Nonradioactive in Situ Nick Translation: A Useful Molecular Histochemical Tool to Detect Single-Stranded DNA Breaks.

Koji, Takehiko

doi:10.1267/ahc.29.71

Cited by 16 publications

(11 citation statements)

References 33 publications

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“…To identify nuclei with DNA strand breaks as a marker of apoptosis, ISNT was performed according to the protocol described by Koji et al 21 with modifications. Tissue sections were deparaffinized with xylene and rehydrated in serial ethanol solutions.…”

Section: In Situ Nick Translationmentioning

confidence: 99%

“…Despite the evidence of ISNT as a selective apoptotic marker, 23,24 and as single strand breaks have also been implicated in the regulation of cell proliferation, 21 we performed IHC for the cell proliferating antigen Ki-67 on a lymph node with follicular hyperplasia and six melanoma biopsies, to discard false-positive staining. 25 Discrimination of false-positive staining is more straightforward in lymph nodes with follicular hyperplasia, because apoptosis and proliferation are common features and their histoarchitecture allows differentiation of them.…”

Section: Validation Of In Situ Nick Translation As An Apoptotic Markermentioning

confidence: 99%

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Galectin-3 Expression Correlates with Apoptosis of Tumor-Associated Lymphocytes in Human Melanoma Biopsies

Zubieta

Furman

Barrio

et al. 2006

The American Journal of Pathology

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The immune system recognizes diverse melanoma antigens. However , tumors can evade the immune response , therefore growing and progressing. It has been reported that galectin-3 and galectin-1 can induce apoptosis of activated lymphocytes. However , there is strong evidence indicating that the regulation of galectins function in the human tumor microenvironment is a complex process that is influenced by diverse biological circumstances. Here , we have investigated 33 biopsies (eight primary and 25 metastases) from 24 melanoma patients (15-72 years old) and describe the correlation between the expression of galectin-3 or galectin-1 and the level of apoptosis of tumor-associated lymphocytes using immunohistochemistry and an in situ nick translation assay. The range of galectin-3-positive tumor cells varied between 0% and 93% and that of galectin-1-positive tumor cells varied between 5% and 97%. In addition, 23 ؎ 27% of tumor-associated lymphocytes were apoptotic. Although our results show a correlation between galectin-3 expression and apoptosis of tumorassociated lymphocytes, we could not find such correlation with galectin-1. Considering the complex process of cancer immunoediting, various interacting factors must be considered.

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Section: In Situ Nick Translationmentioning

confidence: 99%

Section: Validation Of In Situ Nick Translation As An Apoptotic Markermentioning

confidence: 99%

Galectin-3 Expression Correlates with Apoptosis of Tumor-Associated Lymphocytes in Human Melanoma Biopsies

Zubieta

Furman

Barrio

et al. 2006

The American Journal of Pathology

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“…To identify nuclei with DNA strand breaks at a cellular level, in situ nick translation (ISNT) was performed (16,17). Briefly, paraffin-embedded sections (4 m) were cut onto silane-coated glass slides and deparaffinized in a routine manner.…”

Section: In Situ Detection Of Dna Strand Breaksmentioning

confidence: 99%

Expression of Tumor-Associated Membrane Antigen, RCAS1, in Human Colorectal Carcinomas and Possible Role in Apoptosis of Tumor-Infiltrating Lymphocytes

Okada

Nakashima²,

Komuta

et al. 2003

Modern Pathology

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RCAS1, a novel tumor-associated antigen, is expressed in advanced human neoplasias including uterine and ovarian carcinomas. RCAS1 protein was indicated to induce cell cycle arrest and apoptosis of cultured human lymphoid and myeloid cell lines and normal lymphocytes. In the present study, we investigated the expression and prognostic value of RCAS1 in 58 patients with colorectal carcinomas. RCAS1 protein was detected by immunoperoxidase staining using a mouse monoclonal anti-RCAS1 antibody (22-1-1 antibody). Immunohistochemical examination showed expression of RCAS1 in 75% of colorectal carcinomas with lymph node metastases (n ‫؍‬ 24), whereas it was present in only 41% of tumors without metastases (n ‫؍‬ 34, P < .05). Patients with RCAS1-positive tumors showed a significantly poorer prognosis than those negative for RCAS1 (P < .05). Multivariate analysis using the Cox regression model indicated that RCAS1 positivity was an independent negative predictor for survival (P ‫؍‬ .0300; risk ratio, 0.496). In addition, apoptotic cells of tumor-infiltrating lymphocytes were examined using nonradioactive in situ nick translation in paraffin-embedded sections. The proportion of apoptotic tumor-infiltrating lymphocytes was significantly higher in RCAS1-positive colorectal carcinomas (11.2 ؎ 1.0) than in RCAS1-negative tumors (7.9 ؎ 1.0, P < .05). Our results suggest that overexpression of RCAS1 may negatively affect the prognosis of human colorectal carcinomas and that RCAS1 may play a role in tumor immune privilege in vivo.

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“…The ISNT method detects single-stranded breaks on the double-stranded DNA stretches [12,34]. Escherichia coli DNA polymerase I (Toyobo, Osaka) elongates the DNA strand with 5' to 3' direction, starting at the 3' single-stranded break point.…”

Section: Isnt Methodsmentioning

confidence: 99%

“…In general, artifactual false positivity can be seen in the DNA fragmentation-based techniques, including TUNEL, ISNT and ssDNA immunostaining [10,12,23,26]. Im- proper fixation and autolysis may yield random breaks in the DNA coils (Fig.…”

Section: Comparison Among the Methodologies And Notes For Precautmentioning

confidence: 99%

Pitfalls and Caveats in Histochemically Demonstrating Apoptosis

Tsutsumi

Kamoshida

2003

Acta Histochem. Cytochem

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For investigating the biological significance of apoptosis, the exact and sensitive histochemical identification of apoptosis and apoptotic cells is essential. However, we need to recognize both the pitfalls and caveats in performing histochemical staining and in interpreting the findings obtained. DNA fragmentation-based approaches, such as TdTmediated dUTP-biotin nick end-labeling (TUNEL), in situ nick translation (ISNT) and immunostaining for single-stranded DNA, represent DNA alterations in the apoptotic cell, but they are technically unstable and occasionally give false positive and false negative findings. In contrast, immunostaining for intracellular proteins cleaved and activated by caspases, including cleaved caspase 3, cleaved poly(ADP-ribose) polymerase, cleaved cytokeratin 18 and cleaved actin (fractin), is technically reproducible, but the intracellular accumulation of the activated proteins is not necessarily synchronized. The present review focuses on the pretreatments for enhancing the sensitivity of these techniques, as well as their limitations and comparisons in histochemically demonstrating apoptosis and apoptotic cells.

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Nonradioactive in Situ Nick Translation: A Useful Molecular Histochemical Tool to Detect Single-Stranded DNA Breaks.

Cited by 16 publications

References 33 publications

Galectin-3 Expression Correlates with Apoptosis of Tumor-Associated Lymphocytes in Human Melanoma Biopsies

Galectin-3 Expression Correlates with Apoptosis of Tumor-Associated Lymphocytes in Human Melanoma Biopsies

Expression of Tumor-Associated Membrane Antigen, RCAS1, in Human Colorectal Carcinomas and Possible Role in Apoptosis of Tumor-Infiltrating Lymphocytes

Pitfalls and Caveats in Histochemically Demonstrating Apoptosis

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