Constitutive p53 heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors of BCL-xL.

Proapoptotic molecules directly targeting the BCL-2 family network are promising anticancer therapeutics, but an understanding of the cellular stress signals that render them effective is still elusive. We show here that the tumor suppressor p53, at least in part by transcription independent mechanisms, contributes to cell death induction and full activation of BAX by BH3 mimetic inhibitors of BCL-xL. In addition to mildly facilitating the ability of compounds to derepress BAX from BCL-xL, p53 also provides a death signal downstream of anti-apoptotic proteins inhibition. This death signal cooperates with BH3-induced activation of BAX and it is independent from PUMA, as enhanced p53 can substitute for PUMA to promote BAX activation in response to BH3 mimetics. The acute sensitivity of mitochondrial priming to p53 revealed here is likely to be critical for the clinical use of BH3 mimetics.

Bcl-xL controls a switch between cell death modes during mitotic arrest.

Antimitotic agents such as microtubule inhibitors (paclitaxel) are widely used in cancer therapy while new agents blocking mitosis onset are currently in development. All these agents impose a prolonged mitotic arrest in cancer cells that relies on sustained activation of the spindle assembly checkpoint and may lead to subsequent cell death by incompletely understood molecular events. We have investigated the role played by anti-apoptotic Bcl-2 family members in the fate of mitotically arrested mammary tumor cells treated with paclitaxel, or depleted in Cdc20, the activator of the anaphase promoting complex. Under these conditions, a weak and delayed mitotic cell death occurs that is caspase- and Bax/Bak-independent. Moreover, BH3 profiling assays indicate that viable cells during mitotic arrest are primed to die by apoptosis and that Bcl-xL is required to maintain mitochondrial integrity. Consistently, Bcl-xL depletion, or treatment with its inhibitor ABT-737 (but not with the specific Bcl-2 inhibitor ABT-199), during mitotic arrest converts cell response to antimitotics to efficient caspase and Bax-dependent apoptosis. Apoptotic priming under conditions of mitotic arrest relies, at least in part, on the phosphorylation on serine 62 of Bcl-xL, which modulates its interaction with Bax and its sensitivity to ABT-737. The phospho-mimetic S62D-Bcl-xL mutant is indeed less efficient than the corresponding phospho-deficient S62A-Bcl-xL mutant in sequestrating Bax and in protecting cancer cells from mitotic cell death or yeast cells from Bax-induced growth inhibition. Our results provide a rationale for combining Bcl-xL targeting to antimitotic agents to improve clinical efficacy of antimitotic strategy in cancer therapy.

[Neonatal outcome of fetal hyperechogenic bowel]. / Devenir néonatal des fœtus présentant un intestin hyperéchogène.

OBJECTIVE: Echogenic bowel (EB) represents 1 % of pregnancy and is a risk factor of fetal pathology (infection, cystic fibrosis, aneuploidy). The aim of our study was to determine the fetuses' outcomes with isolated EB. PATIENTS AND METHODS: This is a retrospective study of all patients who presented singleton gestations with a fetal isolated echogenic bowel between 2004 and 2011 in two prenatal diagnosis centers. Search of aneuploidy, infection and cystic fibrosis was systematically proposed as well as an ultrasound monitoring. RESULTS: On 109 fetus addressed for isolate echogenic bowel five had other signs associated and 74 had a real isolated echogenic bowel (without dilatation, calcification, intrauterine growth restriction). In 30 cases, the EB was not found. Eighty-five percent of the patients had in the first trimester a screening for trisomy 21. None fetus with isolated EB had trisomy, infection or cystic fibrosis. One fetus died in utero and one newborn died of a metabolic disease without digestive repercussions. DISCUSSION AND CONCLUSION: The risk of trisomy 21 and the risk to have a serious disease appear low for the fetus with EB. It does not seem necessary to propose a systematic amniocentesis in case of isolated echogenic bowel.

[Spontaneous regression of breast cancer after biopsy. About two cases]. / Régression spontanée de cancers mammaires après biopsie : à propos de deux cas.

We report two cases of spontaneous regression of breast cancer occurred in our institution. It was about a 34- and an 82-year-old patient. The examination revealed a palpable nodule. In both cases, biopsy revealed a negative immunohistochemical staining for estrogen and progesterone receptors, and HER-2 negative. Histological analysis of the surgical specimen found fibrous tissue rearrangement without carcinoma. In the first case, a sarcoidosis was diagnosed at the same time, the mediastinal nodes mimicked a metastatic cancer. These cases illustrate a rare phenomenon. The main hypothesis is a carcinoma- directed immune response triggered by the biopsy.

[Evaluation of endometrectomy by radiofrequency for premenopausal women: a retrospective study]. / Évaluation de l'endométrectomie par radiofréquence chez les femmes préménopausiques : étude rétrospective sur 90 cas.

