The Potential Role of DFNA5, a Hearing Impairment Gene, in P53-Mediated Cellular Response to DNA Damage
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(2006) 51:652–664 DOI 10.1007/s10038-006-0004-6 ORIGINAL ARTICLE The potential role of DFNA5, a hearing impairment gene, in p53-mediated cellular response to DNA damage Yoshiko Masuda Æ Manabu Futamura Æ Hiroki Kamino Æ Yasuyuki Nakamura Æ Noriaki Kitamura Æ Shiho Ohnishi Æ Yuji Miyamoto Æ Hitoshi Ichikawa Æ Tsutomu Ohta Æ Misao Ohki Æ Tohru Kiyono Æ Hiroshi Egami Æ Hideo Baba Æ Hirofumi Arakawa Received: 17 March 2006 / Accepted: 21 April 2006 / Published online: 2 August 2006 Ó The Japan Society of Human Genetics and Springer-Verlag 2006 Abstract The tumor suppressor p53 plays a crucial gene was strongly induced by exogenous and endoge- role in the cellular response to DNA damage by tran- nous p53. The chromatin immunoprecipitation assay scriptional activation of numerous downstream genes. indicated that a potential p53-binding sequence is lo- Although a considerable number of p53 target genes cated in intron 1 of the DFNA5 gene. Furthermore, the have been reported, the precise mechanism of p53- reporter gene assay revealed that the sequence displays regulated tumor suppression still remains to be eluci- p53-dependent transcriptional activity. The ectopic dated. Here, we report a novel role of the DFNA5 gene expression of DFNA5 enhanced etoposide-induced cell in p53-mediated etoposide-induced cell death. The death in the presence of p53; however, it was inhibited DFNA5 gene has been previously reported to be in the absence of p53. Finally, the expression of responsible for autosomal-dominant, nonsyndromic DFNA5 mRNA was remarkably induced by gamma- hearing impairment. The expression of the DFNA5 ray irradiation in the colon of p53(+/+) mice but not in that of p53(–/–) mice. These results suggest that DFNA5 plays a role in the p53-regulated cellular re- Y. Masuda Æ M. Futamura Æ H. Kamino Æ Y. Nakamura Æ sponse to genotoxic stress probably by cooperating N. Kitamura Æ S. Ohnishi Æ Y. Miyamoto Æ H. Arakawa (&) with p53. Cancer Medicine and Biophysics Division, National Cancer Center Research Institute, Keywords p53 Æ Tumor suppressor gene Æ Hearing 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan e-mail: [email protected] impairment Æ DNA damage Æ Cell death Æ Apoptosis Æ Etoposide Æ p53 target gene H. Ichikawa Cancer Transcriptome Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Introduction T. Ohta Æ M. Ohki Mutations in the p53 gene are the most common genetic Center for Medical Genomics, alterations found in human cancers. A normal cell ex- National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan presses a low level of p53 protein because of its short half-life. However, the expression level and functional T. Kiyono activity of p53 increases in response to various cellular Virology Division, stresses. The activated p53 then functions as a tran- National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan scription factor that activates the transcription of many target genes; this leads to the expression of multiple H. Egami Æ H. Baba Æ Y. Masuda physiological functions of p53 (Vogelstein et al. 2000; Department of Gastroenterological Surgery, Vousden 2002; Nakamura 2004; Arakawa 2005). Thus Graduate School of Medical Science, Kumamoto University, 1-1-1 Honjo, far, a number of p53 transcriptional targets have been Kumamoto 860-8556, Japan identified. These include cell-cycle-regulating proteins 123 (2006) 51:652–664 653 (p21/WAF1, El-Deiry et al.1993; and hCDC4b, Kimura mechanism of the p53-dependent pathway for sup- et al. 2003a), apoptosis-regulating proteins (BAX, Mi- pressing cancer. yashita and Reed 1995; p53AIP1, Oda et al. 2000; Matsuda et al. 2002; Yoshida et al. 2004; and STAG1 Anazawa et al. 2004), DNA damage-repair proteins Materials and methods (p53R2, Tanaka et al.2000; Yamaguchi et al. 2001; Kimura et al. 2003; and GADD45, Kastan et al. 1992), Cell lines and transfections negative regulators of p53 (MDM2, Barak et al. 1993; and Pirh2, Leng et al. 2003), positive regulators of p53 Human cancer cell lines HepG2 (hepatoblastoma), (p53DINP1, Okamura et al. 2001), and inhibitors of H1299 (lung cancer), and COS7 (monkey kidney angiogenesis (TSP-1, Dameron et al. 1994; and BAI1, fibroblast) were purchased from American Type Cul- Nishimori et al. 1997). On the other hand, the analysis ture Collection. Human cancer cell line T98G (glio- of a microarray including 6,000 human genes indicated blastoma) was purchased from Human Science that 107 genes were up-regulated and 54 were down- Research Resource Bank (HSRRB, Japan). All cell regulated in cells infected with the adenovirus vector lines were cultured under conditions recommended by designed to express p53 (Zhao et al. 2000). These their respective depositors. observations clearly suggested that a considerable For transfection, cells were seeded at 2·105 cells per number of p53 target genes have yet to be isolated. well of six-well