Glandular Epithelial Cells From Mice: A Method for Selective Cultivation2

Owens, Robert B.

doi:10.1093/jnci/52.4.1375

Cited by 91 publications

(41 citation statements)

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“…Because of the higher degree of binding in the two-hybrid assay between L9 and MMTV(C3H) Gag compared to MMT-V(HP/Mtv1), we tested whether L9 overexpression would change the localization of Gag derived from Mtv1. In fact, L9 overexpression also induced localization of a subpopulation of Mtv1 Gag to nucleoli (data not shown), indicating that the difference in binding affinity observed in the quantitative yeast two-hybrid assay was not apparent in NMuMG cells, which are derived from Namru mice (37)(38)(39). It is possible that Namru mice, like BALB/cJ mice, do not exhibit differences in the rates of tumor formation with variants of MMTV tested by Swanson et al (34) (Fig.…”

Section: Resultsmentioning

confidence: 80%

Nucleolar Trafficking of the Mouse Mammary Tumor Virus Gag Protein Induced by Interaction with Ribosomal Protein L9

Beyer

Bann

Rice

et al. 2013

J Virol

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fThe mouse mammary tumor virus (MMTV) Gag protein directs the assembly in the cytoplasm of immature viral capsids, which subsequently bud from the plasma membranes of infected cells. MMTV Gag localizes to discrete cytoplasmic foci in mouse mammary epithelial cells, consistent with the formation of cytosolic capsids. Unexpectedly, we also observed an accumulation of Gag in the nucleoli of infected cells derived from mammary gland tumors. To detect Gag-interacting proteins that might influence its subcellular localization, a yeast two-hybrid screen was performed. Ribosomal protein L9 (RPL9 or L9), an essential component of the large ribosomal subunit and a putative tumor suppressor, was identified as a Gag binding partner. Overexpression of L9 in cells expressing the MMTV(C3H) provirus resulted in specific, robust accumulation of Gag in nucleoli. Förster resonance energy transfer (FRET) and coimmunoprecipitation analyses demonstrated that Gag and L9 interact within the nucleolus, and the CA domain was the major site of interaction. In addition, the isolated NC domain of Gag localized to the nucleolus, suggesting that it contains a nucleolar localization signal (NoLS). To determine whether L9 plays a role in virus assembly, small interfering RNA (siRNA)-mediated knockdown was performed. Although Gag expression was not reduced with L9 knockdown, virus production was significantly impaired. Thus, our data support the hypothesis that efficient MMTV particle assembly is dependent upon the interaction of Gag and L9 in the nucleoli of infected cells. Since its discovery as a milk-transmitted agent in the 1930s, the oncogenic retrovirus mouse mammary tumor virus (MMTV) has served as an important model in breast cancer research and immunology (1). However, little is known about the molecular mechanisms that govern MMTV assembly. The 9-kb MMTV RNA genome consists of the common retroviral elements gag, pro, pol, and env, as well as dut (dUTPase) (2), sag (superantigen) (3), and rem (regulator of export of MMTV mRNA) (4, 5). Like all retroviruses, MMTV uses full-length viral RNA to transcribe the viral structural proteins Gag, Gag-Pro, and Gag-Pro-Pol. The Gag protein directs assembly of complete, immature viral capsids in the cytoplasm, which are subsequently transported to the plasma membrane for release by budding.Unlike acutely transforming retroviruses like Rous sarcoma virus (RSV), MMTV does not carry an oncogene and instead induces tumors primarily by integrating near cellular oncogenes and disrupting their regulation. In addition, the MMTV Gag and Env proteins also promote tumorigenesis independently of the proviral integration site (6, 7). Moreover, differences in pathogenesis between the highly tumorigenic MMTV(C3H) strain and the tumor-attenuated MMTV hybrid provirus (HP) strain map to the CA and NC regions of the Gag protein (6), which led us to hypothesize that the Gag proteins from the C3H and HP strains might differentially interact with cellular proteins to promote malignant transformation.The eukaryotic ribos...

