7 results on '"Patient tissue"'
Search Results
2. Phospholipase D inhibitors reduce human prostate cancer cell proliferation and colony formation
- Author
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Noble, Amanda R, Maitland, Norman J, Berney, Daniel M, and Rumsby, Martin G
- Published
- 2018
- Full Text
- View/download PDF
3. Phospholipase D inhibitors reduce human prostate cancer cell proliferation and colony formation
- Author
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Amanda R. Noble, Martin G. Rumsby, Norman J. Maitland, and Daniel M. Berney
- Subjects
Male ,0301 basic medicine ,Oncology ,Cancer Research ,medicine.medical_specialty ,Indoles ,patient tissue ,education ,Tumor cells ,Biology ,urologic and male genital diseases ,Human prostate ,03 medical and health sciences ,0302 clinical medicine ,Piperidines ,Cell Line, Tumor ,Internal medicine ,expression ,Phospholipase D ,Tumor Cells, Cultured ,medicine ,Humans ,Enzyme Inhibitors ,phospholipase D1 ,Cell Proliferation ,cellular localisation ,activity ,Cancer cell proliferation ,Prostatic Neoplasms ,prostate cancer ,Immunohistochemistry ,inhibition ,Domperidone ,Prostatic Neoplasms, Castration-Resistant ,030104 developmental biology ,Colony formation ,Tissue Array Analysis ,Cell culture ,030220 oncology & carcinogenesis ,Neoplastic Stem Cells ,Cancer research ,Benzimidazoles ,Translational Therapeutics - Abstract
BACKGROUND: Phospholipases D1 and D2 (PLD1/2) hydrolyse cell membrane glycerophospholipids to generate phosphatidic acid, a signalling lipid, which regulates cell growth and cancer progression through effects on mTOR and PKB/Akt. PLD expression and/or activity is raised in breast, colorectal, gastric, kidney and thyroid carcinomas but its role in prostate cancer (PCa), the major cancer of men in the western world, is unclear. METHODS: PLD1 protein expression in cultured PNT2C2, PNT1A, P4E6, LNCaP, PC3, PC3M, VCaP, 22RV1 cell lines and patient-derived PCa cells was analysed by western blotting. PLD1 protein localisation in normal, benign prostatic hyperplasia (BPH), and castrate-resistant prostate cancer (CRPC) tissue sections and in a PCa tissue microarray (TMA) was examined by immunohistochemistry. PLD activity in PCa tissue was assayed using an Amplex Red method. The effect of PLD inhibitors on PCa cell viability was measured using MTS and colony forming assays. RESULTS: PLD1 protein expression was low in the luminal prostate cell lines (LNCaP, VCaP, 22RV1) compared with basal lines (PC3 and PC3M). PLD1 protein expression was elevated in BPH biopsy tissue relative to normal and PCa samples. In normal and BPH tissue, PLD1 was predominantly detected in basal cells as well in some stromal cells, rather than in luminal cells. In PCa tissue, luminal cells expressed PLD1. In a PCa TMA, the mean peroxidase intensity per DAB-stained Gleason 6 and 7 tissue section was significantly higher than in sections graded Gleason 9. In CRPC tissue, PLD1 was expressed prominently in the stromal compartment, in luminal cells in occasional glands and in an expanding population of cells that co-expressed chromogranin A and neurone-specific enolase. Levels of PLD activity in normal and PCa tissue samples were similar. A specific PLD1 inhibitor markedly reduced the survival of both prostate cell lines and patient-derived PCa cells compared with two dual PLD1/PLD2 inhibitors. Short-term exposure of PCa cells to the same specific PLD1 inhibitor significantly reduced colony formation. CONCLUSIONS: A new specific inhibitor of PLD1, which is well tolerated in mice, reduces PCa cell survival and thus has potential as a novel therapeutic agent to reduce prostate cancer progression. Increased PLD1 expression may contribute to the hyperplasia characteristic of BPH and in the progression of castrate-resistant PCa, where an expanding population of neuroendocrine-like cells express PLD1.British Journal of Cancer advance online publication, 14 November 2017; doi:10.1038/bjc.2017.391 www.bjcancer.com.
