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Activation of Cre recombinase alone can induce complete tumor regression.


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Activation of Cre Recombinase Alone Can Induce

Complete Tumor Regression

Yulin Li, Peter S. Choi, Stephanie C. Casey, Dean W. Felsher*

Department of Medicine, Division of Oncology, School of Medicine, Stanford University, Stanford, California, United States of America


The Cre/loxP system is a powerful tool for generating conditional genomic recombination and is often used to examine the mechanistic role of specific genes in tumorigenesis. However, Cre toxicity due to its non-specific endonuclease activity has been a concern. Here, we report that tamoxifen-mediated Cre activation in vivo induced the regression of primary lymphomas in p532/2 mice. Our findings illustrate that Cre activation alone can induce the regression of established tumors.

Citation:Li Y, Choi PS, Casey SC, Felsher DW (2014) Activation of Cre Recombinase Alone Can Induce Complete Tumor Regression. PLoS ONE 9(9): e107589. doi:10.1371/journal.pone.0107589

Editor:Robert Oshima, Sanford Burnham Medical Research Institute, United States of America ReceivedJune 13, 2014;AcceptedAugust 13, 2014;PublishedSeptember 10, 2014

Copyright:ß2014 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability:The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper. Funding:The research was supported by grants from National Cancer Institute (R01CA170378, U54CA149145) and the Leukemia and Lymphoma Society Translational Research grant R6223-07 (DF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests:The authors have declared that no competing interests exist. * Email: dfelsher@stanford.edu


The Cre/loxP system has been widely used to induce tissue- and developmental stage- specific genomic recombination [1,2]. Cre/ loxP is a particularly tractable strategy to examine the role of gene activation or inactivation in the initiation and maintenance of tumorigenesis. However, Cre activation can also induce non-specific genomic recombination. In some cases, Cre activation has been shown to induce significant cellular toxicity associated with marked cell death in cell culture [3–5]. This toxicity is not seen in Cre mutants that do not have endonuclease activities [3].

Similarly, Cre activationin vivo in mouse models can induce toxicity in normal cellular lineages. For example, in mouse lines with Cre expressed in neuronal progenitors, defects have been observed in brain development [6]. Myocardial-specific Cre activation with tamoxifen in the aMHC-MERCreMER mice

induces cardiac fibrosis and heart failure [7,8]. Furthermore, systemic Cre activation with tamoxifen in theRosa26-CreERT2

mice results in thymic atrophy and severe hematological toxicity [9].

The Cre/loxP system has been used to interrogate the role of specific genes in tumorigenesis [2]. Although most studies have presumed that Cre does not have effects on tumorigenesis, one report described that Cre expression blocked tumor formation in a mouse model of lymphoma transplantation [10]. Here we report that Cre activation resulted in the regression of primary lymphoma induced by p53 deficiency. Our results have implica-tions for the use of the Cre/loxP system for tumorigenesis studies.


Lymphoma model

All animal work was approved by the Stanford IACUC committee (protocol number 14045) and follows AAALAC guidelines. The p53 deficient mice (p532/2) were used as the spontaneous lymphoma model. The p532/2 mice were main-tained in the FVB/N background and the majority of them developed malignant lymphoma within 6 months of age [11]. The

UBC-Cre-ERT2mice carrying the transgenicCre-ERT2controlled by the Ubiquitin C (UBC) promoter were maintained in the 129S6 background [12]. Thep532/2mice were crossed with the UBC-Cre-ERT2mice to derive theUBC-Cre-ERT2; p532/2mice.

Conditional Cre activation

For in vivo activation of Cre, the mice were gavaged with tamoxifen (200 mg/kg, once daily, with one day off after 4 consecutive doses) for 9 days. Forin vitro activation of Cre, the

p532/2lymphoma cell lines carrying eitherMSCVempty vector or MSCV-Cre-ERT2 were treated with 1 micromolar of 4-hydroxytamoxifen (Sigma, H7904) for 48 hours.

