A human severe combined immunodeficiency (SCID) condition with increased sensitivity to ionizing radiations and impaired V(D)J rearrangements defines a new DNA recombination/repair deficiency.

The products of recombination activating gene (RAG)1 and RAG2 initiate the lymphoid-specific phase of the V(D)J recombination by creating a DNA double-strand break (dsb), leaving hairpin-sealed coding ends. The next step uses the general DNA repair machinery of the cells to resolve this dsb. Several genes involved in both V(D)J recombination and DNA repair have been identified through the analysis of in vitro mutants (Chinese hamster ovary cells) and in vivo situations of murine and equine severe combined immunodeficiency (scid). These studies lead to the description of the Ku-DNA-dependent protein kinase complex and the XRCC4 factor. A human SCID condition is characterized by an absence of B and T lymphocytes. One subset of these patients also demonstrates an increased sensitivity to the ionizing radiation of their fibroblasts and bone marrow precursor cells. This phenotype is accompanied by a profound defect in V(D)J recombination with a lack of coding joint formation, whereas signal joints are normal. Functional and genetic analyses distinguish these patients from the other recombination/repair mutants, and thus define a new group of mutants whose affected gene(s) is involved in sensitivity to ionizing radiation and V(D)J recombination.

Increased radiosensitivity of granulocyte macrophage colony-forming units and skin fibroblasts in human autosomal recessive severe combined immunodeficiency.

We studied the radiosensitivity of granulocyte macrophage colony-forming units (GM-CFU) in patients with a severe combined immunodeficiency (SCID). Three patients lacking both mature T and B cells showed a twofold higher GM-CFU radiosensitivity calculated as the DO value (dose required to reduce survival to 37%), and an identical observation was made with fibroblasts from one of these patients. A patient with an SCID with hypereosinophilia, i.e., Omenn's syndrome characterized by extremely restricted T cell heterogeneity and a lack of B cells, also showed abnormal GM-CFU radiosensitivity. In contrast, GM-CFU from a patient lacking only T cells (X-linked form of SCID) showed normal GM-CFU radiosensitivity. These data further support the similarity between human T(-) B(-) SCID and the murine acid mutation characterized by a defect in T cell receptor and immunoglobulin gene rearrangement, and by an abnormal double-strand DNA break repair function. In addition, they strongly suggest that the Omenn's immunodeficiency syndrome may be a leaky T(-)B(-) SCID phenotype as previously indicated by the coexistence of the two phenotypes in siblings.

The influence of DNA double-strand break structure on end-joining in human cells.

DNA end-joining is the major repair pathway for double-strand breaks (DSBs) in higher eukaryotes. To understand how DSB structure affects the end-joining process in human cells, we have examined the in vivo repair of linearized plasmids containing complementary as well as several different configurations of non-complementary DNA ends. Our results demonstrate that, while complementary and blunt termini display comparable levels of error-free rejoining, end-joining fidelity is decreased to varying extents among mismatched non-complementary ends. End structure also influences the kinetics of repair, accurately recircularized substrates for blunt and complementary termini being detected significantly earlier than for mismatched non-complementary ends. These results suggest that the end-joining process is composed of an early component, capable of efficiently repairing substrates requiring a single ligation event, and a late component, involved in the rejoining of complex substrates requiring multiple processing steps. Finally, these two types of repair events may have different genetic requirements as suggested by the finding that exposure of cells to wortmannin, a potent inhibitor of phosphatidylinositol 3-related kinases (PI 3-related kinases), blocks the repair of complex substrates while having little or no effect on those requiring a simple ligation event.

Repair of 4,5',8-trimethylpsoralen plus light-induced DNA damage in normal and Fanconi's anemia cell lines.

In conditions in which solely monoadducts (MA) are induced in DNA, i.e., treatment with 4,5',8-trimethylpsoralen and 405 nm radiation, Fanconi's anemia cells (FA) appear to be more sensitive than normal human fibroblasts (1 BR/3) to cytotoxicity. The repair of such induced MA is impaired in FA compared to normal cells. When increasing the proportion of DNA interstrand cross-links (CL) over MA using a reirradiation protocol, the differential sensitivity between FA and normal human cells increases. Moreover, for a constant number of total adducts or at different ratios of CL over MA, the repair of CL is systematically hampered in FA as compared to normal cells. Incision of CL being progressively diminished by increasing amounts of MA in normal cells (D. Papadopoulo, D. Averbeck, and E. Moustacchi, Photochem. Photobiol., 47:321-326, 1988), we show here that it is even more so for FA cells.

