Spontaneous and Radiation-Induced Genomic Instability in Human Cell Lines Differing in Cellular TP53 Status

Abstract

Structural chromosomal rearrangements are commonly observed in tumor karyotypes and in radiation-induced genomic instability. Here we report the effects of TP53 deficiency on karyotypic stability before and after irradiation using related cells and clones differing in cellular TP53 status. The parental cell line, TK6, is a TP53 wild-type human B-lymphoblastoid line with a highly stable karyotype. In the two TK6 derivatives used here, TP53 has been inactivated by biochemical means (expression of HPV16 E6; TK6-5E) or genetic means (allelic inactivation; NH32). Biochemical inactivation of TP53 (TK6-5E) had little effect on the spontaneous karyotype, whereas allelic inactivation of TP53 (NH32) resulted in a modest increase in spontaneous karyotypic instability. After 2 Gy gamma irradiation, the number of unstable clones derived from TP53-deficient cells was significantly elevated compared to the TP53 wild-type counterpart. Extensively destabilized clones were common after irradiation in the set of clones derived from NH32 cells, and one was observed in the set of TK6-5E clones; however, they were never observed in TK6-derived clones. In two of the irradiated NH32 clones, whole chromosomes or chromosome bands were preferentially involved in alterations. These results suggest that genomic instability may differ both quantitatively and qualitatively as a consequence of altered TP53 expression. Some of the results showing repeated and preferential chromosome involvement in aberrations support a model in which instability may be driven by cis mechanisms.

Karyotypic Instability and Centrosome Aberrations in the Progeny of Finite Life-Span Human Mammary Epithelial Cells Exposed to Sparsely or Densely Ionizing Radiation

“…Here, unirradiated finite life-span HMEC clones never showed severe karyotypic instability. This agrees with the lack of chromosomal instability in unirradiated clones of immortalized human-hamster hybrid cells (44) and results for unirradiated human TK6 lymphoblast clones evaluated by G-banding (43). In work by others, TK6 cells had a higher background incidence of chromosome end-to-end fusions than primary cells, but radiationinduced genomic instability was still seen in the clonal descendants of TK6 cells exposed to either ␥ rays or 1 GeV/ nucleon iron ions (45,46).…”

“…Our definition of mild karyotypic instability is similar to the description of karyotypic heterogeneity used for irradiated immortalized human lymphoblasts (8,43), and our definition of severe karyotypic instability is similar to the description of extensive instability used in studies of irradiated normal diploid human fibroblasts (14) and immortalized human-hamster hybrid cells (38). Here, unirradiated finite life-span HMEC clones never showed severe karyotypic instability.…”

Suppression of Nonhomologous End Joining Repair by Overexpression of HMGA2

“…Ku is a heterodimer that has DNA end binding activity and is required for proper DSB repair by NHEJ (20). To explore the mechanisms associated with HMGA2 and DNA-PKcs hyper-phosphorylation, we utilized an in vitro Ku70/Ku80 DNA repair assay to monitor whether or not HMGA2 or HMGA2(4P/A), a mutant form that carries 4 Pro→Ala substitutions at amino acid residues 48, 52 (second AT-hook), 76 and 80 (third AT-hook), altered Ku DNA end-binding.…”

“…Lentiviral vectors pRRLsin.hCMV-HMGA2, pΔ8.7, and pVSV-G were constructed and used for lentiviral production in HEK 293T cells as previously described (20). WI-38 cells were infected with lentiviruses encoding HMGA2 or vector alone, and the transduced WI-38 cells were maintained in culture for three passages, at 1:3 dilutions, to allow time for the genome to stabilize before further analysis was performed.…”

“…WI-38 cells were infected with lentiviruses encoding HMGA2 or vector alone, and the transduced WI-38 cells were maintained in culture for three passages, at 1:3 dilutions, to allow time for the genome to stabilize before further analysis was performed. For cytogenetic analysis, the HMGA2- and vector-transduced WI-38 cells were harvested according to established protocol (20). HMGA2 was knocked down by retroviruses produced using pSin-Puro-miR30-shHMGA2 vector generously provided by Dr. Scott Lowe (Cold Spring Harbor Laboratory).…”