1.- COMPLEMENTATION ANALYSIS BY CELL FUSION IN FANCONI'S ANEMIA. SEARCH FOR COMPLEMENTATION OF DEFECTIVE REPAIR FUNCTIONS BY DNA TRANSFECTION.
2.- INDUCIBILITY OF REPAIR FUNCTIONS IN FA FIBROBLASTS.
Fanconi's anaemia (FA) cells ar hypersensitive to the lethal effect of agents which can form deoxyribonucleic acid (DNA) interstrand crosslinks (CL). The increased susceptibility of FA cells can be due to an abnormality in the processing of CL. In vitro studies demonstrate that the successive pathways of CL repair involve several enzymatic activities, including the strand transferase activity of the Rec A protein. Therefore it was logical to study various parameters of recombinational repair in human cells and to focus our work on FA cells. This aim led to the combination of an in vivo study using a nuclear replicating double stranded DNA virus (herpes simplex virus (HSV)) as a probe and an in vitro study allowing us to determine the characteristics of recombination and recombinational repair catalysed by human nuclear extracts.
The extent of multiplicity reactivation (MR) of HSV containing CL in its DNA was tested because MR has been demonstrated to involve recombination between coinfecting viruses and that 1 CL in viral DNA constitutes a lethal lesion in condition of single infection. The main result obtained is the fact that MR of crosslinked HSV is greater in FA than in normal cells. We interpret this as follows: CL in DNA gives rise to double strand breaks (DSB) following replication across it; recombination between cotransfected viral DNA is strongly stimulated by DSB. The increased MR of HSV in FA cells can reflect an accumulation of DSBs resulting from slowed down removal of CL from damaged DNA. The fact that the initial step of CL repair (ie incision) is slower and finally less efficient in FA than in normal cells supports this possibility.
In addition, mutagenesis studies based upon measurements of the mutation rate of HSV towards a tk- phenotype show that the mutation rate is lower for HSV replicated in FA cells than for that replicated in normal cells, and that, in normal cells, the mutation rate is greater in the progeny from damaged HSV repaired by MR than in that from undamaged virus. In FA cells, it remains unchanged.
In accord with data obtained in our group for an endogenous gene such as hypoxanthine guanine phosphoribosyltransferase (HGPRT), FA cells exhibit an hypomutator effect towards replicating HSV or reactivating by multiplicity psoralen damaged HSV. Since recombination appears to be an error free pathway in human cell extracts, this may account for the hypomutator state toward HSV in FA cells in as much as recombination is relatively more involved in these cells than in the normal cells.
Our in vitro data show that human nuclear extracts contain proteins promoting recombination between 2 duplex DNAs via single strand exchange which can be reciprocal and also recombinational repair of DSB. Extracts from normal fibroblast cells, SV-40 transformed cells, lymphoblastoid cells and also cell lines derived from human carcinoma (lung, cervix) exhibit comparable levels of activities promoting either recombination or recombinational repair of DSBs.
A comparison of these activities in different cell lines (including FA) and according to their genetic origin is planned. However, it seems to be still premature at this stage. Indeed results indicate that numerous enzymes and proteins are involved in either recombination process. For instance, the catalysis of DSB recombinational repair uses at least endonuclease, helicase, exonuclease (polarity?), possibly strand transferase, DNA polymerase, resolvase and ligase. We do not exactly know the precise participation of the recipient bacteria in the complete process. In this context, it is important to point out that it was suggested that the misrepair of DSB could be due to an excess of exonucleolytic activity in X-rays hypersensitive ataxia telangiectasia cells. Indeed the mere imbalance between the relative amounts of these enzymatic activities could heavily perturb either recombinational process.
In conclusion the availability of more refined tests is required to enable precise comparison of the physiological status of recombination enzymatic machineries in human cell strains expected to present recombination alterations such as FA cells. Progress has been achieved in this direction by determining a method and then developing a device according to which the different proteins of a human nuclear extract promoting the strand exchange reaction can be separated by gel electrophoresis and detected.
