Selection of transfored cells in serum-free media

Chiang, Lin-Chang; Silnutzer, Janet; Pipas, James M.; Barnes, David W.

doi:10.1007/bf02620926

Cited by 17 publications

(9 citation statements)

References 38 publications

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“…We have previously demonstrated that either EGF or aTGF alone is ineffective in promoting the soft agar growth of primary cultures of normal mouse or rat mammary epithelial cells, although both growth factors are mitogenic for these cells in monolayer culture (Zwiebel et al, 1982). Since the NMuMG cell line utilized in the present study represents a spontaneously immortalized population of mouse mammary epithelial cells, it is certainly possible that immortalization has further sensitized these cells to EGF or aTGF so as to permit their growth in soft agar in response to these growth factors (Kaplan and Ozanne, 1983;Marshall, 1984;Chiang et al, 1985). This may be the case since rat embryo fibroblasts that have been experimentally immortalized by transfection with a c-myc-containing plasmid acquire the ability to grow in soft agar in response to only EGF (Stern et al, 1986).…”

Section: Discussionmentioning

confidence: 99%

Loss of growth responsiveness to epidermal growth factor and enhanced production of alpha‐transforming growth factors in ras‐transformed mouse mammary epithelial cells

Salomon

Perroteau

Kidwell

et al. 1987

Journal Cellular Physiology

101

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A mouse mammary epithelial cell line, NMuMG, exhibits a low capacity to grow in semisolid medium as colonies and it is not tumorigenic in nude mice. In contrast, NMuMG cells which have been transformed by an activated c-Harvey ras proto-oncogene, NMuMG/rasH, or by the polyoma middle T-transforming gene, NMuMG/pyt, are able to grow in soft agar and, when injected into nude mice, produce undifferentiated carcinomas. Human epidermal growth factor (EGF) or human alpha-transforming growth factor (alpha TGF) can stimulate, in a dose-dependent fashion, the anchorage-independent growth of NMuMG and NMuMG/pyt cells in soft agar but fail to enhance the anchorage-independent growth of the NMuMGrasH cells. Likewise, human EGF or human alpha TGF is also able to stimulate the anchorage-dependent growth of normal NMuMG cells and NMuMG/pyt cells in a serum-free medium supplemented with insulin, transferrin, fetuin, and laminin, or in medium containing low concentrations of serum, whereas these same growth factors under comparable culture conditions have little or no effect upon the anchorage-dependent growth of the ras-transformed NMuMG-rasH cells. The biological refractoriness of the NMuMG/rasH cells to human EGF or human alpha TGF is reflected by a reduction in the total number of cell surface receptors for EGF and by an absence of a high-affinity population of binding sites for mouse [125l]EGF on these cells as compared to the NMuMG or NMuMG/pyt cells. In addition, concentrated conditioned medium (CM) obtained from NMuMG/rasH and NMuMG/pyt cells contains a relatively higher amount of biologically active TGFs than CM obtained from comparably treated NMuMG cells as measured by the ability to induce the anchorage-independent growth of normal rat kidney cells in soft agar. The higher levels of biologically active TGFs found in the CM from the transformed cells relative to the NMuMG cells is paralleled by a corresponding increase in the CM from these cells in the amount of immunoreactive alpha TGF, by an increase in the amount of EGF receptor-competing activity, and by an increase in the levels of alpha TGF mRNA in the NMuMG/rasH cells. These results demonstrate that mammary epithelial cells which have been transformed by an activated ras proto-oncogene, but not by the polyoma middle T-transforming gene, become unresponsive to exogenous EGF or alpha TGF.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Discussionmentioning

confidence: 99%

Loss of growth responsiveness to epidermal growth factor and enhanced production of alpha‐transforming growth factors in ras‐transformed mouse mammary epithelial cells

Salomon

Perroteau

Kidwell

et al. 1987

Journal Cellular Physiology

101

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“…After 12 h, cells were washed twice with phosphate-buffered saline and cultured in either serum-free medium or serum-free medium supplemented with 50 ng of selenous acid per ml (26). Serum-free medium consisted of a 1:1 (vol/vol) mixture of Ham's F-12 (Life Technologies) and DMEM plus 25 g of bovine holo-transferrin per ml, 10 g of bovine insulin per ml, 10 ng of mouse epidermal growth factor per ml, and 25 g of human high-density lipoprotein per ml (17). Cells were harvested after another 48 h.…”

Section: Animals and Dietsmentioning

confidence: 99%

Selenium Deficiency Reduces the Abundance of mRNA for Se-Dependent Glutathione Peroxidase 1 by a UGA-Dependent Mechanism Likely To Be Nonsense Codon-Mediated Decay of Cytoplasmic mRNA