- Published
- 2017
4. Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases
- Author
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Yasukawa, Takehiro, Kirino, Yohei, Ishii, Norie, Holt, Ian J., Jacobs, Howard T., Makifuchi, Takao, Fukuhara, Nobuyoshi, Ohta, Shigeo, Suzuki, Tsutomu, and Watanabe, Kimitsuna
- Subjects
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TRANSFER RNA , *AMINOACYL-tRNA , *ORGANS (Anatomy) , *PRESERVATION of organs, tissues, etc. - Abstract
Abstract: Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNALeu(UUR) with a MELAS A3243G mutation and mt tRNALys with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine-containing modifications [τm5(s2)U; 5-taurinomethyl-(2-thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF. [Copyright &y& Elsevier]
- Published
- 2005
- Full Text
- View/download PDF
5. Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue
- Author
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Hembrough Todd, Thyparambil Sheeno, Liao Wei-Li, Darfler Marlene M, Abdo Joseph, Bengali Kathleen M, Taylor Paul, Tong Jiefei, Lara-Guerra Humberto, Waddell Thomas K, Moran Michael F, Tsao Ming-Sound, Krizman David B, and Burrows Jon
- Subjects
Formalin fixed ,FFPE ,EGFR ,Gefitinib ,Targeted therapy ,Patient tissue ,Quantitative ,Personalized medicine ,Molecular diagnostics ,Non-small cell lung cancer ,Medicine - Abstract
Abstract Background Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies. Methods A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel. Results The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and are consistent with EGFR protein levels in these tumors as previously-reported by western blot and SRM analysis of the matched frozen tissue. In addition, the SRM assay was applied to a collection of histologically-characterized FFPE NSCLC patient tumor tissue where EGFR levels were quantitated from not detected (ND) to 670amol/μg. Conclusions This report describes and evaluates the performance of a robust and reproducible SRM assay designed for measuring EGFR directly in FFPE patient tumor tissue with accuracy at extremely low (attomolar) levels. This assay can be used as part of a complementary or companion diagnostic strategy to support novel therapies currently under development and demonstrates the potential to identify candidates for EGFR-inhibitor therapy, predict treatment outcome, and reveal mechanisms of therapeutic resistance.
- Published
- 2012
- Full Text
- View/download PDF
6. Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases
- Author
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Kimitsuna Watanabe, Howard T. Jacobs, Yohei Kirino, Shigeo Ohta, Ian J. Holt, Takao Makifuchi, Norie Ishii, Nobuyoshi Fukuhara, Takehiro Yasukawa, and Tsutomu Suzuki
- Subjects
Mitochondrial Diseases ,Taurine ,Post-transcriptional modification ,Mitochondrial disease ,Molecular Sequence Data ,Mutant ,Biophysics ,Wobble base pair ,Biology ,medicine.disease_cause ,Biochemistry ,RNA, Transfer ,Structural Biology ,Genetics ,medicine ,Humans ,Molecular Biology ,Mutation ,Base Sequence ,Point mutation ,Translation (biology) ,RNA Probes ,Cell Biology ,medicine.disease ,Transfer RNA ,Nucleic Acid Conformation ,Patient tissue ,Mitochondrial tRNA ,HeLa Cells - Abstract
Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNA(Leu(UUR)) with a MELAS A3243G mutation and mt tRNA(Lys) with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine-containing modifications [taum(5)(s(2))U; 5-taurinomethyl-(2-thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF.
- Published
- 2005
7. Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue
- Author
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Todd Hembrough, Jiefei Tong, Paul J. Taylor, Ming-Sound Tsao, Thomas K. Waddell, Humberto Lara-Guerra, Michael F. Moran, Wei-Li Liao, David B. Krizman, Sheeno Thyparambil, Joseph Abdo, Kathleen Bengali, Marlene Darfler, and Jon Burrows
- Subjects
Pathology ,medicine.medical_specialty ,medicine.medical_treatment ,EGFR ,Clinical Biochemistry ,lcsh:Medicine ,Proteomics ,FFPE ,Targeted therapy ,03 medical and health sciences ,0302 clinical medicine ,Gefitinib ,Non-small cell lung cancer ,medicine ,Molecular diagnostics ,Epidermal growth factor receptor ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,biology ,business.industry ,Research ,Selected reaction monitoring ,lcsh:R ,General Medicine ,Personalized medicine ,3. Good health ,030220 oncology & carcinogenesis ,Cancer research ,biology.protein ,Molecular Medicine ,Immunohistochemistry ,Formalin fixed ,Patient tissue ,business ,medicine.drug ,Quantitative - Abstract
Background Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies. Methods A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel. Results The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/μg to 12,860amol/μg and are consistent with EGFR protein levels in these tumors as previously-reported by western blot and SRM analysis of the matched frozen tissue. In addition, the SRM assay was applied to a collection of histologically-characterized FFPE NSCLC patient tumor tissue where EGFR levels were quantitated from not detected (ND) to 670amol/μg. Conclusions This report describes and evaluates the performance of a robust and reproducible SRM assay designed for measuring EGFR directly in FFPE patient tumor tissue with accuracy at extremely low (attomolar) levels. This assay can be used as part of a complementary or companion diagnostic strategy to support novel therapies currently under development and demonstrates the potential to identify candidates for EGFR-inhibitor therapy, predict treatment outcome, and reveal mechanisms of therapeutic resistance.
- Full Text
- View/download PDF
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