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[Cancer Research 61, 4990-4993, July 1, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

DNA Damage-activated Kinase Chk2 Is Independent of Proliferation or Differentiation Yet Correlates with Tissue Biology1

Claudia Lukas, Jirina Bartkova, Lucia Latella, Jacob Falck, Niels Mailand, Tine Schroeder, Maxwell Sehested, Jiri Lukas and Jiri Bartek2

Institute of Cancer Biology, Danish Cancer Society [C. L., J. Bartk., L. L., J. F., N. M., T. S., J. L., J. Barte.], and Department of Pathology, University Hospital [J. F., M. S.], DK-2100 Copenhagen, Denmark


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
The Chk2 kinase is a tumor suppressor and key transducer of DNA-damage checkpoints. We show that the human Chk2 protein is relatively stable, nuclear, and responding to {gamma}-radiation throughout the cell cycle. Contrary to the retinoblastoma protein-regulated, labile Chk1 kinase restricted to S-G2 phases, Chk2 remains activatable even in quiescent and differentiating cells. In human tissues, Chk2 is homogeneously expressed in renewing cell populations such as epidermis or intestine, heterogeneous in conditionally renewing tissues, and absent or cytoplasmic in static tissues such as muscle or brain. These data highlight striking differences between Chk2 and Chk1 and show unexpected correlation of Chk2 expression with tissue biology.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
Maintenance of genomic integrity and prevention of tumor development depend, at least in part, on functional cell cycle checkpoints, surveillance cascades activated in response to DNA damage, or stalled DNA replication (1) . Among the intricate network of sensors, transducers, and effectors of DNA damage response pathways in mammalian cells, the Chk2/Cds1 (Chk2) kinase (2, 3, 4, 5) has recently attracted considerable attention because of its emerging roles in cell cycle checkpoint mechanisms capable of blocking the cell cycle in G1 (6 , 7) , S (8) , and G2-M phases (2 , 9) , as well as stimulating DNA repair (10) . To achieve these various functions, Chk2 becomes activated by the upstream kinase ataxia telangiectasia mutated upon DNA damage (11 , 12) , and it phosphorylates and regulates such key cellular targets as the p53 (6 , 7 , 9) and BRCA1 (10) tumor suppressors or the cell cycle-promoting phosphatases Cdc25A (8) and Cdc25C (2 , 4) . Consistent with these multiple roles in protecting genetic stability, Chk2 qualifies as a tumor suppressor, the loss-of-function mutations of which predispose to a wide spectrum of malignancies characteristic of the Li-Fraumeni syndrome (13) and occur also in subsets of sporadic tumors including carcinomas of the colon and lung (13 , 14) . Despite the rapid progress in the DNA damage field, there is only very limited information available on the expression, regulation, or function of mammalian Chk2 in relation to various cell cycle phases and in proliferating versus differentiated cells and tissues, critical aspects of Chk2 biology, which have been addressed in this study.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Plasmids and Mutagenesis.
Wild-type, full-length human Chk2 cDNA (2) was cloned in frame with the gene for glutathione transferase into pGEX 2TK plasmid. Purification of the glutathione S-transferase-Chk2 protein from bacteria was performed according to the manufacturer’s instructions (Pharmacia Upjohn). The pcDNA3 expression plasmid containing Myc-tagged wild-type human Chk2 was described (8) . Additional mutagenesis was performed by Quick-Change site-directed mutagenesis kit (Stratagene). In vitro transcription/translation reactions in rabbit reticulocyte lysate followed the procedure recommended by the manufacturer (Promega).

Antibodies and Immunochemical Methods.
Mouse MAbs3 DCS-270 and DCS-273 specific for Chk2 were raised using established hybridoma technology. DCS-270 and DCS-273 are of IgG2{alpha} and IgG1 isotypes, respectively. They show no cross-reactions with any other proteins on immunoblots of total cell lysates, and their applicability in diverse immunochemical methods is specified in the "Results and Discussion" section. Other antibodies included: MAb DCS-6 to cyclin D1 (15) , SC-8408 to Chk1 from Santa Cruz Biotechnology, and MAb 245 to pRb from PharMingen. Procedures for gel electrophoresis and immunoblotting were described previously, as was the immunofluorescence staining of cells cultured on coverslips (15, 16, 17) .

