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 November 1, 2010; First published October 11, 2010
Citation Information: J Clin Invest. 2010;120(11):3820-3833. https://doi.org/10.1172/JCI41959.
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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

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Figure 3

In vitro differentiation of progenitor cells recapitulates the in vivo phenotype.

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In vitro differentiation of progenitor cells recapitulates the in vivo p...
(A) RT-PCR analysis of progenitor cell– and hepatocyte-specific markers in asynchronous (as) cells and at different time points during differentiation (Diff.). Noncontiguous lanes from the same gel were spliced. (B) Quantitation of γ-H2AX staining by immunofluorescence at the indicated time points after the induction of differentiation. At least 3 independent experiments were analyzed. Asynchronously growing cells were set as 100%. (C) Quantitation of cells in S phase by BrdU uptake (experiments were performed in triplicate). (D) Hepatic progenitor cells induce senescence upon differentiation. Western blot analysis during differentiation shows a strong accumulation of the senescence-associated marker p15. Progenitor cells from Cul3loxP/loxP AlfpCre mice stain positive for β-galactosidase at pH 5.8 at 96 hours after the induction of differentiation. All experiments were repeated 3–4 times. Actin was used as a loading control and analysis performed as noted in Methods. Noncontiguous lanes from the same blot were spliced. (E) Analysis of proliferation of CD34-positive cells during differentiation by quantification of CD34/BrdU. Proliferation of CD34-positive cells in asynchronously growing culture is also shown (n = 2). (F) Immunofluorescence γ-H2AX staining of CD34-positive cells at 96 hours of differentiation. Squares indicate CD34-positive cells, showing that these do not accumulate DNA damage. Scale bars: 50 μm. *P < 0.05, **P < 0.005.