Abstract
Background:
We recently identified matrix extracellular phosphoglycoprotein/osteoblast factor 45 (MEPE/OF45) as a new cofactor of CHK1 in rat cells. The aim of this study was to determine the role of human MEPE/OF45 (hMEPE/OF45 has ∼50% homology with rat MEPE/OF45 (rMEPE/OF45)) in affecting the sensitivity of human tumour cells to DNA damage.
Methods:
hMEPE/OF45 expression in different human tumour cell lines and its relevance to the resistance of cell lines to DNA damage inducers such as ionising radiation (IR) or camptothecin (CPT) were assessed. Cells lines stably expressing wild-type MEPE/OF45 or mutant MEPE/OF45 (with the CHK1 interactive key domain (amino acids 488–507) deleted) were established. Cell survival, G2 accumulation, CHK1 half-life and the CHK1 level in ligase 3 complexes were examined.
Results:
hMEPE/OF45 expression correlates with the resistance of cell lines to IR or CPT. Upregulating wild-type hMEPE/OF45 (but not mutant hMEPE/OF45) could stabilize CHK1 by reducing CHK1 interaction for its E3 ligases Cul1 or Cula4A; it increases the G2 checkpoint response and increases the resistance of tumour cells to IR or CPT treatment.
Conclusion:
hMEPE/OF45 could be a new target for sensitizing tumour cells to radiotherapy or chemotherapy.
Similar content being viewed by others
Main
Matrix extracellular phosphoglycoprotein/osteoblast factor 45 (MEPE/OF45) was originally cloned from a human oncogenetic hypophosphataemia tumour (Rowe et al, 2000) and then identified in rat bone-marrow-derived osteoblasts (Petersen et al, 2000). The murine homologue of MEPE/OF45 was reported the following year (Argiro et al, 2001). Since the identification of MEPE/OF45, its function related to bone metabolism has been widely investigated. We previously identified that MEPE/OF45, as a cofactor of CHK1, protects cells from DNA damage-induced killing in rat embryo fibroblast cells (Liu et al, 2009). However, because human MEPE/OF45 (hMEPE/OF45) has only ∼50% homology with rat MEPE/OF45 (rMEPE/OF45), it remained unclear whether hMEPE/OF45 has similar function to rMEPE/OF45.
CHK1 is one of the essential checkpoint proteins involved in cellular response to multiple DNA damage inducers (Liu et al, 2000; Zhao et al, 2002). It is believed that upregulated CHK1 protects cells from DNA damage-induced cell killing, including that induced by ionising radiation (IR) and camptothecin (CPT) (Hu et al, 2001, 2005a; Zhao et al, 2002; Wang et al, 2002b; Brown and Baltimore, 2003), by promoting homologous recombination repair (HRR) (Sorensen et al, 2005; Hu et al, 2005b). Despite the importance of CHK1 in DNA damage response, the regulation of CHK1 in mammalian cells is not well understood, partly because of its essential nature (Liu et al, 2000; Lam et al, 2004).
In this study, on the basis of our previous report about the new role of rMEPE/OF45 in protecting cells from DNA damage-induced killing (Liu et al, 2009), we investigate whether hMEPE/OF45 is generally expressed in different types of tumour cells, whether the expression levels were relevant to the resistance of tumour cells to DNA damage inducers and whether, similar to rMEPE/OF45, hMEPE/OF45 (as a cofactor of CHK1) could be a new target for sensitizing human tumour cells to DNA damage inducers.
Materials and methods
Cell lines and antibodies
All human tumour cell lines used in this study were derived from ATCC. These cell lines were grown in DMEM medium supplemented with 10% calf serum. The antibody against hMEPE/OF45 was as described (Rowe et al, 2000, 2004). Purified HA-tagged JNK2 protein (sc-4062) and antibodies against CHK1 (sc-8408 and sc-7898), HA (sc-805, sc-7392), PCNA (sc-56), α-Tubulin (sc-8035), Cul4 (sc-10782) and ubquitin (Ub) (sc-9133) were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). The antibody against Cul1 was purchased from Zymed Laboratories (San Francisco, CA, USA) and the antibody against H2A (07-146) was purchased from Upstate Biotechnology (Lake Placid, NJ, USA).