Cell Culture and Irradiation.
Human normal diploid fibroblast strain BJ, human osteosarcoma cell line U-2-OS, and its derivative U-2-OS/A5C1 conditionally expressing nonphosphorylatable pRb (17) were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and antibiotics. The murine C2C12 myoblast cell line was cultured in DMEM supplemented with 10% fetal bovine serum. Differentiation was induced by starving the cells in serum-free medium and completed within 3 days, as monitored by morphology and other established characteristics as described (18) . Ionizing radiation was delivered by X-ray generator (RT100; Philips Medico; 100kV, 8 mA, dose-rate 0.92Gy min-1) and cell extracts prepared 1 h later except where indicated otherwise.

Tissues and Immunohistochemistry.
The human tissues were from the tissue banks of the Department of Pathology, University Hospital Copenhagen, the Institute of Cancer Biology, Copenhagen, or obtained commercially as the Checkerboard MultiTissue Blocks (DAKO) containing sections from a wide spectrum of normal human tissues, formalin fixed, paraffin-embedded, and mounted on poly-L-lysine-coated slides. The sensitive immunoperoxidase staining using the Vectastain Elite kit (Vector Laboratories) was performed as described previously (15) . For antigen unmasking, the deparaffinized sections were boiled in a microwave for 10 min in citrate buffer (0.01 M citric acid monohydrate, pH 6.0) before incubation with primary antibodies. Parallel sections stained with the DCS-270 and DCS-273 antibodies (against distinct epitopes of Chk2, see below) showed identical staining patterns, whereas nonimmune mouse immunoglobulin served as a negative control. Nuclear counterstaining was omitted to allow unbiased detection of the nuclear positivity of Chk2 and photographic documentation.


   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
To study the Chk2 protein in mammalian cells and tissues, we raised and characterized two MAbs, designated DCS-270 and DCS-273, applicable in various assays including immunoblotting and immunohistochemistry. Immunoblotting analysis of a series of in vitro translated mutant versions of Chk2 showed that the target epitope recognized by DCS-270 localizes within the SQ domain because even the truncated polypeptide restricted to the 110 NH2-terminal amino acid residues of Chk2 reacts with the antibody (Fig. 1, A and B)Citation . The NH2-terminal SQ domain has a regulatory function, and it contains multiple serine residues phosphorylated after DNA damage by the upstream ataxia telangiectasia-mutated kinase (11 , 12) . The DCS-273 epitope, on the other hand, resides in the putative protein-protein interaction FHA domain (1 , 2) , as documented by loss of DCS-273 reactivity with Chk2 selectively deleted for FHA sequences and the ability of a single amino acid substitution (R117A) to entirely abolish the reactivity with DCS-273 but not DCS-270 (Fig. 1, A and B)Citation . The R117A missense mutation of Chk2 targets a highly conserved amino acid residue within the FHA domain (2) . Because also tumor-associated mutations of Chk2 may target the FHA domain (13) , and because both DCS-270 and DCS-273 recognize the wild-type protein in diverse human cell types without any detectable cross-reaction (Fig. 1C)Citation , the selective lack of DCS-273 reactivity with tumor-derived Chk2 may help identify at least a subset of mutations in this tumor suppressor.



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  		Fig. 1. Immunochemical analysis of human Chk2 using novel MAbs. A, wild type and the indicated mutants of Chk2 were transcribed and translated in a rabbit reticulocyte system, electrophoretically separated, and either exposed on phosphorimager to reveal the presence and size of the in vitro translated Chk2 proteins (IVT) or immunoblotted with antibodies DCS-270 and DCS-273 as indicated. B, schematic representation of the major Chk2 domains and location of the epitopes recognized by DCS-270 and DCS-273. SQ, regulatory domain; FHA, forkhead-homology region presumably involved in phosphorylation-dependent protein-protein interactions; Kinase, the kinase domain (numbers indicate the amino acids). C, cell lysates prepared from the BJ fibroblasts and U-2-OS cells (25 µg per lane) were immunoblotted with the DCS-270 and DCS-273 antibodies as indicated. As a negative control, the primary antibodies were replaced by the mouse preimmune serum (PI). D, human diploid BJ fibroblasts were synchronized in G1 and S-phase by release from contact inhibition for 8 and 22 h, respectively and irradiated (+) or mock-treated (-). After 2 h, the cells were analyzed by immunoblotting for the presence and mobility of the endogenous Chk2 protein with MAbs DCS-270 and DCS-273. Note the progressive loss of recognition of the activated, slower-migrating Chk2 by DCS-270, especially in G1 cells. E, U-2-OS cells were either grown exponentially (Exp.) or synchronized in G1 by treatment with lovastatin (Lova.; 40 µM) for 36 h, then irradiated and analyzed by immunoblotting using MAb DCS-270. Two different exposures are provided documenting loss of DCS-270 ability to detect activated Chk2, especially in G1 cells. F, human BJ fibroblasts growing on glass coverslips were exposed to IR (10 Gy) or mock-treated as indicated, and 2 h later the cells were fixed and stained with MAb DCS-270 against Chk2 using immunofluorescence. Nuclear DNA in the matching fields was counterstained by Hoechst 33258.

