BRIEF REPORT |
Correspondence to: Daniel R. Ciocca, LARLAC, Casilla de Correo 855, 5500 Mendoza, Argentina.
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Summary |
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We are in the process of assessing the response of cancer tissues to chemotherapy, evaluating, among other points, the proportion of cancer cells undergoing apoptosis. However, the apoptotic index obtained with the original TUNEL technique was lower than that obtained by evaluation of apoptosis on H&E-stained sections. Here we describe a small modification of the TUNEL technique that significantly increases the sensitivity of the assay. In the nonmodified TUNEL technique, a digoxigenin-labeled probe is detected using a direct peroxidase-conjugated system, whereas here we report the advantage of using a streptavidinbiotinimmunoperoxidase system. This, in conjunction with pretreatment of tissue sections with proteinase K and microwave irradiation, improved the detection of apoptotic cells. (J Histochem Cytochem 47:837839, 1999)
Key Words: apoptosis, TUNEL, sensitivity
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Introduction |
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Although uncontrolled neoplastic growth is believed to result from increased cell proliferation, a diminished rate of cell death, resulting from the failure of cells to undergo apoptosis in response to different signals, has also been implicated in tumor growth (
Ideally, apoptosis should be assessed by more than one method. In the small biopsies that we have been studying, apoptosis was evaluated by morphological characteristics and by the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) technique. This technique has become the most widely used histochemical method to detect apoptotic cells. However, it is somewhat disappointing because of its limited sensitivity.
In this study we have developed a slight modification of the detection system using the ApopTag Plus kit (Oncor, Gaithersburg, MD; S7101-KIT), which enabled us to significantly increase the sensitivity of the technique. We replaced the direct immunoperoxidase detection of digoxigenin-labeled genomic DNA with an amplification system. Briefly, tissue sections 56 µm thick were mounted on 3-aminopropyltrietoxysilane (Sigma Chemical; St Louis, MO)-coated slides. Thereafter, the sections were deparaffinized and rehydrated through graded alcohols to water. The sections were then incubated with 20 µg/ml of proteinase K (Promega; Madison, WI) in PBS at pH 7.4 for 15 min at room temperature (RT). Endogenous peroxidase activity was quenched by incubation with 2% H2O2 in PBS for 5 min at RT. We did not observe decreased activity of TdT when the samples were pretreated with H2O2 to block endogenous peroxidase, contrary to what has been reported by
For positive control, we used paraffin sections from involuting prostates of castrated rats (n = 2). In this tissue apoptosis is well characterized and easy to detect on H&E-stained sections. We also evaluated the apoptotic index (AI) in tumor cells from postchemotherapy biopsies from breast (n = 2) and cervical (n = 2) cancer patients. In each case, a total of eight slides were examined and the AI was obtained in 1000 cells from each slide. Table 1 shows the AI obtained on H&E-stained sections and on serial sections treated with the TUNEL technique. The best results were obtained when the samples were subjected to proteolytic digestion with proteinase K (20 µg/ml) for 15 min at RT followed by microwave irradiation with 0.01 M citrate buffer, pH 3 (
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Acknowledgments |
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Supported by a grant from the Agencia Nacional de Promoción Científica y Tecnológica of Argentina (BID802/OC-AR PID:05-00000-00766).
We wish to thank Dr Andrei Laszlo (Washington University School of Medicine, St Louis, MO) for editing the manuscript.
Received for publication December 7, 1998; accepted February 9, 1999.
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Literature Cited |
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Carson DA, Ribeiro JM (1993) Apoptosis and disease. Lancet 341:1251-1254[Medline]
LabatMoleur F, Guillermet C, Lorimier P, Robert C, Lantuejoul S, Brambilla E, Negoescu A (1998) TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement. J Histochem Cytochem 46:327-334
Migheli A, Atanasio A, Schifer D (1995) Ultraestructural detection of DNA strand breaks in apoptotic neural cells by in situ end-labelling techniques. J Pathol 176:27-35[Medline]
VargasRoig LM, Gago FE, Tello O, Aznar JC, Ciocca DR (1998) Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy. Int J Cancer [Pred Oncol] 79:468-475[Medline]