The Parkinson's gene PINK1 regulates cell cycle progression and promotes cancer-associated phenotypes.

PINK1 (phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced kinase 1), a Parkinson's disease-associated gene, was identified originally because of its induction by the tumor-suppressor PTEN. PINK1 promotes cell survival and potentially metastatic functions and protects against cell stressors including chemotherapeutic agents. However, the mechanisms underlying PINK1 function in cancer cell biology are unclear. Here, using several model systems, we show that PINK1 deletion significantly reduced cancer-associated phenotypes including cell proliferation, colony formation and invasiveness, which were restored by human PINK1 overexpression. Results show that PINK1 deletion causes major defects in cell cycle progression in immortalized mouse embryonic fibroblasts (MEFs) from PINK1(-/-) mice, and in BE(2)-M17 cells stably transduced with short hairpin RNA against PINK1. Detailed cell cycle analyses of MEF cell lines from several PINK1(-/-) mice demonstrate an increased proportion of cells in G2/M and decreased number of cells in G1 following release from nocodazole block. This was concomitant with increased double and multi-nucleated cells, a reduced ability to undergo cytokinesis and to re-enter G1, and significant alterations in cell cycle markers, including failure to increase cyclin D1, all indicative of mitotic arrest. PINK1(-/-) cells also demonstrated ineffective cell cycle exit following serum deprivation. Cell cycle defects associated with PINK1 deficiency occur at points critical for cell division, growth and stress resistance in cancer cells were rescued by ectopic expression of human PINK1 and demonstrated PINK1 kinase dependence. The importance of PINK1 for cell cycle control is further supported by results showing that cell cycle deficits induced by PINK1 deletion were linked mechanistically to aberrant mitochondrial fission and its regulation by dynamin-related protein-1 (Drp1), known to be critical for progression of mitosis. Our data indicate that PINK1 has tumor-promoting properties and demonstrates a new function for PINK1 as a regulator of the cell cycle.

Co-regulation of intragenic microRNA miR-153 and its host gene Ia-2 ß: identification of miR-153 target genes with functions related to IA-2ß in pancreas and brain.

AIMS/HYPOTHESIS: We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2ß. We also identified miR-153 target genes that correlated with IA-2ß localisation and function. METHODS: A bioinformatics approach was used to identify miR-153's genomic organisation. To analyse the co-regulation of miR-153 and IA-2ß, quantitative PCR analysis of miR-153 and Ia-2ß (also known as Ptprn2) was performed after a glucose stimulation assay in MIN6B cells and isolated murine pancreatic islets, and also in wild-type Ia-2 (also known as Ptprn), Ia-2ß single knockout and Ia-2/Ia-2ß double knockout mouse brain and pancreatic islets. Bioinformatics identification of miR-153 target genes and validation via luciferase reporter assays, western blotting and quantitative PCR were also carried out. RESULTS: Two copies of miR-153, miR-153-1 and miR-153-2, are localised in intron 19 of Ia-2 and Ia-2ß, respectively. In rodents, only miR-153-2 is conserved. We demonstrated that expression of miR-153-2 and Ia-2ß in rodents is partially co-regulated as demonstrated by a strong reduction of miR-153 expression levels in Ia-2ß knockout and Ia-2/Ia-2ß double knockout mice. miR-153 levels were unaffected in Ia-2 knockout mice. In addition, glucose stimulation, which increases Ia-2 and Ia-2ß expression, also significantly increased expression of miR-153. Several predicted targets of miR-153 were reduced after glucose stimulation in vitro, correlating with the increase in miR-153 levels. CONCLUSIONS/INTERPRETATION: This study suggests the involvement of miR-153, IA-2ß and miR-153 target genes in a regulatory network, which is potentially relevant to insulin and neurotransmitter release.

Reduction of aflatoxin B1 in stored peanuts (Arachis hypogaea L.) using Saccharomyces cerevisiae.

