Radiation Research

Published by: Radiation Research Society

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Radiation Research 170(2):192-200. 2008
doi: 10.1667/RR1359.1

Cell Membrane is a More Sensitive Target than Cytoplasm to Dense Ionization Produced by Auger Electrons

Jean-Pierre Pouget1ab, Lore Santorob, Laure Raymondb, Nicolas Chouinc, Manuel Bardièsc, Caroline Bascoul-Mollevid, Helena Huguetd, David Azriab, Pierre-Olivier Kotzkib, Monique Pèlegrinbe, Eric Vivèsb, and André Pèlegrinb

aDirection de la Radioprotection de l'Homme, Institut de Radioprotection et de Sûreté Nucléaire, Fontenay-aux-Roses, F-92262 France;

bIRCM, Institut de Recherche en Cancérologie de Montpellier; INSERM, U896; Université Montpellier1; CRLC Val d'Aurelle Paul Lamarque, Montpellier, F-34298, France;

cU892, Nantes, F-44093 France;

dUnité de Biostatistique, CRLC Val d'Aurelle-Paul Lamarque, F-34298 Montpellier, France

eService de Médecine Nucléaire, CHU de Nîmes, Nîmes F-30029, France

1Address for correspondence: INSERM, U896, Institut de Recherche en Cancérologie de Montpellier, CRLC Val d'Aurelle, 34298 Montpellier Cedex 5, France;

Abstract

Pouget, J-P., Santoro, L., Raymond, L., Chouin, N., Bardiès, M., Bascoul-Mollevi, C., Huguet, H., Azria, D., Kotzki, P-O., Pè legrin, M., Vivès, E. and Pèlegrin, A. Cell Membrane is a More Sensitive Target than Cytoplasm to Dense Ionization Produced by Auger Electrons. Radiat. Res. 170, 192–200 (2008).

To improve radioimmunotherapy with Auger electron emitters, we assessed whether the biological efficiency of 125I varied according to its localization. A-431 and SK-OV-3 carcinoma cells were incubated with increasing activities (0–4 MBq/ml) of 125I-labeled vectors targeting the cell membrane, the cytoplasm or the nucleus. We then measured cell survival by clonogenic assay and the mean radiation dose to the nucleus by assessing the cellular medical internal radiation dose (MIRD). The relationship between survival and the radiation dose delivered was investigated with a linear mixed regression model. For each cell line, we obtained dose–response curves for the three targets and the reference values (i.e., the dose leading to 75, 50 or 37% survival). When cell survival was expressed as a function of the total cumulative decays, nuclear 125I disintegrations were more harmful than disintegrations in the cytoplasm or at the cell membrane. However, when survival was expressed as a function of the mean radiation dose to the nucleus, toxicity was significantly higher when 125I was targeted to the cell membrane than to the cytoplasm. These findings indicate that the membrane is a more sensitive target than the cytoplasm for the dense ionization produced by Auger electrons. Moreover, cell membrane targeting is as cytotoxic as nuclear targeting in SK-OV-3 cells. We suggest that targeting the membrane rather than the cytoplasm may contribute to the development of more efficient radioimmunotherapies based on Auger electron radiation, also because most of the available vectors are directed against cell surface antigens.

Received: February 1, 2008; Accepted: April 8, 2008



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FIG. 1. Clonogenic survival as a function of test activity. Clonogenic survival was assessed in cells that were exposed for 2 days to the 125I-labeled vectors with different activities. A-431 (panel A) and SK-OV-3 (panel B) cells were both targeted with non-internalizing 125I-labeled mAb (bold line), internalizing 125I-labeled mAb (dotted line), the 125I-Tat peptide (dashed line), or the irrelevant 125I-PX mAb (thin line). Results are the means of four experiments done in triplicate

FIG. 2. Uptake of radioactivity per cell (Bq/cell). Cells were exposed for 2 days to 125I-labeled vectors and activities were measured over a 6-day period. A-431 cells were targeted with the non-internalizing 125I-labeled mAb (panel A), the internalizing 125I-labeled mAb (panel B), or the 125I-Tat peptide (panel C). SK-OV-3 cells were targeted with the non-internalizing 125I-labeled mAb (panel D), the internalizing 125I-labeled mAb (panel E), or the 125I-Tat peptide (panel F). Results are the means of three experiments done six times

FIG. 3. Clonogenic survival as a function of cumulated uptake of radioactivity (Ãrh). The survival data shown in Fig. 1 were plotted as a function the cumulated uptake of radioactivity (Ãrh) for all targeting models. A-431 (panel A) and SK-OV-3 (panel B) cells were both targeted with non-internalizing 125I-labeled mAb (solid line), internalizing 125I-labeled mAb (dotted line), and the 125I-Tat peptide (dashed line)

FIG. 4. Clonogenic survival as a function of calculated mean nuclear radiation dose. The mean radiation doses delivered to the nucleus were calculated using the MIRD approach and by assuming that cells were isolated. The survival data shown in Fig. 1 were plotted as a function of the calculated radiation dose (Gy) for all targeting models. A-431 (panel A) and SK-OV-3 (panel B) cells were both targeted with non-internalizing 125I-labeled mAb (solid line), internalizing 125I-labeled mAb (dotted line), and the 125I-Tat peptide (dashed line)

FIG. 5. Micronucleus frequency. The yield of micronuclei in 500 binucleated cells were determined on days 1, 2 and 3 after incubation of A-431 and SK-OV-3 cells with the appropriate 125I-labeled vectors. The cumulative frequency was then determined over 3 days. A-431 (panel A) and SK-OV-3 (panel B) cells were both targeted with non-internalizing 125I-labeled mAb (squares), internalizing 125I-labeled mAb (circles), and the 125I-Tat peptide (triangles). Results are the means of three experiments

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TABLE 1 S Values

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TABLE 2 Reference Doses

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TABLE 3 Reference Doses for Internalizing mAbs

 
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