Inhibition of Single Minded 2 gene expression mediates tumor-selective apoptosis and differentiation in human colon cancer cells Mireille J. Aleman*†‡§, Maurice Phil DeYoung*†§¶, Matthew Tress*†, Patricia Keating*†, Gary W. Perryʈ, and Ramaswamy Narayanan*†** *Center for Molecular Biology and Biotechnology, Departments of †Biology and ‡Chemistry, and ¶Center for Complex System and Brain Sciences, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431 Communicated by Herbert Weissbach, Florida Atlantic University, Boca Raton, FL, July 21, 2005 (received for review April 4, 2005) A Down’s syndrome associated gene, Single Minded 2 gene short bound AhR͞ARNT complex (12) and hence prevent carcinogen form (SIM2-s), is specifically expressed in colon tumors but not in metabolism, leading to cumulative DNA damage and cancer. the normal colon. Antisense inhibition of SIM2-s in a RKO-derived The growth arrest and DNA damage (GADD) family of genes colon carcinoma cell line causes growth inhibition, apoptosis, and was originally isolated from UV radiation-treated cells and subse- inhibition of tumor growth in a nude mouse tumoriginicity model. quently grouped according to their coordinate regulation by growth The mechanism of cell death in tumor cells is unclear. In the present arrest and DNA damage (13). The GADD family members include study, we investigated the pathways underlying apoptosis. Apo- GADD34,-45␣,-45␤,-45␥, and -153 (14, 15). These are stress- ptosis was seen in a tumor cell-specific manner in RKO cells but not response genes induced by both genotoxic and nongenotoxic in normal renal epithelial cells, despite inhibition of SIM2-s expres- stresses (16–18). GADD45␣ is the most extensively studied mem- sion in both of these cells by the antisense. Apoptosis was de- ber of the family and is regulated in both a p53-dependent and pended on WT p53 status and was caspase-dependent; it was -independent manner (13, 19). GADD45␣-mediated apoptosis may inhibited by a pharmacological inhibitor of mitogen-activated involve activation of JNK and͞or p38 mitogen-activated protein protein kinase activity. Expression of a key stress response gene, kinase (MAPK) signaling pathways (14, 20). CELL BIOLOGY growth arrest and DNA damage gene (GADD)45␣, was up-regu- We have recently demonstrated that the SIM2 short-form lated in antisense-treated tumor cells but not in normal cells. In an (SIM2-s) gene is specifically expressed in distinct solid tumors, isogenic RKO cell line expressing stable antisense RNA to including colon, pancreas, and prostate, but not in the correspond- GADD45␣, a significant protection of the antisense-induced apo- ing normal tissues (21–23). Antisense inhibition of SIM2-s expres- ptosis was seen. Whereas antisense-treated RKO cells did not sion caused apoptosis in colon and pancreatic cancer-derived cells. undergo cell cycle arrest, several markers of differentiation were In an effort to understand the molecular mechanism underlying the deregulated, including alkaline phosphatase activity, a marker of role of SIM2-s in tumor cell growth, we have embarked on mapping terminal differentiation. Protection of apoptosis and block of the key pathways linked to SIM2-s. We show that apoptosis is tumor differentiation showed a correlation in the RKO model. Our results cell-selective and requires GADD45␣ function. Further, we dem- support the tumor cell-selective nature of SIM2-s gene function, onstrate that key pathways, including caspase, p38 MAPK activities, provide a direct link between SIM2-s and differentiation, and may and WT p53 status, are critical to apoptosis. Our results link provide a model to identify SIM2-s targets. apoptosis to induction of tumor cell-selective differentiation. Down’s syndrome ͉ antisense ͉ p53 Methods Cell Culture. Isogenic colon cancer-derived cell lines (RKO, RKO- ␣ he Single Minded 2 (SIM2) gene is present in chromosome 21 E6-p53- knockout, and RKO-AS45.1-stable GADD45 antisense Tat the Down’s syndrome critical region, which in triplication is clone) and OM-1 and SW 480 colon carcinoma-derived cells were associated with diverse phenotypic characteristics of Down’s syn- obtained from American Type Culture Collection and were main- drome (1). Patients with Down’s syndrome show various neurolog- tained as recommended by American Type Culture Collection. ical symptoms and a high incidence of leukemia (1, 2). Members of Early-passage primary human renal epithelial (HRE) cells were the SIM family include SIM1 and SIM2, which map to 6q16.3-q21 obtained from Cambrex (Walkersville, MD) and were cultured and 21q22.2, respectively (3), and belong to a family of transcription following the manufacturer’s instructions. factors containing a basic helix–loop–helix motif, two period ho- mologue (PER)͞ARNT͞SIM (ARNT, aryl hydrocarbon receptor Inhibitors and Antibodies. Antisense SIM2-s was synthesized as nuclear translocator) domains, and the HIF1␣͞SIM͞TRH domains described (22). Methylmethane sulfonate was obtained from Sigma (4–6). In Drosophila, SIM is a master regulator of fruit-fly neuro- Aldrich. Caspase-family inhibitor set IV was obtained from Biovi- genesis, regulating midline gene expression (6, 7). The SIM2 gene sion (Mountain View, CA). p38 kinase inhibitor (SB202190) was exists in two distinct forms, the long and short forms (SIM2-l and obtained from A. G. Scientific (San Diego). General tyrosine SIM2-s), due to alternative splicing (3). A putative cancer-related kinase inhibitor (Genistein) was from Sigma Aldrich. Antibodies role of the SIM family of genes is their ability to transcriptionally were obtained from Santa Cruz Biotechnology. regulate key metabolic enzymes to inactivate carcinogens (8). Binding of carcinogens to the aryl hydrocarbon receptor (AhR), Abbreviations: ALP, alkaline phosphatase; GADD, growth arrest and DNA damage gene; which is kept sequestered in the cytoplasm by heat-shock protein MAPK, mitogen-activated protein kinase; SIM2, Single Minded 2; SIM2-s, SIM2 short form; (HSP) 90 (9), dissociates HSP 90. The ligand-bound AhR is then HRE, human renal epithelial. translocated into the nucleus, where it can dimerize with ARNT §M.J.A. and M.P.D. contributed equally to this work. (10). This complex binds to the xenobiotic response element, ¶Present address: Massachusetts General Hospital and Harvard Medical School, Boston, present in the promoters of key oxidative enzymes, and activates MA 02114. gene transcription (8, 11), thus causing inactivation of the carcin- **To whom correspondence should be addressed. E-mail: [email protected]. ogen. The SIM proteins can inhibit the dimerization of the ligand- © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0505484102 PNAS ͉ September 6, 2005 ͉ vol. 102 ͉ no. 36 ͉ 12765–12770 Downloaded by guest on September 29, 2021 RT-PCR. End-point RT-PCR analysis and primers used were de- To further clarify the apoptotic mechanism, we next used specific scribed previously (22, 23). Quantitative real-time PCR was done by pathway inhibitors. The RKO cells were significantly protected using ␤2 microglobulin standards (Roche Applied Sciences, Indi- from apoptosis induced by SIM2-s antisense by both general anapolis). Exon-specific RT-PCR primers were used (see Table 1, (Z-VAD-FMK) and specific caspase inhibitors (caspase 9 and 10). which is published as supporting information on the PNAS This effect was not seen when caspase 2 or 8 inhibitors were used web site). (Fig. 1D). Inhibitors of other caspases (caspases 3, 4, 5, 6, and 13) showed only marginal protection (10–15%) against apoptosis (data Cell Cycle and FACS Analysis of GADD45␣ Protein. Cells were har- not shown). To further map the signal transduction involved in vested after 100 nM antisense or control oligo treatment and fixed apoptosis, we next used key kinase pathway pharmacological in- at 4°C overnight in 70% ethanol. The fixed cells were resuspended hibitors. Significant protection from apoptosis was seen when the in PBS and treated with 20 ␮g͞ml RNaseA for 30 min at 37°C, RKO cells were pretreated with a p38 MAPK pathway inhibitor stained with 60 ␮g͞ml propidium iodide at room temperature for (SB 202190). As a control, we used Genistein, a nonspecific tyrosine 30 min, and scanned by an Epics XL-MCL Flow Cytometer kinase inhibitor, which did not cause such a protection (Fig. 1E). (Beckman Coulter). Cell cycle distribution was calculated by using Treatment of RKO cells with these inhibitors alone did not cause MULTICYCLE V.3.1.1 software (Phoenix Flow Systems, San Diego). apoptosis (data not shown). GADD45␣ protein expression was analyzed in antisense-treated We next addressed whether apoptosis mediated by the inhibition RKO or HRE cells by using either GADD45␣-phycoerythrin of SIM2-s expression depends on p53 tumor suppressor gene (PE)-conjugated antibody or its IgG1 isotype control PE- function. Isogenic RKO (WT p53) or RKO-E6 (p53 functional conjugated antibody by using an Epics XL-MCL Flow Cytometer knockout by HPV-E6 oncogene) cells were treated either with the (Beckman Coulter). control oligo or SIM2-s antisense and at the indicated time analyzed by apoptosis ELISA (Fig. 1F). In the isogenic RKO E6 cells, SIM2-s Caspase and p38 Kinase Inhibitor Experiments. For inhibitor studies, antisense-induced apoptosis was 4- to 5-fold reduced. the cells were pretreated with 20 ␮M caspase inhibitors, 20 ␮M SB202190 (p38 MAPK inhibitor), or 10 ␮M Genistein (general Identification of GADD45␣ as a Critical Mediator of SIM2-s Antisense tyrosine kinase inhibitor) for 1 h before transfection with the Mechanism. Up-regulation of GADD genes is often seen in tumor oligonucleotides and then continuously incubated with the inhibi- cells undergoing apoptosis. Hence we investigated the regulation of tors. At select time points, apoptosis was measured by using the Cell GADD family members in the SIM2-s antisense-treated RKO cells Death Detection ELISAPlus (Roche Applied Science), as described (Fig. 2A). The inhibition of SIM2-s by antisense in RKO cells (23).