Plasmid construction and transfection
The full-length cDNA of wild-type hMEPE/OF45 or mutant hMEPE/OF45 (with the sequence encoding 488–507 amino acids (aa) deleted) was ligated with HA tag at the C terminus by PCR. hMEPE/OF45 was then cloned into pLenti6/V5-D-TOPO vector (Invitrogen), according to the manufacturer's protocol. The HA tag was inserted into the plasmid. The primers used for wild-type hMEPE/OF45 are: forward, 5′-CACCATGGGAGTTTTCTGTGTGGGACTACTCCTTTTCAGTGTGACCTG-3′ and reverse, 5′-AGGTCCTCCCAGGCTGGCATAGTCAGGCACGTCATAAGGATAGTCACCATCGCTCTCACTTGAAC-3′. The procedure to construct mutant hMEPE/OF45 has been described in Supplementary Figure S1. The primers for fragment 1 (1–477 aa) are F1, ACGGATCCATGCGAGTTTTCTGTGTGGGACTACTCC and R1, TACTGTCATCCTTATTCCGTGTAGA, and for fragment 2 (507–514 aa) are F2, ACGGAATAAGGATGACAGTAGTGAGTCATC and R2, AAGAATTCGTCACCATCGCTCTCACTTG. For lentivirus production, pLenti6/V5-D-TOPO was co-transfected with packaging vectors into human 293 cells and the supernatant was harvested after 48 h. The virus was filtered through a Millex-HV PVDF filter (hole diameter: 0.45 μ M). Titres were determined by infecting 293 cells with a serial dilution of the concentrated virus. For a typical preparation, the titre was approximately 2- to 7 × 104 TU ml−1. For infection of 293 cells, 30–50% confluent cells were incubated with a suitable amount of virus particles and 10 μg ml−1 polybrene for 8–16 h at 37°C. A volume of 10 μg ml−1 of blasticidin (Invitrogen) was used to select stably expressed cells.
Immunoprecipitation
Immunoprecipitation was performed as described previously (Wang et al, 2002a). The samples were washed with buffer (0.5% NP-40, 1 mM Na3VO4, 5 mM NaF and 0.2 mM PMSF in PBS buffer) and boiled in 30 μl of protein loading buffer. They were loaded onto 10% Tris-PAGE gel, followed by the standard western blot.
Cell sensitivity to DNA damage inducers and flow cytometry measurement
Cell sensitivity to IR or to CPT was determined by clonogenic assay as described previously (Hu et al, 2001; Wang et al, 2002b). The G2 accumulation of cells after radiation was detected by flow cytometry as described previously (Hu et al, 2001). Briefly, 2 × 105 cells were plated in 60 mm dishes, with 3 ml of growth medium. After 30 h, the cells were exposed to 6 Gy and returned to 37°C. At different times thereafter, the cells were trypsinised and fixed in 70% ethanol. Cells were stained in solution (62 μg ml−1 RNase A, 40 μg ml−1 propidium iodide and 0.1% Triton X-100 in PBS buffer) at room temperature for 1 h. The distribution of cells in the cell cycle was measured using a flow cytometer (Coulter Epics Elite, Beckman Coulter Inc., Atlanta, GA, USA).
Statistical analysis
Differences between treatment groups were analysed using statistical software (t-test). P-values <0.05 were regarded as significant.
