Antiapoptosis chemicals prolong productive lifetimes of mammalian cells upon Sindbis virus vector infection

Viral expression systems allow for the rapid production of large amounts of recombinant protein in cell culture. In particular, Sindbis virus vectors now exist that make possible the expression of a variety of heterologous pr oteins in mammalian culture systems. Unfortunately, infection of cultured c ells with Sindbis virus vectors typically results in apoptotic cell death, as demonstrated in the current study by DNA laddering and fluorescence micr oscopy. Fortunately, it has recently been demonstrated that apoptosis can b e inhibited in vitro by certain chemical reagents that are capable of block ing specific steps during the cell death cascade. In this study, a rat pros tate carcinomal cell line, AT3-neo, was infected with a Sindbis virus vecto r containing the gene for chloramphenicol acetyltransferase (dsSV-CAT) in t he presence of several representative antiapoptotic chemicals and analyzed for cell viability as well as recombinant protein production. N-acetylcyste ine (NAC), pyrrolidine dithiocarbamate (PDTC), bongkrekic acid (BA), and N- benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) all exhibited the capacity to limit apoptosis in the infected cells, in fact, after just 1 day, percentage viabilities of the cells exposed to chemical reagents wer e between 72% and 91%, compared with 44% for the untreated controls. Furthe rmore, cells maintained on these agents were able to survive the infection from 1 to 3 days longer than the control samples. In addition to providing gains in cell viability, chemical treatment allowed for higher levels of re combinant protein production in most cases. Maximum chloramphenicol acetylt ransferase (CAT) productivities in cells maintained on BA, NAG, and Z-VAD.f mk were 1.7-, 2.2-, and 3.9-fold higher than those obtained from the untrea ted cultures. Consequently, the addition of chemical reagents to culture me dia as a means of inhibiting apoptosis may be a valuable tool in the cell c ulture industry, where cell death severely limits productivity levels and a dds significantly to production costs. (C) 1999 John Wiley & Sons, Inc.