Carbohydrate-binding Protein 35: Levels of transcription and mRNA accumulation in quiescent and proliferating cells

Abstract

In previous studies, we observed proliferation-dependent expression and nuclear localization of the lectin, designated carbohydrate-binding protein 35 (CBP35), in 3T3 fibroblasts at the polypeptide level by Western blot and immunofluorescence analysis. In the present study, we have compared the expression of the CBP35 gene in quiescent and proliferative 3T3 cells at the levels of (a) accumulated mRNA by Northern blotting and (b) nuclear transcription by run-off assays. When serum-starved, quiescent cultures of 3T3 cells were stimulated by the addition of serum, there was an increase in the nuclear transcription of the CBP35 gene and in the accumulation of its mRNA early (1–3 h) in the activation process, well before the first wave of DNA synthesis. These increases were not dependent on de novo protein synthesis inasmuch as they occurred even in the presence of cycloheximide. There was also an elevated transcription rate and mRNA level in transformed cells when compared to their normal counterparts. Finally, the expression of CBP35 was compared between sparse, proliferating cultures of 3T3 cells and density-inhibited confluent monolayers of the same cells. Although the rate of transcription of the CBP35 gene was approximately the same in the two cultures, there was a higher level of CBP35 mRNA in the dense cells. Thus, it appears that posttranscriptional mechanisms may be involved in the accumulation of mRNA.