Transformation of quail embryo fibroblasts by a retrovirus carrying a normal human c-myc gene.

We have constructed avian retroviruses expressing the human c-myc oncogene. These viruses morphologically transformed primary quail embryo fibroblasts upon transfection and infection. Transformed cells produced viruses harboring a spliced c-myc gene and contained high levels of p64-67c-myc protein. One of these infectious viruses, vSX-AHM, was molecularly cloned and the nucleotide sequence of the spliced c-myc insert determined. No mutation was found within the c-myc coding sequence of this transforming clone when compared to the normal genomic progenitor. Thus, we concluded that no mutation within the human c-myc gene is required to induce primary avian embryo fibroblast transformation.     

c-myc gene expression in human cells is controlled by glucose

The c-myc oncogene is implicated in normal growth and differentiation processes. Human cell lines IM9 and HepG2 stably cultured at "low" glucose concentrations (5.5 mM) show c-myc mRNA levels 3-4 times higher than cells cultured at "high" glucose concentrations (25 nM). D-fructose (a metabolizable exose) substitutes for D-glucose in reducing c-myc expression while 3-ortho-methylglucose (a non metabolizable exose) is uneffective. c-myc expression is up-regulated (by PMA) or down-regulated (by dexamethasone and long-term exposure to FCS) in human cells cultured at "low" glucose but not in cells cultured at "high" glucose. We previously demonstrated that insulin receptor gene expression in human cell lines in enhanced by glucose. Therefore, glucose controls in an opposite way the expression of two genes important in the regulation of eukaryotic cell growth and differentiation.     

Basal levels of max are sufficient for the cotransformation of C3H10T1/2 cells by ras and myc.

The ras and myc oncoproteins cooperate to transform the established murine fibroblast cell line C3H10T1/2. To determine the impact of overexpression of the myc oncoprotein on the phenotype of C3H10T1/2 cells, two C3H10T1/2-myc clonal cell lines, SVc-myc 11A and myc neo 13A, were isolated and characterized. Although both C3H10T1/2-myc cell lines are morphologically indistinguishable from wild-type C3H10T1/2 cells and possess growth properties similar to those of C3H10T1/2 cells, each displays a predisposition to transformation following transfection with the activated form of the human H-ras gene. In C3H10T1/2 cells overexpressing the v-myc or H-ras oncogenes, the levels of mRNA encoding max, the recently identified oligomerization partner of myc, remain unchanged, suggesting that the endogenous level of max in C3H10T1/2 cells is sufficient for a high frequency of transformation by ras and myc. Based on these studies, the C3H10T1/2-myc clonal cell lines we describe are suitable model systems for examining the molecular role of the myc protein in transformation and for characterizing additional factors that synergize with myc in multistep transformation.     

L-myc cooperates with ras to transform primary rat embryo fibroblasts.

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.     

Human smooth muscle myosin light chain-2 gene expression is repressed in ras transformed fibroblast cells.

We have previously characterized human smooth muscle myosin light chain (MLC)-2 isoform by complementary DNA cloning and have shown that this isoform is expressed in a number of nonmuscle cells such as fibroblast cells. In this report, we show that when human osteosarcoma derived clonal cells (TE 85 clone F-5) (HOS), which are immortalized and nontumorigenic, undergo transformation following infection by Kirsten murine sarcoma virus (K-HOS) or by a chemical carcinogen [N-methyl-N-nitro-N-nitrosoguanidine (MNNG-HOS)], the smooth muscle MLC-2 mRNA is repressed. Revertants of transformed K-HOS cells (K-HOS312H) show normal levels of smooth muscle MLC-2 mRNA. Transformation of HOS cells by Ha-ras oncogene sequences, either by retroviral infection or by transfection followed by selection for tumorigenic cells in nude mice, results in complete repression of smooth muscle MLC-2 mRNA level. Treatment of HOS cells with tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in repression of smooth muscle MLC-2 mRNA. Smooth muscle MLC-2 mRNA level is repressed in many, but not all, transformed cell lines, suggesting that it is not an indirect consequence of transformation but is specific to the agent that brings about transformation. HOS cells synthesize three MLC-2 protein species resolved by the two-dimensional gel electrophoretic system. The identity of the smooth muscle MLC-2 isoform was established by coelectrophoresis of the in vitro synthesized MLC-2 protein corresponding to the cloned complementary DNA in the two-dimensional gel system along with total [35S]methionine labeled HOS cell proteins. Quantitative analysis of MLC-2 isoforms in different HOS cells indicates that the synthesis of smooth muscle MLC-2 isoform is specifically repressed to an undetectable level in ras transformed and MNNG transformed cells and also following treatment with 12-O-tetradecanoylphorbol-13-acetate.     

