Molecular Cloning and Biological Activity of a Novel Ha-Ras Suppressor Gene Predominantly Expressed in Skeletal Muscle, Heart, Brain, and Bone Marrow by Differential Display Using Clonal Mouse EC Cells, ATDC5

Abstract

We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 consists of 167 amino acids and shows 46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.The mammalian ras protooncogenes, Ha-, Ki-, and N-ras, are expressed in a variety of tissues (1). For example, Ha-ras is highly expressed in skin and skeletal muscle; Ki-ras in gut and thymus; and N-ras in testis and thymus. Ras proteins bind guanine nucleotides and possess intrinsic GTPase activity, serving as transducers of diverse physiological signals including those controlling cellular proliferation and differentiation (2). Some of the biological activities of Ras proteins are known to be modulated by other proteins, including Krev-1 (3) and Ha-rev107 (4). However, physiological functions of Ras proteins and these regulatory proteins in mammalian cells remain largely unknown.Chondrogenesis is a key event in skeletal development in vertebrates. We previously reported that chondrogenesis could be induced in chondroprogenitor-like EC cells, ATDC5, at a high incidence when cultured in the presence of insulin and that ATDC5 cells keep track of the overt chondrogenesis in vitro (5-9). Clonal mouse embryonic fibroblast cells, C3H10T1/2, retain the properties of pluripotent mesodermal progenitors and have been shown to differentiate into adipocytes, myoblasts, osteoblasts, as well as chondrocytes (10) under distinct cultural conditions, including the presence of 5-azacytidine or high dose bone morphogenetic protein-2 (10, 11). In this study, comparison by differential display of mRNAs expressed in undifferentiated ATDC5 cells with those in undifferentiated C3H10T1/2 cells led us to isolate a novel cDNA clone encoding a Ha-rev107-related protein predominantly expressed in skeletal muscle, heart, hippocampus, and bone marrow as well as ATDC5 cells. EXPERIMENTAL PROCEDURESCells and Culture Conditions-ATDC5 cells were plated in six-multiwell plates at an initial cell density of 6 ϫ 10 4 cells/well and cu...

Discovery and Characterization of a Ca2+-independent Phosphatidylethanolamine N-Acyltransferase Generating the Anandamide Precursor and Its Congeners

“…H-REV107 has been reported to be a class II tumor suppressor because it shows a growth-inhibiting activity in H-rastransformed cell lines (42,43). Similarly, A-C1, found as an H-REV107-related protein, significantly inhibits proliferation of H-ras-transformed NIH3T3 cells (44). These results suggest that members of the H-REV107-related protein family are involved in the control of cell proliferation and act as negative regulators of the oncogenic Ras signal.…”

“…These rat genes appear to be homologs of human genes (HRLP5 (H-REV107-like protein 5), A-C1, and H-REV107, respectively). H-REV107 (42,43) and A-C1 (44) have been shown to act as tumor suppressors, but have not been reported as enzymes. The function of HRLP5 remains unclear.…”

Molecular Cloning and Biological Activity of a Novel Lysyl Oxidase-related Gene Expressed in Cartilage

“…Section 1734 solely to indicate this fact. ϩ RNA of calcified ATDC5 cells was constructed in ZAP Express vector (Stratagene, La Jolla, CA), and 1 ϫ 10 6 plaques were screened with the cDNA fragment as a probe (12,16). Plaques were transferred to the membranes (137-mm nylon membrane, PerkinElmer Life Sciences), and the 600-bp fragment was 32 P-labeled (BcaBEST labeling kit, Takara, Otsu, Japan), and hybridization was performed as described previously (12).…”

“…On day 21, the culture medium was switched to ␣-minimum essential medium (ICN Pharmaceuticals, Inc.) containing 5% fetal bovine serum, 10 M human transferrin, 30 nM sodium selenite, and 10 M bovine insulin, and the CO 2 concentration was shifted to 3% for facilitating cellular hypertrophy and mineralization in culture. Clonal mouse embryonic fibroblast C3H10T1/2 cells, clonal mouse osteogenic MC3T3-E1 cells, clonal mouse fibroblast NIH3T3 cells, and clonal mouse myoblastic C2C12 cells (RIKEN Cell Bank, Tsukuba, Japan) were plated in 6-multiwell plates at an initial cell density of 6 ϫ 10 4 cells/well and cultured as described previously (16). …”

Accumulation of DNA methylation is associated with tumor stage in gastric cancer

“…38 HRASLS is a human homologue of mouse A-C1, which has been reported to inhibit growth of ras-transformed NIH3T3 cells. 39 HAND1 encodes a basic helix-loop-helix transcriptional factor, which is essential for placental development and cardiac morphogenesis. 40 We examined whether CIMP and the number of methylated genes correlates with clinical features such as age, sex, histology, and tumor stage to clarify the difference between the GC with a high number of methylated genes and CIMP-positive GC.…”