Chemiluminescence detection of proteins from single cells.

Gillespie, Peter G.; Hudspeth, A. J.

doi:10.1073/pnas.88.6.2563

Cited by 93 publications

(66 citation statements)

References 37 publications

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“…Proteins were separated in one dimension by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 8% gels with 4% stacking gels using a minigel apparatus (Mini Protean II System; BioRad, Richmond, Calif.). Immunoblotting, incubation, and developing procedures followed the protocol for chemiluminescence detection of proteins (5) as modified by Gillespie and Hudspeth (13). Briefly, after electrophoresis, gels were transferred to charged nylon (Nytran from Schleicher & Schuell, Keene, N.H.) or polyvinylidene difluoride membranes (Immobilon P from Millipore, Bedford, Mass.)…”

Section: Methodsmentioning

confidence: 99%

Spontaneous In Vitro Immortalization of Breast Epithelial Cells from a Patient with Li-Fraumeni Syndrome

Shay

Tomlinson

Piatyszek

et al. 1995

Molecular and Cellular Biology

143

102

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Individuals with germ line mutations in the p53 gene, such as Li-Fraumeni syndrome (LFS), have an increased occurrence of many types of cancer, including an unusually high incidence of breast cancer. This report documents that normal breast epithelial cells obtained from a patient with LFS (with a mutation at codon 133 of the p53 gene) spontaneously immortalized in cell culture while the breast stromal fibroblasts from this same patient did not. Spontaneous immortalization of human cells in vitro is an extremely rare event. This is the first documented case of the spontaneous immortalization of breast epithelial cells from a patient with LFS in culture. LFS patient breast stromal fibroblasts infected with a retroviral vector containing human papillomavirus type 16 E7 alone were able to immortalize, whereas stromal cells obtained from patients with wild-type p53, similarly infected with human papillomavirus type 16 E7, did not. The present results indicate a protective role of normal pRb-like functions in breast stromal fibroblasts but not in breast epithelial cells and reinforces an important role of wild-type p53 in the regulation of the normal growth and development of breast epithelial tissue.Familial cancer syndromes with germ line mutations, such as the dominantly inherited p53 mutations present in Li-Fraumeni syndrome (LFS), have helped to illustrate the important role of tumor suppressor genes in the development of human cancers (3,30,31,35,36,37,52). The p53 gene is presently considered to be one of the most frequently mutated genes in human cancer (19,22,28,29,47,56,58), and the functional effects of mutations in evolutionarily conserved regions of the p53 phosphoprotein are currently a subject of intense study (24,25,28,43,46,(59)(60)(61)(62)(63). While a complete understanding of wild-type p53 function is not yet known, it is generally believed that perturbations of wild-type p53 function may lead to genomic instability and permit the expansion of the pool of proliferating cells, which leads to a cascade of additional mutations, increasing the probability of neoplastic transformation. The discovery of the importance of the tumor suppressor gene p53, and the identification of germ line mutations in p53 in LFS-affected families, has led to a growing awareness of the cancer risk to such families. Even though rare bone and soft tissue sarcomas are relatively common in families affected by LFS, other, more frequently occurring forms of cancer other than breast cancer (such as colorectal carcinoma) are not overrepresented. Among women in families affected by LFS, breast tumors are the most prevalent cancer (afflicting at least 50%), with 28% of the breast cancers diagnosed before age 30 and 89% diagnosed before age 50 (21, 31, 37). A molecular explanation for the specifically increased incidence of breast cancer, particularly early-onset breast cancer, in families affected by LFS relative to other forms of cancer has not yet been elucidated (20, 41).We and others have shown that spontaneous immortalization ...

