A stable propidium iodide staining procedure for flow cytometry.

Deitch, Arline D.; Law, Hung-Fai S; White, Ralph deVere

doi:10.1177/30.9.6182188

Cited by 152 publications

(65 citation statements)

References 4 publications

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“…During analysis, cells were pelleted and resuspended in PBS. These ethanol-fixed cells were incubated in a staining solution containing 50 g/ml propidium iodide and 100 g/ml RNase A (Stratagene) in PBS as described previously (53). The cells were analyzed after 1 h of incubation in darkness at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Subcellular Localization and Mechanisms of Nucleocytoplasmic Trafficking of Steroid Receptor Coactivator-1

Amazit¹,

Alj²,

Tyagi³

et al. 2003

Journal of Biological Chemistry

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Steroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation.

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Section: Methodsmentioning

confidence: 99%

Subcellular Localization and Mechanisms of Nucleocytoplasmic Trafficking of Steroid Receptor Coactivator-1

Amazit¹,

Alj²,

Tyagi³

et al. 2003

Journal of Biological Chemistry

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“…Flow cytometry was performed on representative fresh-frozen samples of tumour tissue obtained at surgical resection (16). After mechanical disaggregation with scalpels to obtain a monodispersed cell suspension, the samples were treated with 0.05% Nonidet P40 non-ionic detergent (Sigma, USA) for cell lysis, and stained with a propidium iodide solution (Sigma; 50 μg/ml in Tris-MgCl 2 buffer), including ribonuclease (Sigma; 1 mg/ml in phosphate- …”

Section: Methodsmentioning

confidence: 99%

Aneuploidy and high S-phase as biomarkers of poor clinical outcome in poorly differentiated and anaplastic thyroid carcinoma

Pinto

Silva²,

Banito³

et al. 1994

Oncol Rep

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Abstract. This study aimed to investigate the prognostic influence of DNA flow cytometry and RAS gene mutations in patients with poorly differentiated (PDTC) and anaplastic thyroid carcinoma (ATC). The series consisted of 26 patients with PDTC and ATC, and a median follow-up of 10 months (range 1-138). DNA ploidy and S-phase fraction (SPF) were assessed by flow cytometry on frozen samples. RAS point mutations were detected using PCR techniques. Disease staging and tumour angioinvasion were included as prognostic parameters for survival analysis. Nineteen patients (73.1%) succumbed to the disease (median time 5 months; range 1-45). Eighteen tumours (69.2%) were classified as DNA aneuploid. Median SPF was 5.6% (range 1.9-23.1), which was used as a cut-off value to distinguish between low versus high cell proliferation. Three of 20 (15%) patients presented N-RAS gene mutations in codon 61. DNA aneuploidy was most frequently found in female patients (p=0.034). Kaplan-Meier and Cox regression analyses showed that only DNA aneuploidy (p=0.044 and p=0.055, respectively) and high SPF (p=0.001 and p=0.006, respectively) significantly correlated with worse survival. The results indicate that aneuploidy and high SPF are biomarkers of poor clinical outcome in PDTC and ATC, which may provide useful prognostic information with a potentially therapeutic impact in patient management.

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“…The samples were processed according to a previously reported procedure (Deitch et al, 1982), but slightly modified by us. Briefly, samples were thawed and complete cell lysis was assured with NP-40 treatment (final concentration 0.5%) and the isolated nuclei were washed once with ice-cold phosphate-buffered saline.…”

Section: Tumor Specimensmentioning

confidence: 99%

H-RAS 81 polymorphism is significantly associated with aneuploidy in follicular tumors of the thyroid

et al. 2006

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Follicular thyroid tumors are often aneuploid. It was advanced that chromosomal instability is closely associated to RAS mutations, but such association remains unproven. H-RAS can be alternatively spliced in two different proteins, p21 and p19, the former being the active protein. In order to investigate the relationship between RAS mutational status and ploidy in thyroid tumors, we analysed RAS genes in a series of 99 follicular lesions (14 nodular goiters, 70 follicular adenomas and 15 follicular carcinomas), eight thyroid carcinoma cell lines and a control group of 102 blood donors, correlating the presence of RAS mutations with the ploidy of the tumors and evaluating the two spliced forms of H-RAS. Overall, 20% of the follicular tumors harbored RAS mutations and 62% of the patients with follicular tumors (and 51% of blood donors) harbored the H-RAS 81T-C polymorphism. The presence of RAS mutations was not associated with aneuploidy. The H-RAS polymorphism did not seem to confer a higher propensity for neoplastic transformation as it was also found in hyperplastic lesions, but was strongly associated with aneuploidy (Po0.0001). The presence of the H-RAS 81T-C polymorphism was associated with significantly higher amounts of total H-RAS mRNA expression, higher amounts of p21 isoform and a higher fraction of neoplastic cells in S phase. Our results suggest that the H-RAS 81T-C polymorphism may induce aneuploidy through overexpression of the active p21 isoform of H-RAS.

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A stable propidium iodide staining procedure for flow cytometry.

Abstract: A propidium iodide (P1) staining procedure is described in which 50 pg/mI P1 in 102 M Tris, pH 7.0, with 5 mM incubation time at 37#{176}Cmust be doubled to obtain the

Cited by 152 publications

References 4 publications

Subcellular Localization and Mechanisms of Nucleocytoplasmic Trafficking of Steroid Receptor Coactivator-1

Subcellular Localization and Mechanisms of Nucleocytoplasmic Trafficking of Steroid Receptor Coactivator-1

Aneuploidy and high S-phase as biomarkers of poor clinical outcome in poorly differentiated and anaplastic thyroid carcinoma

H-RAS 81 polymorphism is significantly associated with aneuploidy in follicular tumors of the thyroid

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