Cellular and molecular characteristics of an immortalized ataxia-telangiectasia (group AB) cell line.
Ataxia-telangiectasia (A-T) is a multisystem hereditary disease featuring neurodegeneration, immunodeficiency, extreme cancer proneness, chromosomal instability, and radiosensitivity. A-T is found in many ethnic groups, and is genetically heterogeneous: four complementation groups have been identified in A-T so far. Attempts to isolate the A-T gene are based in part on gene transfer experiments, using permanent A-T fibroblast lines, obtained by transformation with SV40. "Immortalization" of A-T primary diploid fibroblasts using SV40 is difficult, possibly because of the chromosomal instability of these cells. The number of currently available permanent A-T fibroblast lines is small, and not all of them have been assigned to specific complementation groups. Using the assay of X-ray induced inhibition of DNA synthesis, we have assigned the A-T strain AT22IJE to complementation group AB. Origin-defective SV40 was used to transfect these cells, and one transformant (AT22IJE-T), which survived crisis, was found to have the typical characteristics of permanent cell lines obtained in this way. "In-gel renaturation" analysis did not show any DNA amplification of high degree in AT22IJE-T. Cytogenetic analysis showed considerable chromosomal instability in the new cell line, and medium conditioned by these cells contained the clastogenic activity which is characteristic of the parental strain as well. Other parameters of the "cellular A-T phenotype" have also been retained in the immortalized cells: hypersensitivity to the lethal effects of X-rays and neocarzinostatin, as well as "radioresistant" DNA synthesis. However, the sensitivity of AT22IJE-T to both DNA-damaging agents is less pronounced than that of the parental cells. The capacity of the cells for uptake of foreign DNA was tested by introducing into them the plasmid pRSVneo, using three different transfection methods. Satisfactory frequency of G418-resistant transfectants (0.66%) was achieved using a protocol recently published by Chen and Okayama (Mol. Cell Biol., 7: 2745-2752, 1987), which was found to be superior to the traditional calcium phosphate transfection method and to the polybrene-based method.
Ziv
Y
Y
Department of Human Genetics, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Israel.
Jaspers
N G
NG
Etkin
S
S
Danieli
T
T
Trakhtenbrot
L
L
Amiel
A
A
Ravia
Y
Y
Shiloh
Y
Y
D001260:Ataxia Telangiectasia / Q000235:genetics*; D002460:Cell Line; D002869:Chromosome Aberrations; D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage; D005784:Gene Amplification; D006801:Humans; D007621:Karyotyping; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
D004247:DNA