Moriarty

Reddy

Maquat

1998

Molecular and Cellular Biology

206

187

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The mammalian mRNA for selenium-dependent glutathione peroxidase 1 (Se-GPx1) contains a UGA codon that is recognized as a codon for the nonstandard amino acid selenocysteine (Sec). Inadequate concentrations of selenium (Se) result in a decrease in Se-GPx1 mRNA abundance by an uncharacterized mechanism that may be dependent on translation, independent of translation, or both. In this study, we have begun to elucidate this mechanism. We demonstrate using hepatocytes from rats fed either a Se-supplemented or Se-deficient diet for 9 to 13 weeks that Se deprivation results in an ϳ50-fold reduction in Se-GPx1 activity and an ϳ20-fold reduction in Se-GPx1 mRNA abundance. Reverse transcription-PCR analyses of nuclear and cytoplasmic fractions revealed that Se deprivation has no effect on the levels of either nuclear pre-mRNA or nuclear mRNA but reduces the level of cytoplasmic mRNA. The regulation of Se-GPx1 gene expression by Se was recapitulated in transient transfections of NIH 3T3 cells, and experiments were extended to examine the consequences of converting the Sec codon (TGA) to either a termination codon (TAA) or a cysteine codon (TGC). Regardless of the type of codon, an alteration in the Se concentration was of no consequence to the ratio of nuclear Se-GPx1 mRNA to nuclear Se-GPx1 pre-mRNA. The ratio of cytoplasmic Se-GPx1 mRNA to nuclear Se-GPx1 mRNA from the wild-type (TGA-containing) allele was reduced twofold when cells were deprived of Se for 48 h after transfection, which has been shown to be the extent of the reduction for the endogenous Se-GPx1 mRNA of cultured cells incubated as long as 20 days in Se-deficient medium. In contrast to the TGA allele, Se had no effect on expression of either the TAA allele or the TGC allele. Under Se-deficient conditions, the TAA and TGC alleles generated, respectively, 1.7-fold-less and 3-fold-more cytoplasmic Se-GPx1 mRNA relative to the amount of nuclear Se-GPx1 mRNA than the TGA allele. These results indicate that (i) under conditions of Se deprivation, the Sec codon reduces the abundance of cytoplasmic Se-GPx1 mRNA by a translation-dependent mechanism and (ii) there is no additional mechanism by which Se regulates Se-GPx1 mRNA production. These data suggest that the inefficient incorporation of Sec at the UGA codon during mRNA translation augments the nonsense-codon-mediated decay of cytoplasmic Se-GPx1 mRNA.Many organisms, including bacteria, Saccharomyces cerevisiae, and vertebrates, appear to have established mechanisms that eliminate the production of mRNAs that prematurely terminate translation because of a frameshift or nonsense mutation (reviewed in references 32, 33, 37, and 42). Therefore, the discovery of mRNAs in both bacterial and mammalian cells in which one or more UGA codons are purposefully used as selenocysteine (Sec) codons rather than as nonsense codons (reviewed in references 10 and 30) raises the interesting issue of whether these mRNAs are reduced in abundance when the UGA codon is recognized as nonsense.Cues from (i) biochemical and genetic stu...

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“…In initial attempts to formulate a serum-free medium for salmon embryo cells analogous to previous work with rodent embryo cells (26,27), we were unsuccessful in formulating a combination ofmedium supplements from known hormones, attachment proteins, binding proteins, and nutritional factors that would allow sustained growth of CHSE-214 chinook salmon embryo cells. These results led us to explore other potential sources of mitogenic activity for salmonid cells in vitro.…”

Section: Resultsmentioning

confidence: 99%

Mitogenic activity from trout embryos.

Collodi

Barnes

1990

Proc. Natl. Acad. Sci. U.S.A.

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An extract of 21-day rainbow trout embryos stimulated growth of several piscine cell lines in the absence of added serum. Established lines from trout (RTG-2 and STE-137), salmon (CHSE-214), carp (EPC), and goldfish (CAR) and early-passage cells initiated from trout embryos grew in serumfree medium containing the embryo extract. In addition the extract was sufficient for maintaining long-term cultures of CHSE-214 cells for several months through a minimum of 20 passages (-50 population doublings) in the absence of serum. Optimal response was achieved with 100 ,ug of extract protein per ml, but a significant growth-promoting effect was observed with as little as 2.5 jg/ml. The activity was nondialyzable, protease-sensitive, and stable in 200 mM acetic acid. The level of mitogenic response induced by the extract could not be duplicated with purified mammalian growth factors added individually or in combination, and the extract did not stimulate DNA synthesis in quiescent mouse fibroblasts. These results suggest that trout embryo extract may contain a novel growth-promoting activity for fish cells.

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Selection of transfored cells in serum-free media

Cited by 17 publications

References 38 publications

Loss of growth responsiveness to epidermal growth factor and enhanced production of alpha‐transforming growth factors in ras‐transformed mouse mammary epithelial cells

Loss of growth responsiveness to epidermal growth factor and enhanced production of alpha‐transforming growth factors in ras‐transformed mouse mammary epithelial cells

Selenium Deficiency Reduces the Abundance of mRNA for Se-Dependent Glutathione Peroxidase 1 by a UGA-Dependent Mechanism Likely To Be Nonsense Codon-Mediated Decay of Cytoplasmic mRNA

Mitogenic activity from trout embryos.

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