Mutagenic response of Fanconi's anemia cells from a defined complementation group after treatment with photoactivated bifunctional psoralens.

The induction of mutants at the hypoxanthine-guanine phosphoribosyltransferase and Na+/K+ ATPase loci by photoaddition of two bifunctional psoralens was compared in normal and in Fanconi's anemia lymphoblasts from the genetic complementation group A. For the two loci, the frequency of mutants was significantly lower in Fanconi's anemia than in normal cells. This is true whether the data are expressed as a function of dose or as a function of survival level. It is suggested that the chromosomal instability characteristic of Fanconi's anemia is responsible for the cancer proneness rather than the mutability at the gene level.

The mutagenic processing of psoralen photolesions leaves a highly specific signature at an endogenous human locus.

To assess the role of a given genotoxic agent in the etiology of human cancers, it is useful to establish the mutational specificity of this agent. The aim of this study was to investigate whether the processing of psoralen photolesions, interstrand cross-links (CL) and monoadducts (MA), leaves a specific molecular signature in the mutational events produced at an endogenous locus, HPRT. Human lymphoblasts were treated by 4,5',8-trimethylpsoralen (Me3Pso) in association with a double irradiation protocol (365 plus 365 nm) which allows us to increase the proportion of CL for a given constant number of total photoadducts. The molecular spectrum of mutations at the HPRT locus induced in these conditions was compared to the previously reported spectra of mutations induced by the same psoralen in combination with a single irradiation of either 365 nm (induction of MA and a low proportion of CL) or 405 nm (producing almost exclusively MA). In all treatment conditions, base substitutions constitute the major type of Me3Pso photoinduced mutations. The majority of base substitutions involve a T residue preferably within a 5'-TpA sequence which corresponds to the favoured sites of psoralen photoadducts. In other words, the Me3Pso photolesions induce at the endogenous HPRT locus a high specific signature. Moreover, base substitutions have been essentially found in the non-transcribed strand of the HPRT gene suggesting that the psoralen photolesions are preferentially removed from the transcribed strand. In spite of the considerable difference between the proportion of lesions of both types (CL or MA) induced in different treatment conditions, the kind of mutations and their sequence distribution are similar suggesting that the mutagenic processing of psoralen CL and MA is similar at least for the steps resulting in base substitutions.

Fanconi anemia C gene product plays a role in the fidelity of blunt DNA end-joining.

Mutations in genes controlling the correct functioning of the replicative, repair and recombination machineries may lead to genomic instability. A high level of spontaneous chromosomal aberrations amplified by treatment with DNA cross-linking agents is the hallmark of Fanconi anemia (FA), an inherited chromosomal instability syndrome associated with cancer proneness. Two of the eight FA genes have been cloned (FAA and FAC), but their function has not yet been defined. The lack of homology with known genes suggests the involvement of FA genes in a novel pathway specific to vertebrates. Using a DNA end-joining assay in cultured cells, we studied the processing of both blunt and cohesive-ended double strand breaks (DSB) in normal and FA cells. The results show that: (i) the overall ligation efficiency is normal in FA lymphoblasts; (ii) in FA-C, error-free processing of blunt-ended DSB is markedly decreased, resulting in a higher deletion frequency and larger deletion size; (iii) the fidelity of processing of blunt-DSB is completely restored in FACC cells (complemented with wild-type FAC gene) and the deletion size shifted to values similar to that observed in normal cells; (iv) the fidelity of cohesive end-joining is not affected in FA cells; (v) activities and/or expression of known factors involved in DSB processing, such as the components of the DNA-PK complex and XRCC4, are normal in FA cells. Our results provide strong evidence that the lack of a functional FAC gene results in loss of fidelity of end-joining, which likely accounts for the FA-C phenotype of chromosome instability. We conclude that FAC, and perhaps all FA gene products, are likely to play a role in the fidelity of end-joining of specific DSB.