Results and discussion
MEPE/OF45 presents in all kinds of human dividable cell lines tested in our laboratory
We previously identified that rMEPE/OF45 could protect rat cells from DNA damage-induced killing (Liu et al, 2009). However, because the amino-acid sequence of hMEPE/OF45 (525 amino acid) has ∼50% homology compared with that of rMEPE/OF45 (435 amino acid) (Argiro et al, 2001), it remained unclear whether hMEPE/OF45 has a function similar to rMEPE/OF45. To investigate whether MEPE/OF45 was important for human cells to respond to DNA damage, we examined the MEPE/OF45 expression level in different human cell lines, including primary fibroblast cells, transformed fibroblast cells, transformed lymphoblast cells and tumour cell lines from different human tissues. These cell lines all showed a positive expression of MEPE/OF45, although the levels of expression varied (Figure 1A), indicating that MEPE/OF45 expresses in replicating cells. The signals of human MEPE/OF45 showed two bands (Figure 1A), which might be due to alternatively spliced MEPE/OF45s. Our group recently identified a new exon of hMEPE/OF45 between formerly identified exons 3 and 4 (GenBank accession no. DQ854717). The functions of the different forms of MEPE/OF45 need to be investigated further. Interestingly, the levels of MEPE/OF45 in different human tumour cell lines broadly correlate with the resistance of cell lines to DNA damage inducers (Figure 1B). The higher the level of MEPE/OF45 was in the cell line, the more resistant the cell line was to IR or CPT. There were 293 cells with the lowest level of MEPE/OF45, and they were most sensitive to IR or CPT. In addition, we reported that knocking down MEPE/OF45 could sensitise HeLa cells to DNA damage inducers (Liu et al, 2009). These results indicate that MEPE/OF45 protects human cells from DNA damage-induced killing.
Human MEPE/OF45 interacts with CHK1 and stabilises CHK1
We previously reported that rMEPE/OF45 protected cells from DNA damage-induced killing through its interaction with CHK1 (Liu et al, 2009). We wanted to determine whether similar to rMEPE/OF45, hMEPE/OF45 could also interact with CHK1. To study the relationship of hMEPE/OF45 with CHK1, we first synchronised HeLa cells and examined the expression of MEPE/OF45 at different phases of the cell cycle, as CHK1 expression depends on the cell cycle (Kaneko et al, 1999). The results showed that similar to CHK1, MEPE/OF45 expressed low in the G1 phase and high in the S and G2 phases (Supplementary Figure S2), suggesting a correlation between these two proteins. We further examined the interaction between hMEPE/OF45 and CHK1 using an immunoprecipitation (IP) approach in HeLa cells. The results showed that, similar to rMEPE/OF45, hMEPE/OF45 also interacted with CHK1 (Figure 2A). The key domain of rMEPE/OF45 was 400–418 amino acids (Liu et al, 2009). The corresponding amino-acid sequence in hMEPE/OF45 is 490–507 (Argiro et al, 2001; Figure 2B, left). To study whether CHK1 was stabilised when interacting with hMEPE/OF45, as it was when interacting with rMEPE/OF45, we generated the constructs encoding HA-tagged either wild-type hMEPE/OF45 or mutant hMEPE/OF45 with amino acid 488–507 deleted (Supplementary Figure S1). We expressed the wild-type hMEPE/OF45 or mutant hMEPE/OF45 in 293 cells, as this cell line had the lowest level of MEPE/OF45 in the human tumour cell lines tested in our laboratory (Figure 1A). The results showed that wild-type hMEPE/OF45 but not mutant hMEPE/OF45 could interact with CHK1 (Figure 2B, right), indicating that the 488–507 amino acids are the key domain for hMEPE/OF45 to interact with CHK1.
We were then interested in determining whether similar to the key domain of rMEPE/OF45, the key domain of hMEPE/OF45 also had an essential role in stabilising CHK1 by reducing the interaction of CHK1 for its E3 ligases, Cul1 or Cul4A. For this, we first compared the half-life of CHK1 between wild-type hMEPE/OF45-expressing cells and mutant hMEPE/OF45-expressing cells.
The results showed that the half-life of CHK1 in the wild-type hMEPE/OF45-expressing cells (W1) is longer than that in parental cells (293), the 293 cells transfected with the vector alone (V1) and the 293 cells transfected with mutant hMEPE/OF45 (D1) (Figure 2C). These results indicate that the interaction domain is essential for hMEPE/OF45 to stabilise CHK1. We then compared the CHK1 levels in its E3 ligase complexes with wild-type hMEPE/OF45-expressing cells and mutant hMEPE/OF45-expressing cells. Results showed that although the total levels of CHK1 in either cytoplasmic extracts (CE) or nuclear extracts (NE) are similar to all types of cells (293, V1, D1 and W1), the CHK1 levels in Cul1 or Cul4A IP complexes in W1 cells were less than that in 293, V1 or D1 cells (Figure 2D). Additional data showed that when hMEPE/OF45 was knocked down, ubiquitinated CHK1 was increased (Supplementary Figure S3A and B). These results indicate that although hMEPE/OF45 has ∼50% homology with rMEPE/OF45, its C-terminal amino-acid sequence, including the key domain to interact with CHK1, is relatively conserved, and similarly functions to stabilise CHK1.
