Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescence-activated cell sorting

Grove, Jeffrey; Dieckmann, B; Schroer, Trina A.; Ringold, Gordon M.

doi:10.1016/0092-8674(80)90113-0

Cited by 61 publications

(25 citation statements)

References 12 publications

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“…In contrast, the vast majority of previously described mutants lacking certain glucocorticoid responses carry defects in one or more of the receptor properties tested here (13,27,46). Nevertheless, it is intriguing to consider that the receptors in CR1 or CR4 (or both) in fact contain lesions not detected in the commonly used assays.…”

Section: Resultsmentioning

confidence: 59%

“…For example, subtle changes in the receptor might preclude specific intermolecular interactions or might inhibit receptor-associated functions required for some, but not all, glucocorticoid responses in M1.54. It would be useful to carry out complementation tests in pairwise cell hybrid combinations of CR1, CR4, and known receptor mutants, such as those described in a line of MTV-infected HTC cells closely related to M1.54 (13). If these genetic tests suggest that the lesions may reside in the receptor protein, more detailed analyses, such as sequence-specific interactions with MTV DNA (25; F. Payvar, G. L. Firestone, S. R. Ross, V. L. Chandler, 0.…”

Section: Resultsmentioning

confidence: 99%

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Two classes of mutant mammary tumor virus-infected HTC cell with defects in glucocorticoid-regulated gene expression.

Firestone¹,

Yamamoto²

1983

Mol. Cell. Biol.

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We have isolated mutant derivatives of M1.54 (a mammary tumor virus [MTV]infected rat hepatoma [HTC] cell line containing multiple integrated proviruses) that fail to express hormone-inducible cell surface viral glycoproteins. In wildtype M1.54, the synthetic glucocorticoid dexamethasone selectively stimulates the rate of synthesis of MTV RNA. In addition, dexamethasone is essential for posttranslational maturation of three of the four cell surface viral glycoproteins processed from the MTV glycosylated precursor polyprotein; the fourth mature species is produced constitutively. Two mutant phenotypes are described; each contains glucocorticoid receptors that are indistinguishable from the wild-type receptor with respect to hormone affinity, intracellular concentration, nuclear translocation efficiency, DNA-cellulose chromatography, and sedimentation rate. In one class, represented by the mutant line CR1, dexamethasone fails to stimulate the low basal rate of MTV gene transcription; surprisingly, hormonal regulation of tyrosine aminotransferase activity is also defective in CR1, whereas several other cellular responses to dexamethasone are normal. In the second class of mutants, represented by CR4, dexamethasone stimulates synthesis of MTV transcripts indistinguishable from those produced in M1.54, but only the constitutive cell surface viral glycoprotein is expressed. Thus, these mutants define two distinct and novel aspects of glucocorticoid regulated gene expression in HTC cells: CR4 contains a defect in a hormone inducible protein maturation pathway that acts on specific viral (and presumably cellular) precursor polypeptides, whereas the lesion in CR1 appears to affect the expression of a subset of the gene products normally under glucocorticoid control in M1.54.Among vertebrates, glucocorticoid hormones affect the expression of specific genes in virtually every tissue; interestingly, the set of gene products whose synthesis or activities are altered, i.e., the glucocorticoid domain (16), is highly cell type specific (2). The effects of glucocorticoids and other steroids appear to be mediated by hormone-specific intracellular receptor proteins; the hormone-receptor interaction potentiates a change in the receptor (activation) that results in its increased affinity for DNAcontaining sites in the cell nucleus (see reference 44 for review). Purified glucocorticoid-receptor complexes bind in vitro with high affinity to specific sites on cloned mouse mammary tumor virus (MTV) DNA (25), whose expression in vivo is glucocorticoid regulated (see reference 29 for review); this is consistent with the view that selective receptor-DNA interactions participate in defining the stringent gene selectivity of hormone responses.The glucocorticoid-receptor complex stimulates the rate of MTV gene transcription in infected cells (31,41,49) by increasing the efficiency of MTV promoter utilization

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Section: Resultsmentioning

confidence: 59%

Section: Resultsmentioning

confidence: 99%

Two classes of mutant mammary tumor virus-infected HTC cell with defects in glucocorticoid-regulated gene expression.