Abnormal rearrangements associated with V(D)J recombination in Fanconi anemia.

The hallmark of Fanconi anemia (FA), a rare inherited cancer prone disorder, is a high level of chromosome breakage, spontaneous and induced by cross-linking agents. The increased genomic instability of FA is reflected at the gene level by an overproduction of intragenic deletions. Two of the eight FA genes have been cloned, however, their function remains unknown. We recently demonstrated that the lack of functional FA genes lead to a marked decrease in the fidelity of non-homologous end-joining, a pathway that mammalian cells predominantly use to repair DNA double-strand breaks (DSB). Knowing that specific DSB are generated during V(D)J recombination, here we have examined the molecular features of V(D)J rearrangements in normal and FA lymphoblasts belonging to complementation groups C and D. Using appropriate extrachromosomal recombination substrates, V(D)J coding and signal joint formation have been analysed quantitatively and qualitatively. Our results show that the frequency of coding and signal joint formation was not significantly different in normal and FA cells. However, when the fidelity of the V(D)J reaction was examined, we found that in normal human lymphoblasts V(D)J recombination proceeds with high precision, whereas, in FA cells a several fold increase in the frequency of aberrant rearrangements is associated with V(D)J coding joint formation. The abnormal recombinants that we recovered in FA are consistent with excessive degradation of DNA ends generated during the V(D)J reaction. On the basis of these findings, we propose a working model in which FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion proneness.

Photochemotherapy using pyridopsoralens.

Aiming to decrease the acute side effects and genotoxic hazards of PUVA, pyrido (3,4-C) psoralen (PP) and 7-methyl pyrido (3,4-C) psoralen (MPP) were synthesized and studied. Their UVA maximum absorption lies at 325 and 330 nm, respectively. Their photostability is comparable to that of 8-MOP. They complex to DNA in the dark, and, in the presence of UVA, produce only monoadditions to DNA, as shown by fluorescence and DNA denaturation-renaturation studies. In diploid eukaryotic yeast they are more effective than 8-MOP for the induction of lethal effects and mitochondrial damage. Their mutagenic activity per unit dose of UVA is in the same range as that of 8-MOP. However, per viable cell they are clearly less mutagenic than 8-MOP. This difference is also observed for recombinogenic activity. No oxygen effect is observed. In mammalian cells the following ranges of effectiveness are found: inhibition of DNA synthesis in human fibroblasts: MPP greater than PP greater than 8-MOP; mutagenic activity in V79 Chinese hamster cells: MPP greater than PP greater than 8-MOP; cell transforming ability in C3H embryonic mouse cells: MPP greater than 8-MOP greater than PP as a function of UVA dose, and: 8-MOP greater than MPP greater than PP as a function of survival; induction of sister chromatic exchanges (SCE) per unit dose: MPP greater than PP greater than 8-MOP in the linear part of the induction curve, and : 8-MOP greater than PP greater than MPP at the maximum level of SCE obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased deletion mutation in radioadapted human lymphoblasts.

Survival and HPRT- mutant frequency were measured in human lymphoblastoid cells preexposed or not to a low dose of 0.02 Gy gamma rays and then treated with a high dose of 4.0 Gy. When compared to the high dose alone, the low-dose preexposure induced a 70% reduction of the mutant frequency, whereas cell survival was not affected. To understand the mechanisms underlying this phenomenon, the molecular nature of an HPRT- mutant collection was established using Southern hybridization analysis. Among mutants induced by irradiation with the high dose alone, 78% (21/27) had detectable alterations of the HPRT gene. In contrast, only 42% (15/26) of mutants which were "adapted" by the low-dose exposure were of this type. Moreover, the fraction of mutants with only partial deletions of the HPRT gene was significantly reduced (52 and 19% for the 4.0 Gy and 0.02 Gy plus 4.0 Gy induced mutant sets, respectively). In other words, the mutational specificity differed, depending on whether or not the cells were adapted. We suggest that low-dose preexposure leads to a reduced susceptibility to the mutagenic effect of a high dose of gamma rays by inducing an error-free repair system. Our data indicate that this putative system acts preferentially on the class of premutagenic lesions which produce deletions.

