Intrinsic susceptibility to radiation-induced apoptosis of human lymphocyte subpopulations

doi:10.1016/S0360-3016(03)00637-0

International Journal of Radiation OncologyBiologyPhysics

Volume 57, Issue 3, 1 November 2003, Pages 769-778

https://doi.org/10.1016/S0360-3016(03)00637-0 Get rights and content

Abstract

Purpose

With the aim to evaluate intrinsic radiosensitivity, the susceptibility of lymphocyte subpopulations to radiation-induced apoptosis was determined. The investigated parameters included measurement reliability, phenotypic variance, intra- and inter-individual variability, and correlations between radiation-induced and spontaneous apoptosis.

Methods and materials

Quiescent lymphocytes of 63 healthy volunteers, sampled up to four times over a 1-year period were γ-irradiated in vitro. Subsequent apoptosis (annexin V) was measured for T4-, T8-, and B-lymphocyte subpopulations using 6-color flow cytometry. Spontaneous apoptosis was measured and radiosensitivity was quantified from the dose–effect curves.

Results

After thawing and short-term culture, both spontaneous apoptosis as well as radiation-induced apoptosis (radiosensitivity) differed among the three lymphocyte subpopulations, with T4 being most resistant, and B most sensitive. Spontaneous and radiation-induced apoptosis were correlated in all cell types, and variance between individuals was considerably higher than variance within individuals for both. A small but highly significant increase of both spontaneous and radiation-induced apoptosis was observed with age for T8, but not for T4 and B. Radiosensitivity of T8 and B proved to be sex-independent, whereas female T4 lymphocytes were less radiosensitive than those from males. T4 and T8 radiosensitivities were loosely correlated, and neither of them was related to B radiosensitivity.

Conclusions

Tendency to spontaneous and radiation-induced apoptosis of lymphocyte subpopulations differs among individuals. In addition, depending on the cell types, age and sex are factors influencing these parameters.

Introduction

Extreme radiosensitivity has been associated with severe cases of morbidity after radiotherapy (1). Also, although seldom documented, it has been generally proposed that smaller differences in radiation sensitivity would explain the variability between patients in the severity of normal-tissue complications after radiotherapy 2, 3. In a study with 402 breast cancer patients, Turesson et al. estimated that physical factors such as volume of normal tissues in the irradiated area and total dose or fractionation regimen, would account for only 30% of the total patient-to-patient variability (4). The remaining variability would be more specifically related to cellular sensitivity, as determined in unknown proportions, by genetic variations and epigenetic factors. Phenotypic factors under close genetic control such as hair, eyes, and skin colors have been advocated to be used to modulate the radiotherapy protocol 4, 5, 6.

In vitro evaluation of intrinsic radiosensitivity has been proposed for over 25 years, to allow individualization of radiotherapy treatment schemes. To this end, different assays have been studied, measuring either short-term radiation effects such as DNA strand-breaks, nucleotide dimmers, or apoptosis, or long-term radiation effects such as surviving fraction (SF2), clonogenic potential, chromosomal damage, or micronuclei (7). In all of these approaches, however, important experimental variation has hindered the general recognition of a possible correlation between in vitro radiosensitivity of lymphocytes (8) or fibroblasts 9, 10 and in vivo radiosensitivity. A major drawback of many of the published studies is the lack of statistical power due to insufficient numbers of individuals, and often, the absence of repeated sampling of a large enough group of individuals (11).

We have studied the parameters that may influence the radiosensitivity of several lymphocyte subpopulations in a group of healthy donors who provided blood samples on multiple occasions. Multicolor flow cytometry was used to achieve precise identification of lymphocyte subpopulations and simultaneously detect early stigma of apoptosis on large numbers of cells and individuals, thus ensuring adequate statistical power for the analysis of the data.

Section snippets

Samples—study population

Venous blood was drawn from healthy normal volunteers between 9:00 am and 11:00 am in 7-mL Vacutainer tubes containing Lithium Heparin as an anticoagulant (Becton Dickinson, San Jose, CA), at the Etablissement Français du Sang, Hôpital Saint Louis, Paris. One hundred mL of venous blood from an arbitrarily chosen reference individual was drawn for use as internal control in different experiments. Informed, written consent was obtained from all donors, in accordance with local ethical guidelines.

