Quantitative Amplification of Genomic DNA from Histological Tissue Sections after Staining with Nuclear Dyes and Laser Capture Microdissection

Ehrig, Torsten; Abdulkadir, Sarki A.; Dintzis, Suzanne M.; Milbrandt, Jeffrey; Watson, Mark A.

doi:10.1016/s1525-1578(10)60645-9

Cited by 65 publications

(47 citation statements)

References 8 publications

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“…3B, arrows) in human ZZ liver via laser capture-microdissection (LCM) and then subjecting the product to the insoluble isolation technique and immunoblot for ␣1AT. 32,33 The result showed that the ␣1AT isolated was entirely insoluble. Furthermore, total protein stain of the gel from an identical analysis revealed no significant protein outside of the ␣1AT band.…”

Section: Isolation Of ␣1atz Protein Polymers From Livermentioning

confidence: 98%

Quantitative isolation of α1AT mutant Z protein polymers from human and mouse livers and the effect of heat

Blomenkamp

Lindblad

et al. 2005

Hepatology

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Alpha-1-antitrypsin (␣1AT) deficiency in its most common form is caused by homozygosity for the ␣1AT mutant Z gene. This gene encodes a mutant Z secretory protein, primarily synthesized in the liver, that assumes an abnormal conformation and accumulates within hepatocytes causing liver cell injury. Studies have shown that mutant ␣1ATZ protein molecules form unique protein polymers. These Z protein polymers have been hypothesized to play a critical role in the pathophysiology of liver injury in this disease, although a lack of quantitative methods to isolate the polymers from whole liver has hampered further analysis. In this study, we demonstrate a quantitative ␣1ATZ polymer isolation technique from whole liver and show that the hepatocellular periodic acid-Schiff-positive globular inclusions that are the histopathological hallmark of this disease are composed almost entirely of the polymerized ␣1ATZ protein. Furthermore, we examine the previously proposed but untested hypothesis that induction of ␣1ATZ polymerization by the heat of physiological fever is part of the mechanism of hepatic ␣1ATZ protein accumulation. The results, however, show that fever-range temperature elevations have no detectable effect on steady-state levels of intrahepatic Z protein polymer in a model in vivo system. In conclusion, methods to separate insoluble protein aggregates from liver can be used for quantitative isolation of ␣1ATZ T he most common form of alpha-1-antitrypsin (␣1AT) deficiency is caused by homozygosity for the ␣1AT mutant Z gene. [1][2][3][4][5][6][7][8][9][10][11] The Z mutation confers an abnormal conformation on the nascent polypeptide, which is synthesized in the liver, resulting in accumulation within the endoplasmic reticulum (ER) of hepatocytes. ZZ individuals have an increased risk of chronic liver disease and hepatocellular carcinoma resulting from this intracellular accumulation of mutant ␣1ATZ protein.Studies of the mutant ␣1ATZ protein molecule have shown that the nascent polypeptide has the tendency to form unique protein homopolymers. 2,[12][13][14][15][16] Although the "loop sheet" insertion mechanism of ␣1ATZ polymerization-in which the reactive site loop of one molecule inserts into a surface groove in a neighboring moleculedoes not involve the formation of covalent bonds, physical/chemical studies of these polymers suggest that this conformation is highly favored. It has been widely hypothesized that liver injury in humans with ␣1AT deficiency is directly related to the hepatic accumulation of the polymerized ␣1ATZ protein, rather than to the monomeric form. Furthermore, in vitro studies of ␣1ATZ molecules show that the equilibrium favoring formation of polymers increases as temperature increases. 13 These data have been used to propose that progressive liver disease in ZZ humans is directly related to increased ␣1ATZ polymers formed during episodes of physiological fever. 1,8 Our laboratory has reported a series of studies that have begun to examine the mechanism of liver cell injury in ␣1AT deficie...

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Section: Isolation Of ␣1atz Protein Polymers From Livermentioning

confidence: 98%

Quantitative isolation of α1AT mutant Z protein polymers from human and mouse livers and the effect of heat

Blomenkamp

Lindblad

et al. 2005

Hepatology

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“…The slides were then air-dried at RT and used for micro-dissection. Incubation and staining times were kept as short as possible to enhance DNA recovery and proteinase K digestion (Godfrey et al, 2000;Ehrig et al, 2001).…”