Genotoxic effects and DNA photoadducts induced in Chinese hamster V79 cells by 5-methoxypsoralen and 8-methoxypsoralen.

The induction of lethal effects and 6-thioguanine-resistant (6-TGr) mutants were studied in Chinese hamster V79 cells after treatment with the two bifunctional furocoumarins 5- and 8-methoxypsoralens (5-MOP, 8-MOP) in the presence of 365-nm radiation (UVA). The in vivo DNA-photobinding capacity of these two compounds was measured and in parallel the cross-linking capacities of 5-MOP and 8-MOP were determined using the alkaline elution technique. The results show that 5-MOP plus UVA was about 2.5 times more effective than 8-MOP plus UVA for inhibiting cell survival and for inducing the same frequency of 6-TGr mutants (10(-4]. The total number of photoinduced lesions by 5-MOP plus UVA was about 6 times higher than that induced by 8-MOP plus UVA. However, the cross-linking capacities of 5-MOP and 8-MOP were found to be within the same range at equal doses of UVA. At equal number of DNA photoadducts produced, the lesions induced by 5-MOP appeared to be less genetically active than those induced by 8-MOP. The apparently weaker genotoxicity of 5-MOP-induced lesions is likely to be due to the induction of a lower proportion of cross-links by 5-MOP at a given number of photoadducts.

Mutagenic effects photoinduced in normal human lymphoblasts by a monofunctional pyridopsoralen in comparison to 8-methoxypsoralen.

The photobiological effects induced by the monofuctional 7-methylpyrido[3,4-c]psoralen (MePyPs) in comparison to the bifunctional furocoumarin 8-methoxypsoralen (8-MOP) have been studied in a human lymphoblast cell line TK6. We report that, in human lymphoblasts, the cytotoxic effect of MePyPs plus UVA (365 nm) is much higher than that of 8-MOP plus 365-nm irradiation. The dose-modifying factor at the 37% survival level between the 2 compounds equals 120. Mutation induction by photoactivated MePyPs and 8-MOP has been studied in 2 genetic loci, hypoxanthine phosphoribosyl transferase (HPRT) and Na+/K+ ATPase. For equal UVA doses, the mutagenic effectiveness of MePyPs was higher than that of 8-MOP. However at equal survival levels, the mononfuctional psoralen MePyPs was less efficient than the bifunctional 8-MOP. In other words, compared to 8-MOP, the monofunctional agent MePyPs is more cytotoxic than mutagenic. This higher phototoxic and mutagenic efficiency of MePyPs in comparison to 8-MOP is likely to be related to the chemical nature of MePyPs-induced lesions which may be responsible for a reduced recognition and/or accessibility of the repair enzymes to damaged DNA.

Chromosomal aberrations induced by ethylene oxide in a human amniotic cell line in vitro.

Ethylene oxide was found to be a potent inducer of chromatid-type aberrations in a human amniotic cell line. This induction was dose-dependent. At doses corresponding to about 9% survival, the number of chromatid exchanges was increased about 20 times, and the number of induced breaks increased 50 times, above that observed in the controls.

The fate of 8-methoxypsoralen-photoinduced DNA interstrand crosslinks in Fanconi's anemia cells of defined genetic complementation groups.

The fate of 8-methoxypsoralen (8-MOP)-photoinduced DNA interstrand crosslinks was followed by alkaline elution in Fanconi's anemia (FA) fibroblasts belonging to complementation groups A (FA 150 and FA 402) and B (FA 145) in comparison to a normal (1 BR/3) and a heterozygote (F 311) cell line. Clonogenic cell survival to 8-MOP photoaddition was established in parallel for all cell lines. In comparison to normal cells, group A FA cells demonstrated a higher photosensitivity than group B cells (sensitivity index 2.3 and 1.5, respectively), the heterozygote cell line being only slightly more sensitive. FA cells from both groups A and B demonstrated an incision capacity of crosslinks, the kinetics and extent of which being, however, different from that of normal or heterozygote cells. The incision is slower in FA cells and, at 24 h of post-treatment incubation, the amount of crosslinks incised is clearly lower than that observed in normal cells for group A cells, whereas in group B cells incision approaches the level of normal cells. These results correlate with survival as well as with rates of DNA semi-conservative synthesis after 8-MOP photoaddition.

