gammaH2AX and cleaved PARP-1 as apoptotic markers in irradiated breast cancer BT474 cellular spheroids.

Abstract:

:Chemo- and radiotherapy induce apoptosis in tumours and surrounding tissues. In a search for robust and reliable apoptosis markers, we have evaluated immunostaining patterns of gammaH2AX and cleaved PARP-1 in paraffin-embedded cellular spheroids. Breast cancer BT474 cells were grown as cell spheroids to diameters of 700-800 microm. The spheroids contained an outer cell layer with proliferative cells, a deeper region with quiescent cells and a central area with necrosis. They were irradiated with 5 Gy and the frequency of apoptotic cells was determined at several time points (0-144 h) and distances (0-150 microm) from the spheroids surface. gammaH2AX and cleaved PARP-1 were quantified independently. Apoptotic frequencies for the two markers agreed both temporally and spatially in the proliferative regions of the spheroids. The gammaH2AX signal was stronger and had lower background compared to cleaved PARP-1. The central necrotic region was intensely stained with cleaved PARP-1, whereas no gammaH2AX could be detected. The apoptotic frequency increased with distance from surface for all time points. However, apoptotic frequencies, above unirradiated control levels, could only be detected for the last time point, 144 h after irradiation. We have shown that the spheroid model is a practical system for evaluation of staining patterns and specificities of apoptosis markers. Also, the radial gradient provides the opportunity to study apoptosis under a range of physiological conditions within the same system. We have further shown that gammaH2AX and cleaved PARP-1 are applicable markers for apoptosis in the proliferative regions of the spheroids. However, the more intense and clear staining patterns of gammaH2AX suggests that this marker is preferable for quantification of apoptosis in spheroids and similar paraffin-embedded materials.

journal_name

Int J Oncol

authors

Qvarnström OF,Simonsson M,Eriksson V,Turesson I,Carlsson J

doi

10.3892/ijo_00000311

subject

Has Abstract

pub_date

2009-07-01 00:00:00

pages

41-7

issue

1

eissn

1019-6439

issn

1791-2423

journal_volume

35

pub_type

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