 
Previous studies have established that mammalian Chk1, a checkpoint kinase structurally unrelated to Chk2 but performing an analogous role in propagating signals from damaged or unreplicated DNA (1) , is expressed and active only in S-G2 phases of the cell cycle (19) . Chk2, on the other hand, was found expressed throughout the cell cycle in HeLa cells (2) . However, it remained formally possible that the latter observation was restricted to transformed cell lines or influenced by chemical-based synchronization protocols. We therefore compared Chk2 protein levels and response to IR in human normal diploid fibroblasts synchronized by contact inhibition and release, followed by examination at different time points, correlated with flow cytometry. No significant change in Chk2 abundance was detected by either of the two antibodies in different cell cycle phases, and exposure to IR always resulted in a Chk2 protein with slower electrophoretic mobility, a characteristic phosphorylation-induced mobility shift firmly associated with the activation of the kinase (2 , 5) . Interestingly, whereas DCS-273 recognized both the inactive and the shifted forms of Chk2 with similar intensity, the reactivity of the activated, retarded Chk2 with antibody DCS-270 was considerably reduced, particularly in cells in G1 phase (Fig. 1D)Citation . To additionally verify the latter effect of cell-phase position, an independent model of human U-2-OS cells was examined growing exponentially or synchronized in G1 by lovastatin. Irradiation of U-2-OS cells gave rise to a slower-migrating species of the Chk2 protein, although the extent of the shift in this tumor cell line was reproducibly less pronounced (Fig. 1E)Citation compared with diploid cells (Fig. 1D)Citation . This may indicate that the extent of Chk2 modification and/or the kinetics of its dephosphorylation could be altered in some transformed cells, an issue currently under investigation in our laboratory. Nevertheless, the slightly retarded form of Chk2 in U-2-OS cells was well recognized in the exponentially growing but poorly recognized in the G1-synchronized cells (Fig. 1E)Citation , consistent with the data obtained in fibroblasts (Fig. 1D)Citation . The selective loss of the DCS-270 epitope reflects its location in the SQ domain, which becomes phosphorylated after IR (11 , 12) , and the observation that this phenomenon is more pronounced in G1 suggests potential qualitative (or quantitative) differences in phosphorylating this domain in G1 versus S-phase cells. Immunofluorescence analysis of exponentially growing BJ or U-2-OS cells showed nuclear localization of Chk2 when stained with the DCS-270 antibody (Fig. 1F)Citation . Consistent with the immunoblotting data, the epitope recognized by DCS-270 was hardly detectable after irradiation also by this type of analysis in situ (Fig. 1F)Citation . DCS-273 appeared to stain cell nuclei irrespective of irradiation, but the signal was generally much weaker when compared with DCS-270 and required specific epitope unmasking and more sensitive immunoperoxidase-based staining procedures for additional intensity amplification (see Fig. 3Citation and "Materials and Methods"). This feature of DCS-273 could possibly be explained by its epitope localization within the FHA domain of Chk2 which, at least under nondenaturing conditions, could be masked by interacting proteins. Collectively, these results show that the MAbs DCS-270 and DCS-273 are complementary tools recognizing distinct domains of Chk2 with their target epitopes differentially sensitive to the activator modification of Chk2 upon DNA damage and to the tumor-associated mutations in the FHA domain, respectively.



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  		Fig. 3. Immunohistochemical detection of Chk2 in normal human tissues. A, nuclear Chk2 detectable in multiple epithelial layers of exocervix. B, in the section adjacent to A, the Ki67 marker of proliferating cells is limited to the second and third suprabasal epithelial layers. Arrows in A and B mark basement membrane. C, homogeneous expression of Chk2 in colon mucosa including the quiescent differentiated epithelium (arrow). D, heterogeneous (mosaic) pattern of Chk2 in the thyroid gland. E, brain tissue shows variable Chk2 in many neurons localized in the cytoplasm (arrows). F, Chk2 is undetectable in skeletal muscle tissue in contrast with positive blood vessel endothelium (arrow).

