Cell Growth & Differentiation, Vol 1, Issue 11 511-518, Copyright © 1990 by American Association of Cancer Research

ARTICLES

Isolation of a gene sequence induced later by tumor-promoting 12-O-tetradecanoylphorbol-13-acetate in mouse osteoblastic cells (MC3T3-E1) and expressed constitutively in ras-transformed cells

K Nose, H Saito and T Kuroki
Department of Cancer Cell Research, University of Tokyo, Japan.

Growth factors and tumor-promoting phorbol esters induce various types of "early response" genes, most of which encode transcription factors. To identify possible target genes for these early response gene products, we isolated complementary DNA clones of genes that were induced later by 12-O-tetradecanoylphorbol-13-acetate. A complementary DNA library was constructed using mRNA from mouse osteoblastic cells (MC3T3-E1) treated with 12-O-tetradecanoylphorbol-13-acetate for 4 h, and the library was screened by differential hybridization. Five independent clones were isolated. All of these genes were induced maximally 4 to 8 h after 12-O-tetradecanoylphorbol-13-acetate addition. Sequencing showed that three of them (OTS-1, -3, and -4) were those of the metallothionein II gene, and one (OTS-5) was identical to the osteopontin/2ar gene. The other (OTS-8) had no significant homology with known sequences and was considered to be a novel gene. The induction of OTS-5 and OTS-8 was decreased in the presence of cycloheximide. In mice, OTS-8 mRNA was detected in the lungs but not at appreciable levels in other organs. Open reading frame of OTS-8 complementary DNA and its in vitro transcription/translation product suggested that this gene encodes an Mr 26,000 protein. OTS-8 mRNA was induced by epidermal growth factor but not by serum. The steady-state levels of OTS-5 and OTS-8 mRNAs were significantly elevated in v-ras-transformed MC3T3 cells.

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