strand breaks in mammalian cells

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Cyclin A overexpression induces chromosomal doublestrand breaks in mammalian cells Shoji Tane & Taku Chibazakura Published online: 01 Dec 2009.

To cite this article: Shoji Tane & Taku Chibazakura (2009) Cyclin A overexpression induces chromosomal double-strand breaks in mammalian cells, Cell Cycle, 8:23, 3900-3903, DOI: 10.4161/cc.8.23.10071 To link to this article: http://dx.doi.org/10.4161/cc.8.23.10071

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Cell Cycle 8:23, 3900-3903; December 1, 2009; © 2009 Landes Bioscience

Cyclin A overexpression induces chromosomal double-strand breaks in mammalian cells Shoji Tane and Taku Chibazakura* Department of Bioscience; Tokyo University of Agriculture; Setagaya-ku, Tokyo Japan

Keywords: cyclin A, Cdk2, γ-H2AX, DNA double-strand breaks, Cdk activity

Downloaded by [93.183.155.39] at 16:26 26 August 2015

Cyclin A is a major regulator in vertebrate cell cycle, associated with cyclin-dependent kinase (Cdk), and involved in Sphase progression and entry into mitosis. It has been known that cyclin A overexpression not only causes premature Sphase entry but also induces prolongation of S phase. Here we show that ectopic expression of cyclin A leads to extensive γ-H2AX focus formation, which is indicative of DNA double-strand breaks. Likewise, cyclin E, but not cyclin B1 and cyclin D1, also induced the γ-H2AX focus formation, suggesting that these DNA lesions may be induced via aberrant DNA replication process. Moreover, the γ-H2AX focus formation was suppressed by co-expressing p21Cip1/Waf1 or dominant-negative Cdk2 mutant, suggesting that aberrant cyclin A-Cdk2 activation induces the chromosomal double-strand breaks.

Introduction

Results

Cyclins are the major regulators in the mammalian cell cycle, together with their catalytic subunits, cyclin-dependent kinases (Cdks). To ensure the accurate cell division and genomic integrity, the cyclin proteins are controlled by transcriptional and post-translational events. The balance between transcription and protein turnover gives a unique periodicity to each cyclin expression and enables characteristic events to occur in each cell cycle phase. Disruption of the balance is harmful, and induces many disorders including carcinogenesis, abnormal proliferation,1 aneuploidy2-4 and DNA rereplication.5 Although deregulation of the positive regulators for cell division often contributes to cellular transformation, enforced expression of an oncogene in normal diploid cells occasionally induces growth arrest, apoptosis or senescence rather than transformation. Similar phenotypes have been observed in precancerous lesions,6-9 thus they act partly as anti-cancer barriers to protect cells from tumorigenesis. It has been known that the anti-cancer barriers include the activation of DNA damage checkpoint which is triggered by DNA double-strand breaks (DSBs) or the generation of extensive single-stranded DNAs (ssDNAs).6-9 However, little is known how oncogenes induce DNA lesions. Although deregulated G1 cyclins have been known to cause aberrant DNA replicative machinery-mediated genetic stresses,5,10 DNA abnormalities by enforced expression of cyclin A has not been investigated despite its effect on replicative components during S phase11,12 and genomic instability.13 Here, we show that excess cyclin A induces DSBs, which are detected as the γ-H2AX foci. Also, the γ-H2AX focus formation depends on Cdk activity, suggesting that the cyclin A-induced DNA lesions are mediated by the unscheduled phosphorylation of Cdk target protein(s).

Overexpression of cyclin A induces phosphorylation of histone H2AX. To monitor whether overexpression of cyclin A induces DNA damage, several mammalian cultured cells were transiently transfected with the cytomegalovirus (CMV) promoterdriven expression vector carrying myc-tagged LacZ or cyclin A, and co-immunostained with the antibodies against myc tag and γ-H2AX, the phosphorylated form of histone H2AX, which is known as a good marker for DNA lesions.14 Since recent reports have revealed that histone H2AX is phosphorylated without DNA damage during M phase,15,16 we excluded mitotic cells (judged from their morphology) from these assays. In contrast to myc-tagged LacZ-positive cells and untransfected cells, myctagged cyclin A-positive cells exhibited remarkable increase in the γ-H2AX foci (Fig. 1A). Since similar results were observed in both mouse and human cell lines (Fig. 1B), we suggest that increased γ-H2AX focus formation by cyclin A overexpression is a general effect in mammalian cells. Next, to ascertain whether γ-H2AX focus formation via cyclin overexpression is a cyclin A-specific effect, we performed the same experiment as above with other major cyclins in mouse embryonic fibroblasts (MEFs). As reported previously,17 the exogeneous cyclin B1 mainly remained in cytoplasm in interphase cells, and did not induce γ-H2AX focus formation (Fig. 2). Likewise, cyclin D1-overexpressing cells exhibited no increase in the γ-H2AX foci.5 By contrast, enforced expression of cyclin E induced the extensive γ-H2AX focus formation on the chromatin, as observed for the cyclin A overexpression (Fig. 2). Requirement of cyclin A-associated Cdk activity for induction of γ-H2AX focus formation. A recent work has revealed that cyclin E promotes the recruitment of MCM complex onto