Developmental interactions of cells mutant for Strong's luxoid gene with normal cells in chimeric mice

Mouse chimeras were made by fusing embryos from the albino BALB/cFo normal skeleton strain producing a slow variant isozyme of glucose phosphate isomerase (GPI) with embryos from the black pigmented SH strain carrying Strong's luxoid gene (symbol: 1st) for skeletal anomalies and producing a fast GPI variant. All chimeras were estimated to bALB/cFo mice to determine the mosaic status of their gonads. In addition, the quantitative proportions of BALB/cFo and SH cells in skin and limb muscles of chimeras were determined by visual estimation of the degree of coat pigmentation and by a serial dilution method applied to electrophoresis and GPI isozyme reaction of limb muscle homogenates. Skeletons of all chimeras and of representative samples of BALB/cFo and SH mice were examined and graded for expression of a number of normal and mutant skeletal characteristics. The most important conclusion of this study is that there was a definite quantitative effect on the development of skeletal characteristics exerted by the relative amount of BALB/ cFo and SH cells present in a chimera such that a structure could vary from normal to entirely mutant, depending on the proportion of each type of cell present.     

Secretion of Mr 46,000 protein from human hepatoma cells treated with tumor promotors is regulated by c-myc gene expression

Previously an Mr 46,000 protein (named p46) was shown to be induced in the culture medium of human hepatoblastoma cells, Huh-6 Cl-5, treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA). For further characterization of p46, Huh-7 Cl-4, another line of well differentiated liver cells, was incubated in the presence and absence of TPA, and proteins were labeled with [35S]methionine, and the proteins secreted into the medium were analyzed by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. A protein of Mr 46,000 was observed in the culture medium of Huh-7 Cl-4 cells treated by TPA or plated at low density, but only slightly in the culture medium of Huh-7 Cl-4 cells plated at high density. As these results suggested that the expression of p46 was regulated by a factor that was activated in the growth state and by TPA treatment, the effect of introduction of the competence gene c-myc into Huh-7 Cl-4 cells was examined. All 37 independent transfected colonies obtained showed enhanced expression of p46. As a control, the c-Ha-ras gene was introduced into Huh-7 Cl-4 cells and shown not to enhance expression of p46. These results strongly suggested that the expression of p46 was regulated by the competence gene c-myc.     

c-myc expression is down-regulated by cell-cell and cell-extracellular matrix contacts in normal hepatocytes, but not in hepatoma cells.

Primary culture of adult rat hepatocytes resulted in marked increase of c-myc expression within a few hours. The high level of c-myc mRNA was maintained throughout culture on collagen-coated dishes, but decreased greatly with time during culture on collagen-gel or matrigel. Expression of c-myc was also down-regulated at high cell density. The decrease in its expression appeared closely related to inhibitions of DNA synthesis and cell spreading. In contrast, hepatoma H4TG cells showed a high level of c-myc expression which was not affected by culture on any extracellular matrices examined or by the cell density. These results suggest that up-regulation of expression of the c-myc gene is linked to G0 to G1 transition during cell cycle progression, which in normal hepatocytes is strictly regulated by cell-cell and cell-extracellular matrix interactions, but that this control mechanism is defective in malignant hepatic tumor cells.     

The V1a vasopressin receptor is necessary and sufficient for normal social recognition: a gene replacement study

Vasopressin modulates many social and nonsocial behaviors, including emotionality. We have previously reported that male mice with a null mutation in the V1a receptor (V1aR) exhibit a profound impairment in social recognition and changes in anxiety-like behavior. Using site-specific injections of a V1aR-specific antagonist, we demonstrate that the lateral septum, but not the medial amygdala, is critical for social recognition. Reexpressing V1aR in the lateral septum of V1aR knockout mice (V1aRKO) using a viral vector resulted in a complete rescue of social recognition. Furthermore, overexpression of the V1aR in the lateral septum of wild-type (wt) mice resulted in a potentiation of social recognition behavior and a mild increase in anxiety-related behavior. These results demonstrate that the V1aR in the lateral septum plays a critical role in the neural processing of social stimuli required for complex social behavior.