Jean Feunteun

Association of BRCA1 with Rad51 in Mitotic and Meiotic Cells

BRCA1 immunostaining reveals discrete, nuclear foci during S phase of the cell cycle. Human Rad51, a homolog of bacterial RecA, behaves similarly. The two proteins were found to colocalize in vivo and to coimmunoprecipitate. BRCA1 residues 758-1064 alone formed Rad51-containing complexes in vitro. Rad51 is also specifically associated with developing synaptonemal complexes in meiotic cells, and BRCA1 and Rad51 were both detected on asynapsed (axial) elements of human synaptonemal complexes. These findings suggest a functional interaction between BRCA1 and Rad51 in the meiotic and mitotic cell cycles, which, in turn, suggests a role for BRCA1 in the control of recombination and of genome integrity.

Dynamic Changes of BRCA1 Subnuclear Location and Phosphorylation State Are Initiated by DNA Damage

BRCA1 localizes to discrete nuclear foci (dots) during S phase. Hydroxyurea-mediated DNA synthesis arrest of S phase MCF7 cells led to a loss of BRCA1 from these structures. Ultraviolet light, mitomycin C, or gamma irradiation produced a similar effect but with no concurrent arrest of DNA synthesis. BARD1 and Rad51, two proteins associated with the BRCA1 dots, behaved similarly. Loss of the BRCA1 foci was accompanied by a specific, dose-dependent change(s) in the state of BRCA1 phosphorylation. Three distinct DNA damaging agents preferentially induced this change in S phase. The S phase BRCA1 phosphorylation response to DNA damage occurred in cells lacking, respectively, two DNA damage-sensing protein kinases, DNA-PK and Atm, implying that neither plays a prime role in this process. Finally, after BRCA1 dot dispersal, BRCA1, BARD1, and Rad51 accumulated, focally, on PCNA+ replication structures, implying an interaction of BRCA1/BARD1/Rad51 containing complexes with damaged, replicating DNA. Taken together, the data imply that the BRCA1 S phase foci are dynamic physiological elements, responsive to DNA damage, and that BRCA1-containing multiprotein complexes participate in a replication checkpoint response.

PML induces a novel caspase-independent death process

PML nuclear bodies (NBs) are nuclear matrix-associated structures altered by viruses and oncogenes. We show here that PML overexpression induces rapid cell death, independent of de novo transcription and cell cycling. PML death involves cytoplasmic features of apoptosis in the absence of caspase-3 activation, and caspase inhibitors such as zVAD accelerate PML death. zVAD also accelerates interferon (IFN)-induced death, suggesting that PML contributes to IFN-induced apoptosis. The death effector BAX and the cdk inhibitor p27KIP1 are novel NB-associated proteins recruited by PML to these nuclear domains, whereas the acute promyelocytic leukaemia (APL) PML/RARα oncoprotein delocalizes them. Arsenic enhances targeting of PML, BAX and p27KIP1 to NBs and synergizes with PML and IFN to induce cell death. Thus, cell death susceptibility correlates with NB recruitment of NB proteins. These findings reveal a novel cell death pathway that neither requires nor induces caspase-3 activation, and suggest that NBs participate in the control of cell survival.

P53 germline mutations in childhood cancers and cancer risk for carrier individuals

The family history of cancer in children treated for a solid malignant tumour in the Paediatric Oncology Department at Institute Gustave-Roussy, has been investigated. In order to determine the role of germline p53 mutations in genetic predisposition to childhood cancer, germline p53 mutations were sought in individuals with at least one relative (first- or second-degree relative or first cousin) affected by any cancer before 46 years of age, or affected by multiple cancers. Screening for germline p53 mutation was possible in 268 index cases among individuals fulfilling selection criteria. Seventeen (6.3%) mutations were identified, of which 13 were inherited and four were de novo. Using maximum likelihood methods that incorporate retrospective family data and correct for ascertainment bias, the lifetime risk of cancer for mutation carriers was estimated to be 73% for males and nearly 100% for females with a high risk of breast cancer accounting for the difference. The risk of cancer associated with such mutations is very high and no evidence of low penetrance mutation was found. These mutations are frequently inherited but de novo mutations are not rare.

BRCA1 Supports XIST RNA Concentration on the Inactive X Chromosome

BRCA1, a breast and ovarian tumor suppressor, colocalizes with markers of the inactive X chromosome (Xi) on Xi in female somatic cells and associates with XIST RNA, as detected by chromatin immunoprecipitation. Breast and ovarian carcinoma cells lacking BRCA1 show evidence of defects in Xi chromatin structure. Reconstitution of BRCA1-deficient cells with wt BRCA1 led to the appearance of focal XIST RNA staining without altering XIST abundance. Inhibiting BRCA1 synthesis in a suitable reporter line led to increased expression of an otherwise silenced Xi-located GFP transgene. These observations suggest that loss of BRCA1 in female cells may lead to Xi perturbation and destabilization of its silenced state.