plates. After 24 h, cells were trans- Moreover, the identification of these additional p53 fected with 1 lg of plasmid mixtures pre-incubated for target genes is likely to be indispensable for under- 15 min with 6 ll of Fugene6 transfection reagent standing the mechanism of the p53-regulated cellular (Roche). response in order to prevent tumor formation and progression. DNA-damaging treatments The DFNA5 gene was initially identified as one of the genes whose expression is inversely correlated with Cells were seeded 12 h before treatment and were 60– that of the estrogen receptor in breast carcinomas, and 70% confluent at the time of treatment. To examine therefore, it was initially designated as ICERE-1 (in- the expression of DFNA5 in response to genotoxic versely correlated with estrogen receptor expression) stresses, HepG2 cells were continuously treated with (Thompson and Weigel 1998). However, later on, a 1.0 lg/ml adriamycin for 2 h, UV-irradiated at 30 J/m2 mutation in the DFNA5 gene was reported to be using a UV cross-linker (Stratagene), or c-irradiated at associated with autosomal-dominant, nonsyndromic 50 Gy using a 60Co source. hearing impairment in an extended Dutch family (Van Laer et al. 1998). An insertion/deletion mutation in DNA microarray intron 7 of this gene caused the skipping of exon 8, which resulted in the premature termination of the Total cellular RNA was extracted at the indicated time open reading frame (Van Laer et al. 1998). Thus far, from HepG2 cells infected with adenovirus vector at two additional mutations in DFNA5 have been re- 30 moi, which was designed to express wild-type p53 ported in a Chinese family and another Dutch family. (Ad-p53-WT), a mutant p53 (Ad-p53-46F) or EGFP Interestingly, these mutations also cause skipping of (Ad-EGFP) (Nakamura et al. 2006). For gene-expres- exon 8, thereby resulting in the generation of the same sion profiling, GeneChip Human Genome U133A and aberrant transcript as in the case of the first Dutch U133B microarrays (Affymetrix, Santa Clara, CA, family identified with the mutations (Yu et al. 2003; USA) were used that contain 22,215 and 22,577 probe Bischoff et al. 2004). Therefore, it was speculated that sets, respectively, to examine a total of about 39,000 hearing impairment may not be due to haploinsuffi- transcripts. Target cRNA for microarray hybridization ciency but rather due to a gain-of-function mutation. was prepared from 5 lg of total RNA according to the Furthermore, mutant DFNA5 may have a new dele- manufacturer’s instructions using a BioArray RNA terious function. Despite these observations, the transcript labeling kit (Enzo Diagnostics, Farmingdale, physiological function of the DFNA5 protein still re- NY, USA). Hybridization to the microarrays, washing mains to be elucidated. and staining with the antibody amplification procedure, Here, we report an unexpected role of the DFNA5 and scanning were also carried out according to the gene in the p53-mediated cellular response to DNA manufacturer’s instructions. The expression value of damage; this implies the involvement of DFNA5 in each gene was calculated and normalized using Af- tumorigenesis. These findings will shed light on the fymetrix Microarray Suite software version 5.0. 123 654 (2006) 51:652–664 RNA interference performed on immunoprecipitated chromatin using specific primers: 5¢-TCTGTGATTGAGTATCCTC We established the p53 knock-down (HepG2-p53-KD) GT-3¢ (p53BS1, forward) and 5¢-CCTGGTGTTGCT and the control (HepG2-p53-Cont) cell lines as de- GACCATG-3¢ (p53BS1, reverse), or 5¢-GATGCCC scribed previously (Hara et al. 2004), which were de- CTCTGTTCTAATT-3¢ (p53BS2, forward) and 5¢-GC rived from a hepatoblastoma cell line (HepG2 that CTCATATGACTCATTCTGT-3¢ (p53BS2, reverse), contains wild-type p53). In brief, HepG2 cells were or 5¢-GCCAGGCTGGTTTCAAACTC-3¢ (p53BS3, infected with SI-MSCV-puro-H1R-p53Ri retrovirus forward) and 5¢-ATGAAGGATCTAGAACTCAA for down-regulation of p53 expression and with SI- GT-3¢ (p53BS3, reverse), or 5¢-ATTCTGATCACAG MSCV-puro-H1R retrovirus for negative control. Then ATCCTCCA-3¢ (p53BS4, forward) and 5¢-GAT the infected cells were selected with 1 lg/ml puromycin GCTGTAGCTGAGGAGC-3¢ (p53BS4, reverse), or for 2 weeks, and the single clones were isolated. 5¢-ACCTTTCACCATTCCCCTAC-3¢ (p21/WAF1, forward) and 5¢-GCCCAAGGACAAAATAGCCA-3¢ Northern blotting (p21/WAF1, reverse). To ensure that PCR was per- formed in linear range, template DNA was amplified Using TRIZOL reagent (GIBCO BRL), total RNAs for a maximum of 30 cycles. were extracted at various time points from HepG2 infected with Ad-p53-WT, Ad-p53-46F or Ad-EGFP, Gene reporter assay and also form HepG2-p53-KD and HepG2-p53-Cont cells subjected to DNA damaging treatments. Total A DNA fragment, including potential p53-binding sites RNAs were further purified to poly(A)+ RNA using of DFNA5, was amplified by PCR, and cloned into the mRNA purification kits (TAKARA) according to the pGL3-promoter vector (Promega, Madison, WI, manufacturer’s instructions.