OBJECTIVE: In present study, we are assessing the efficiency of endometrial ablation by radiofrequency (Novasure(®)) for the treatment of abnormal uterine bleeding. MATERIAL AND METHODS: A total of 90 patients underwent an endometrial ablation by radiofrequency for uterine bleeding between 2009 and 2012. For the postoperative follow-up, symptoms amelioration and eventual adverse-events were evaluated by a self-administered questionnaire given to all patients after the surgery. RESULT: Sixty-five patients (74%) responded to the questionnaire with an average of 17.5 months. Among them, endometrial bleeding decreased in 92% of the cases (IC 95%; 86-99). The amenorrhea rate was 55% (IC 95%; 43-67) and 36% of the patients presented a diminution of menstrual bleeding after treatment. Thirty-two patients (36%) presented dysmenorrhea before the radiofrequency and 78% of them experienced an amelioration of the symptoms after treatment (IC 95%; 64-93). In 19 patients (21%), the cause of uterine bleeding was adenomyosis, among them, bleeding decreased in 84% of the cases (IC 95%; 71-98) and dysmenorrhea in 70%. (IC 95%; 41-97). Finally, 84% of the patients were satisfied with the result of the treatment. CONCLUSION: Our findings suggest that endometrial radiofrequency is effective for the treatment of menometrorrhagia, dysmenorrhea and also adenomyosis.

pRb/E2F-1-mediated caspase-dependent induction of Noxa amplifies the apoptotic effects of the Bcl-2/Bcl-xL inhibitor ABT-737.

Although Bcl-2 family members control caspase activity by regulating mitochondrial permeability, caspases can, in turn, amplify the apoptotic process upstream of mitochondria by ill-characterized mechanisms. We herein show that treatment with a potent inhibitor of Bcl-2 and Bcl-xL, ABT-737, triggers caspase-dependent induction of the BH3-only protein, Mcl-1 inhibitor, Noxa. RNA interference experiments reveal that induction of Noxa, and subsequent cell death, rely not only on the transcription factor E2F-1 but also on its regulator pRb. In response to ABT-737, pRb is cleaved by caspases into a p68Rb form that still interacts with E2F-1. Moreover, pRb occupies the noxa promoter together with E2F-1, in a caspase-dependent manner upon ABT-737 treatment. Thus, caspases contribute to trigger the mitochondrial apoptotic pathway by coupling Bcl-2/Bcl-xL inhibition to that of Mcl-1, via the pRb/E2F-1-dependent induction of Noxa.

Ku-deficient yeast strains exhibit alternative states of silencing competence.

In Saccharomyces cerevisiae, efficient silencer function requires telomere proximity, i.e. compartments of the nucleoplasm enriched in silencing factors. Accordingly, silencers located far from telomeres function inefficiently. We show here that cells lacking yKu balance between two mitotically stable states of silencing competence. In one, a partial delocalization of telomeres and silencing factors throughout the nucleoplasm correlates with enhanced silencing at a non-telomeric locus, while in the other, telomeres retain their focal pattern of distribution and there is no repression at the non-telomeric locus, as observed in wild-type cells. The two states also differ in their level of residual telomeric silencing. These findings indicate the existence of a yKu-independent pathway of telomere clustering and Sir localization. Interestingly, this pathway appears to be under epigenetic control.

The essential function of Not1 lies within the Ccr4-Not complex.

The five Saccharomyces cerevisiae Not proteins are associated with the Ccr4 and Caf1 proteins in 1.2 MDa and 2 MDa complexes. The Not proteins have been proposed to repress transcription of promoters that do not contain a canonical TATA sequence, while the Ccr4 and Caf1 proteins are required for non-fermentative gene expression. The mechanism of transcriptional regulation by the Ccr4-Not complex is unknown and the role of its different components is unclear. Only Not1p is essential for yeast viability.Here, we show that most strains carrying combinations of two null alleles of the non-essential CCR4-NOT genes are non-viable. This would suggest that the Ccr4-Not complex is essential. We find that Not1p consists of at least two domains, a C-terminal domain that is essential for yeast viability, and a N-terminal domain that is dispensable but required for yeast wild-type growth. The essential C-terminal domain of Not1p can associate with Not5p, and both proteins are present in 1.2 and 2 MDa complexes in the absence of the N-terminal Not1p domain. In contrast, in the absence of the N-terminal domain of Not1p, Ccr4p does not efficiently associate in large complexes nor with the C-terminal domain of Not1p. Healthy growth is observed when both domains of Not1p are expressed in trans, and is correlated with their physical association, together with Ccr4p, in large complexes. These results are consistent with the essential function of Not1p lying within the Ccr4-Not complex.

The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae.

We have developed a novel technique for combined immunofluorescence/in situ hybridization on fixed budding yeast cells that maintains the three-dimensional structure of the nucleus as monitored by focal sections of cells labeled with fluorescent probes and by staining with a nuclear pore antibody. Within the resolution of these immunodetection techniques, we show that proteins encoded by the SIR3, SIR4, and RAP1 genes colocalize in a statistically significant manner with Y' telomere-associated DNA sequences. In wild-type cells the Y' in situ hybridization signals can be resolved by light microscopy into fewer than ten foci per diploid nucleus. This suggests that telomeres are clustered in vegetatively growing cells, and that proteins essential for telomeric silencing are concentrated at their sites of action, i.e., at telomeres and/or subtelomeric regions. As observed for Rap1, the Sir4p staining is diffuse in a sir3- strain, and similarly, Sir3p staining is no longer punctate in a sir4- strain, although the derivatized Y' probe continues to label discrete sites in these strains. Nonetheless, the Y' FISH is altered in a qualitative manner in sir3 and sir4 mutant strains, consistent with the previously reported phenotypes of shortened telomeric repeats and loss of telomeric silencing.

Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression.

Transcriptional repression at the silent mating-type loci in yeast requires the targeting of silent information regulator (Sir) proteins through specific interactions formed at cis-acting silencer elements. We show here that a reporter gene flanked by two functional silencers is not repressed when integrated at >200 kb from a telomere. Repression is restored by creation of a new telomere 13 kb from the integrated reporter or by elevated expression of SIR1, SIR3, and/or SIR4. Coupled expression represses in an additive manner, suggesting that all three factors are in limiting concentrations. When overexpressed, Sir3 and Sir4 are dispersed throughout the nucleoplasm, in contrast to wild-type cells where they are clustered in a limited number of foci together with telomeres. Efficient silencer function thus seems to require either proximity to a pool of concentrated Sir proteins, that is, proximity to telomeres, or delocalization of the silencing factors.

Cooperation at a distance between silencers and proto-silencers at the yeast HML locus.

Transcriptional repression at the silent yeast mating type loci is achieved through the formation of a particular nucleoprotein complex at specific cis-acting elements called silencers. This complex in turn appears to initiate the spreading of a histone binding protein complex into the surrounding chromatin, which restricts accessibility of the region to the transcription machinery. We have investigated long-range, cooperative effects between silencers by studying the repression of a reporter gene integrated at the HML locus flanked by various combinations of wild-type and mutated silencer sequences. Two silencers can cooperate over >4000 bp to repress transcription efficiently. More importantly, a single binding site for either the repressor activator protein 1 (Rap1), the autonomous replicating sequence (ARS) binding factor 1 (Abf1) or the origin recognition complex (ORC) can enhance the action of a distant silencer without acting as a silencer on its own. Functional cooperativity is demonstrated using a quantitative assay for repression, and varies with the affinity of the binding sites used. Since the repression mechanism is Sir dependent, the Rap1, ORC and/or Abf1 proteins bound to distant DNA elements may interact to create an interface of sufficiently high affinity such that Sir-containing complexes bind, nucleating the silent chromatin state.

Sequence analysis of murine cytokeratin endo A (no. 8) cDNA. Evidence for mRNA species initiated upstream of the normal 5' end in PCC4 cells.

Keratin 8 (Endo A) is expressed in simple epithelia, together with keratin 18 (Endo B). Filaments formed by this keratin pair are the first cytoskeletal elements induced during mouse embryogenesis. We have isolated Endo A cDNA clones from lambda gt11 libraries prepared with mRNA isolated from PCC4 embryonal carcinoma (EC) cells. Sequencing of three overlapping cDNAs and of a genomic clone allowed us to determine the complete sequence of the Endo A message. Analysis of the protein sequence deduced showed that the Endo A protein presented all the characteristics of intermediate filaments, including an alpha-helical central rod domain and nonhelical N- and C-termini. In the rod domain, the degree of similarity to the other members of the basic keratin family was high. A high degree of homology to keratin 8 of other species was observed, even in the non-helical domains. During these analyses, we found clones extending upstream of the normal 5' end of the mRNA. Sequence comparison between these cDNAs and the 5' upstream region of the Endo A gene suggested that they corresponded to transcripts initiated at an upstream alternative promoter. These observations supported previous results showing the presence of Endo A transcripts initiated upstream of the normal 5' end in mouse morulae and blastocysts.

Retroviral characteristics of the long terminal repeat of murine E.Tn sequences.

E.Tn sequences form a family of long moderately repeated sequences which are abundantly transcribed in the pluripotent cell lineage between day 3.5 and 7.5 of early mouse embryogenesis. The structure of the long terminal repeat (LTR) bordering the E.Tn sequences has been investigated by nucleotide sequencing, primer extension and S1 mapping experiments. This has allowed the identification of U3, R and U5 domains, and of several other structural features all of which are characteristics of retroviral LTRs.

Modulation of specific protein expression in teratocarcinoma cell aggregates by antibodies affecting cell-cell interactions.

Decompacting Fab were used as tools to modify cell interactions during embryoid body formation and differentiation of the embryonal carcinoma (EC) PSMB. Two kinds of Fab were used: anti-uvomorulin Fab which recognize only the adhesion glycoprotein uvomorulin and Fab from an anti-EC serum which recognizes a series of molecules including uvomorulin. Both types of Fab led to decompaction and formation of loose aggregates with abnormal deposition of extracellular matrix (ECM) components. Anti-uvomorulin Fab did not affect the pattern of embryoid body differentiation. In contrast Fab anti-EC induced a differentiation of most of the cells in the aggregate into an endoderm-like phenotype. This effect was observed only when anti-EC Fab were added to aggregates and not to monolayer cultures.