Aflatoxin B(1) is a toxigenic and carcinogenic compound produced by Aspergillus flavus and Aspergillus parasiticus. To inhibit aflatoxin contamination of peanuts, seeds of two peanut breeds, IAC Caiapó and IAC Runner 886, were inoculated with A. parasiticus (1.0 × 10(6) spores per ml) and the yeast Saccharomyces cerevisiae (3.2 × 10(7) cells per ml) and incubated at 25°C for 7 and 15 days. Two experiments were conducted for each incubation period separately. The treatments were completely randomized, with three replications per treatment. Treatments included the two cultivars and three types of inoculation (pathogen alone, yeast and pathogen, and yeast 3 h before pathogen). Aflatoxin B(1) was quantified with a densitometer at 366 nm after thin layer chromatography. Aflatoxin B(1) contamination in peanuts was reduced after the addition of S. cerevisiae. The concentration of aflatoxin B(1) decreased by 74.4 and 55.9% after 7 and 15 days, respectively. The greatest aflatoxin reduction was observed when S. cerevisiae was inoculated 3 h before the pathogen in IAC Caiapó seeds and incubated for 7 days at 25°C. The use of S. cerevisiae is a promising strategy for biological control of aflatoxin contamination in peanuts.

Stable expression of recombinant human alpha3/4 fucosyltransferase III in Spodoptera frugiperda Sf9 cells.

Human alpha3/4 fucosyltransferase III (FT3; EC 2.4.1.65) synthesizes fucosylated glycoconjugates, namely the Lewis (Le) determinants. FT3 is detected in milk, gastric mucosa, kidney and other organs, but is found in very low amounts in these native tissues. In this work, we describe the expression of a soluble secretory form of FT3 (SFT3) in Spodoptera frugiperda (Sf9) insect cells using a non-lytic vector system. The coding sequence was cloned into the expression vector pIB/V5-His-TOPO which contains the transcriptional control of the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 (OpIE2) promoter. Transfected cells were selected using blasticidin-HCl. It was observed that the secreted activity SFT3 increased until the sixth day of culture when it reached the value 1.9 mU x 10(-6) cells and 13.4 mg/l, whereas only 5% of activity was retained inside the cells. Western blot analysis of secreted and intracellularly retained SFT3 had a similar variation. Comparison of the stable with the lytic baculovirus expression system showed that the former yielded approx. 13-fold more active SFT3, which was possibly due to a lower accumulation of intracellular SFT3.

Expression and characterization of recombinant human alpha-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system.

The human alpha-3/4-fucosyltransferase III (Fuc-TIII) participates in the synthesis of Lewis determinants. The enzyme from human sources is scarce and heterogeneous. In this paper we describe the expression of a secreted form of Fuc-TIII (SFT3) in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn), using the baculovirus expression system. The Sf9 cells secreted approx. 0.4 unit/l (1 mg/l) of the enzyme. The Tn cells secreted approx. 3-fold this amount. A large proportion of active protein was accumulated in the two cell lines (50 and 75% respectively for Sf9 and Tn cells, on the fourth day after infection) indicating a possible limitation not only of the folding machinery, but also a saturation of the secretory pathway. SFT3 was purified by cation-exchange chromatography followed by affinity chromatography. The enzyme from the Tn cell line had a lower global charge, possibly due to post-translational modifications, such as phosphorylation or sulphation. The two glycosylation sites from SFT3 were occupied. SFT3 secreted by Sf9 cells was completely deglycosylated by peptide-N-glycanase F, whereas 50% of SFT3 secreted by Tn cells was resistant to deglycosylation by this enzyme. The apparent kinetic parameters determined with the type I acceptor were k(cat)=0.4 s(-1) and K(m)=0.87 mM for the SFT3 secreted by Tn cells, and k(cat)=0.09 s(-1) and K(m)=0.76 mM for the SFT3 secreted by Sf9 cells, indicating that the enzymes had substrate affinities within the same order of magnitude as their mammalian counterpart. Furthermore, SFT3 secreted by either cell type showed a clear preference for type 1 carbohydrate acceptors, similarly to human Fuc-TIII.