Genotoxic potential of psoralen cross-links versus monoadducts in normal human lymphoblasts.

Using the 4,5',8-trimethylpsoralen in combination with the reirradiation protocol, we show that, in normal human lymphoblasts, the cytotoxic potential of photoinduced cross-links (CL) is higher than that of monoadducts (MA). In contrast to cytotoxicity, the significant increase in the proportion of CL, at a constant level of total adducts, had no effect on the induction of mutations at the HPRT locus. Comparison with the data obtained in yeast and rodent cells using the same double irradiation protocol shows that the mutagenic potential of CL versus MA varies between species. This suggests that the equilibrium between the excision, the recombinational and the mutagenic components of the repair pathways which probably determine the mutagenic efficiency of CL versus MA is likely to be species-dependent.

Mutagenic effects of 3-carbethoxypsoralen and 8-methoxypsoralen plus 365-nm irradiation in mammalian cells.

Cell survival, i.e. colony-forming ability, and the induction of 6-thioguanine-resistant (6-TGr) mutants were determined in Chinese hamster V79 cells by using two photoreactive furocoumarins of photochemotherapeutic interest: the bifunctional compound 8-methoxypsoralen (8-MOP) and the monofunctional compound 3-carbethoxypsoralen (3-CPs). To quantify the mutation induction in V79 cells mutants deficient in the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were selected with the purine analogue 6-thioguanine (6-TG). The effects of the compounds alone at 50 microM in the absence of light and those of 365-nm radiation (UVA) at doses of up to 6 kJm-2 were negligible. When exposed to equimolar concentrations of the compounds together with UVA, V79 cells were about 8 times more sensitive to 8-MOP-plus-UVA than to 3-CPs-plus-UVA. Per unit dose of UVA, 8-MOP was about 7 times more effective than 3-CPs for the induction of 6-TGr mutants. The induction followed about one-hit kinetics for 3-CPs and about two-hit kinetics for 8-MOP. At 50% survival the frequency of 6-TGr mutants induced by 8-MOP plus UVA and 3-CPs plus UVA differed by a factor of about 3.5. These results show a marked concordance with those obtained in the yeast Saccharomyces cerevisiae: both compound exhibited lethal and mutagenic activities but the monofunctional compound 3-CPs was less lethal and mutagenic than the bifunctional compound 8-MOP.

Molecular spectrum of mutations induced at the HPRT locus by a cross-linking agent in human cell lines with different repair capacities.

Molecular characterization of mutations photoinduced by a cross-linking agent, 4,5',8-trimethylpsoralen (Me3Pso), in normal human lymphoblasts was conducted in parallel with lymphoblasts derived from Fanconi anemia patients. Such cells have been previously described to be impaired in repair of psoralen photolesions. The endogenous HPRT locus was used as a target gene. The treatment of cells with Me3Pso in combination with 365 nm irradiation leads to the formation of interstrand cross-links, and specific monoadducts. Our analysis revealed that the mutagenic processing of Me3Pso photoadducts in normal human cells results essentially in base substitutions (84%). These are localized to sequences shown previously to be favored for the formation of Me3Pso monoadducts. The mutagenic processing of the same lesions in Fanconi anemia cells results in fewer base substitutions (22%), with deletions (66%) being the predominant class of mutation. In contrast to prokaryotic systems, frameshifts are poorly represented among Me3Pso induced mutations in human cells. In spite of important differences between the kinds of mutations observed in the two cell lines, our analysis reveals similarities in the type of base substitutions and their sequence distribution. In both normal and Fanconi anemia cell lines mutations, mostly targeted on thymine residues, are preferentially located on the non-transcribed strand.

Frequencies of HPRT- lymphocytes and glycophorin A variants erythrocytes in Fanconi anemia patients, their parents and control donors.