 
The observation that Chk2 protein levels did not change between G1 and S-phase (Fig. 1D)Citation raised the question of whether even quiescent cells express this kinase and, if yes, whether it can be activated by DNA damage. Comparative immunoblotting analysis of extracts prepared from S-phase fibroblasts versus quiescent, contact-inhibited (Fig. 2A)Citation or serum-starved (not shown) cells demonstrated that Chk2 was as abundant in the G0 cells as in S-phase cells and was subjected to activator modification upon IR, in contrast with Chk1, which was undetectable in the quiescent cells (Fig. 2A)Citation . Judging from the loss of recognition by the DCS-270 antibody, the extent of the Chk2-activating modification was again more robust in quiescent cells (Fig. 2ACitation , DCS-270 panel), reminiscent of what we observed in the BJ fibroblasts and U-2-OS cells synchronized in G1 (Fig. 1, D and E)Citation . To examine also whether cells quiescent because of induced differentiation express Chk2, a widely used model of cultured mouse C2C12 myoblasts capable of experimentally induced differentiation (18) were examined in a time course experiment over 3 days. Skeletal muscle fibers are prototypic terminally differentiated cells of which the differentiation process can be recapitulated in vitro. Murine C2C12 myoblasts can be propagated in culture in the presence of growth factors, and mitogen withdrawal triggers their differentiation, which begins with irreversible exit from the cell cycle. Representative examples of the data obtained on day 0 (exponentially proliferating myoblasts) and 3 (quiescent, differentiated myotubes) show that unlike Chk1, which declined dramatically with advanced differentiation, the Chk2 kinase remained detectable at similar levels, and it also responded to IR by the mobility shift, regardless of the proliferation or differentiation state of these cells (Fig. 2B)Citation .



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  		Fig. 2. Expression and activation of the Chk2 but not Chk1 kinase in nonproliferating cells. A, BJ fibroblasts were arrested by contact inhibition in G0-G1 and either kept arrested (Contact inhib.) or released to cycle by replating in a 1:3 ratio into fresh medium for 22 h (S phase). Before harvesting (2 h), the cells were irradiated (+) or mock-treated (-) and the Chk2 and Chk1 proteins analyzed by immunoblotting with the DCS-270, DCS-273, and SC-8408 antibodies as indicated. B, mouse C2C12 myoblasts grown exponentially (0) or induced to differentiate into myotubes for 3 days (3) were irradiated (+) or not (-) as indicated and abundance and status of Chk2 and Chk1 examined by immunoblotting as in A, except that a mixture (1:1) of DCS-270 and DCS-273 was used to maximize the detectability of the mouse Chk2 protein. C, stability of the Chk2 and Chk1 proteins in exponentially growing, nonirradiated BJ cells, measured by immunoblotting at the indicated time points after addition of the translation inhibitor cycloheximide (CHX; 25 µg/ml) into the culture medium. Cyclin D1 was analyzed in parallel (by MAb DCS-6) as an example of an unstable protein. D, U-2-OS/A5C1 cells conditionally expressing mutant pRb{Delta}Cdk were kept uninduced (off) or induced for 24 h (on) then irradiated (+) or not (-) and Chk2 and Chk1 analyzed by immunoblotting.

 
As oscillating cell cycle regulatory proteins are often short-lived, and there is no data on protein turnover of either Chk1 or Chk2, we next assessed their stability in exponentially growing BJ fibroblasts in a time course immunoblotting experiment upon addition of a protein synthesis inhibitor, cycloheximide. This method was chosen to compensate for the lack of antibodies suitable for quantitative immunoprecipitation of Chk1. The results shown in Fig. 2CCitation indicate that Chk2 is a stable protein of which the level remained virtually unchanged throughout the entire 6-h experiment. In contrast, the abundance of the Chk1 protein declined progressively, with the estimated half-life of ~1 h (Fig. 2C)Citation . Thus, the nonoscillating nature of Chk2 is consistent with the stable protein, whereas the relatively fast protein turnover of Chk1, together with its reported variation at the mRNA level (19) , may jointly contribute to its oscillation with its appearance at the G1-S boundary and peak levels in S-G2. The latter mode of regulation is reminiscent of genes regulated by the pRb-E2F pathway, a notion confirmed by conditional expression of a nonphosphorylatable, constitutively active allele of pRb (pRb{Delta}Cdk) in U-2-OS/A5C1 cells (17) , which resulted in rapid down-regulation of Chk1, but not Chk2, in cells entering S-phase (Fig. 2D)Citation .

