B-cell chronic lymphocytic leukemia (B-CLL IntroductionThe tumor suppressor TP53 plays an important role in the control of key genes involved in the regulation of DNA repair, cell cycle, and apoptosis. 1,2 p53 is activated in response to DNA damage or other forms of stress, protecting cells from malignant transformation. This is the reason why p53 is frequently inactivated in human cancer. p53 is a short-lived protein, and its cellular level is controlled by the rate at which it is degraded. Although several U3 ubiquitin ligases have been implicated in p53 ubiquitylation and degradation, MDM2 appears to function as a master regulator of p53. 3,4 MDM2 not only facilitates p53 degradation, but it also binds p53 and inhibits its transcriptional activity. Therefore, inhibitors of p53-MDM2 binding are expected to stabilize and activate p53. Recently, the first potent and selective small-molecule antagonists of MDM2, the nutlins, have been shown to activate the p53 pathway in cancer cells with wild-type p53 in vitro and in vivo. 5 B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5 ϩ B lymphocytes. 6 TP53 is mutated in only 5% to 10% of B-CLL cases at diagnosis, but in nearly 30% in chemotherapy-resistant tumors. TP53 mutation is associated with poor clinical outcome, shorter survival, and lack of response to therapy with purine nucleoside analogs or alkylating agents. [7][8][9][10][11] In fact, alterations in the TP53 gene are among the worst prognostic indicators for B-CLL. [12][13][14] Most of the chemotherapeutic drugs currently used induce cell cycle arrest or apoptosis through activation of p53, and p53 inactivation leads to chemoresistance. 1,2 Chemotherapeutic drugs, including purine analogs, topoisomerase inhibitors, and alkylating agents, have been shown to effectively increase p53 levels in B-CLL. 15,16 Thus, p53 activation is considered among the critical molecular events in chemotherapy-induced apoptosis in B-CLL cells. Although TP53 is mutated in only 5% to 10% of patients, the p53 pathway could be altered at a higher frequency, thus effectively attenuating p53 function. One of the mechanisms involved in p53 stabilization in response to DNA damage is its phosphorylation by ataxia telangiectasia mutated (ATM) protein. 1,2 Interestingly, ATM is inactivated in 10% to 20% of B-CLL cases, thus providing an alternative way to disable p53 function. [17][18][19][20] Tumors with alterations upstream of p53 would not respond adequately to genotoxic chemotherapeutics that act through the p53 pathway (eg, alkylating agents such as chlorambucil and cyclophosphamide; purine nucleosides such as fludarabine and cladribine; or topoisomerase inhibitors such as doxorubicin and mitoxantrone). Therefore, new therapies that overcome these For personal use only. on May 11, 2018. by guest www.bloodjournal.org From defects by acting directly on p53 stability may benefit these patients. Nutlins activate p53 by releasing it from MDM2-mediated negative control and thus compensate for d...

Gamete failure-derived infertility affects millions of people worldwide; for many patients, gamete donation by unrelated donors is the only available treatment. Embryonic stem cells (ESCs) can differentiate in vitro into germ-like cells, but they are genetically unrelated to the patient. Using an in vitro protocol that aims at recapitulating development, we have achieved, for the first time, complete differentiation of human induced pluripotent stem cells (hiPSCs) to postmeiotic cells. Unlike previous reports using human ESCs, postmeiotic cells arose without the over-expression of germline related transcription factors. Moreover, we consistently obtained haploid cells from hiPSCs of different origin (keratinocytes and cord blood), produced with a different number of transcription factors, and of both genetic sexes, suggesting the independence of our approach from the epigenetic memory of the reprogrammed somatic cells. Our work brings us closer to the production of personalized human gametes in vitro.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5+ B lymphocytes. TPA (12-O-tetradecanoylphorbol 13- acetate) and interleukin-4 (IL-4) inhibit apoptosis of B-CLL lymphocytes ex vivo. We used specific inhibitors of protein kinase C (PKC), extracellular-regulated kinase (ERK), and phosphatidylinositol 3–kinase (PI3-kinase) to study their involvement in TPA- and IL-4–induced survival of B-CLL lymphocytes. BisI, a specific inhibitor of PKC, induced apoptosis and inhibited the antiapoptotic activity of TPA and IL-4. B-CLL cells have a basal PKC activity that was increased by TPA but not by IL-4. TPA, but not IL-4, induced ERK activation. However, the inhibition of ERK activation did not affect the viability of B-CLL lymphocytes, demonstrating that this pathway is not involved in their survival. Inhibition of PI3-kinase by LY294002 induced apoptosis of B-CLL cells and inhibited the survival effect of IL-4 and TPA. In addition, Akt, a downstream effector of PI3-kinase activity, was phosphorylated by TPA and IL-4 in B-CLL cells, though PI3-kinase had no effect on PKC-dependent phosphorylation of Akt. Furthermore, the inhibition of PKC or PI3-kinase increased dexamethasone- and fludarabine-induced apoptosis ex vivo in the presence of survival factors. These results demonstrate that PKC and PI3-kinase are involved in the survival of B-CLL cells and suggest that inhibitors of these pathways could be combined with the drugs used in the treatment of B-CLL.

