ARKO Human Prostate Cancer CWR22R Cells

The advantage of using gene targeting in human somatic cells is that it provides a tool to study the roles of the signaling of interest in human cells instead of ARKO animal model. For this purpose, we attempted to disrupt AR signaling in human androgen-refractory prostate cancer cells in order

TRAMP 3/3 tumor at 20th week

ARKO-TRAMP 1/3 tumor at

TRAMP 2/2 big tumor at

ARKO-TRAMP 2/2 small tumor at 28th week

20t week

TRAMP 3/3 tumor at 20th week

ARKO-TRAMP 1/3 tumor at

TRAMP 2/2 big tumor at

ARKO-TRAMP 2/2 small tumor at 28th week

20t week

Fig. 2. Model for development of inducible ARKO-TRAMP mice.

to investigate the biological importance of AR signaling in those prostate cancers. Human prostate cancer CWR22R cells were transfected with the AR targeting vector using Superfect transfection kit (Qiagen) and selected with 400 pg/ml neomycin reagent.18 The genotypes of the surviving clones were detected by Southern blotting using a DNA probe containing the 5'-UTR sequences of the AR gene. The untargeted and targeted loci produced approximately 9.0-kb and 3.46-kb bands, respectively. AR expression was reduced in those heterozygous clones compared to that in the parental CWR22R cells19 confirming that one of the AR genes is targeted by homologous recombination. Therefore, this method provides a promising result and indicates that it is possible that we could target AR genes in any other cell lines of interest. Efforts to obtain homologous AR-null CWR22R cells failed after extensive screening, although homologous

AR-null MCF cells were successfully established. This suggests that AR is an essential survival factor in prostate cancer cells. The essential role of AR in androgen-refractory prostate cancer has been depicted in many other studies.20'21 In order to knockout both AR genes, we are trying to stably transfect the tetracycline-inducible AR construct into heterozygous CWR22R cells and then target the second allele of AR in the cells. We believe this will provide a powerful tool for us to manipulate AR expression in the prostate cancer studies.

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