The differential expression of Chk2 versus Chk1 in cycling, quiescent and differentiating cells in culture suggested that Chk2 might operate in a wide range of tissues in vivo. As the first step toward the ultimate goal to understand and explore the function of the DNA-damage-activated kinases in vivo, we next performed an immunohistochemical analysis of normal human tissues with the MAbs DCS-270 and DCS-273, which showed that the staining patterns of Chk2 fall into four categories. A homogeneous nuclear staining for Chk2 in the proliferating as well as quiescent, differentiated cells was seen in stratified epithelia including epidermis, exocervix (Fig. 3, A and B)Citation , esophagus, and rectum; in transitional epithelium of the urinary bladder; and in simple epithelia of the stomach, duodenum, small intestine, and colon (Fig. 3C)Citation . Additionally, a more heterogeneous pattern of a mosaic of positive and negative nuclei was detected among the majority of tissues, such as epithelia of the lung, breast, kidney, salivary, thyroid (Fig. 3D)Citation , parathyroid, and adrenal glands; pancreas, prostate, epididymis, sweat glands, and endometrium; stromal mesenchymal cells; blood vessels; lymphoid tissues including lymph nodes, tonsils, and the bone marrow; smooth and cardiac muscle tissues; and peripheral nerves. Also, in contrast to its nuclear staining in other tissues, brain neuronal tissue displayed prominent cytoplasmic staining of Chk2 (Fig. 3E)Citation . Finally, skeletal muscle (Fig. 3F)Citation and cartilage apparently lacked any Chk2 protein despite the fact that positive cells such as blood vessel endothelium in these tissues were positive and served as internal controls.

Collectively, our findings indicate that Chk2 is a long-lived, predominantly nuclear protein that remains expressed and can become activated upon DNA damage in all phases of the mammalian cell cycle as well as in nonproliferating and terminally differentiated cells. The striking difference between the broad expression pattern of Chk2 (2 and this study) compared with the S-G2 phase-restricted expression of Chk1 (19 , 20 and this study) appears conceptually important, suggesting that Chk2 may mediate at least a limited response to DNA damage even in quiescent cells. Whereas Cdc25 phosphatases could hardly be targeted by Chk2 in nonproliferating cells because of their cell cycle-restricted expression, p53 is a feasible candidate for a putative Chk2 substrate in G1 and quiescence, possibly activating some form of DNA repair such as nonhomologous end-joining of double-strand DNA breaks, which is known to occur also in G1 cells (1) . In addition, so-far unidentified substrates and/or additional cellular functions might be targeted by the Chk2 kinase, a challenge to be resolved by future studies. The tumor-suppressor role of Chk2 (1 , 8 , 13 , 14) , as a gene mutated in sporadic cancers and some Li-Fraumeni syndrome families characterized by a range of tumors in diverse organs, is consistent with our present finding of a wide expression of Chk2 in normal human tissues. It is interesting to note that the highest degree of homogeneous expression of Chk2 was seen in the cancer-prone, continuously renewing epithelial cell populations exposed to environmental carcinogens, such as epidermis and the epithelia of the alimentary canal. Also the conditionally renewing cell populations such as those of the breast and endocrine glands showed a rather high degree of Chk2 expression regardless of the proliferation status. The cytoplasmic localization of Chk2 in postmitotic neurons is intriguing and warrants future studies into potential subcellular trafficking of Chk2, particularly in neuronal cell models and brain malignancies. Another aspect documenting that the Chk2 expression patterns broadly correspond to the biology of the diverse cell populations is the lack of Chk2 in only a few terminally differentiated, static cell populations such as adult skeletal muscle and cartilage. In such tissues, the function(s) of Chk2 seem dispensable only during the long-term maintenance of their irreversibly quiescent state in adults, whereas Chk2 is still present and functional during the myoblast differentiation into myotubes (Fig. 2B)Citation and in fetal skeletal muscle in situ.4 The results and tools presented in this study should help elucidate the role(s) of Chk2 in diverse mammalian cell and tissue types and differentiation stages and assist the search for cancer-associated aberrations of this tumor suppressor and key mediator of cellular responses to genotoxic stress.


   ACKNOWLEDGMENTS
 
We thank M. Crescenzi, S. Elledge, K. Helin, and J. Shay for some of the reagents and Clæs Lindeneg and A. A. Kjerulff for technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Danish Cancer Society, Danish Medical Research Council, Eva and Henry Frænkels Fund, Director Jens Aage Sørensen and Edith Ingeborg Sørensens Fund, and the European Commission. Back

2 To whom requests for reprints should be addressed, at Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark. Phone: (45) 35 25 73 57; Fax: (45) 35 25 77 21; E-mail: bartek{at}biobase.dk Back

3 The abbreviations used are: MAb, monoclonal antibody; SQ, SQ-rich; pRb, retinoblastoma protein; IR, ionizing radiation; FHA, forkhead-associated. Back

4 Unpublished observations. Back

Received 3/22/01. Accepted 5/ 7/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 

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