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Section: Methodsmentioning

confidence: 99%

Spontaneous In Vitro Immortalization of Breast Epithelial Cells from a Patient with Li-Fraumeni Syndrome

Shay

Tomlinson

Piatyszek

et al. 1995

Molecular and Cellular Biology

143

102

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“…Binding reaction mixtures were identical to those described above except that the labelled NS-1 was not included. Proteins present in the eluted fractions were biotinylated by procedures outlined previously (16). Briefly, 1 l of 5 N NaOH was added to the 500 l of eluate to raise the pH above 8.0.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional Activation by the Parvoviral Nonstructural Protein NS-1 Is Mediated via a Direct Interaction with Sp1

Krady

Ward

1995

Molecular and Cellular Biology

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The nonstructural protein NS-1, encoded by the parvovirus minute virus of mice, is a potent regulator of viral gene expression. NS-1 does not bind DNA in a sequence-specific manner, and the mechanism by which it modulates viral promoter function is unclear. We have used Gal4-NS-1 fusion protein constructs to identify and characterize an activating domain encoded within the C-terminal 88 amino acids of NS-1 which competes effectively with the acidic activator domain of the herpes simplex virus VP16 protein. DNA affinity chromatography and immunoprecipitation experiments demonstrate that protein-protein interactions between the transcription factor Sp1 and NS-1 are required to bind NS-1 to promoter DNA in vitro. Cotransfection of Gal4-NS-1 and Sp1-VP16 acidic activator constructs into Drosophila melanogaster Schneider cells, which lack endogenous Sp1, stimulates transcription from a minimal promoter containing five Gal4 binding sites, while single-construct transfections do not. Cotransfection of Schneider cells with wild-type NS-1 and Sp1 constructs activates transcription from a simian virus 40 promoter 10-to 30-fold over that of either construct alone. Thus, Sp1-NS-1 interactions in vivo can stimulate transcription from a heterologous promoter containing Sp1 binding sites.NS-1, an 83-kDa nuclear phosphoprotein, is the major nonstructural protein encoded by the parvovirus minute virus of mice (MVM). In addition to prominent roles in viral replication (32, 45) and the cytopathic effects associated with parvoviral infection (3, 5), NS-1 is an important regulator of viral gene expression. The MVM genome contains two transcription units; a promoter at map unit 4, termed P4, drives expression of the nonstructural proteins, including NS-1, while the downstream promoter, P38, controls expression of the structural capsid proteins (9). Regulatory effects on transcription from both promoters have been ascribed to NS-1. NS-1 transactivates the expression of viral structural proteins from the downstream P38 promoter (12, 31), enhancing expression approximately 1,000-fold over the low basal level (2). Effects of NS-1 on expression from its own promoter (P4) have been shown to range from activation to inhibition, presumably in a concentration-dependent manner (1,11,17,33). NS-1 also exhibits a trans-inhibitory effect on expression from a number of heterologous promoters (21, 33), most notably the Rous sarcoma virus long terminal repeat promoter.The mechanism by which NS-1 exerts this transcriptional regulation is currently not understood. Cellular and viral transcriptional regulatory proteins have generally been shown to contain two functionally separate domains (4, 30, 43). A binding domain, responsible for linking the protein to the appropriate promoter, allows an activation domain to interact with the transcriptional machinery to stimulate transcription. Activation domains tend to be acidic in nature although domains rich in glutamine or proline residues have also been described (26). Binding domains can interact either di...

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“…C/EBP ␤ was then purified on nickel-chelating columns using imidazole elution (pRSET Xpress; Invitrogen). Protein purity was assessed by Western immunoblot analysis and silver staining (7,8,15,34).…”

Section: Figmentioning

confidence: 99%

Interaction between CCAAT/Enhancer Binding Protein and Cyclic AMP Response Element Binding Protein 1 Regulates Human Immunodeficiency Virus Type 1 Transcription in Cells of the Monocyte/Macrophage Lineage

Ross

Nonnemacher

Hogan

et al. 2001

J Virol

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Chemiluminescence detection of proteins from single cells.

Abstract: The analysis of proteins from single cells

Cited by 93 publications

References 37 publications

Spontaneous In Vitro Immortalization of Breast Epithelial Cells from a Patient with Li-Fraumeni Syndrome

Spontaneous In Vitro Immortalization of Breast Epithelial Cells from a Patient with Li-Fraumeni Syndrome

Transcriptional Activation by the Parvoviral Nonstructural Protein NS-1 Is Mediated via a Direct Interaction with Sp1

Interaction between CCAAT/Enhancer Binding Protein and Cyclic AMP Response Element Binding Protein 1 Regulates Human Immunodeficiency Virus Type 1 Transcription in Cells of the Monocyte/Macrophage Lineage

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