For the PCR, genomic DNA was extracted from the cell lin es and from SKOV3 and MCF7 (positive controls). PCR was performed on three variable regions which were then sequenced: AR exon 1 (CAG repeat polymorph ism), and two areas of microsatellite instability (MSI): AR exon 8 and hypo xanthine-guanine phosphoribosyl transferase (HPRT) exon 3. Results All cell lines were CK-8/18 positive and most also expressed CK-7 a nd -19. Heterogeneous CK-20 expression was detected for the first time in p rostatic cell lines. All lines were positive for vimentin and negative for desmin. MUC1 was expressed in one malignant (DU-145) and all immortalized c ell lines. E-cadherin expression was low or absent in three lines: PNT1A, 1 B and PC-3. Only PC-3 failed to express alpha-catenin; beta- and gamma-cate nin were expressed by all lines. PSA, PAP and AR were only expressed by LNC aP and LNCaP-r. On PCR, the CAG repeat lengths in exon 1 of the AR ranged f rom 19 to 27. Three pairs of cell lines had the same exon 1 CAG repeat leng th: LNCaP/PC-3 (26 repeats), BPH-1/DU-145 (19 repeats) and PNT1 A/1B (20 re peats). Exon 8 sequences were identical except for LNCaP, which showed a si ngle base mutation, and HPRT exon 3 sequences were all identical. There was no evidence of generalized MSI in any of the cell lines examined. Conclusions The cell lines studied fell into three broad groups according t o their phenotypic characteristics: (i) prostatic marker positive (LNCaP an d LNCaP-r); (ii) high expression of most antigens (DU-145, PC-3 and BPH-1); and (iii) low or absent expression of most antigens (PNT1 A and 1B). Each of the cell lines derived from PC could be identified on the basis of exon 1 and 8 AR sequence variability. DU145 and BPH-1 had identical profiles of the three areas studied, but these cell lines are easily distinguished by t heir different phenotypic characteristics. PNT1A and 1B had identical genet ic and similar phenotypic profiles, which is unsurprising given that they a re subclones derived from the same parental line. Even so, these were separ able on the basis of CK-19 immunostaining. Using a combination of geno- and phenotypic markers it was possible to derive a 'fingerprint' for each of t he cell lines assessed, which will allow meaningful comparison between simi lar cell lines held in other laboratories.