Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells.

Resveratrol, a polyphenol found in grapes and other fruit and vegetables, is a powerful chemopreventive and chemotherapeutic molecule potentially of interest for the treatment of breast cancer. The human breast cancer cell line MCF-7, which is devoid of caspase-3 activity, is refractory to apoptotic cell death after incubation with resveratrol. Here we show that resveratrol arrests cell proliferation, triggers death and decreases the number of colonies of cells that are sensitive to caspase-3-dependent apoptosis (MCF-7 casp-3) and also those that are unresponsive to it (MCF-7vc). We demonstrate that resveratrol (i) acts via multiple pathways to trigger cell death, (ii) induces caspase-dependent and caspase-independent cell death in MCF-7 casp-3 cells, (iii) induces only caspase-independent cell death in MCF-7vc cells and (iv) stimulates macroautophagy. Using BECN1 and hVPS34 (human vacuolar protein sorting 34) small interfering RNAs, we demonstrate that resveratrol activates Beclin 1-independent autophagy in both cell lines, whereas cell death via this uncommon form of autophagy occurs only in MCF-7vc cells. We also show that this variant form of autophagic cell death is blocked by the expression of caspase-3, but not by its enzymatic activity. In conclusion, this study reveals that non-canonical autophagy induced by resveratrol can act as a caspase-independent cell death mechanism in breast cancer cells.

Gene expression profiling of acute promyelocytic leukaemia identifies two subtypes mainly associated with flt3 mutational status.

Acute promyelocytic leukaemia (APL) is a well-defined disease characterized by a typical morphology of leukaemic cells, the presence of t(15;17) translocation and the unique sensitivity to the differentiating effect of all-trans retinoic acid. Nevertheless, some aspects are variable among APL patients, with differences substantially related to morphological variants, peripheral leukocytes count, the presence of a disseminated intravascular coagulopathy, different PML/RARalpha isoforms (long, variable or short) and Fms-like tyrosine kinase 3 (Flt3) mutations. In order to better define this variability, we investigated the gene expression profiles of 18 APL cases revealing, besides a high uniformity in gene expression pattern, the presence of few robust differences among patients able to identify, by an unsupervised analysis, two major clusters of patients characterized by different phenotypes (hypogranular M3v vs classical M3) and by the presence or absence of Flt3 internal tandem duplications (ITDs). Further supervised analysis confirmed that Flt3 status was the APL parameter best associated with these two subgroups. We identified, between Flt3 wild-type and Flt3-ITDs subsets, 147 differentially expressed genes that were involved in the cytoskeleton organization, in the cell adhesion and migration, in the proliferation and the coagulation/inflammation pathways as well as in differentiation and myeloid granules constitution suggesting a role of Flt3 mutations in the pathogenesis and clinical manifestations of APL.

Identification of the B-cell tumor-specific molecular fingerprint using non-radiolabelled PCR consensus primers.

BACKGROUND: The complementarity determining region 3 (CDR3) of the immunoglobulin (Ig) heavy chain variable region (VH) is the most reliable molecular fingerprint for most if not all human B cells. The nucleotide sequence encoding for any B-cell tumor-specific VH CDR3 is currently identified by PCR sequencing based on procedures involving the usage of either radioactive materials, patient/family-specific primers, or bacterial cloning. PATIENTS AND METHODS: In six consecutive patients with follicular lymphoma we assessed the feasibility of a method that allows for identification of the tumor-specific VH CDR3 using consensus primers while avoiding both radioactive materials and bacterial cloning procedures. RESULTS: The tumor-specific VH CDR3 was successfully identified in all six patients in nearly half the time typically required by any other method currently utilized. The feasibility of the proposed method was not significantly affected either by the tumor-specific Ig isotype, or by the tumor infiltration in the original biopsy specimen. In the three patients for whom tumor specimen-derived hybridomas were available, the tumor-specific VH CDR3 was also found in at least 8 of 10 of them. CONCLUSIONS: The proposed method allows the ability to quickly identify the B-cell tumor-specific VH CDR3 using consensus primers while avoiding radioactive materials and bacterial cloning procedures.

