We describe the molecular identification of a gene, designated T1, whose expression in mouse NIH 3T3 cells is strongly induced by the Ha-ras(EJ) and v-mos oncogenes and by serum. The T1 gene encodes a 38.5-kDa protein, as predicted from its primary sequence and shown by in vitro translation. The protein was processed at its amino terminus and extensively modified by N-linked glycosylation in vitro in the presence of microsomal vesicles. Sequence comparison of T1 with the MIPSX data base (Max-Planck-Institut fir Biochemie, Martinsried/Munich) revealed similarity to the human carcinoembryonic antigen, a tumor marker which is overexpressed in colon adenocarcinomas and in fetal tissues.Considerable sequence similarity has also been observed to the short conserved region of other proteins which, like carcinoembryonic antigen, are encoded by members of the immunoglobulin gene superfamily.The binding of growth factors to their cell surface receptors induces a cascade ofevents which culminates in the induction of DNA synthesis and mitosis. The initiation of a mitogenic stimulus requires the expression of genes which presumably control these events (1). Oncogenically transformed cells exhibit a reduced requirement for growth factors. One might therefore expect oncogene expression to lead to the activation of those genes whose expression is also induced through the action of growth factors. Other genes are selectively induced by oncoproteins (ref. 2 and references therein), and their products are thought to bring about the transformed phenotype. We have tried to identify specific alterations in gene expression mediated by the conditional expression of the Ha-ras(EJ) and the v-mos oncogenes. We have previously isolated a gene, designated T1, whose expression is transiently stimulated by the two oncogene products as well as by serum growth factors in quiescent cells (32). Here we describe the molecular identification of the T1 gene. § It encodes a glycoprotein with sequence similarity to the carcinoembryonic antigen (CEA) and it is probably a member of the immunoglobulin gene superfamily. METHODSCell Lines and Culture Conditions. The NIH 3T3 cell line, stably transfected with the viral mos oncogene under the control of the mouse mammary tumor virus (MMTV) promoter (NIH[LTR v-mos]) has been described (3). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Growth arrest was induced by the addition of 1.5% fetal calf serum.RNA Preparation and Northern Blots. For the isolation of total RNA, cells were directly lysed in a suspension of 4 ml of 150 mM Tris-HCI, pH 9.0/5 mM EDTA/1% sodium dodecyl sulfate (SDS) and 8 ml of water-saturated, neutralized phenol on a 15-cm culture dish. This phenol extraction was followed by two phenol/chloroform extractions at 40C. The RNA was precipitated once in 2.5 M LiCl and once in 70% (vol/vol) ethanol. Total RNA (3 Ag) was glyoxylated, electrophoresed (33), blotted onto nitrocellulose membranes, prehybridized, and hybridized to a probe labeled...

A cDNA clone, T1, has been isolated whose corresponding mRNA was transiently expressed at highly elevated levels after conditional expression of the Ha-ras(EJ) gene and after mitogenic activation of quiescent NIH 3T3 cells. Glucocorticoid hormone stimulated substantial T1 expression as well but only in proliferating cells. At least two different signaling pathways participate in the regulation of the T1 gene: a protein kinase C-dependent signal is involved in the response of proliferating NIH 3T3 cells to glucocorticoid in the absence but not the presence of p21ras, whereas a protein kinase C-independent mechanism mediates the response to serum factors. Treatment of cells with the protein kinase inhibitor 2-aminopurine blocked induction of expression of the T1 gene. T1 mRNA accumulation is regulated at the transcriptional level.

A cDNA clone, T1, has been isolated whose corresponding mRNA was transiently expressed at highly elevated levels after conditional expression of the Ha-ras(EJ) gene and after mitogenic activation of quiescent NIH 3T3 cells. Glucocorticoid hormone stimulated substantial T1 expression as well but only in proliferating cells. At least two different signaling pathways participate in the regulation of the T1 gene: a protein kinase C-dependent signal is involved in the response of proliferating NIH 3T3 cells to glucocorticoid in the absence but not the presence of p21ras, whereas a protein kinase C-independent mechanism mediates the response to serum factors. Treatment of cells with the protein kinase inhibitor 2-aminopurine blocked induction of expression of the T1 gene. T1 mRNA accumulation is regulated at the transcriptional level.

The conditional expression of the v-mos and Ha-ras(EJ) oncogenes in NIH 3T3 cells leads to the accumulation of a 23-kDa protein (p23) (R. Klemenz, S. Hoffmann, R. Jaggi, and A.-K. Werenskiold, Oncogene 4:799-803, 1989). We purified p23 to homogeneity and determined part of the amino acid sequence. The obtained sequence is identical with that of the eye lens protein alpha B crystallin. Northern (RNA) blot and Western immunoblot experiments were performed to demonstrate that alpha B crystallin mRNA and protein do indeed accumulate as a consequence of v-mos and Ha-ras oncogene expression. Comparison of cDNA clones obtained from the mRNA of eye lenses and of oncogene-expressing fibroblasts revealed identity between them. The major transcription initiation site of the alpha B crystallin gene in our experimental system was shown by primer extension experiments to be identical with the one used in eye epithelial cells. In addition, we identified a second minor initiation site 49 nucleotides further upstream. Serum growth factors did not stimulate alpha B crystallin expression in growth-arrested cells.