*Correspondence to: Taku Chibazakura; Email: [email protected] Submitted: 08/24/09; Accepted: 09/14/09 Previously published online: www.landesbioscience.com/journals/cc/article/10071 3900

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experiments to suppress cyclin A or E-associated kinase activity. First, we checked the γ-H2AX focus formation in the NIH3T3 cells co-transfected with cyclin A or cyclin E, with or without p21Cip1/Waf1, an inhibitor of cyclin-Cdk complex.19 With increasing amounts of the p21Cip1/Waf1-expressing vector, the γ-H2AX focus formation in the cells overexpressing myc-tagged cyclin A or E was suppressed (Fig. 3A). By contrast, the cells overexpressing myc-tagged LacZ exhibited only background focus intensities both in the presence and absence of p21Cip1/Waf1. Secondly, we examined the effect of dominant-negative Cdk2 mutant (dn-Cdk2), which is able to bind to cyclins but is incapable of catalyzing the phosphotransfer reaction.20 NIH3T3 cells were transiently co-transfected with the LacZ, cyclin A or cyclin E expression vector, together with wild-type Cdk2 or the dn-Cdk2 expression vector, and then immunostained for the γ-H2AX signals. As expected, approximately 70% of the myc-tagged cyclin A- and cyclin E-positive cells exhibited γ-H2AX signals when co-transfected with the empty vector or the wildtype Cdk2-expressing vector (Fig. 3B). By contrast, when co-transfected with dn-Cdk2, the γ-H2AX focus formation induced by cyclin A or cyclin E was drastically reduced to the background level. Taken together, these results suggest that overexpression of cyclin A or E induces the γ-H2AX focus formation in a Cdk activity-dependent manner. Discussion Ectopic overexpression of cyclin A or E accelerates the transition from G1 to S phase and increases the S-phase population.13,21,22 Previous works have also shown that the cells overexpressing these cyclins exhibit prolonged S phase duration as well as premature S phase entry, suggesting that overexpression of cyclin A or E causes a delay in the S phase progression.21,22 Here we demonstrate that overexpression of cyclin A or E, but not B1 or D1, induces the γ-H2AX focus formation. Thus, the delay in S phase progression could partly be due to the increase in DNA Figure 1. Ectopic expression of cyclin A induces γ-H2AX focus formation. (A) lesions caused by the cyclin overexpression. WI-38 cells (a), mouse embryonic fibroblasts (MEFs, b) and NIH3T3 cells (c) were Cyclin E has been reported to facilitate MCM transiently transfected with pCSMTlacZ (LacZ) or pCSMTcycA (CycA) plasmid and coimmunostained with DAPI and antibodies against myc tag (red) and γ-H2AX loading onto chromatin during the G0-to-S transi(green). (B) Indicated cells were transfected and treated as in (A), and approximately tion via its Cdk-independent function.18 In contrast 200 each of the transfected (Myc tag +) and non-transfected (Myc tag -) cells were to that, the γ-H2AX focus formation induced by counted for the γ-H2AX signal. The mean ± standard derivations of the percentages cyclin A or E overexpression depends on the Cdk of γ-H2AX-positive cells from triplicate experiments are shown. activity since it was suppressed under the conditions compromising the Cdk activity (Fig. 3). We chromatin during re-entry into cell cycle from G0 phase inde- also confirmed that purified histone H2AX protein is not phospendently of Cdk activity.18 However, it is not clear whether any phorylated in vitro by cyclin A-associated Cdk (data not shown), Cdk-independent function of cyclin is involved in other cyclin- indicating that histone H2AX is not a direct substrate for Cdk. induced events. To ask if Cdk activity is required for inducing Thus, it is likely that overexpression of cyclin A or E induces the the γ-H2AX focus formation, we performed the following two chromosomal DSBs via unscheduled phosphorylation of certain

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Figure 2. Overexpression of cyclin E, but not cyclin B1 or D1, induces γ-H2AX focus formation. MEFs were transiently transfected with the expression plasmids for the indicated cyclins and examined for immunofluorescence as described in Figure 1A.

