A balance between proliferation and cell death is critical for achieving desirable high cell densities in mammalian cell culture. In this study, we evaluate a recently discovered anti-apoptotic gene, aven, and examine its effectiveness alone and in combination with a member of the Bcl-2 family, bcl-x(L). The commercially popular cell line, Chinese hamster ovary (CHO), was genetically modified to constitutively express aven, bcl-x(L), and the two genes in combination. Cells were exposed to several model insults that simulate severe bioreactor environments, including serum deprivation, spent medium, and Sindbis virus infection, as well as staurosporine, a known chemical inducer of apoptosis. CHO cells exhibited DNA fragmentation, a hallmark of apoptosis, after exposure to these model insults. After exposure to serum deprivation, 4- and 5-day spent medium, and staurosporine, cells expressing Aven provided limited protection against cell death when compared with the protection afforded by cells expressing Bcl-x(L) alone. However, the highest survival levels for all insults were achieved when Aven was expressed in combination with Bcl-x(L), In fact, Aven appeared to act synergistically to enhance the protective function of Bcl-x(L) for several insults, because the protective function of the two genes expressed together in one cell line often exceeded the additive protective levels of each anti-apoptosis gene expressed alone. Surprisingly, Aven expression provided a mildly proapoptotic response in CHO isolates infected with Sindbis virus. However, CHO cells expressing both Bcl-x(L) and Aven showed protection against Sindbis virus infection due to the inhibitory properties of the bxl-x(L) anti-apoptosis gene. This study shows that combinatorial anti-apoptosis cell engineering strategies may be the most effective mechanisms for providing extended protection against cell death in mammalian cell culture. (C) 2004 Wiley Periodicals, Inc.