MRI imaging

Lymphoma development in the mice was monitored using magnetic resonance imaging (MRI). Once the lymphoma was established, mice were treated with tamoxifen. The tumors were imaged before treatment and 12–16 days after the start of treatment with a 7T MRI system (T2-weighed fast spin echo) at Stanford small animal imaging facility. The MRI image stacks were analyzed with the Osirix image application to derive the tumor volumes.


Flow cytometric analysis of apoptosis

For detection of apoptosis, the lymphoma cells were stained with Annexin V and 7-AAD and analyzed with a FACSCalibur (BD Biosciences). The apoptotic cell populations were visualized with FlowJo (Treestar).

Immunohistochemical analysis of apoptosis

For detection of apoptosisin vivo, the thymic lymphomas from mice treated with tamoxifen were fixed, paraffin-embedded and sectioned. Tissue slides were stained with a cleaved-Caspase 3 antibody (Cell Signaling#9661) following manufacturer’s instruc-tions. The slides were developed with DAB (Vector Laboratories) and also counterstained with hematoxylin.

Results and Discussion

The p53 deficient mice (p532/2) were used as the spontaneous lymphoma model. As previously reported, the p532/2 mice developed malignant lymphoma within 6 months of age [11]. The

UBC-Cre-ERT2was introduced into thep532/2background by crossing. Upon Cre activation with tamoxifen treatment, the temporal regression ofUBC-Cre-ERT2; p532/2lymphoma was observed in multiple independent tumors as measured by MRI imaging (Figure 1). Tumor volume quantification using MRI image stacks showed that the size of theUBC-Cre-ERT2; p532/2 lymphoma was reduced to 0%–30% of the pretreatment level (Figure 2). The regression of the tumors was also visually Figure 1. Changes in tumor volume upon tamoxifen treatment

in controlp532/2andUBC-Cre-ERT2; p53

2/2mice as shown by MRI imaging. The coronal sections of the thymic lymphoma were shown with tumors labeled with white asterisks. The letter H denotes the location of heart. Post-treatment scans were performed 14 days after starting tamoxifen treatment.


Figure 2. Summary of tumor volume changes upon tamoxifen treatment. Relative tumor volume was calculated by dividing post-treatment tumor volume by pre-post-treatment tumor volume. Unpaired t test, p,0.001. The code number of each mouse is labeled on the x-axis. doi:10.1371/journal.pone.0107589.g002

Figure 3.In vitroCre-ERT2activation with tamoxifen inp532/2

lymphoma cells results in apoptosis. Cells were treated in triplicates with 1 micromolar 4-hydroxytamoxifen for 48 hours and analyzed with flow cytometry after Annexin V/7-AAD staining. * Paired t test, p,0.005.


Figure 4.In vivoCre-ERT2activation with tamoxifen results in apoptosis in primary p532/2 lymphoma. Mice with thymic lymphomas were treated with tamoxifen. Tumor sections were stained with a cleaved-Caspase 3 antibody. Apoptotic cells were stained brown color. The numbers in the top right corners represent the percentage of apoptotic cells. Data are presented as mean + standard deviation. Paired t test p,0.01, for Cre-ERT2post-treatment versus pre-treatment.


Activation of Cre Recombinase Induces Tumor Regression


confirmed by postmortem dissection. In contrast, the volume of the p532/2 lymphoma without UBC-Cre-ERT2 transgene increased 4–6 fold despite tamoxifen treatment (Figure 2).