Long-term follow up of a randomized, controlled trial on prophylactic sclerotherapy of small oesophageal varices in liver cirrhosis.

BACKGROUND: In order to evaluate the prophylactic impact of sclerotherapy of small varices in patients with cirrhosis and no endoscopic signs suggesting risk of haemorrhage, a randomized clinical trial was performed. METHODS: Seventy-one hospitalized patients met the inclusion criteria of diagnosis of cirrhosis with no previous bleeding and small varices. Due to exclusion criteria of: gastroduodenal ulcers (n = 5), diverticulosis (n = 1), hepatic insufficiency (n = 10), hepatocellular carcinoma (n = 4), death before randomization (n = 6), age over 70 (n = 1) and denial of consent to participate in the study (n = 1), 43 patients could be randomized, 21 for sclerotherapy and 22 for the control group. Two patients (one in each group) were lost to follow up, and another patient, although not lost, refused sclerotherapy after randomization. Ethanolamine oleate was used as the sclerosing agent. All patients were followed up for a mean time of 60 months, initially every 2 months for the first 2 years and clinical and endoscopic controls were performed each 6-12 months thereafter. RESULTS AND CONCLUSIONS: During the first 2 years clinical assessment showed that there were five bleedings in the sclerotherapy group and none in the control group, but mortality was similar in both groups. Long-term follow up continued to show a higher prevalence of bleeding in the sclerotherapy group but that mortality was not different from the control group.

A novel mutation (8342G-->A) in the mitochondrial tRNA(Lys) gene associated with progressive external ophthalmoplegia and myoclonus.

We describe a patient who suffered from impaired ocular motility from age 10 years and at 16 years developed ptosis, proximal weakness and progressive fatigability. At 35 years she developed massive myoclonic jerks, and head and distal tremor. A muscle biopsy showed a high percentage of cytochrome c oxidase negative fibers but no ragged-red fibers. A novel heteroplasmic mutation (8342G-->A) was found in the mitochondrial transfer RNA(Lys) gene by single-strand conformation polymorphism screening, followed by sequence and restriction fragment length polymorphism analysis. Approximately 80% of muscle mitochondrial DNA (mtDNA) harbored the mutation, while the mutation was absent in lymphocyte DNA of the proband, as well as of her mother, daughter and a maternal aunt. However, the pathogenicity of the mutation was confirmed by restriction fragment length polymorphism analysis of single muscle fibers, which revealed a significantly greater level of mutant mtDNA in cytochrome c oxidase negative over cytochrome c oxidase positive fibers.

Double-blind randomized clinical trial comparing neomycin and placebo in the treatment of exogenous hepatic encephalopathy.

Hepatic encephalopathy due to cirrhosis is frequently precipitated by exogenous factors, and the effectiveness of a specific treatment with neomycin sulfate has so far not been submitted to clinical trials. Over a period of five years, 102 cirrhotic patients developed hepatic encephalopathy at admission or during hospitalization, and 39 were randomized for treatment with either neomycin sulfate or placebo. Exclusion criteria were: 1. current usage of specific treatment for hepatic encephalopathy, 2. chronic hepatic encephalopathy and 3. multiple organ failure syndrome associated with hepatic encephalopathy. The group of excluded patients (n = 63) was compared with the randomized group (n = 39), and no statistical differences were found regarding sex and age distributions, Child-Pugh classification, etiology of cirrhosis, percipitating factors and grade of hepatic encephalopathy. These same parameters were also comparable among the 20 patients who received active neomycin and the 19 who were treated with placebo. The therapy for hepatic encephalopathy consisted in the control of precipitating factors associated with 6 g of neomycin sulfate "per os" or placebo. Therapeutic failure and death by the fifth day of treatment, occurred in four patients (10.2%), two in each of the randomized groups. The time elapsed between the initiation of the therapeutic procedure and regression to grade zero of hepatic encephalopathy was 39.11 +/- 23.04 hours for the group of active neomycin, and 49.47 +/- 21.92 hours for the placebo group, but this difference did not achieve statistical significance.