Fanconi's anaemia (FA) cells are hypersensitive to deoxyribonucleic acid (DNA) crosslinking (CL) agents, including psoralen derivatives in combination with near ultraviolet light (UVA). Although incision of CL following posttreatment incubation takes place in FA cells, the processing of these lesions is hampered compared to normal cells; the incision kinetics is slower and the final amounts of CL incised are lower in FA relative to normal cells. FA complementation group A cells are more affected than group B cells both in terms of cell survival and in terms of CL repair. To our surprise in the absence of CL, FA cells showed a higher sensitivity than normal cells to the cytotoxicity of monoadducts (MA) induced by trimethylpsoralen (TMP) and 405 nm, in this case, group B cells being more sensitive than group A cells. Both cell lines have in fact a reduced incision capacity of MA relative to normal cells, this is especially true for monoadducts on the furan side (MAf). Thus, FA cells are altered in the processing of both CL and MA of a specific structural type. In the presence of high amounts of MAf, the incision of CL is blocked and it is only when the proportion of MAf declines the incision of CL can take place. This phenomenon is even more pronounced in FA cells. Such alteration in processing of DNA lesions is associated with hypomutability which suggests a defect in an error prone pathway. By attempting to clone the normal sequences correcting the FA sensitivity to CL agents and by characterising the correcting diffusible factor identified by cocultivation, we hope to unravel the nature of this pathway.
THE PROJECT WILL LAST FOR FOUR YEARS AND CONSIST OF TWO PARALLEL PARTS. THE FIRST PART WILL STUDY THE GENETIC CONTROL OF FRANCONI ' S ANEMIA (FA) AND THE CLONING OF THE GENE OR GENES RESPONSIBLE FOR REPAIR IN THIS DISEASE. OUR PRELIMINARY EXPERIMENTS AND RECENT EVIDENCE IN THE LITERATURE INDICATE THAT, AS IN OTHER DNA REPAIR DEFECTIVE DISEASES, THERE IS GENETIC HETEROGENEITY. WE INTEND TO DEFINE AND CHARACTERISE THE NUMBER OF COMPLEMENTATION GROUPS IN FA:
-BY SOMATIC HYBRIDIZATION OF DIFFERENT FA LINES FROM DIVERSE GEOGRAPHICAL ORIGINS, TRANSFORMED OR NOT BY SV40.
-BY SOMATIC HYBRIDIZATION USING HUMAN AND MOUSE CELLS.
-CLONING BY TRANSFECTION OF HUMAN GENES INVOLVED IN FA.
-THE DEGREE OF HOMOLOGY BETWEEN NORMAL HUMAN REPAIR GENES AND THOSE OF MICE (MCS) AND YEAST (PSO 2) WILL BE TESTED BY TRANSFECTION AND ATTEMPTS AT COMPLEMENTATION OF FA LINES.
THE SECOND PART WILL CONSIST OF RESEARCH INTO THE POSSIBLE INDUCTION OF REPAIR FUNCTIONS IN NORMAL HUMAN CELLS IN COMPARISON WITH CELLS DERIVED FROM PATIENTS SUFFERING FROM FA. THE PROBE MADE UP OF THE HERPES SIMPLEX VIRUS (HSV) CARRYING DNA LESIONS WILL BE USED FOR THIS STUDY. SEVERAL PARAMETERS WILL BE ANALYSED.
-KINETICS OF VIRAL REACTIVATION AFTER TREATMENT WITH RADIATION AND DNA CROSS-LINKING AGENTS, THE ROLE OF MULTIPLE INFECTION AND THE GROWTH PHASE OF THE HOST CELLS
-EFFECTS OF INHIBITORS OF DNA POLYMERASES AND TOPOISOMERASES
-SYNTHESIS OF DNA LIGASES AND THE STIMULATION OF PLASMINOGEN
-MUTAGENESIS AND INDUCED RECOMBINATION ACTIVITY BY COMPARISON OF NORMAL AND FA CELLS.