A comparison of methods for the analysis of low abundance proteins in desmoid tumor cells

Picariello, Lucia; Sala, Sílvia Carbonell; Martineti, Valentina; Gozzini, Alessia; Aragona, Pasquale; Tognarini, Isabella; Paglierani, Milena; Nesi, Gabriella; Brandi, Maria Luisa; Tonelli, Francesco

doi:10.1016/j.ab.2006.03.047

Cited by 23 publications

(20 citation statements)

References 19 publications

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“…Indeed, evidence of phosphorylation/activation is more specific and predictive than overexpression, and the proportion of cases with protein expression is higher using frozen section than fixed materials, particularly as concerns KIT protein. 17 It has recently been emphasized that autocrine/ paracrine oncogenic mechanisms can be associated with markedly low levels of RTK activation that can nonetheless be successfully inhibited by targeted treatment, providing the tumor cells depend on the signaling mechanism in question. 14 We still do not know whether the autocrine paracrine oncogenic effect of KIT, PDGFRa, and PDGFRb play a vital part in ESFT, but our findings, coupled with the results obtained on ESFT cell lines 4,7 and in vivo, 18 support this hypothesis.…”

Section: Discussionmentioning

confidence: 99%

“…No activating mutations were revealed by direct PCR product sequences in the tyrosine kinase domain of c-KIT (exon 11), in the juxtamembrane regions, or in the tyrosine kinase domains of either PDGFRa (exons 10-18) or PDGFRb (exons [10][11][12][13][14][15][16][17][18][19][20]. In PDGFRa sequences, 1 case had a silent mutation at base 2203 (G to A) corresponding to residue 603 and 1 at base 2866 (C to T) corresponding to residue 824.…”

Section: Esft Fusion Transcript Detectionmentioning

confidence: 99%

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Evidence for activation of KIT, PDGFRα, and PDGFRβ receptors in the Ewing sarcoma family of tumors

Bozzi

Tamborini

Negri

et al. 2007

Cancer

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BACKGROUND. The Ewing sarcoma family of tumors (ESFT) is one of the most common malignant neoplasms of children and adolescents, characterized by nonrandom translocations involving the Ewing sarcoma (EWS) gene. Over the years the adoption of intensive multimodality treatment approaches has led to a gradual improvement in the survival of patients with ESFT. The prognosis is still unsatisfactory for high‐risk patients, however, and novel therapeutic approaches are desirable. The aim of the study was to investigate the expression/activation of KIT, PDGFRα, and PDGFRβ receptor tyrosine kinases (RTKs) as potential therapeutic targets in ESFT. METHODS. RNA and proteins were extracted from 20 frozen ESFT specimens to ascertain the state activation of KIT, PDGFRα, and PDGFRβ. RESULTS. No mutations were found, whereas the cognate ligands were detected in all cases by polymerase chain reaction (PCR). The expression and activation of KIT, PDGFRα, and PDGFRβ were confirmed by quantitative PCR, immunohistochemistry, and immunoprecipitation and/or Western blot analysis. In particular, when compared with a protein pool obtained from normal adult tissues, PDGFRβ showed a greater protein expression and/or a stronger phosphorylation signal. CONCLUSIONS. The results are consistent with an autocrine/paracrine loop activation of the KIT, PDGFRα, and PDGFRβ receptors and suggest a rationale for the use of RTK inhibitors, either alone or in combination with chemotherapy. Cancer 2007. © 2007 American Cancer Society.

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Section: Discussionmentioning

confidence: 99%

Section: Esft Fusion Transcript Detectionmentioning

confidence: 99%

Evidence for activation of KIT, PDGFRα, and PDGFRβ receptors in the Ewing sarcoma family of tumors

Bozzi

Tamborini

Negri

et al. 2007

Cancer

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“…On the other hand, one also has to invest in relatively expensive epifluorescence microscopes for manual cell counts of DAPI-or Hoechststained nuclei. Despite the initial cost, the Odyssey Imager is a multipurpose machine that also measures Western immunoblots in a quantitative manner 47,48 . We have adapted use of the Odyssey for quantifications of immunostaining in macroscopic brain structures such as the rat or mouse striatum or telencephalic cortex.…”

Section: Discussionmentioning

confidence: 99%

Viability Assays for Cells in Culture

Posimo

Unnithan

Gleixner

et al. 2014

JoVE

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Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function. Video LinkThe video component of this article can be found at

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“…It should be noted that the ER positive results of the highly sensitive RT-PCR and WB methods may be considered as functionally negative with respect to the minimum of 10% ER+ generally accepted as the limit of a successful endocrine therapy. ER negativity does not necessarily mean that the tumor is not affected by anti-estrogens, in fact even in ER negative disease there are additional pathways through which SERM may be theraputic (2,7,72,84,117,193). A functional interaction of the Wnt/APC/β-catenin and estrogen signaling pathways has been reported in both Drosophila and human tumor cell lines, but the evidence is circumstantial (71).…”

Section: Assessment Of the Expression Of Ers And Prs In Sporadical Dementioning

confidence: 99%

“…Today, virtually all ER determinations are performed with specific monoclonal antibody -based methods for ER-α and ER-β (72,83,84). In an IHC study of 40 cases of extraabdominal fibromatoses, (193). Table 26.…”

Section: Assessment Of the Expression Of Ers And Prs In Sporadical Dementioning

confidence: 99%

Diagnosis and multidisciplinary treatment of sporadical desmoid tumors

Mátrai¹

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A comparison of methods for the analysis of low abundance proteins in desmoid tumor cells

Cited by 23 publications

References 19 publications

Evidence for activation of KIT, PDGFRα, and PDGFRβ receptors in the Ewing sarcoma family of tumors

Evidence for activation of KIT, PDGFRα, and PDGFRβ receptors in the Ewing sarcoma family of tumors

Viability Assays for Cells in Culture

Diagnosis and multidisciplinary treatment of sporadical desmoid tumors

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