Wild-type but not mutant hMEPE/OF45 renders tumour cells resistant to DNA damage inducers
To further examine whether hMEPE/OF45 was similar to rMEPE/OF45, and could protect cells from DNA damage-induced killing through interaction with CHK1, we compared the effects of wild-type hMEPE/OF45 and mutant hMEPE/OF45 on the G2 checkpoint response and the sensitivity of 293 cells to DNA damage inducers. The results showed that wild-type hMEPE/OF45 could increase the G2/M accumulation of cells after IR, but not mutant hMEPE/OF45 (Figure 3A). Similarly, knocking down hMEPE/OF45 reduced IR-induced G2 accumulation (Supplementary Figure S3A and C). These results further support that hMEPE/OF45 functions through the CHK1 pathway to affect cell response to DNA damage, because this kind of checkpoint response, G2-phase cell accumulation, is ATM independent (Xu et al, 2002) but CHK1 dependent (Wang et al, 2003). More importantly, the results showed that wild-type hMEPE/OF45 could increase resistance of the cells to either IR or CPT treatment, but not mutant hMEPE/OF45 (Figure 3B). The P-values between 293W (including 1 and 2) and other groups are <0.01. These results indicate that hMEPE/OF45 protects cells from DNA damage-induced killing depending on its interaction with CHK1. It is known that CHK1 promotes HRR through regulation of checkpoint activation (Sorensen et al, 2005; Hu et al, 2005b). We believe that hMEPE/OF45 increases the CHK1 level by stabilising CHK1, which promotes HRR and protects cells from DNA damage-induced killing.
Conclusion
Taken together, the results of our study indicate that MEPE/OF45 protecting human cells from IR- or CPT-induced killing mainly depends on its interaction with CHK1. The specific molecular mechanism for this protective role of MEPE/OF45 could be linked to MEPE/OF45 stabilising CHK1 by reducing CHK1 degradation. Because CHK1 is an essential gene (Liu et al, 2000; Lam et al, 2004) and MEPE/OF45 is not (Gowen et al, 2003), our study results indicate that MEPE/OF45 is a new cofactor of CHK1, which could be a new target with less side effects for sensitizing tumour cells to DNA damage inducers and could benefit cancer treatment.
Accession codes
Change history
16 November 2011
This paper was modified 12 months after initial publication to switch to Creative Commons licence terms, as noted at publication
References
Argiro L, Desbarats M, Glorieux FH, Ecarot B (2001) MEPE, the gene encoding a tumor-secreted protein in oncogenic hypophosphatemic osteomalacia, is expressed in bone. Genomics 74: 342–351
Brown EJ, Baltimore D (2003) Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes Dev 17: 615–628
Gowen LC, Petersen DN, Mansolf AL, Qi H, Stock JL, Tkalcevic GT, Simmons HA, Crawford DT, Chidsey-Frink KL, Ke HZ, McNeish JD, Brown TA (2003) Targeted disruption of the osteoblast/osteocyte factor 45 gene (OF45) results in increased bone formation and bone mass. J Biol Chem 278: 1998–2007
Hu B, Han S-Y, Wang X, Ottey M, Potoczek MB, Dicker A, Huebner K, Wang Y (2005a) Involvement of the Fhit gene in the ionizing radiation-activated ATR/CHK1 pathway. J Cell Physiol 202: 518–523
Hu B, Wang H, Wang X, Lu H-R, Huang C, Powell SN, Huebner K, Wang Y (2005b) Fhit and CHK1 have opposing effects on homologous recombination repair. Cancer Res 65: 8613–8616
Hu B, Zhou XY, Wang X, Zeng ZC, Iliakis G, Wang Y (2001) The radioresistance to killing of A1-5 cells derives from activation of the Chk1 pathway. J Biol Chem 276: 17693–17698
