Abstract
Three complete cDNA clones encoding Id-related helix-loop-helix (HLH) proteins lacking a basic region were isolated from a pcD2 cDNA expression library prepared from TIG-3 human diploid fibroblasts (HDF). Of these cDNAs (Id-1H, Id-1H', and Id-2H), two (Id-1H and Id-1H') appeared to be derived by alternative RNA splicing. Id-1H and Id-2H seem to be human homologues of mouse Id-1 and Id-2, respectively, and have potential to encode 154 and 135 amino acid proteins. The Id-1H and Id-2H mRNAs were barely detectable in quiescent early passage HDF; serum coordinately induced both mRNAs, with two peaks of expression, in early and late in G1. Antisense oligomers complementary to Id-1H and Id-2H mRNA prevented early passage HDF from entering the S phase of the cell cycle. The treatment of serum-stimulated early passage cells with the antisense Id-1H oligomer completely abolished Id-1H. In senescent cells, serum barely induced the Id-1H and Id-2H mRNAs, although the levels of c-myc expression induced were similar in early passage and senescent cells. The expression levels of these Id genes vary among immortal human cell lines. Both genes were overexpressed in VA4 SV40-transformed lung fibroblasts and EJ-1 bladder carcinoma cells, while these genes were expressed at a very low level in SVts8 cells derived from SV40 tsA-transformed TIG-3 cells. SVts8 cells may acquire some function redundant to Id proteins. HT1080 fibrosarcoma cells expressed the Id-1H gene but not the Id-2H gene, suggesting these Id genes may subserve redundant functions.
Highlights
From the Departments of $Applied Biological Science and $Biological Science and Technology, Science University of Ibkyo, Noda 278, Japan, the Wesearch Institute for Microbial Disease, Osaka University, Suita 565, Japan, the IDepartment of Cellular and Molecular Biology, Hiroshima University School of Medicine, Hiroshima 734, Japan, and the **Department of Cell and Molecular Biology, Lawrence Berkeley Laboratory, Berkeley, California 94720
Althoughmost pared from TIG-3 human diploid fibroblasts (HDF)
Because antisense oligomers inhibited DNA synthesis in early passage Human diploid fibroblasts (HDF), we suggest that Id-1H and Id-2H expression is required for the Go to S phase transition and that re
Summary
Cipitates were collected on protein A-agarose beads and washed four times with lysis buffer. The precipitate was dissolved in 25 pl of SDS. Cell Culture-TIG-3 human embryonic lung fibroblasts(31)were a sample buffer, boiled for 10 min, and subjected to electrophoresison a gift from the Japanese Cancer Research Resources Bank, received a t 15% SDS-polyacrylamidegel. The cells were cultivataedt 37 "C in Dulbecco's modified Eagle's medium containing 10% fetal calf serum
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