Research Article The Early Response to DNA Damage Can Lead to Activation of Alternative Splicing Activity Resulting in CD44 Splice Pattern Changes Valery Filippov, Maria Filippova, and Penelope J. Duerksen-Hughes Department of Biochemistry and Microbiology, Loma Linda University School of Medicine, Loma Linda, California Abstract in these cells, p53-independent pathways must be engaged. This is Expression of the human papillomavirus 16 E6 oncogene particularly true in cervical carcinoma cases caused by human interferes with several vital cellular processes, including the papillomavirus (HPV) 16 infection, as the viral E6 protein p53-dependent response to DNA damage. To assess the effectively eliminates cellular p53 by accelerating its proteolysis influence of E6 on the early response to DNA damage, we (6). E6-expressing cells, therefore, can function as a model system analyzed gene expression following mitomycin C–induced for discovering and studying p53-independent, DNA damage– genotoxic stress in human E6–expressing U2OS cells triggered apoptotic pathways. (U2OSE64b) as well as in p53-expressing control cells In contrast to the well-established role of transcriptional (U2OSE6AS) by comparative global expression profiling. As regulation, alternative splicing is only now emerging as an expected, genes involved in p53-dependent pathways were important mechanism for regulating gene function. It has been estimated that at least 74% of multi-exon genes are alternatively activated in p53-expressing cells. In the U2OSE64b cells, however, a largely nonoverlapping group of genes was spliced (7), and these isoforms frequently have opposing functions. identified, including two splicing factors of the SR family. This may have a significant effect on a variety of cell processes, Immunoblot analysis revealed increased expression of several including apoptosis (8). SR proteins during the early response to DNA damage, which There is now ample evidence that aberrations of alternative was accompanied by activation of alternative splicing activity. splicing are widespread in tumors (9). Although the relationship Disruption of splicing activity by treatment with small between changes in splicing and tumor progression has not yet been well-defined, condition-dependent splicing alterations in such interfering RNA directed against splicing factor SRp55 FAS, RBM9, CD44, hnRNPA/B, APLP2 MYL6 resulted in the increased viability of p53-deficient cells genes as , and were following DNA damage. To determine whether the transient found in breast cancer cells by using splicing-sensitive microarrays activation of splicing activity was due to E6-mediated (10). Despite growing interest in splicing regulation and the degradation of p53, or was due to some other activity of E6, discovery of a large number of genes involved in pre–RNA splicing, we compared the early response of the p53 wild-type and the mechanisms governing this process are not well-understood (11). It is believed that alternative splicing activity is regulated by p53À/À isogenic HCT116 cell lines, and found that the increase in splicing activity was observed only in the absence the combinatorial actions of a fairly large number of regulatory of p53. Finally, both the U2OSE64b and the p53À/À cells factors, including two families of proteins that generally function in showed altered splicing patterns for the CD44 receptor. an antagonistic manner, heterogeneous ribonucleoproteins and SR Together, these data show that cells lacking p53 can activate (serine/arginine-rich) proteins (12). SR proteins participate in both constitutive and alternative alternative splicing following DNA damage. [Cancer Res 2007;67(16):7621–30] splicing. Although they seem to be functionally redundant in constitutive splicing, a growing body of evidence shows that they have distinctive and not fully overlapping functions in alternative Introduction splicing. Each particular SR protein is able to bind to a specific Genotoxic stresses from chemical, physical, or viral agents splicing enhancer and activate splicing of weak splicing sites, initiate damage response pathways that lead to cell cycle arrest, leading to altered splicing patterns of target genes (12). DNA repair, and apoptosis (1, 2). The mechanisms by which these In this report, we have investigated the early response of the cellular responses are triggered are not fully understood, but it is osteosarcoma U2OSE64b (E6-expressing) and colon carcinoma clear that the p53 tumor repressor is one of the most important HCT116 p53À/À cell lines to DNA damage caused by the genotoxic checkpoint proteins, influencing multiple response pathways to a drug mitomycin C. The results from this study provide strong diversity of stressors (3). evidence that alternative splicing activity can be involved in However, p53 is not indispensable for the induction of apoptosis regulating the stress response in the absence of the classical p53- (4, 5). This has clinical relevance, as more than half of human dependent pathways. tumors lack wild-type p53. Therefore, if apoptosis is to be induced Materials and Methods Cell lines and cell culture. U2OS cells, derived from a human Requests for reprints: Penelope J. Duerksen-Hughes, Department of Biochemistry osteosarcoma, were obtained from the American Type Culture Collection. and Microbiology, 121 Mortensen Hall, Loma Linda University School of Medicine, HCT116 human colorectal cancer cells and p53-null derivatives (13) were Loma Linda, CA 92354. Phone: 909-558-4300, ext. 81361; Fax: 909-558-0177; E-mail: supplied by Dr. B. Vogelstein. They were cultured in McCoy’s 5A medium [email protected]. I2007American Association for Cancer Research. (Invitrogen) supplemented to contain 10% fetal bovine serum (Invitrogen), doi:10.1158/0008-5472.CAN-07-0145 penicillin (100 Ag/mL), and streptomycin (100 Ag/mL; Sigma). Construction www.aacrjournals.org 7621 Cancer Res 2007; 67: (16). August 15, 2007 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Cancer Research of the pHA-E6S and pHA-E6AS plasmids, which respectively, contain either minigene fused to luciferase (16) as described previously (17) and split into the sense or the antisense versions of epitope-tagged E6 (HA-E6) under the a 96-well plate 24 h later. At 48 h posttransfection, cells were treated with control of the CMV promoter, and establishment of the U2OSE64b and 2 Ag/mL of mitomycin C for the indicated amount of time and harvested in U2OSE6AS cell lines by stable transfection with these plasmids, have been luciferase lysis buffer (Promega). Luciferase activity was quantified using a described previously (14). MicroLumatPlus microplate reader (Berthold Technology). Three indepen- Measurement of mitomycin C cytotoxicity. To measure cell survival dent experiments, were each conducted in triplicate, and significance was following mitomycin C treatment, cells in exponential growth were exposed analyzed by one-way ANOVA. Differences were considered significant at a to mitomycin C (Roche) at the concentrations noted for the individual 0.05 level of confidence. experiments. After incubation for the indicated time, the number of viable Inhibition of SFRS6 gene expression by small interfering RNA. Cells cells was quantified by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- were transfected with predesigned small interfering RNAs (siRNA) for the lium bromide (MTT) assay as described earlier (14). Three independent human SFRS6 gene from Ambion or from Santa Cruz Biotechnology, or with experiments were each conducted in triplicate and significance was control siRNA using the siRNA transfection kit as recommended by the analyzed by Student’s t test. manufacturer (Santa Cruz Biotechnology). Levels of silencing of the SFRS6 Immunoblot analysis. Proteins were extracted from cells (106) in 100 AL gene were evaluated by RT-PCR and immunoblot analyses as described of Laemmli’s buffer. Lysates were sonicated and protein concentration was above. Twenty-four hours posttransfection, cells were treated with D A determined by the C protein assay (Bio-Rad). Lysates (15 g) were mitomycin C for 24 h, followed by quantification of viable cells by the separated on SDS-polyacrylamide gels and transferred onto Immobilon MTT assay (14). Three independent experiments were done, each was P membranes (Millipore). Membranes were probed with primary antibodies conducted in quadruplicate, and significance was analyzed by one-way directed against p53 (NCL-p53-DO7; Novocastra), poly(ADP-ribose) poly- ANOVA. Differences were considered significant at a 0.05 level of merase-1 (PARP-1; Ab-2; EMD Biosciences), phosphorylated Chk1 (phospho- confidence. Chk1-Ser345; Cell Signaling Technology), h-actin (Sigma), or pan-SR (1H4; American Type Culture Collection). After washing, membranes were incubated with the appropriate horseradish peroxidase–conjugated sec- Results ondary antibodies (Sigma), and bands were detected using the SuperSignal E6 decreases the level of cellular p53, but does not inhibit West Substrate System (Pierce). Densitometry measurements were done cell death following DNA damage. Our laboratory has previously using the ChemImager Imaging System and processed with the AlphaEase shown that in U2OS cells in which E6 expression is regulated by a software package (Alpha Innotech). tetracycline-responsive promoter, increased amounts of E6 led to a Semiquantitative reverse transcriptase-PCR. Reverse transcriptase- dose-dependent decrease in p53 as measured