Most of the circulating cells appear to be nondividing and the clonal excess of B cells is mainly caused by defects that prevent programmed cell death rather than by alterations in cell cycle regulation. 3 Glucocorticoids and other chemotherapeutic agents used clinically, including the nucleoside analogues 2-chloro-2Ј-deoxyadenosine and fludarabine, induce apoptosis in B-CLL lymphocytes, [4][5][6][7][8] suggesting that apoptosis is the mechanism of their therapeutic action. Several signaling pathways regulate apoptosis induced by chemotherapy. Thus, we recently demonstrated that phosphatidylinositol 3-kinase and protein kinase C play important roles in the survival of B-CLL cells. Furthermore, inhibition of these kinases increases glucocorticoid-and fludarabine-induced apoptosis ex vivo in the presence of survival factors. 9 The precursor of nucleotide biosynthesis acadesine or 5-aminoimidazole-4-carboxamide (AICA) riboside has various effects in several types of eukaryotic cells. These effects include inhibition of growth and depletion of pyrimidine nucleotide pools in fibroblasts, 10,11 accelerated repletion of purine nucleotide pools in heart, 12 reduction of endurance in skeletal muscle, 13 inhibition of fatty acid, sterol synthesis, and gluconeogenesis in hepatocytes, and increase in glucose uptake in muscle. 14 Acadesine is phosphorylated to AICA ribotide (ZMP), which mimics 5Ј-adenosine monophosphate (AMP) and activates both AMP-activated protein kinase (AMPK) and AMPK kinase (AMPKK). 14,15 The effects on glucose and lipid metabolism are mediated through activation of the AMPK cascade. 14 Previous studies report that acadesine inhibits glucocorticoidinduced apoptosis in quiescent thymocytes, 16 apoptosis caused by serum deprivation in fibroblasts overproducing fructose 2,6-bisphosphate, 17 and ceramide-induced apoptosis in primary astrocytes. 18 To contribute to the understanding of glucocorticoidinduced apoptosis in B-CLL cells, we attempted to block apoptosis with acadesine and, surprisingly, we found that acadesine induced apoptosis in B-CLL cells, whereas T cells from these patients were not affected. Here we study the mechanism of acadesine-induced apoptosis and propose a new pathway involving AMPK and AMPKK in the control of apoptosis in B-CLL cells. Reprints: Joan Gil, Departament de Ciè ncies Fisiolò giques II, Campus de Bellvitge, Universitat de Barcelona, c/ Feixa Llarga s/n, E-08907 L'Hospitalet de Llobregat, Spain; e-mail: joangil@bellvitge.bvg.ub.es.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C. section 1734. Patients, materials, and methods Patients with B-CLL and cell isolationSeventy samples from patients with B-CLL who had not received treatment in the previous 6 months were studied. B-CLL was diagnosed according to standard clinical and laboratory criteria. Cells were obtained from the Hospital Clinic, Barcelona, Spain. Written inf...

Aspirin and other non-steroidal anti-inflammatory drugs induce apoptosis in many cell types. Although the involvement of caspases has been demonstrated, the mechanism leading to caspase activation remains unknown. We have studied the role of the mitochondrial pathway in aspirin-induced apoptosis. The apoptotic effect of aspirin was analyzed in different cell lines (Jurkat, MOLT-4, Raji and HL-60) showing induction of mitochondrial cytochrome c release and caspases 9, 3 and 8 processing. Furthermore, early aspirin-induced cytochrome c release was not affected by the caspase inhibitor Z-VADW Wfmk and preceded loss of mitochondrial membrane potential. Therefore, aspirin-induced apoptosis involves caspase activation through cytochrome c release. ß

