The cyclin E regulator cullin 3 prevents mouse hepatic progenitor cells from becoming tumor-initiating cells
Uta Kossatz, … , Jeffrey D. Singer, Nisar P. Malek
Uta Kossatz, … , Jeffrey D. Singer, Nisar P. Malek
Published October 11, 2010
Citation Information: J Clin Invest. 2010;120(11):3820-3833. https://doi.org/10.1172/JCI41959.
View: Text | PDF
Research Article Oncology

The cyclin E regulator cullin 3 prevents mouse hepatic progenitor cells from becoming tumor-initiating cells

Abstract

Cyclin E is often overexpressed in cancer tissue, leading to genetic instability and aneuploidy. Cullin 3 (Cul3) is a component of the BTB-Cul3-Rbx1 (BCR) ubiquitin ligase that is involved in the turnover of cyclin E. Here we show that liver-specific ablation of Cul3 in mice results in the persistence and massive expansion of hepatic progenitor cells. Upon induction of differentiation, Cul3-deficient progenitor cells underwent substantial DNA damage in vivo and in vitro, thereby triggering the activation of a cellular senescence response that selectively blocked the expansion of the differentiated offspring. Positive selection of undifferentiated progenitor cells required the expression of the tumor suppressor protein p53. Simultaneous loss of Cul3 and p53 in hepatic progenitors turned these cells into highly malignant tumor-initiating cells that formed largely undifferentiated tumors in nude mice. In addition, loss of Cul3 and p53 led to the formation of primary hepatocellular carcinomas. Importantly, loss of Cul3 expression was also detected in a large series of human liver cancers and correlated directly with tumor de-differentiation. The expression of Cul3 during hepatic differentiation therefore safeguards against the formation of progenitor cells that carry a great potential for transformation into tumor-initiating cells.

Authors

Uta Kossatz, Kai Breuhahn, Benita Wolf, Matthias Hardtke-Wolenski, Ludwig Wilkens, Doris Steinemann, Stephan Singer, Felicitas Brass, Stefan Kubicka, Brigitte Schlegelberger, Peter Schirmacher, Michael P. Manns, Jeffrey D. Singer, Nisar P. Malek

×

Figure 4

Accumulation of cyclin E in differentiating hepatic progenitor cells is responsible for the accumulation of DNA damage.

Options: View larger image (or click on image) Download as PowerPoint
Accumulation of cyclin E in differentiating hepatic progenitor cells is ...
(A) Cyclin E half-lives during differentiation and in asynchronously proliferating cells. At 24 hours of differentiation, cyclin E accumulates (experiments were performed in triplicate). (B) Overexpression of myc-tagged cyclin E in asynchronously growing cells from Cul3loxP/loxP AlfpCre mice and quantitation of DNA damage (γ-H2AX). Immunofluorescence for myc tagged (MT)-cyclin E, γ-H2AX, and DAPI. Arrows indicate cyclin E–overexpressing cell positive for γ-H2AX (pink arrow) and an untransfected cell (white arrow) (n = 3). (C) Treatment of cells from Cul3loxP/loxP AlfpCre mice with H2O2 and analysis of DNA damage by γ-H2AX and pChk1 Western blots. (D) Measurement of cyclin E half-lives upon siRNA-mediated knockdown of Fbw7 and Cul1 in asynchronously proliferating cells. Loss of Fbw7 and Cul1 increases cyclin E protein stability (n = 3/experiment). CHX, cycloheximide. (E) Confirmation of siRNA-mediated knockdown by RT-PCR. GAPDH was used as control. Noncontiguous lanes from the same gel were spliced. (F) Increased DNA damage as measured by γ-H2AX staining upon knockdown of Fbw7 and Cul1. For identification of transfected cells, a labeled siRNA control was cotransfected. (G) siRNA-mediated knockdown of cyclin E during differentiation and determination of DNA damage (γ-H2AX). Knockdown of cyclin E was confirmed by Western blotting. The arrow indicates the cyclin E band. c., scrambled siRNA control. Actin was used as a loading control and analysis performed as noted in Methods. Noncontiguous lanes from the same blot were spliced. The graph shows quantification of γ-H2AX–positive cells transfected with the indicated siRNAs 96 hours after the induction of differentiation (n = 3). (H) RT-PCR analysis of Fbw7 and Cul3 Δ3–7 expression during differentiation of progenitor cells into hepatocytes at indicated time points. GAPDH was used as a control. *P < 0.05.