[CANCER RESEARCH 63, 8768 – 8776, December 15, 2003] IG20, a MADD Splice Variant, Increases Cell Susceptibility to -Irradiation and Induces Soluble Mediators That Suppress Tumor Cell Growth Elena Efimova, 1 Osvaldo Martinez, 1 Anna Lokshin, 2 Takayasu Arima, 1 and Bellur S. Prabhakar 1 1 Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, Illinois, and 2 LUMINEX Core Facility, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania ABSTRACT The IG20 gene encodes at least four splice variants, including DENN-SV and IG20. DENN-SV is constitutively expressed at higher levels in tumor tissues. Cells transfected with the DENN-SV cDNA show in- creased resistance to tumor necrosis factor  (TNF), TNF-related apo- ptosis-inducing ligand (TRAIL), etoposide, and vinblastine treatment, whereas overexpression of IG20 enhanced susceptibility to both intrinsic (drugs) and extrinsic (e.g., TNF and TRAIL) death signals. In this study, we investigated whether expression of the IG20 can render cells suscep- tible to -irradiation. Consistent with previous results, overexpression of DENN-SV and IG20 in HeLa cells conferred resistance and susceptibility, respectively, to the effects of -irradiation. HeLa IG20 cell susceptibility was attributable to enhanced apoptosis and reduced cell growth. This growth suppression was mediated by secreted soluble factors. Although HeLa DENN-SV cells grew more rapidly than control cells, replenishment with conditioned media from HeLa IG20 cells suppressed their growth. In addition, the conditioned media from HeLa IG20 cells stopped the growth of ovarian PA-1 cancer cells in the G 1 -G 0 cell cycle stage. Among an array of cytokines tested, interleukin 6 (IL-6) was found at the highest levels in HeLa IG20 culture supernatants, and IL-6 neutralization showed that it was, in part, responsible for the cell growth suppression. HeLa IG20 cells had elevated basal nuclear factor B levels, a known regulator of IL-6 transcription. Finally, IG20 overexpression enhanced the combined apo- ptotic effects of TRAIL and -irradiation on HeLa cells. These results suggest that understanding further the mechanism of action of the IG20 splice variant may help in the advancement of cancer therapies. INTRODUCTION To date, we and others (1– 4) have identified seven putative splice variants of the IG20 gene. The differences between the splice variants are limited to alternative splicing of exons 13L, 16, 21, 26, and 34. However, extensive analyses of expression of these splice variants using 57 human normal and tumor tissues and 14 different cell lines revealed that only four of the seven splice variants (namely IG20, MADD, IG20-SV2, and DENN-SV) are expressed. Interestingly, the IG20 gene is overexpressed in tumor tissues compared with normal tissues (2, 3). Additional examination of the gene expression patterns in normal and tumor tissues revealed that this overexpression was mostly represented by DENN-SV (4). When the IG20 variants were stably expressed in HeLa cells, it was observed that only two of the four variants could readily influence cell proliferation and induced cell death. Overexpression of DENN-SV and IG20 in transfected cells con- ferred significant resistance and susceptibility, respectively, to tumor necrosis factor  (TNF), and TNF-related apoptosis-inducing ligand (TRAIL) induced cell death by increasing caspase and nuclear factor B (NFB) activity, respectively. Furthermore, IG20 could potentiate TRAIL-induced apoptosis by increasing the recruitment of Fas-asso- ciated death domain and caspase-8 to the death-inducing signaling complex. 3 The contrasting effects of DENN-SV and IG20 on susceptibility to death-inducing stimuli suggest that the eventual outcome of these signaling pathways in tumor cells is determined, at least in part, by a balance in the expression levels of these two proteins. Indeed, HeLa cells that normally express all four variants on treatment with TNF undergo apoptosis; however, approximately only one-half of the cells die. When dying cells were separated, on the basis of expression of apoptotic markers, from living cells and tested for expression of various splice variants, it was noted that whereas cells undergoing apoptosis expressed higher levels of IG20 (4), the viable cells ex- pressed higher levels of DENN-SV. The high level of expression of DENN-SV and the consequent imbalance in the relative levels of DENN-SV and IG20 found in human tumors suggest a role in tumor formation and/or propagation. It is already known that IG20 and DENN-SV can increase or decrease apoptosis, respectively, in stably transfected HeLa cells treated with etoposide or vinblastine (4). How IG20 and DENN-SV influence apoptosis activated by the intrinsic pathway remains unresolved. It is also not yet established whether these variants can similarly modulate the effects of -irradiation. Radiation therapy takes advantage of the inherently unstable nature of tumors. The DNA lesions induced by -irradiation activate an intrinsic cellular pathway for dealing with DNA damage (5). Cells initiate a set of physiological responses thought to facilitate DNA repair processes that include cell cycle arrest in G 1 , S, and G 2 (a slowing of DNA replication) phases and increased transcription of genes encoding proteins that participate in DNA replication and repair. If the degree of damage suffered by a cell is extensive, then the apoptotic pathway is activated, leading to cell death. At the molecular level, several pathways have been studied, including p53-dependent and p53-independent pathways (6, 7). Other molecules involved in the response to DNA damage include ataxia telangiectasia mutated, ataxia telangiectasia mutated and Rad3 related, DNA-protein kinase, hCds1/ Chk2, and p21. Although there is considerable insight on the molec- ular mechanisms that regulate DNA damage, the process remains unclear. In this study, we examined the effects of IG20 on tumor cell growth and -irradiation-induced apoptosis. We demonstrate that the expres- sion of IG20 enhances the effects of -irradiation, whereas DENN-SV promotes cancer cell survival. We further demonstrate that HeLa cells stably transfected with IG20 produce soluble factors that can function in an autocrine or paracrine manner and markedly slow cell growth. We show that conditioned media (CM) from HeLa IG20 cells could cause growth arrest of PA-1 ovarian cancer cells, specifically at the G 0 -G 1 stage of the cell cycle. Testing for a variety of soluble medi- ators showed a significant increase in interleukin 6 (IL-6) production by the HeLa IG20 cells. The induction of IL-6 was probably due to an increase in basal levels of NFB activity. Neutralization of IL-6 Received 7/10/03; revised 10/13/03; accepted 10/20/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Bellur S. Prabhakar, Department of Microbiology and Immu- nology (M/C 790), Room E-709, Building 935, 835 South Wolcott Avenue, Chicago, IL 60612. Phone: (312) 996-1831; Fax: (312) 996-6415; E-mail: bprabhak@uic.edu. 3 M. Ramaswamy, E. V. Efimova, O. Martinez, N. U. Mulherkar, S. P. Singh, and B. S. Prabhakar. IG20 (MADD splice variant-5), a pro-apoptotic protein, interacts with DR4/DR5 and enhances TRAIL induced apoptosis by increasing recruitment of FADD and caspase-8 to the DISC, submitted for publication. 8768 Research. on July 1, 2015. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from