Abstract
HeLaM is a variant cell line in which the transcriptional induction of many genes by alpha interferon has special characteristics (Tiwari et al., Mol. Cell. Biol. 8:4289-4294, 1988). The same characteristics were also displayed for induced transcription of a permanently transfected chimeric gene containing the interferon- stimulated response element of gene 561. For understanding the molecular basis of the special requirements of HeLaM cells, an analysis of the interferon-stimulated gene factors (ISGF) was undertaken. By using gel shift assays, it was shown that the activation of ISGF3 by alpha interferon treatment of HeLaM cells had characteristics identical to those of induced transcription: inhibition by 2-aminopurine and the need for ongoing protein synthesis which was obviated by pretreating the cells with gamma interferon. Upon mixing in vitro the cytoplasmic fraction of gamma interferon-treated HeLaM cells with that of cells treated with alpha interferon and cycloheximide, active ISGF3 was reconstituted, presumably through complementation of two components, ISGF3γ and ISGF3α, present in the two respective fractions. Because, unlike other cells, untreated HeLaM cells did not contain detectable levels of either component, we could induce them individually and study their independent properties. Induction of ISGF3γ but not of ISGF3α needed ongoing protein synthesis and was blocked by 2-aminopurine. Once induced, ISGF3γ was active for 24 h and was present in both the nuclear and cytoplasmic fractions. Activated ISGF3α, on the other hand, did not translocate to the nucleus in the absence of ISGF3γ, and in the cytoplasm its activity decayed within 2 h of its activation. In reference to our working model, all of the above observations indicate that ISGF3γ is the product of signal 1 and ISGF3a is the product of signal 2.