To investigate the mechanism of lymphoma regression, we tested whether Cre activation could induce apoptosis in ap532/2 lymphoma cell line derived from the mouse model.Cre-ERT2was overexpressed in the p532/2 lymphoma cell line with the retroviral Murine Stem Cell Virus (MSCV-Cre-ERT2). Upon Cre activation in cell culture with one micromolar of 4-hydroxytamoxifen, there was a four-fold increase of apoptotic cells (11% to 42%) as shown by flow cytometric analysis after Annexin V/7-AAD staining. In contrast, the control p532/2 lymphoma cell line carrying an emptyMSCVvector did not show significant changes in apoptosis rate (Figure 3). To further test whether Cre activation could induce apoptosisin vivo, we stained the p532/2 thymic lymphomas using a cleaved-Caspase 3 antibody. Tamoxifen treatment induced a significant increase in cleaved-Caspase 3 staining (1% to 18%) in the p532/2; UBC-Cre-ERT2tumors but only moderate changes in the controlp532/

2tumors (Figure 4). Hence, Cre activation induced significant cell death which can contribute to thein vivotumor regression in the

p532/2mouse model.

Our results are the first to demonstrate that Cre activation alone can induce the regression of established primary tumorsin vivo. We found that Cre activation can induce marked apoptosis in

p532/2lymphoma. Our results are consistent with prior reports that Cre activation can result in toxicity in normal tissues [6–9]. The most likely explanation for our finding is that Cre activation induces genomic rearrangement associated with the cryptic loxP sites within the mouse genome [9]. We recognize that the effects of Cre are likely dose- and duration-dependent.

Our findings reinforced the previous notion that, for studies with the Cre/loxP system, an experimental control using mice with Cre expression but without the loxP sites should always be included for the possibility that Cre activation can markedly perturb the initiation and progression of tumorigenesis and even induce the regression of established tumors [10].

Author Contributions

Conceived and designed the experiments: YL DF. Performed the experiments: YL PC SC. Analyzed the data: YL SC. Contributed to the writing of the manuscript: YL DF.


1. Gu H, Zou YR, Rajewsky K (1993) Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73: 1155–1164.

2. Rajewsky K, Gu H, Kuhn R, Betz UA, Muller W, et al. (1996) Conditional gene targeting. J Clin Invest 98: 600–603.

3. Loonstra A, Vooijs M, Beverloo HB, Allak BA, van Drunen E, et al. (2001) Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci U S A 98: 9209–9214.

4. Pfeifer A, Brandon EP, Kootstra N, Gage FH, Verma IM (2001) Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo. Proc Natl Acad Sci U S A 98: 11450–11455.

5. Baba Y, Nakano M, Yamada Y, Saito I, Kanegae Y (2005) Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells. Microbiol Immunol 49: 559–570.

6. Forni PE, Scuoppo C, Imayoshi I, Taulli R, Dastru W, et al. (2006) High levels of Cre expression in neuronal progenitors cause defects in brain development leading to microencephaly and hydrocephaly. J Neurosci 26: 9593–9602.

7. Bersell K, Choudhury S, Mollova M, Polizzotti BD, Ganapathy B, et al. (2013) Moderate and high amounts of tamoxifen in alphaMHC-MerCreMer mice induce a DNA damage response, leading to heart failure and death. Dis Model Mech 6: 1459–1469.

8. Lexow J, Poggioli T, Sarathchandra P, Santini MP, Rosenthal N (2013) Cardiac fibrosis in mice expressing an inducible myocardial-specific Cre driver. Dis Model Mech 6: 1470–1476.

9. Higashi AY, Ikawa T, Muramatsu M, Economides AN, Niwa A, et al. (2009) Direct hematological toxicity and illegitimate chromosomal recombination caused by the systemic activation of CreERT2. J Immunol 182: 5633–5640. 10. Schmidt-Supprian M, Rajewsky K (2007) Vagaries of conditional gene targeting.

Nat Immunol 8: 665–668.

11. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA Jr, et al. (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356: 215–221.

12. Ruzankina Y, Pinzon-Guzman C, Asare A, Ong T, Pontano L, et al. (2007) Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss. Cell Stem Cell 1: 113–126.

Activation of Cre Recombinase Induces Tumor Regression


Figure 3. In vitro Cre-ER T2 activation with tamoxifen in p53 2 / 2 lymphoma cells results in apoptosis


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