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Increased Tumorigenicity and Sensitivity to Ionizing Radiation Upon Loss of Chromosomal Protein HMGN1

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Increased Tumorigenicity and Sensitivity to Ionizing Radiation Upon Loss of Chromosomal Protein HMGN1 Research Article Increased Tumorigenicity and Sensitivity to Ionizing Radiation upon Loss of Chromosomal Protein HMGN1 Yehudit Birger,1 Fre´de´ric Catez,1 Takashi Furusawa,1 Jae-Hwan Lim,1 Marta Prymakowska-Bosak,1 Katherine L. West,1 Yuri V. Postnikov,1 Diana C. Haines,2 and Michael Bustin1 1Protein Section, Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland and 2Pathology/Histotechnology Laboratory, Science Applications International Corporation, National Cancer Institute, Frederick, Maryland Abstract which plays a key role in determining cellular stability and transformation, is dependent on specific binding to chromatin We report that loss of HMGN1, a nucleosome-binding (6); moreover, chromatin remodeling complexes, such as BRG1 and protein that alters the compaction of the chromatin fiber, BRM, play a role in genomic stability (7). Perturbation in the higher- increases the cellular sensitivity to ionizing radiation and order chromatin structure has also been linked to the activation of the tumor burden of mice. The mortality and tumor burden À/À the ATM, a protein kinase that plays a key role in the cellular of ionizing radiation–treated Hmgn1 mice is higher than +/+ À/À response to ionizing radiation and the prevention of tumor that of their Hmgn1 littermates. Hmgn1 fibroblasts formation (8, 9). Likewise, numerous studies have linked the have an altered G -M checkpoint activation and are 2 correct repair of UV damage to the ability of nucleotide excision hypersensitive to ionizing radiation. The ionizing radiation repair complexes to reach the UV-damaged sites in chromatin hypersensitivity and the aberrant G2-M checkpoint activation / (10–12). Thus, the structure of chromatin and the ability of nuclear of Hmgn1À À fibroblasts can be reverted by transfections complexes that sense and repair DNA damage to reach their with plasmids expressing wild-type HMGN1, but not with target sites in chromatin play an important role in DNA repair, in plasmids expressing mutant HMGN proteins that do not bind maintaining genomic stability, and in tumor susceptibility (13). to chromatin. Transformed Hmgn1À/À fibroblasts grow in Given that access of regulatory factors and DNA repair soft agar and produce tumors in nude mice with a complexes to their target sites in chromatin is important, it is significantly higher efficiency than Hmgn1+/+ fibroblasts, possible that nuclear proteins, such as the nonhistone HMGN suggesting that loss of HMGN1 protein disrupts cellular proteins (14, 15), that are known to affect the stability of the higher- events controlling proliferation and growth. Hmgn1À/À mice order chromatin structure may play a role in DNA repair processes have a higher incidence of multiple malignant tumors and and in tumorigenicity. The high mobility group N (HMGN) is a metastases than their Hmgn1+/+ littermates. We suggest that family of structural proteins present in the nuclei of all mammalian HMGN1 optimizes the cellular response to ionizing radiation cells that binds specifically to nucleosomes, the building block and to other tumorigenic events; therefore, loss of this of the chromatin fiber (14, 15). HMGN proteins move rapidly protein increases the tumor burden in mice. (Cancer Res throughout the nucleus, bind to nucleosomes transiently (16, 17), 2005; 65(15): 6711-8) and reduce the compaction of the chromatin fiber (14, 15). The binding of HMGN proteins to nucleosomes affects the levels of Introduction posttranslational modification in core histones and alters DNA- Chromatin plays a central role in regulating the fidelity of gene related nuclear processes such as transcription (18–20) and expression, in maintaining genomic stability, and in mounting replication (21). Hmgn1À/À mice are hypersensitive to UV most proper responses to various cellular stresses, including UV and likely because loss of HMGN1 protein alters the accessibility of the ionizing radiation (1). For example, the repair of irradiation- damaged sites to the nucleotide excision repair machinery and induced double-strand breaks (DSB) is linked to the phosphoryla- decreases the rate of removal of UV-induced lesions from tion of the histone variant H2AX (2), and the loss of a single H2ax transcriptionally active chromatin (22). These findings, and the allele decreases genomic stability and increases tumor suscepti- growing evidence that chromatin plays an important role in DSB bility (3, 4). Additional histone modifications, such as methylation repair, led us to test whether HMGN1 affects the cellular response and acetylation, were also found to play important roles in DNA to ionizing radiation. repair processes (1) and inhibitors of histone deacetylases are being Using Hmgn1À/À mice and cells, we find that HMGN1 protein tested as potential anticancer therapeutic agents (5). The plays a role in the cellular