An Ionizing Radiation-Sensitive CHO Mutant Cell Line: irs-20. IV. Genetic Complementation, V(D)J Recombination and the scid Phenotype

Abstract

The genetic defect responsible for hypersensitivity of Chinese hamster ovary (CHO) irs-20 cells to ionizing radiation was found to be recessive in nature and could be complemented to produce wild-type radiosensitivity in irs-20/human hybrids. The radiosensitivities of six hybrid clones were determined based on their colony-forming ability under continuous irradiation at 6 cGy/h. A parallel cytogenetic analysis revealed a concordance between the presence or absence of human chromosome 8 and the resistant or sensitive phenotype. Confirming evidence was obtained using human chromosome 8-specific PCR primers. Positive amplification was obtained in hybrids with wild-type radiosensitivity, while no amplification was obtained in sensitive hybrids. Complementation analysis between radiosensitive CHO irs-20 and murine scid cell lines was carried out to determine whether the defects leading to their ionizing radiation hypersensitivity could be corrected by genetic complementation in the hybrids. Complementation did not occur. A transient V(D)J recombination assay after the introduction of the RAG1 and RAG2 genes indicated that the V(D)J recombination ability of the CHO irs-20 cells was about 10% of that for the CHO wild-type cells for signal join formation with an 80% joining fidelity and only 3% of the parental level for coding join formation. These data show that murine scid and irs-20 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination.

Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20

“…g These results have been normalised to the controls used in the original experiments. The data presented has been taken from references 15,16,28,29,[32][33][34]38,39,56,58,59. The mutational analysis of V-3 cells are our unpublished observations.…”

“…In this case no residual DNA-PKcs protein can be detected (14,16,31). Recently irs-20, a radio-sensitive mutant derived from another CHO cell line, CHO 10B2, has been shown to belong to the same complementation group (32)(33)(34). Irs-20 differs from V-3 and scid cells in showing less sensitivity to ionising radiation, indicating that it may retain some residual protein function.…”

The C Terminus of Ku80 Activates the DNA-Dependent Protein Kinase Catalytic Subunit

“…Clones expressing Hs Ku80⌬554 cDNA resemble cell lines defective in DNA-PKcs (e.g., the mouse SCID cell line) in having DNA end-binding activity but lacking DNA-PK activity (2, 13). DNA-PKcs-defective cell lines (V-3, irs20, and the mouse SCID line) differ from Ku80-defective lines in the nature of their V(D)J recombination defect (26,27,34,40,42). Ku80-defective mutants have major defects in both signal joint and coding-joint formation, whereas the DNA-PKcs-defective lines can rejoin signal sequences with only a slightly impaired frequency.…”

Deficiency in the Catalytic Subunit of DNA-Dependent Protein Kinase Causes Down-Regulation of ATM

“…There are, in fact, many differences in the genetically unstable M059J tumor cell lines relative to its sister cell line, M059K, derived from the same human tumor, and further differences relative to normal cells. Although no PRKDC mutants other than the M059J line are available for humans, there are several Prkdc mutant rodent cell lines (24)(25)(26)(27)(28)(29)(30)(31)(32). Rodent cells normally express DNA-PKcs at levels that are only about 2% to 4% of that for human cells, although there are large differences in expression levels among cells from different tissues (31).…”