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Section: Resultsmentioning

confidence: 80%

Nucleolar Trafficking of the Mouse Mammary Tumor Virus Gag Protein Induced by Interaction with Ribosomal Protein L9

Beyer

Bann

Rice

et al. 2013

J Virol

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“…Cells and Materials-NMuMg nontransformed mouse mammary epithelial cells were originally derived from normal glandular tissue of an adult NAMRU mouse (37). Cells were regularly cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 g/ml insulin, 50 units/ml penicillin, and 50 g/ml streptomycin.…”

Section: Methodsmentioning

confidence: 99%

Hyperosmotic Stress Stimulates Promoter Activity and Regulates Cellular Utilization of the Serum- and Glucocorticoid-inducible Protein Kinase (Sgk) by a p38 MAPK-dependent Pathway

Bell¹,

Leong²,

Kim³

et al. 2000

Journal of Biological Chemistry

143

149

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We have established that the serum-and glucocorticoid-inducible protein kinase (Sgk) is a new component of the hyperosmotic stress response. Treatment of NMuMg mammary epithelial cells with the organic osmolyte, sorbitol, caused the stable accumulation of Sgk transcripts and protein after an approximately 4-h lag. Transient transfection of a series of sgk-CAT reporter plasmids containing either 5 deletions or continuous 6-base pair substitutions identified a hyperosmotic stress-regulated element that is GC-rich and is necessary for the sorbitol stimulation of sgk gene promoter activity. Gel shift analysis identified four major DNAprotein complexes in the hyperosmotic stress-regulated element that, by competition with excess consensus wild type and mutant oligonucleotides and by antibody supershifts, contains the Sp1 transcription factor. Several lines of evidence suggest that the p38 MAPK signaling pathway mediates the hyperosmotic stress stimulation of sgk gene expression. Treatment with pharmacological inhibitors of p38 MAPK or with a dominant negative form of MKK3, an upstream regulator of p38 MAPK, significantly reduced or ablated the sorbitol induction of sgk promoter activity or protein production. Using an in vitro peptide transphosphorylation assay, sorbitol treatment activates either endogenous or exogenous Sgk that is localized to the cytoplasmic compartment. Thus, we propose that the stimulated expression of enzymatically active Sgk after sorbitol treatment is a newly defined component of the p38 MAPK-mediated response to hyperosmotic stress.

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“…The human cell lines used in this study are listed in Table I (7)(8)(9)(10)(11)(12)(13). All Hs-prefixed cell lines were isolated by the Owens differential trypsinization technique (14). All cell lines were propagated in antibiotic-free medium, either Dulbecco modified medium (Grand Island Biological Co., Grand Island, N.…”

Section: Methodsmentioning

confidence: 99%

Tumor-specific cell-mediated immunity in household contacts of cancer patients..

Byers¹,

Levin²,

Hackett³

et al. 1975

J. Clin. Invest.

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A B S T R A C T Patients with osteogenic sarcoma (and related tumors), hypernephroma, and breast carcinoma, and their household contacts were tested for tumorspecific cell-mediated immunity against these tumors with the use of a short-term chromium-51 release assay. This assay, reproducible over many months and well-correlated with the clinical course of the patients, was used to demonstrate that household contacts of patients with osteogenic sarcoma and breast carcinoma have specific immunity against the tumor type with which they have been in contact. In both types of tumors, the range of cytotoxicity values produced by lymphocytes from the household contacts was significantly higher than that of the normal population. The incidence of immunity was much higher in household contacts of patients with breast carcinoma than in those of patients with osteogenic sarcoma. Immunity was found with equal frequency in men and women, as well as in genetically and nongenetically related household contacts (guardians, adopted children, spouses).

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Glandular Epithelial Cells From Mice: A Method for Selective Cultivation2

Cited by 91 publications

References 0 publications

Nucleolar Trafficking of the Mouse Mammary Tumor Virus Gag Protein Induced by Interaction with Ribosomal Protein L9

Nucleolar Trafficking of the Mouse Mammary Tumor Virus Gag Protein Induced by Interaction with Ribosomal Protein L9

Hyperosmotic Stress Stimulates Promoter Activity and Regulates Cellular Utilization of the Serum- and Glucocorticoid-inducible Protein Kinase (Sgk) by a p38 MAPK-dependent Pathway

Tumor-specific cell-mediated immunity in household contacts of cancer patients..

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