complex and the DNA-PK, so that other factors not yet identified might existthat control the radiosensitivity of these normal human fibroblasts.

Keyword(s): Radiosensitivity; Ku; DNA·PK

Ip IX.6! Negative regulation of DNA-dependent protein kinase ac­tivity In human normal peripheral blood lymphocytes:Expression of a variant form of the Ku 86 protein

Catherine Muller, Caroline Dusseau, Patrick Calsou, Bernard Salles. Institutde Pharmacologie et de BiologieStructurale, UPR9062 CNRS, 205 routedeNarbonne. 3/077 Toulouse cedex, France

The DNA-dependent protein kinase (DNA.PK) plays a critical role in marn­malian double-strand break repair and lymphoid V(D)J recombination. Itsregulatory sub-unit, the Ku heterodimer (composed of two tightly associatedpolypeptides of 72 and 86 kDa) binds to DNA termini and recruits the 46()"kDa serine/threonine kinase catalytic sub-unit (DNA·PKcs).

In light of the role of DNA·PK in repairing DSBs, we analyzed theexpression and function of DNA·PK in human normal peripheral bloodlymphocytes, a cell population described as highly sensitive to ionizingradiation. In this study we demonstrate that B lymphocytes expressed avariant form of the Ku 86 protein with an apparent molecular weight of69 kDa, and not the 86·kDa full length protein. Although the heterodimerKu 70/variant Ku 86 binds to DNA-ends (as shown in electrophoreticmobility assays), this altered form of the Ku 86 heterodimer has a decreasedability to recruit the catalytic component of the complex, DNA·PKcs, whichcontributes to the absence of detectable DNA·PK activity in B-cells. Thesedata provide molecular basis for the increased sensitivity of B-cells toionizing radiation and allow the characterization of a new mechanism thatcontrols the level of DNA·PK activity in fresh human cells.

Keyword(s): DNA-dependent protein kinase; ionizing radiation; human lym­phocyte

Ip IX.71 X-ray-sensltlve Chinese hamster ovary cell mutants, XR­Cl and XR-C2, are defective In the SCIDIDNA-PKcs gene,but only XR-Cl Is complemented by human chromosome

8

Margaret Z. Zdzienicka", Ab. Errami, Anna A. FriedlI, WilhelminaJ.I. Overkamp", Bruno Morelli", Friednke Eckardt-Schupp'', Paul H.M.Lohman', Steven P. Jackson", JDepartment of Radiation Genetics andChemical Mutagenesis, MGC, Leiden University, and JA. Cohen Insti­tute, Interunioersity Research Institute for Radiopathology and RadiationProtection, Leiden, Wassenaarsewg 72, 2333 AL Leiden, The Netherlands;2 GSP; Institutfor Radiation Biology, Neuherberg, Germany; J Wellcome/CRCInstitute, University ofCambridge, Cambridge, UK

Two X-ray·sensitive mutants, XR·CI and XR-C2, were isolated from Chinesehamster ovary cells by the replica plating technique, after treatment ofCH09cells with ethylnitroso urea (ENU). The mutants are about 2-3·fold moresensitive to X·rays than parental CH09 cells. Repair of DNA double strandbreaks after irradiation, by pulsed field gel electrophoresis, was found to bedefective in XR·CI. Genetic complementation analysis, by cell fusion, withthe representatives of different complementation groups of rodent ionizingradiation sensitive mutants, indicates that XR-CI, as well as, XR-C2 belongto the same complementation group 7 (SCID/DNA·PKcsIXRCC7). DNA­PK activity was absent in both mutants, confirming these results. Antibodiesagainst DNA·PKcs, were able to detect this protein in CH09 and XR-C2, butnot in XR·Cl cells. Surprisingly, a single human chromosome 8 introducedinto XR-Cl and XR-C2 cells, by microcell mediated chromosome transfer,complemented the X-ray sensitivity only in XR·CI but not in XR-C2 cells,despite the presence of DNA·PK activity in all the examined microcellhybrid clones. This suggests that in the hybrid clones of XR-C2 with humanchromosome 8, the mutated hamster DNA·PKcs protein might act as an

S·IX: Radiation sensitivIty, recombination and repair S63

inhibitor for normal human DNA·PKcs, while in XR-CI the human proteinis replacing the mutated hamster DNA-PKcs.

Keyword(s): X.ray·sensitive rodent cell mutants; DSB repair; DNA-PKcs

Ip Ix.sl Induction of recombination In DNA repair dlsorden andchromosomal aberrations In hereditary cance....prone syn­dromes

P.J. Abrahams', A. Houweling", F. Darroudi'', C.M. Meijers2, C. Terleth ',

AJ. van der Eb l, A.T. Natarajan". JMGC. Department ofMolecular CellBi­

ology. Laboratoryfor Molecular Carcinogenesis, LeidenUniversity. RG.Box9503, 2300 RA Leiden, The Netherlands; 2MGC, Department ofRadiationGenetics and Chemical Mutagenesis. Leiden University. 2333 AL Leiden,The Netherlands

Normal human and DNA repair deficient cells exhibit transient inductionof SOS·like responses such as Enhanced Reactivation (ER) and EnhancedMutagenesis (EM). With the use of Herpes Simplex Virus mutants, weinvestigated whether recombinogenic activities are also transiently inducedin UV·C·exposed normal human cells and cells from the following DNArepair disorders: xeroderma pigmentosum(XP), trichothiodystrophy (TID),Cockayne's syndrome (CS) and Bloom's syndrome (BS). Normal cellsdisplay recombination activities following the same kinetics as the ER andEM response. NER-deficient XP cells show higher levels of recombinogenicactivity, remaining much longer expressed than in normal cells. XP·variantand CS cells also perform higher levels of recombination, but it's kineticsare the same as those in normal cells. Repair deficient TID cells exhibitexpression of recombinogenic activity during various days, but repair profi­cient TID cells behave like normal cells. In contrast, hardly no induction ofrecombinogenic activity could be detected in Bloom's syndrome cells. Theseresults suggest that persisting DNA damage results in transient expressionof a recombinogenic activity, which is deficient in Bloom's cells.

DNA repair disorders such asXp, BS, Ataxia Telangiectasia and Fanconi'sAnemia are characterized by chromosomal abnormalities (SCEs) and highcancer incidence. Impaired DNA repair capacity has been observed in cellsfrom families with high incidence of breast or breast/ovarian cancer. Wewondered whether that might result in induction of chromosomal abnormal­ities. UV·C-exposed cells from afflicted and even as yet unafflicted offspringfrom members of a family with high incidence of breast/ovarian cancer showinduction of higher levels of SCEs, than those in normal cells.

Keyword(s): Recombination; DNA repair disorders; hereditary cancer-pronesyndromes