Figure 7.
Inhibition of cellular proliferation by WT1 requires association with Hsp70. (A) Inhibition of colony formation in U2OS and Saos-2 cells following transfection of wild-type WT1, the amino-terminal truncation WT1–Δ6–180, DNAJ–WT1, H42Q–DNAJ–WT1 (encoding a point mutation within the DNAJ domain), and DNAJ–WT1–delZ (encoding a truncation of the WT1 DNA binding domain). All WT1 constructs (except DNAJ–WT1–delZ) encoded the (−KTS) splicing variant, with an uninterrupted DNA binding domain, which has been linked to inhibition of cellular proliferation in these osteosarcoma cells. Cells were cotransfected with a construct encoding puromycin resistance and drug resistant colonies were counted after 14 days (U2OS) or 21 days (Saos-2) in culture. Standard deviations are given. (B) Absence of correlation between transcriptional repression or promoter-reporter constructs and inhibition of colony formation by WT1 variants. U2OS cells were transfected with WT1 and DNAJ–WT1 constructs, along with either the GC-rich EGR1–CAT or the TC-rich EGFR–CAT promoter reporters. Representative experiments are shown, with transcriptional repression quantitated by scintillation counting. (C) G1 phase cell cycle arrest induced by wild-type WT1 and DNAJ–WT1. Saos-2 cells were transiently transfected with WT1 and DNAJ–WT1 constructs, along with a construct encoding the cell surface marker CD20. CD20-expressing transfectants were identified by FACS analysis, and their cell cycle distribution was determined by staining with propidium iodide. A representative experiment is shown. (D) Induction of p21 by wild-type WT1 and DNAJ–WT1. Saos-2 cells were transiently transfected with constructs encoding WT1 and DNAJ–WT1 variants. Cellular extracts were isolated 24 hr after transfection, and equal amounts of lysates were analyzed by immunoblotting by use of antibody against p21. Saos-2 cells have a deletion of endogenous p53 and express low levels of endogenous p21; their high transfection efficiency makes it possible to analyze the induction of the native p21 gene following transient transfection of expression constructs. As a control, induction of p21 is shown following transient transfection of CMV-driven p53 (1 μg).