Mitotic catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy

A conflict in cell cycle progression or DNA damage can lead to mitotic catastrophe when the DNA structure checkpoints are inactivated, for instance when the checkpoint kinase Chk2 is inhibited. Here we show that in such conditions, cells die during the metaphase of the cell cycle, as a result of caspase activation and subsequent mitochondrial damage. Molecular ordering of these phenomena reveals that mitotic catastrophe occurs in a p53-independent manner and involves a primary activation of caspase-2, upstream of cytochrome c release, followed by caspase-3 activation and chromatin condensation. Suppression of caspase-2 by RNA interference or pseudosubstrate inhibitors as well as blockade of the mitochondrial membrane permeabilization prevent the mitotic catastrophe and allow cells to further proceed the cell cycle beyond the metaphase, leading to asymmetric cell division. Heterokarya generated by the fusion of nonsynchronized cells can be driven to divide into three or more daughter cells when Chk2 and caspases are simultaneously inhibited. Such multipolar divisions, resulting from suppressed mitotic catastrophe, lead to the asymmetric distribution of cytoplasm (anisocytosis), DNA (anisokaryosis) and chromosomes (aneuploidy). Similarly, in a model of DNA damage-induced mitotic catastrophe, suppression of apoptosis leads to the generation of aneuploid cells. Our findings delineate a molecular pathway through which DNA damage, failure to arrest the cell cycle and inhibition of apoptosis can favor the occurrence of cytogenetic abnormalities that are likely to participate in oncogenesis.

PHD2 Mutation and Congenital Erythrocytosis with Paraganglioma

Prolyl hydroxylase domain (PHD) proteins play a major role in regulating the hypoxia-inducible factor (HIF) that induces expression of genes involved in angiogenesis, erythropoiesis, and cell metabolism, proliferation, and survival. Germ-line mutations in the prolyl hydroxylase domain 2 gene (PHD2) have been reported in patients with familial erythrocytosis but not in association with tumors. We describe a patient with erythrocytosis and recurrent paraganglioma who carries a newly discovered PHD2 mutation. This mutation affects PHD2 function and stabilizes HIF-alpha proteins. In addition, we demonstrate loss of heterozygosity of PHD2 in the tumor, suggesting that PHD2 could be a tumor-suppressor gene.

Sensitivity and predictive value of criteria for p53 germline mutation screening.

Editor—The history of Li-Fraumeni syndrome (LFS) is a good illustration of the delineation of dominantly inherited family cancer syndromes. The identification of this syndrome is the result of the combination of two kinds of evidence, firstly, a number of reports on particular familial aggregations1 ,2 and, secondly, systematic family studies of childhood sarcomas.3-6 Among these studies, the decisive contribution came from Li and Fraumeni3 who were the first to publish the results of a family study on 641 children with rhabdomyosarcoma which led to the identification of four families in which a sib or a cousin was affected by rhabdomyosarcoma or another soft tissue sarcoma (STS). These families also had several members who were affected by diverse types of malignant tumours, in particular sarcomas and breast cancer at a very young age. This prompted the authors to propose the existence of a new familial syndrome.7 A prospective study on these families over 12 years provided evidence of a strong predisposition to cancer with a strikingly high frequency of multiple tumours.8 The term “Li-Fraumeni syndrome” was used for the first time in 19829 and the criteria, which subsequently became the classical definition of the syndrome, were proposed by Li and Fraumeni in 1988.10 These were a proband with a sarcoma before 45 years of age, a first degree relative with cancer before this age, and another close (first or second degree) relative in the lineage with either cancer before this age or a sarcoma at any age. These criteria led to the selection of 24 families which exhibited a wide variety of tumours including bone sarcomas, STS, breast cancer, brain tumours, leukaemia, adrenocortical carcinoma, lymphoma, lung, stomach, pancreas, and prostate cancer, but only the first six types were significantly in excess …

Gamma-rays-induced death of human cells carrying mutations of BRCA1 or BRCA2

There is now evidence to suggest that BRCA1 and BRCA2 are involved in the response of cells to DNA damage and cell cycle checkpoint control. This report examines the death pathways of human cells with various BRCA1 and BRCA2 genotypes after exposure to gamma-rays. A lack of functional BRCA1 and BRCA2 led to defective repair of DNA double-strand breaks in irradiated cells. This impairment resulted in a relaxation of cell cycle checkpoints, production of micronuclei, and a loss of proliferative capacity. Heterozygous BRCA1 and BRCA2 mutations also led to enhanced radiosensitivity, with an impaired proliferative capacity after irradiation. The existence of a phenotype related to radiosensitivity in BRCA1+/− and BRCA2+/− cells raises the question of the response of heterozygous women to radiation.

FLI1 monoallelic expression combined with its hemizygous loss underlies Paris-Trousseau/Jacobsen thrombopenia

Paris-Trousseau syndrome (PTS; also known as Jacobsen syndrome) is characterized by several congenital anomalies including a dysmegakaryopoiesis with two morphologically distinct populations of megakaryocytes (MKs). PTS patients harbor deletions on the long arm of chromosome 11, including the FLI1 gene, which encodes a transcription factor essential for megakaryopoiesis. We show here that lentivirus-mediated overexpression of FLI1 in patient CD34(+) cells restores the megakaryopoiesis in vitro, indicating that FLI1 hemizygous deletion contributes to the PTS hematopoietic defects. FISH analysis on pre-mRNA and single-cell RT-PCR revealed that FLI1 expression is mainly monoallelic in CD41(+)CD42(-) progenitors, while it is predominantly biallelic in the other stages of megakaryopoiesis. In PTS cells, the hemizygous deletion of FLI1 generates a subpopulation of CD41(+)CD42(-) cells completely lacking FLI1 transcription. We propose that the absence of FLI1 expression in these CD41(+)CD42(-) cells might prevent their differentiation, which could explain the segregation of the PTS MKs into two subpopulations: one normal and one composed of small immature MKs undergoing a massive lysis, presumably originating from either FLI1(+) or FLI1(-) CD41(+)CD42(-) cells, respectively. Thus, we point to the role of transient monoallelic expression of a gene essential for differentiation in the genesis of human haploinsufficiency-associated disease and suggest that such a mechanism may be involved in the pathogenesis of other congenital or acquired genetic diseases.