Kaneko Y, Watanabe N, Morisaki H, Akita H, Fujimoto A, Tominaga K, Tachibana K, Ikeda K, Nakanishi M (1999) Cell cycle-dependent and ATM-independent expression of human Chk1 kinase. Oncogene 18: 3673–3681
Lam MH, Liu Q, Elledge SJ, Rosen JM (2004) Chk1 is haploinsufficient for multiple functions critical to tumor suppression. Cancer Cell 6: 45–59
Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ (2000) Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint. Genes Dev 14: 1448–1459
Liu S, Wang H, Wang X, Lu L, Gao N, Rowe P, Hu B, Wang Y (2009) MEPE/OF45 protects cells from DNA damage induced killing via stabilizing CHK1. Nucleic Acids Res 37: 7447–7454
Petersen DN, Tkalcevic GT, Mansolf AL, Rivera-Gonzalez R, Brown TA (2000) Identification of osteoblast/osteocyte factor 45 (OF45), a bone-specific cDNA encoding an RGD-containing protein that is highly expressed in osteoblasts and osteocytes. J Biol Chem 275: 36172–36180
Rowe PSN, de Zoysa PA, Dong R, Wang HR, White KE, Econs MJ, Oudet CL (2000) MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics 67: 54–68
Rowe PSN, Kumagai Y, Gutierrez G, Garrett IR, Blacher R, Rosen D, Cundy J, Navvab S, Chen D, Drezner MK (2004) MEPE has the properties of an osteoblastic phosphatonin and minhibin. Bone 34: 303–319
Sorensen CS, Hansen LT, Dziegielewski J, Syljuasen RG, Lundin C, Bartek J, Helleday T (2005) The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair. Nat Cell Biol 7: 195–201
Wang H, Wang X, Zhou XY, Chen DJ, Li GC, Iliakis G, Wang Y (2002a) Ku affects the ataxia and Rad 3-related/Chk1-dependent S-phase checkpoint response after camptothecin treatment. Cancer Res 62: 2483–2487
Wang J-L, Wang X, Wang H, Iliakis G, Wang Y (2002b) CHK1-regulated S-phase checkpoint response reduces camptothecin cytotoxicity. Cell Cycle 1: 267–272
Wang X, Khadpe J, Hu B, Iliakis G, Wang Y (2003) An over-activated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells. J Biol Chem 278: 30869–30874
Xu B, Kim S-T, Lim D-S, Kastan MB (2002) Two molecularly distinct G2/M checkpoints are induced by ionizing radiation. Mol Cell Biol 22: 1049–1059
Zhao H, Watkins JL, Piwnica-Worms H (2002) Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints. Proc Natl Acad Sci USA 99: 14795–14800
Acknowledgements
We thank Ms Doreen Theune for editing this paper. This work was supported by grants from the National Basic Research Program of China (2005CB522506 and 2007CB914604 to BH), the National Natural Science Foundation of China (30770651, 30670616 and 30370441 to BH) and a grant from the National Institutes of Health of USA (GM080771 to YW).
Author information
Authors and Affiliations
Corresponding author
Additional information
Conflict of interest
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on British Journal of Cancer website (http://www.nature.com/bjc)
Supplementary information
Rights and permissions
From twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
About this article
Cite this article
Zhang, P., Wang, H., Rowe, P. et al. MEPE/OF45 as a new target for sensitizing human tumour cells to DNA damage inducers. Br J Cancer 102, 862–866 (2010). https://doi.org/10.1038/sj.bjc.6605572
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.bjc.6605572
Keywords
This article is cited by
-
Berberine chloride suppresses non-small cell lung cancer by deregulating Sin3A/TOP2B pathway in vitro and in vivo
Cancer Chemotherapy and Pharmacology (2020)
-
Artemisitene suppresses tumorigenesis by inducing DNA damage through deregulating c-Myc-topoisomerase pathway
Oncogene (2018)