Interferon-independent activation of (2'-5') oligoadenylate synthetase in Friend erythroleukemia cell variants exposed to HMBA.

Abstract

To provide evidence for the implication of interferon (IFN)-induced proteins in the regulation of cell growth during differentiation, the activation of (2'-5') oligoadenylate synthetase (2-5A synthetase) as well as of PKR, two IFN-induced proteins, during differentiation of Friend erythroleukemia cells, was studied. Two cell variants were used. The first (FL) was completely susceptible to hexamethylene bis-acetamide (HMBA)-treatment and responded in both growth-retardation and hemoglobin synthesis. The second (R1) failed to synthesize hemoglobin in response to HMBA although cell growth was still inhibited. In both cell variants, 2-5A synthetase enzyme activity was induced in a similar fashion, reaching a peak at 26 hours after treatment with HMBA. However, the down regulation of activity thereafter was not identical in both cases. In R1 cells, the reduction was much slower compared to FL cells. A similar pattern was observed with the appearance of the 43 kDa isoform of 2-5A synthetase in immunoblots. An analysis of 2-5A synthetase gene expression revealed the presence of 1.7 kb transcripts which peaked at 16 hours after HMBA-treatment in both cell variants. Again, the down-regulation in expression was slower in R1 than in FL cells. Addition of anti-murin alpha/beta-IFN antibodies did not reduce the level of either 2-5A synthetase expression or enzyme activity in either cell variant. Interestingly, the presence of antibodies also did not affect the pattern of pRb phosphorylation in the cell variants exposed to HMBA. In both cell variants, an increase in the amount of the phosphorylated form (ppRb) was observed in immunoblots after 4 hours. This form was gradually transformed to the underphosphorylated molecule (pRb) with time in culture, even in the presence of antibodies. This further substantiates the notion that IFN-induced regulation of pRb phosphorylation is mediated by IFN-induced proteins. The basal level of either expression or ezymatic activity of PKR detected in untreated FL or R1 cells, was relatively high. Treatment with HMBA did not result in further induction of PKR in either cell variant.