Quantification of apoptosis

A total of 63 individuals provided multiple blood samples. Blood samples reached the laboratory less than 2 h after venipuncture, and PBL were immediately prepared and frozen in liquid nitrogen. Thawed PBL were placed in culture media for 5 h, and subsequently irradiated.

Among the several experimental schemes available for detection of apoptosis by flow cytometry, we chose the detection of phosphatidyl serine exposure on the external leaflet of the plasma membrane, employing annexin V. This

Discussion

To measure individual intrinsic susceptibility to radiation-induced apoptosis on a large number of individuals, we developed a strategy aiming at the standardization of experimental procedures. Six-color single-tube flow cytometry was employed, allowing evaluation of apoptosis in T4, T8, and B lymphocytes, by simultaneous application of directly conjugated markers CD3, CD4, CD8, and CD19, and annexin V plus Hoechst 33258. This approach allows one to specifically address the radiosensitivity of

Acknowledgements

We warmly acknowledge the contribution of Jean Christophe Beaudoin. We would like to thank Prof. D. Charron and Joelle Treton for the establishment and management of the donor cohort.

References (29)

N.G. Burnet et al.
Normal tissue radiosensitivity—how important is it?
Clin Oncol
(1996)
J. Johansen et al.
Relationship between the in vitro radiosensitivity of skin fibroblasts and the expression of subcutaneous fibrosis, telangiectasia, and skin erythema after radiotherapy
Radiother Oncol
(1996)
I. Turesson et al.
Prognostic factors for acute and late skin reactions in radiotherapy patients
Int J Radiat Oncol Biol Phys
(1996)
I. Turesson
Individual variation and dose dependency in the progression rate of skin telangiectasia
Int J Radiat Oncol Biol Phys
(1990)
S.L. Tucker et al.
Evidence for individual differences in the radiosensitivity of human skin
Eur J Cancer
(1992)
D.N. Floyd et al.
Intrinsic radiosensitivity of adult and cord blood lymphocytes as determined by the micronucleus assay
Eur J Cancer
(1994)
C.M. West et al.
Lymphocyte radiosensitivity is a significant prognostic factor for morbidity in carcinoma of the cervix
Int J Radiat Oncol Biol Phys
(2001)
G. Alsbeih et al.
Correlation between normal tissue complications and in vitro radiosensitivity of skin fibroblasts derived from radiotherapy patients treated for variety of tumors
Int J Radiat Oncol Biol Phys
(2000)
W.A. Brock et al.
Fibroblast radiosensitivity versus acute and late normal skin responses in patients treated for breast cancer
Int J Radiat Oncol Biol Phys
(1995)
M. Ozsahin et al.
Rapid assay of intrinsic radiosensitivity based on apoptosis in human CD4 and CD8 T-lymphocytes
Int J Radiat Oncol Biol Phys
(1997)

H. Seki et al.

Differential protective action of cytokines on radiation-induced apoptosis of peripheral lymphocyte subpopulations

Cell Immunol

(1995)

D. Zarcone et al.

Radiation sensitivity of resting and activated nonspecific cytotoxic cells of T lineage and NK lineage

Blood

(1989)

N.E. Crompton et al.

Sources of variation in patient response to radiation treatment

Int J Radiat Oncol Biol Phys

(2001)

N.E. Crompton et al.

Altered apoptotic profiles in irradiated patients with increased toxicity

Int J Radiat Oncol Biol Phys

(1999)

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^☆: Supported by the “Service de Radioprotection de Electricité de France”, the “Association pour la Recherche sur le Cancer” and “ATC Cohortes et Collections” of INSERM.

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Biology contributionIntrinsic susceptibility to radiation-induced apoptosis of human lymphocyte subpopulations☆

Abstract

Purpose

Methods and materials

Results

Conclusions

Introduction

Section snippets

Samples—study population

Quantification of apoptosis

Discussion

Acknowledgements

Clin Oncol

Radiother Oncol

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Eur J Cancer

Eur J Cancer

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Cell Immunol

Blood

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

Biology contribution
Intrinsic susceptibility to radiation-induced apoptosis of human lymphocyte subpopulations☆