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Two classes of mutant mammary tumor virus-infected HTC cell with defects in glucocorticoid-regulated gene expression
Abstract:
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 wild-type 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.
Insights
Researchers identified two new types of mutations in rat hepatoma cells affecting glucocorticoid regulation. These mutations disrupt the hormone-induced expression of viral glycoproteins and other cellular responses, revealing novel pathways in gene regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- Mammary tumor virus (MTV) infection in rat hepatoma (HTC) cells leads to hormone-inducible expression of viral glycoproteins.
- Glucocorticoids, like dexamethasone, regulate MTV RNA synthesis and glycoprotein maturation.
- Wild-type M1.54 cells exhibit complex glucocorticoid-dependent regulation of viral protein expression.
Purpose of the Study:
- To isolate and characterize mutant HTC cell lines with defects in glucocorticoid-regulated gene expression.
- To elucidate novel mechanisms of glucocorticoid action in MTV-infected cells.
- To identify specific pathways involved in hormone-induced viral glycoprotein maturation and gene transcription.
Main Methods:
- Isolation and characterization of mutant HTC cell lines (CR1, CR4) derived from M1.54.
- Analysis of glucocorticoid receptor properties (hormone affinity, concentration, nuclear translocation, DNA-binding, sedimentation).
- Assessment of MTV RNA synthesis, viral glycoprotein expression, and tyrosine aminotransferase activity in response to dexamethasone.
Main Results:
- Two distinct mutant phenotypes were identified, CR1 and CR4, with normal glucocorticoid receptors.
- CR1 mutants show a failure to induce MTV gene transcription and defective hormonal regulation of tyrosine aminotransferase.
- CR4 mutants synthesize normal MTV transcripts but only express the constitutively produced viral glycoprotein, indicating a maturation defect.
Conclusions:
- Mutant CR4 defines a novel, hormone-inducible protein maturation pathway affecting specific viral and potentially cellular precursors.
- Mutant CR1 harbors a lesion affecting the expression of a subset of glucocorticoid-controlled genes in M1.54 cells.
- These findings reveal distinct, previously unrecognized aspects of glucocorticoid-regulated gene expression in HTC cells.