Firestone¹,

Yamamoto²

1983

Mol. Cell. Biol.

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“…l,-2 is a murine fibroblast line that stably expresses a packaging-deficient retrovirus; it is used, most often, for the production of retroviral vector stocks (13). MSN610.2 is a glucocorticoid receptor-deficient subclone of the mouse mammary tumor virus (MMTV)-infected, rat HTC hepatoma-derived cell line MSC.1 (14). JZ.1 is an HTC hepatoma cell line containing one integrated copy of MMTV (15).…”

Section: Methodsmentioning

confidence: 99%

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

Felgner

Gadek

Holm

et al. 1987

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

4,530

2,483

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A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propylJ-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA. DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5-to >100-fold more effective than either the calcium phosphate or the DEAEdextran transfection technique.

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“…These two groups, designated receptor negative (r-), provided crucial genetic evidence that the cytolytic response (24) and the stimulation of mouse mammary tumor virus (MMTV) gene expression (9) are mediated by the GR. Three other phenotypically distinct types of inactive GR mutants were identified: nuclear transfer-deficient (nt-) receptors; nuclear transfer-increased (nt') receptors (30); and activation-labile (act') receptors with temperature-sensitive hormone-binding capacity (10).…”

mentioning

confidence: 99%

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

1987

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A mouse T-lymphosarcoma cell line stably infected with mouse mammary tumor virus (MMTV) was used as the parent line for a genetic analysis of two glucocorticoid hormone responses, hormone-induced cytolysis and stimulation of viral gene expression. Variants were selected for survival and elevated expression of MMTV proteins in the presence of the steroid. The MMTV marker provided a sensitive test for glucocorticoid receptor (GR) function in the hormone-resistant variants. This strategy resulted in the isolation of two novel types of hormone-resistant variants. One type of variant with only about 25% of the level of GR found in the parent line was resistant to the cytolytic effects of glucocorticoid but produced increased levels of MMTV gene products in response to the hormone. This variant phenotype demonstrated that the MMTV response requires fewer GR than the cytolytic response. Another variant, which required approximately 100-fold higher concentrations of hormone than the wild-type cells for both responses, apparently contained GR with altered hormone-binding properties.Many thymus-derived lymphoma cell lines exhibit a dramatic response to physiological concentrations of glucocorticoid hormones that includes cessation of proliferation within a few hours and cytolysis after 1 to 3 days of continuous exposure (12,14,17). The cytolytic response has provided the basis for a genetic analysis of steroid hormone action via the selection of cell variants that continue to grow in the presence of the hormone. A large number of glucocorticoid-resistant variants have been analyzed in the mouse S49, mouse WEHI7, and human CEM-C7 T-lymphosarcoma cell lines (11,20,23,30). Almost all of these variants have been shown to have either reduced levels or physically altered forms of the glucocorticoid receptor (GR) protein (reviewed in reference 15). These variants have proven to be extremely valuable for defining the structural and functional properties of the GR protein.The largest number of resistant lines have no measurable glucocorticoid-binding activity; a smaller group has less than about 25% of the wild-type binding activity. These two groups, designated receptor negative (r-), provided crucial genetic evidence that the cytolytic response (24) and the stimulation of mouse mammary tumor virus (MMTV) gene expression (9) are mediated by the GR. Three other phenotypically distinct types of inactive GR mutants were identified: nuclear transfer-deficient (nt-) receptors; nuclear transfer-increased (nt') receptors (30); and activation-labile (act') receptors with temperature-sensitive hormone-binding capacity (10). Recent mapping studies of GR mutations in some of these variants have helped to begin the process of defining the functional domains of the GR (4,18,19).Analysis of the hormone-resistant variants and somatic cell hybrids constructed with various quantities of GR (2, 7) indicated that the concentration of glucocorticoid hormones required to evoke the cytolytic response bears an inverse relationship to the number of func...

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Isolation of glucocorticoid-unresponsive rat hepatoma cells by fluorescence-activated cell sorting

Cited by 61 publications

References 12 publications

Two classes of mutant mammary tumor virus-infected HTC cell with defects in glucocorticoid-regulated gene expression.

Two classes of mutant mammary tumor virus-infected HTC cell with defects in glucocorticoid-regulated gene expression.

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

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