ability to mount a proper response to transcription regulatory function of the tumor suppressor p53, ionizing radiation and that loss of this protein increases the tumor susceptibility in mice. Ionizing irradiation leads to accelerated tumor formation and death of mice lacking HMGN1 protein, Note: All animals were cared for and used humanely according to the U.S. Public compared with wild-type mice. Likewise, Hmgn1À/À fibroblasts are Health Service Policy on Humane Care and Use of Animals (2000); the Guide for the Care hypersensitive to ionizing radiation and fail to arrest properly in and Use of Laboratory Animals (1996); and the U.S. Government Principles for Utilization À/À and Care of Vertebrate Animals Used in Testing, Research, and Training (1985). G2-M phase of the cell cycle. SV40-transformed Hmgn1 National Cancer Institute-Frederick animal facilities and animal program are fibroblasts grow in soft agar and produce tumors in nude mice accredited by the Association for Assessment and Accreditation of Laboratory with a significantly higher efficiency than SV40-transformed Animal Care International. +/+ Requests for reprints: Michael Bustin, Protein Section, Laboratory of Metabolism, Hmgn1 fibroblasts, suggesting that loss of HMGN1 protein Center for Cancer Research, National Cancer Institute, NIH, Building 37, Room disrupts cellular events controlling proliferation and growth. 3122B, 9000 Rockville Pike, Bethesda, MD 20892. Phone: 301-49605234; E-mail: À/À [email protected]. Hmgn1 mice have a higher incidence of multiple malignant I2005 American Association for Cancer Research. tumors and metastases than wild-type mice. www.aacrjournals.org 6711 Cancer Res 2005; 65: (15). August 1, 2005 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research Our results identify a chromatin-binding protein that plays a role ice for 5 minutes, and sedimented at 3,000 Â g for 10 minutes. The in the cellular response to ionizing radiation and indicate that loss supernatant was made 25% in TCA by adding 100% TCA, incubated on ice of HMGN1 impairs the cellular response to ionizing radiation and for 15 minutes, and sedimented at 3,000 Â g for 20 minutes. The pellet j increases susceptibility to tumor formation. was stored at À20 C overnight in 100% ethanol, air-dried, and resuspended with 50 to 100 AL of water. Proteins were resolved on 15% Tris-glycine-SDS gels, transferred to a polyvinylidene difluoride membrane, Materials and Methods and subjected to Western blots. Animals and cell lines. Hmgn1À/À mice, primary mouse embryonic Laser scissors. Double-stranded DNA breaks were induced along a fibroblasts (MEF), MEF-derived cell lines, and SV40-transformed MEF cells, defined path essentially as described before (23, 24). Briefly, 20 minutes expressing either wild-type or mutant HMGN1 protein, under the control of before UV exposure, cells were treated with HOECHST 33258 (10 Ag/mL). the Tet promoter, were generated and characterized as described elsewhere UV exposures were set on a Zeiss LSM 510 confocal microscope in a (22). Mice were backcrossed for at least six generations and were over 95% controlled temperature environment. Cells were exposed to a 364 nm laser pure. Primary MEFs were used up to passage 5 because older MEFs had an along a predefined path, under a 40Â C-apo lens. Cells were kept at 37jC for altered phenotype. The longevity study was terminated after 112 weeks at 10 to 30 minutes, fixed with 4% paraformaldehyde, and immunostained (25). which all surviving mice were sacrificed. Statistical analyses. Statistical analyses were done using Fisher’s two- Assessment of genomic instability. Cells were incubated with 0.05 sided test at 95% confidence. Ag/mL colcemid for 1.5 hours, treated with 0.7% KCl for 5 minutes at room temperature, fixed for 16 hours at 4jC with in a solution of 3:1 methanol/ Results acetic acid, spread on slides, and stained with Giemsa as recommended by À/À the manufacturer. Increased carcinogenesis in irradiated Hmgn1 mice. To Irradiation of animals. A total of 40 mice (20 each of Hmgn1À/À test whether loss of HMGN1 protein affects the susceptibility À/À and Hmgn1+/+ littermates), 6 to 8 weeks old, were g-irradiated using a 137Cs to radiation-induced carcinogenesis, we treated Hmgn1 , Shepherd Mark II irradiator, with a cumulative dose of 9 Gy (3 Gy, thrice, on Hmgn1+/+, and Hmgn1+/À littermate mice with a sublethal schedule alternating days). The animals were monitored for 12 months after of g-irradiation (ionizing radiation). Within 1 year of ionizing radi- irradiation for appearance of tumors and survival and then sent to ation treatment, only 45% of the Hmgn1À/À mice, compared with necropsy. over 75% of their Hmgn1+/+ littermates, survived, an indication that Injection of cells into NU/NU mice. Twenty immunodeficient nude loss of HMGN1 protein decreased the survival rate of the irradiated (nu/nu) female mice were injected s.c., in two separate experiments, with +/À 6 À/À 6 +/+ mice (Fig. 1A). The 1-year survival rate of irradiated Hmgn1 either 2 Â 10 Hmgn1 or 2 Â 10 Hmgn1 transformed MEFs or with À/À PBS.
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