Title

Phenotypic and Genotypic characterization of commonly used Human Prostatic Cell lines

Document Type

Article

Department

Pathology and Microbiology

Abstract

Objective: To investigate and catalogue systematically the phenotypic and genotypic characteristics of the commonly used prostatic cell lines using immunocytochemistry and polymerase chain reaction (PCR) of hypervariable sequences within the genome to provide a ‘fingerprint’ characteristic of each cell line.

Materials and Methods: Malignant (LNCaP, LNCaP-r, PC-3, DU-145) and benign immortalized prostatic cell lines (PNT-1A, PNT-1B, BPH-1) were grown on four-well slides, fixed and subjected to indirect streptavidin-biotin immunocytochemistry. Twenty-three antibodies were used in the following groups: cytoskeletal elements: cytokeratins (CK)-5, -7, -8, -14 (two), -16, -18, -19 (three), -20, vimentin and desmin; MUC1 (three); cell adhesion molecules (E-cadherin, α-β-and γ-catenin); and prostatic associated proteins: prostate specific antigen (PSA), prostatic acid phosphatase (PAP) and androgen receptor (AR). For the PCR, genomic DNA was extracted from the cell lines and from SKOV3 and MCF7 (positive controls). PCR was performed on three variable regions which were then sequenced: AR exon 1 (CAG repeat polymorphism), and two areas of microsatellite instability (MSI): AR exon 8 and hypoxanthine-guanine phosphoribosyl transferase (HPRT) exon 3.

Results: All cell lines were CK-8/18 positive and most also expressed CK-7 and -19. Heterogeneous CK-20 expression was detected for the first time in prostatic cell lines. All lines were positive for vimentin and negative for desmin. MUC1 was expressed in one malignant (DU-145) and all immortalized cell lines. E-cadherin expression was low or absent in three lines: PNT1A, 1B and PC-3. Only PC-3 failed to express α-catenin; β- and γ-catenin were expressed by all lines. PSA, PAP and AR were only expressed by LNCaP and LNCaP-r. On PCR, the CAG repeat lengths in exon 1 of the AR ranged from 19 to 27. Three pairs of cell lines had the same exon 1 CAG repeat length: LNCaP/PC-3 (26 repeats), BPH-1/DU-145 (19 repeats) and PNT1 A/1B (20 repeats). Exon 8 sequences were identical except for LNCaP, which showed a single 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 to their phenotypic characteristics: (i) prostatic marker positive (LNCaP and 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 their different phenotypic characteristics. PNT1A and 1B had identical genetic and similar phenotypic profiles, which is unsurprising given that they are subclones derived from the same parental line. Even so, these were separable 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 the cell lines assessed, which will allow meaningful comparison between similar cell lines held in other laboratories.

Publication

BJU International