Increased Chromosomal Aberration and Expression of Specific Breakpoints after Chronic Irradiation of Fibroblasts from a Patient with Non-Hodgkin's Lymphoma

Abstract

Despite the proposed association between DNA repair defects and increased susceptibility to cancer in inherited disorders like ataxia telangiectasia (AT) and xeroderma pigmentosum (XP), the mechanisms underlying this important link have remained poorly understood [1]. Normal body cells from these syndromes, particularly AT and even AT-heterozygotes have been found to exhibit enhanced sensitivity to radiation in vitro [2]. It is believed that the asymptomatic AT-heterozygotes carry at least a 2-3 fold higher risk of dying from a malignant disease and that this potentially "high risk" group alone comprises more than 2-3% of the so-called normal population [3]. These findings stimulated a great deal of interest in exploring the usefulness of in vitro cellular radiosensitivity for the identification of cancer-prone individuals. While cell lines from several cancer-prone disorders other than AT and XP and also some cancer patients have been isolated and their in vitro cellular radiosensitivity demonstrated [4,5] little effort has been made in showing the genetic connection between carcinogen-sensitivity and cancer risks by analyzing chromosomal and mutational alterations in these cells.We have recently reported that skin fibroblast cell lines from most of the Saudi patients with non-Hodgkin's lymphoma (NHL) showed increased sensitivity to chronic gamma irradiation [5]. In the present study, we examined one of these radiosensitive cell lines (L 213725) for chromosomal aberration and specific breakpoints after chronic irradiation compared to the cell lines from a normal subject and an AT patient.Skin fibroblast cell lines developed from cutaneous biopsies obtained from NHL and AT patients as well as normal subjects have been described [5], Early passage (4-10) cells were grown in Ham's F12 medium supplemented with glutamin (2mM), penicillin (100 µg/ml) and 10-15% fetal bovine serum in 100 mm tissue culture plates, and incubated at 37°C in a humidified (80%) atmosphere with 5% CO 2 , 95% air. Cells from the exponential growth phase were harvested, replated in F12 medium and grown to confluence. The plates with confluent cell cultures were irradiated in CO 2 incubator, using a 60 Co medical Gamma source (International Neuronics, Inc., Palo Alto, California, USA), at a dose rate of 0.008 Gy/min. Following irradiation, the growth medium was renewed and the plates were left in a CO 2 incubator overnight. For survival analysis, cells exposed to different doses of radiation were then trypsinized and seeded at a density of 200-20,000 per plate (depending upon radiation dose) together with a feeder layer consisting of radiation (50 Gy) inactivated human fibroblasts (60,000/plate). After further incubation with a weekly change of medium for up to three weeks, the plates were washed with phosphatebuffered saline and stained with crystal violet to count the macroscopic colonies (> 50 cells/colony) as survivors. The percent survival for each cell line was calculated based on the respective unirradiated survivors serving as controls...