Cdk target protein(s). However, physiological consequences of cyclin A and E overexpression in terms of cellular transformation might be different. While a non-degradable mutant of cyclin E causes genomic instability in the absent of p21Cip1/Waf1,2 a stabilized cyclin A mutant induces tetraploidization only when cells lack p21Cip1/Waf1, p27Kip1 and p107.13 It has been known that deregulated cyclin E causes multiple malignant phenomena, via interfering with prereplication complexes (pre-RCs) in G1,10 and activating checkpoint through replicational stress.6-8 The DNAdamaging activity of cyclin A may partly overlap with the activity of cyclin E, especially in these DNA replication-associated events. As noted above, DNA lesions induced by cyclin A overexpression could activate checkpoint response and therefore might function as anti-cancer barriers, similar to certain cases with oncogenes. However, if the checkpoint response is defective, which is widely observed in cancer cells, the aberrant cyclin A-Cdk activation might promote genomic instability as results of accumulated DNA lesions and contribute to tumorigenesis. Regarding this point, it would be very important to check if cyclin A overexpression causes any genetic alterations in the checkpoint-defective (e.g., p53-negative) backgrounds. Materials and Methods Cells. WI-38 cells were obtained from RIKEN Bioresource Center Cell Bank (Tsukuba, Japan). NIH3T3 cells were provided by C. Sherr (St. Jude Children’s Hospital, Memphis, TN). Cells were cultured on 22-mm coverslips in Dulbecco’s Modified Eagles medium (DMEM; Invitrogen, Carlsbad, CA) containing 10% fatal bovine serum (FBS; Tissue Culture Biologicals, Seal Beach, CA), 1% penicillin-streptomycin-glutamine, and 0.2% fungizone (Invitrogen). Plasmids and transfection. Mammalian expression vector for myc-tagged human cyclin A2 (pCSMTcycA) was previously

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Figure 3. Cdk2 activity is required for the induction of γ-H2AX focus formation by overexpressionn of cyclin A or E. (A) Suppression of the cyclin A and cyclin E-induced γ-H2AX focus formation by p21Cip1/Waf1. NIH3T3 cells were transfected with combination of 1 μg of the pCSMTLacZ, -CycA or -CycE vector and indicated amounts of the p21Cip1/Waf1 expression vector. The mean ± standard derivations from triplicate experiments are shown. (B) Suppression of the cyclin A and cyclin E-induced γ-H2AX focus formation by dominant-negative Cdk2 (dn-Cdk2). NIH3T3 cells were transfected with 5 μg of the pCSMT-LacZ, -CycA or -CycE vector and 10 μg of the empty vector, the wild-type Cdk2, or the dn-Cdk2 expression vector. The mean ± standard derivations from triplicate experiments are shown.

described.13 Myc-tagged LacZ expression vector (pCSMTlacZ) was constructed by subcloning the lacZ coding region from pMC1871 (Amersham, Uppsala, Sweden), with addition of NcoI site and initiation ATG codon, into the NcoI site in pCSMT.23 Expression vectors for cyclins B1, D1, E1 and p21Cip1/Waf1 were obtained from B. Clurman (Fred Hutchinson Cancer Research Center, Seattle, WA). And those for HA-tagged wild-type Cdk2 and dn-Cdk2 were provided by S. van den Heuvel (Utrecht University, Netherland).20 Cells were transiently transfected with 5 μg of the indicated plasmids per 60-mm dish using the modified calcium phosphate

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method.24 16 h after transfection, the cells were refed with fresh medium and cultured for 24 h, followed by fixation for immunofluorescent microscopy. Indirect immunofluorescence. To examine fluorescence microscopy, the cells cultured on coverslips were rinsed with Dulbecco’s phosphate-buffered saline (PBS) and immediately fixed with 4% paraformaldehyde in PBS for 10 min in the 6-well plates. After the fixed cells were permeabilized with 0.2% Triton X-100 for 5 min, they were blocked with 1% BSA and 20% heat-inactivated goat serum (Invitrogen) in PBS for 30 min. The blocked cells were labeled with the primary antibodies against myc tag (rabbit polyclonal; MBL, Nagoya, Japan) and γ-H2AX (mouse monoclonal; Upstate Biotechnology, Lake Placid, NY) for 1 h, and washed three times with PBS, each time for 5 min. References


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Then the cells were labeled with FITC-conjugated anti-mouse and rhodamine-conjugated anti-rabbit secondary antibodies (MP Biomedicals, Inc., Aurora, OH), washed three times with PBS, and stained with 0.2 μg/ml DAPI for 5 min. Finally, the coverslips were rinsed once with distilled water and mounted on slide glasses using 10 μl of ProLong Pro Antifade Reagent (Invitrogen) per coverslip. All steps were performed at room temperature. Acknowledgements

We thank B. Clurman, S. van den Heuvel, C. Sherr, and RIKEN Bioresource Center for the materials. This work was supported by Nodai Advanced Research Project (Tokyo University of Agriculture).

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