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3 Acta Biochimica Polonica

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Repair of gamma-ray-induced base damage in L5178Y sublines is damage type-dependent und unrelated to radiation sensitivity

100%

Kruszewski M. , Zastawny T. H. , Szumiel I.

Acta Biochimica Polonica

2001

tom 48

nr 2

The L5178Y (LY) murine lym phoma sublines LY-R and LY-S are dif fer en tially sen si tive to ion iz ing ra di a tion. The high ra di a tion sen si tiv ity of LY-S cells is re lated to impaired rejoining of DNA double strand breaks. We found previously that the g-ray-induced base dam age is higher in the more radiosensitive LY-S subline. Here, we ex am ine the role of the re pair of ion iz ing ra di a tion in duced base dam age in re la tion to the radiosensitivity dif fer ence of these sublines. We used the GS/MS tech nique to es ti mate the re pair rates of six types of base damage in g-irradiated LY cells. All mod i fied DNA bases iden ti fied in the course ofthis study were typ i cal for ir ra di ated chromatin. The to tal amount of ini tial base dam age was higher in the ra di a tion sen si tive LY-S subline than in the ra di a tion re sis tant LY-R subline. The re pair rates of 5-OHMeUra, 5-OHCyt, 8-OHAde were sim i lar in both cell lines, the re pair rates of FapyAde and 8-OHGua were higher in the radiosensitive LY-S cell line, whereas the re pair of 5-OHUra was faster in its radio resistant coun ter part, the LY-R. Al to gether, the re pair rates of the g-ray-induced DNA base dam age in LY sublines are re lated nei ther to the ini tial amounts of the dam aged bases nor to the dif fer en tial le thal or mutagenic ef fects of ion iz ing ra di a tion in these sublines.

Nonhomologous end-joining deficiency of L5178Y-S cells is not associated with mutation in the ABCDE autophosphorylation cluster

84%

Brzoska K. , Kruszewski M. , Szumiel I.

tom 53

nr 1

Cells with mutated autophosphorylation sites in the ABCDE cluster of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are defective in the repair of ionising radiation-induced DSB, but show in an in vitro test the same DNA-PK activity as the cells possessing wild type enzyme. Nevertheless, the mutated DNA-PK is able to undergo ATP-dependent autophosphorylation and inactivation. This characteristics correspond well with the phenotypic features of the L5178Y-S (LY-S) cell line that is defective in DSB repair, shows a pronounced G1 phase radiosensitivity, but in which the level of DNA-PK activity present in total cell extracts is similar to that of its radioresistant counterpart L5178Y-R (LY-R) cell line. The purpose of this work was to examine the possible alterations in the sequence encoding the cluster of autophosphorylation sites in the DNA-dependent protein kinase in LY-S cells. Despite the presence of phenotypic features indicating the possibility of such alterations, no differences were found between the sequences coding for the autophosphorylation sites in L5178Y-R and L5178Y-S cells. In conclusion, the repair defect in LY-S cells is not related to the structure of the DNA-PK autophosphorylation sites (ABCDE casette).

The response of L5178Y lymphoma sublines to oxidative stress: antioxidant defence, iron content and nuclear translocation of the p65 subunit of NF-kappaB

67%

Bouzyk E. , Gradzka I. , Iwanenko T. , Kruszewski M. , Sochanowicz B. , Szumiel I.

nr 4

We examined the response to hydrogen peroxide of two L5178Y (LY) sublines which are inversely cross-sensitive to hydrogen peroxide and X-rays: LY-R cells are radioresistant and hydrogen peroxide-sensitive, whereas LY-S cells are radiosensitive and hydrogen peroxide-resistant. Higher initial DNA breaks and higher iron content (potentially active in the Fenton reaction) were found in the hydrogen peroxide sensitive LY-R cells than in the hydrogen peroxide resistant LY-S cells, whereas the antioxidant defence of LY-R cells was weaker. In particular, catalase activity is twofold higher in LY-S than in LY-R cells. The content of monobromobimane-reactive thiols is 54% higher in LY-S than in LY-R cells. In contrast, the activity of glutathione peroxidase (GPx) is about two times higher in LY-R than in LY-S cells; however, upon induction with selenium the activity increases 15.6-fold in LY-R cells and 50.3-fold in LY-S cells. Altogether, the sensitivity difference is related to the iron content, the amount of the initial DNA damage, as well as to the efficiency of the antioxidant defence system. Differential nuclear translocation of p65-NF-kappaB in LY sublines is due to the more efficient antioxidant defence in LY-S than in LY-R cells.