STUDY QUESTION Are phospholipase C zeta 1 (PLCZ1) mutations associated with fertilization failure (FF) after ICSI? SUMMARY ANSWER New mutations in the PLCZ1 sequence are associated with FFs after ICSI. WHAT IS KNOWN ALREADY FF occurs in 1–3% of ICSI cycles, mainly due to oocyte activation failure (OAF). The sperm PLCζ/PLCZ1 protein hydrolyzes phosphatidylinositol (4, 5)-bisphosphate in the oocyte, leading to intracellular calcium release and oocyte activation. To date, few PLCZ1 point mutations causing decreased protein levels or activity have been linked to FF. However, functional alterations of PLCζ/PLCZ1 in response to both described and novel mutations have not been investigated. STUDY DESIGN, SIZE, DURATION We performed a study including 37 patients presenting total or partial FF (fertilization rate (FR), ≤25%) after ICSI occurring between 2014 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients were divided into two groups based on oocyte evaluation 19 h post ICSI: FF due to a defect in oocyte activation (OAF, n = 22) and FF due to other causes (‘no-OAF’, n = 15). Samples from 13 men with good fertilization (FR, >50%) were used as controls. PLCζ/PLCZ1 protein localization and levels in sperm were evaluated by immunofluorescence and western blot, respectively. Sanger sequencing on genomic DNA was used to identify PLCZ1 mutations in exonic regions. The effect of the mutations on protein functionality was predicted in silico using the MODICT algorithm. Functional assays were performed by cRNA injection of wild-type and mutated forms of PLCZ1 into human in vitro matured metaphase II oocytes, and fertilization outcomes (second polar body extrusion, pronucleus appearance) scored 19 h after injection. MAIN RESULTS AND THE ROLE OF CHANCE In the OAF group, 12 (54.6%) patients carried at least one mutation in the PLCZ1 coding sequence, one patient out of 15 (6.7%) in the no-OAF group (P < 0.05) and none of the 13 controls (P < 0.05). A total of six different mutations were identified. Five of them were single-nucleotide missense mutations: p.I120M, located at the end of the EF-hand domain; p.R197H, p.L224P and p.H233L, located at the X catalytic domain; and p.S500 L, located at the C2 domain. The sixth mutation, a frameshift variant (p.V326K fs*25), generates a truncated protein at the X-Y linker region. In silico analysis with MODICT predicted all the mutations except p.I120M to be potentially deleterious for PLCζ/PLCZ1 activity. After PLCZ1 cRNA injection, a significant decrease in the percentage of activated oocytes was observed for three mutations (p.R197H, p.H233L and p.V326K fs*25), indicating a deleterious effect on enzymatic activity. PLCZ1 protein localization and expression levels in sperm were similar across groups. FRs were restored (to >60%) in patients carrying PLCZ1 mutations (n = 10) after assisted oocyte activation (AOA), with seven patients achieving pregnancy and live birth. LIMITATIONS, REASONS FOR CAUTION Caution should be exerted when comparing the cRNA injection results with fertilization outcomes after ICSI, especially in patients presenting mutations in heterozygosis. WIDER IMPLICATIONS OF THE FINDINGS PLCZ1 mutations were found in high frequency in patients presenting OAF. Functional analysis of three mutations in human oocytes confirms alteration of PLCζ/PLCZ1 activity and their likely involvement in impaired oocyte activation. Our results suggest that PLCZ1 gene sequencing could be useful as a tool for the diagnosis and counseling of couples presenting FF after ICSI due to OAF. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by intramural funding of Clínica EUGIN, by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 049 to M. T.-M. and GENCAT 2015 DI 048 to D. C.-B.) and by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness to A. F.-V. No competing interest declared.

BackgroundCells have the ability to respond and adapt to environmental changes through activation of stress-activated protein kinases (SAPKs). Although p38 SAPK signalling is known to participate in the regulation of gene expression little is known on the molecular mechanisms used by this SAPK to regulate stress-responsive genes and the overall set of genes regulated by p38 in response to different stimuli.ResultsHere, we report a whole genome expression analyses on mouse embryonic fibroblasts (MEFs) treated with three different p38 SAPK activating-stimuli, namely osmostress, the cytokine TNFα and the protein synthesis inhibitor anisomycin. We have found that the activation kinetics of p38α SAPK in response to these insults is different and also leads to a complex gene pattern response specific for a given stress with a restricted set of overlapping genes. In addition, we have analysed the contribution of p38α the major p38 family member present in MEFs, to the overall stress-induced transcriptional response by using both a chemical inhibitor (SB203580) and p38α deficient (p38α-/-) MEFs. We show here that p38 SAPK dependency ranged between 60% and 88% depending on the treatments and that there is a very good overlap between the inhibitor treatment and the ko cells. Furthermore, we have found that the dependency of SAPK varies depending on the time the cells are subjected to osmostress.ConclusionsOur genome-wide transcriptional analyses shows a selective response to specific stimuli and a restricted common response of up to 20% of the stress up-regulated early genes that involves an important set of transcription factors, which might be critical for either cell adaptation or preparation for continuous extra-cellular changes. Interestingly, up to 85% of the up-regulated genes are under the transcriptional control of p38 SAPK. Thus, activation of p38 SAPK is critical to elicit the early gene expression program required for cell adaptation to stress.

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