The mutant frequency of 6-thioguanine resistance (HPRT locus) in circulating T lymphocytes from 23 Fanconi anemia (FA) patients has been determined. The glycophorin A (GPA) in vivo cell mutants assay, which detects allele loss variant phenotypes arising from mutations in erythroid progenitor cells of GPA heterozygous MN individuals, has been applied in parallel to FA patients. No significant difference in frequency of HPRT- mutants was observed in FA compared to age matched healthy donors. In contrast, the mean frequency of GPA variant cells was elevated 31-fold for hemizygous NO variants and 8-fold for homozygous NN variants in FA patients over normal controls. In heterozygous FA parents, HPRT- mutant frequencies and GPA variant frequencies were within the normal range. Molecular analysis of HPRT- mutants has previously shown that FA cells have a high tendency to form deletions. Knowing that the cellular events allowing the detection of mutations at the HPRT and the GPA locus differ, our results emphasize the possible correlation between events of spontaneous loss of heterozygosity and genetic predisposition to cancer as observed in FA.

Molecular spectra of HPRT deletion mutations in circulating T-lymphocytes in Fanconi anemia patients.

The principal cellular feature of Fanconi anemia (FA), an inherited cancer prone disorder, is a high level of chromosomal breakage, amplified after treatment with crosslinking agents. Three of the eight genes involved in FA have been cloned: FANCA, FANCC and FANCG. However, their biological functions remain unknown. We previously observed an excessive production of deletions at the HPRT locus in FA lymphoblasts belonging to the relatively rare complementation group D(1) and an increased frequency of glycophorin A (GPA) variants in erythrocytes derived from FA patients (2). In thi study, we examined the molecular nature of 31 HPRT mutations formed in vivo in circulating T-lymphocytes isolated from 9 FA male patients. The results show that in all FA patients investigated the deletions are by far the most prevalent mutational event in contrast to age matched healthy donors, in which point mutations predominate. The complementation group in the FA patients examined in the present study has not yet been defined. However, knowing that mutations in the FANCA and FANCC gene are found to be involved in at least 70% of the FA patients, it can be expected that the excessive production of deletions is a general feature of the FA phenotype. In addition, the spectrum of HPRT deletions observed in FA patients differs from that of healthy children: there is a high frequency of 3'-terminal deletions and a strikingly low proportion of V(D)J mediated events. Based on previous findings, a decreased fidelity of coding V(D)J joint formation (3) and an inaccurate repair of specific DNA double strand breaks via Non-Homologous End Joining (4), we propose that FA genes play a role in the control of the fidelity of rejoining of specific DNA ends. Such a defect may explain several basic features of FA, such as chromosomal instability and deletion pronenness.

[Chemical cancerization of hamster embryo cells in mass culture]. / Sur la cancérisation chimique de cellules d'embryon de hamster en culture de masse

For periods between 24 and 72 hours mass cultures of hamster embryo cells were put in contact with the following carcinogens: 7,10-dimethylbenz (c) acridine (2 mug/ml), benzo (a) pyrene (0,1 mug/ml), 7-methyl benz (a) anthracene (5 and 10 mug/ml), 3-methyl-cholanthrene (0,1 mum/ml), and 7,12-dimethyl benz (a) anthracene (0,05 mug/ml). The cancerisation of cells thus treated was demonstrated by hamster grafting as soon as 4 1/2 months post-treatment. By contrast neither the cells treated by benzo (e) pyrene (non-carcinogenic in animals) nor those of the two control lines resulted in tumours when grafted into hamsters even after culture for 8 and 15 months. These transformed and malignant cultures differ from the controls by their rapid uncontrolled growth and in their morphology. Moreover the morphological characteristics of transformation are evident as soon as 2 months after treatment and for the majority of the compounds studied well before cancerization is demonstrable by hamster grafts. During the course of the first 2 months toxic effects of the compounds are evident in the partial destruction of the cultures and by the appearance of colonies composed in part of cells in disorderly growth. The cells of these colonies when isolated and returned to culture do not survive beyond 4 or 5 passages. Moreover grafts of cultures at 30 days after exposure to carcinogens has never resulted in tumour in the hamster host.