ISOLATION OF A CDNA-ENCODING A GROWTH-ARREST ASSOCIATED GENE AND CHARACTERIZATION OF ITS REGULATION

We are interested in understanding the molecular events associated wit h the growth-arrest of vascular SMCs. We constructed a subtracted cDNA library enriched in nucleotide sequences associated with quiescent SM Cs. This library was screened with similarly subtracted P-32-labeled c DNAs to identify growth-arrest associated cDNA clones. Characterizatio n of 19 of these cDNA clones revealed that 9 hybridized to mRNAs that exhibited a 2-3-fold increase in growth-arrested SMCs. In addition, tw o other cDNAs hybridized to a 5 Kb mRNA that was elevated approximatel y 10-fold in high density growth-arrested SMCs. Genomic Southern blot hybridization and DNA sequencing analysis indicated that these cDNAs e ncoded the same gene (LG7) and that this gene may be a member of a mul tigene family or that it may contain a sequence shared by other unrela ted genes. Augmented expression of LG7 was associated with both high c ell density and serum deprivation induced growth-arrest. LG7 mRNA expr ession was down-regulated when SMCs were incubated with FBS or with re agents that arrest cells in early S-phase. Additional analysis with ce ll cycle specific inhibitors indicated that LG7 mRNA levels were also low when cells were blocked at the G(2) phase of the cell cycle but bl ockage at mitosis resulted in an elevated level of LG7 mRNA. We furthe r demonstrated that the expression of LG7 was dependent on the presenc e of a relatively labile protein since protein synthesis inhibitors sp ecifically blocked the expression of this mRNA but not the mRNA expres sion of alpha(1)(III) collagen or ferritin H-chain. Finally, we demons trated that Bt(2)cAMP was able to induce mRNA expression of LG7 within 2 h, suggesting that this gene may be directly regulated via the cycl ic-AMP-dependent protein kinase pathway. (C) 1995 Wiley-Liss, Inc.