The Demonstration of Immunohistochemical Biomarkers in Methyl Methacrylate-Embedded Plucked Human Hair Follicles

Randall, Kevin J.; Foster, John R.

doi:10.1080/01926230701748198

Cited by 9 publications

(6 citation statements)

References 41 publications

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“…Hair follicle cells are among the most rapidly proliferating cells in the body and are therefore sensitive to agents that damage DNA. Other studies have demonstrated that antibody-based protein detection methods are effective in hair follicle cells (46, 47). One obvious limitation of our model is that decreased concentrations of the drugs across the surface area of the skin may result in higher doses being required to observe an effect in hair follicles.…”

Section: Discussionmentioning

confidence: 99%

Development of a Validated Immunofluorescence Assay for γH2AX as a Pharmacodynamic Marker of Topoisomerase I Inhibitor Activity

Kinders

Hollingshead

Lawrence

et al. 2010

Clinical Cancer Research

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Purpose: Phosphorylated histone H2AX (γH2AX) serves as a biomarker for formation of DNA doublestrand break repair complexes. A quantitative pharmacodynamic immunofluorescence assay for γH2AX was developed, validated, and tested in human tumor xenograft models with the use of clinically relevant procedures.Experimental Design: The γH2AX immunofluorescence assay uses a novel data quantitation and image processing algorithm to determine the extent of nuclear-specific γH2AX staining in tumor needle biopsies and hair follicles collected from mice bearing topotecan-responsive A375 xenografts. After method validation with the topoisomerase I (Top1) inhibitor topotecan, the assay was used to compare pharmacodynamic properties of three structurally related indenoisoquinoline Top1 inhibitors.Results: γH2AX response to topotecan was quantified over a 60-fold dose range (0.016-1.0 times the murine single-dose maximum tolerated dose), and significant pharmacodynamic response was measured at the mouse equivalent of the 1.5 mg/m 2 clinical dose as well as the lowest dose tested. Responses were within a time window amenable for biopsy collection in clinical trials. These studies enabled characterization of dose and time responses for three indenoisoquinolines, resulting in selection of two for clinical evaluation. γH2AX response to Top1 inhibitors in hair follicles was also observable above a minimal dose threshold. Conclusions: Our γH2AX assay is sufficiently accurate and sensitive to quantify γH2AX in tumor samples and will be used in correlative studies of two indenoisoquinolines in a phase I clinical trial at the National Cancer Institute. Data suggest that hair follicles may potentially serve as a surrogate tissue to evaluate tumor γH2AX response to Top1 inhibitors. Clin Cancer Res; 16(22); 5447-57. ©2010 AACR.

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Section: Discussionmentioning

confidence: 99%

Development of a Validated Immunofluorescence Assay for γH2AX as a Pharmacodynamic Marker of Topoisomerase I Inhibitor Activity

Kinders

Hollingshead

Lawrence

et al. 2010

Clinical Cancer Research

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“…The use of plucked hair follicles allows for the measurement of pharmacodynamic response(s) that is noninvasive and allows for repeated sampling without unduly stressing the subject, although care must be taken to ensure sample quality. Plucked hair follicles can be evaluated by immunohistochemistry or immunofluorescence to identify up-or downregulation of specific markers, such as Ki-67, EGFR, phospho-p27, phosphor-histone H3, phosphor-MAPK, or phosph-Rb (Randall and Foster 2007). Plucked follicles are also amenable to image analysis in order to quantify treatment-related changes of specific markers.…”

Section: Plucked Human Scalp Hair Follicle Analysismentioning

confidence: 99%

In Vitro Skin Models and Their Predictability in Defining Normal and Disease Biology, Pharmacology, and Toxicity

Danilenko¹,

Phillips²,

Diaz³

2016

Toxicol Pathol

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In vitro skin model systems are increasingly being used both in the early evaluation of therapeutic drug candidates and in confirmatory mechanistic studies. The most commonly used of these in vitro model systems are reconstituted human epidermis (RHE) models. These RHE models consist solely of epidermal keratinocytes, which comes with some limitations but also with the advantage of focusing toxicologic and pharmacologic evaluation on keratinocytes alone. RHE models can generally be implemented more quickly, easily, and reproducibly than in vivo models and can thus be used for high throughput compound screening while potentially reducing the need for animal studies. Histologic evaluation of RHE sections can be done quite easily, and the sections are very amenable to quantification via image analysis, including automated analysis. RHE model systems can provide very valuable early indications of therapeutic candidate biology, pharmacology, and toxicity; and early results have demonstrated that RHE models have been quite predictive for in vivo pharmacologic and toxicologic effects on the skin, including clinical skin toxicity.

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“…Immunohistochemistry U87MG tumor xenograft sections (5 mm thick) were dewaxed and rehydrated as described previously (Randall and Foster 2007). The following antigen retrieval techniques were then applied: no pretreatment, incubation for ten minutes with Proteinase K (Dako UK Ltd., Cambridgeshire, UK), or two minutes of microwave treatment at 110 C in either citrate (pH 6.0) or EDTA (pH 8.0) buffer.…”

Section: In Vitro Studiesmentioning

confidence: 99%

The Development of an Immunohistochemical Method to Detect the Autophagy-Associated Protein LC3-II in Human Tumor Xenografts

et al. 2011

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Autophagy is believed to be an important process during tumorgenesis, and in recent years it has been shown to be modulated in response to a number of conventional anticancer agents. Furthermore, the development of targeted small molecule inhibitors, such as those to the PI3K-AKT-mTOR pathway, has presented a molecular link between the disruption of this signalling cascade and the process of autophagy. The cellular consequence of stimulating or inhibiting autophagy in cancer cells is not completely understood, so it is important that this process be monitored, along with antiproliferative and apoptotic biomarkers, in the preclinical setting. The field of autophagy is still evolving, and there is a constantly changing set of criteria for the assessment of the process in cells, tissues, and organs. The gold standard technique for analyzing autophagy in mammalian cells remains transmission electron microscopy, which has many limitations and is often difficult to perform on in vivo tissue including human tumor xenografts. In order to monitor autophagy in human tumor xenogaft tissue, we have taken the approach to develop an immunohistochemical (IHC) method for the detection of the autophagosome-associated protein, microtubule-associated protein 1 light chain 3 (LC3), in human tumor xenografts. After synthesis, LC3 is cleaved to form LC3-I, and upon induction of autophagy, LC3-I is conjugated to the lipid phosphatidylethanolamine to form LC3-II, which is tightly bound to the membrane of the autophagosome. It is thought that detection of endogenous LC3-II by IHC could be difficult because of the relatively low level of expression of the protein. Here we present the validation of an IHC method to detect LC3 in human tumor xenografts that we believe is able to distinguish LC3-I from LC3-II. It is hoped that this assay can become a useful tool for the detection of autophagy in preclinical xenograft models and determine the effects of anticancer therapies on the autophagic process.

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The Demonstration of Immunohistochemical Biomarkers in Methyl Methacrylate-Embedded Plucked Human Hair Follicles

Cited by 9 publications

References 41 publications

Development of a Validated Immunofluorescence Assay for γH2AX as a Pharmacodynamic Marker of Topoisomerase I Inhibitor Activity

Development of a Validated Immunofluorescence Assay for γH2AX as a Pharmacodynamic Marker of Topoisomerase I Inhibitor Activity

In Vitro Skin Models and Their Predictability in Defining Normal and Disease Biology, Pharmacology, and Toxicity

The Development of an Immunohistochemical Method to Detect the Autophagy-Associated Protein LC3-II in Human Tumor Xenografts

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