Section: Tissue Preparation Micro-dissection Dna Preparationmentioning

confidence: 99%

Nuclear localisation of LASP-1 correlates with poor long-term survival in female breast cancer

et al. 2010

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BACKGROUND: LIM and SH3 protein 1 (LASP-1) is a nucleo-cytoplasmatic signalling protein involved in cell proliferation and migration and is upregulated in breast cancer in vitro studies have shown that LASP-1 might be regulated by prostate-derived ETS factor (PDEF), p53 and/or LASP1 gene amplification. This current study analysed the prognostic significance of LASP-1 on overall survival (OS) in 177 breast cancer patients and addressed the suggested mechanisms of LASP-1-regulation. METHODS: Nucleo-cytoplasmatic LASP-1-positivity of breast carcinoma samples was correlated with long-term survival, clinicopathological parameters, Ki67-positivity and PDEF expression. Rate of LASP1 amplification was determined in micro-dissected primary breast cancer cells using quantitative RT -PCR. Cell-phase dependency of nuclear LASP-1-localisation was studied in synchronised cells. In addition, LASP-1, PDEF and p53 expression was compared in cell lines of different tumour entities to define principles for LASP-1-regulation. RESULTS: We showed that LASP-1 overexpression is not due to LASP1 gene amplification. Moreover, no correlation between p53-mutations or PDEF-expression and LASP-1-status was observed. However, nuclear LASP-1-localisation in breast carcinomas is increased during proliferation with peak in G2/M-phase and correlated significantly with Ki67-positivity and poor OS. CONCLUSION: Our results provide evidence that nuclear LASP-1-positivity may serve as a negative prognostic indicator for long-term survival of breast cancer patients.

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“…Tissue (4 -5-m thickness) were used, and foci of choice were dissected as described elsewhere (37,38). DNA was extracted as previously described (39). DNA was analyzed for p53 mutation by the p53 sequencing method.…”

Section: Criteria For Scoring and Evaluation Of Numerical Chromosomalmentioning

confidence: 99%

p53 Alteration and Chromosomal Instability in Prostatic High-Grade Intraepithelial Neoplasia and Concurrent Carcinoma: Analysis by Immunohistochemistry, Interphase In Situ Hybridization, and Sequencing of Laser-Captured Microdissected Specimens

et al. 2001

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p53 mutation has been shown to be associated with chromosomal instability (CI) in many human dysplastic and neoplastic lesions. However, the precise role of p53 in the pathogenesis of prostate carcinoma (Pca) is unknown. Topographic analysis of p53 alteration using immunohistochemistry (IHC) was performed on 35 archived prostatectomy specimens containing Pca foci; high-grade prostrate intraepithelial neoplasia (HPIN) foci intermingled with cancer (HPINI) and situated away (HPINA). Specimens from 2 patients were topographically genotyped using laser capture microdissection, PCR amplification, and direct sequencing of p53 exons 5-9. CI was evaluated in the same tissue foci by interphase in situ hybridization (IFISH) using centromere probes for chromosomes 7, 8, and Y. p53 immunoreactivity was found in 20%, 17%, 0, and 0 in Pca, HPINI, HPINA, and benign epithelium, respectively. p53 molecular analysis in the specimens examined confirmed the IHC findings. IFISH revealed numerical chromosomal alterations in keeping with CI in 71% and 25% of p53؉ and p53؊ Pca, respectively (P ‫؍‬ .1), 67% and 0 of p53؉ and p53؊ HPIN, respectively (P < .02), and in 27% and 0 of HPINI and HPINA, respectively. We concluded that p53 mutation is an early change in at least a subset of Pca. HPINI foci tend to have higher overall p53 immunoreactivity and CI than HPINA. The presence of p53 mutation in HPIN was associated with the presence of CI as determined by IFISH. Our study also provided additional evidence in support of the concept that HPIN might be the earliest precursor of cancer. Furthermore, our studies identify genomic similarities in HPINI and Pca, implying that carcinoma may arise from progression of certain HPIN foci that most likely harbor p53 mutation and/or more CI. KEY WORDS: Chromosomal instability, Immunohistochemistry, In situ hybridization, p53 sequencing, Prostate carcinoma, Prostate intraepithelial neoplasia.

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Quantitative Amplification of Genomic DNA from Histological Tissue Sections after Staining with Nuclear Dyes and Laser Capture Microdissection

Cited by 65 publications

References 8 publications

Quantitative isolation of α1AT mutant Z protein polymers from human and mouse livers and the effect of heat

Quantitative isolation of α1AT mutant Z protein polymers from human and mouse livers and the effect of heat

Nuclear localisation of LASP-1 correlates with poor long-term survival in female breast cancer

p53 Alteration and Chromosomal Instability in Prostatic High-Grade Intraepithelial Neoplasia and Concurrent Carcinoma: Analysis by Immunohistochemistry, Interphase In Situ Hybridization, and Sequencing of Laser-Captured Microdissected Specimens

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