Cell cycle alterations in the mussel Mytilus galloprovincialis hemocytes caused by environmental contamination

Environmental contamination includes a mixture of organic substances that can have detrimental effects on marine organisms and should be evaluated in the quality and risk assessment of investigated marine areas. Marine areas selected for this study are a protected area, a mariculture area, a shipyard and an industrial area. Based on the toxicity of the organic seawater extracts these areas were classified as an undisturbed reference area (S1), an area with the low anthropogenic impact (S2), a potentially endangered area (S3) and an area with high anthropogenic impact (S4) respectively. The organic mixtures present in seawater samples collected at the above defined areas were tested for the induction of DNA damage and cell cycle alterations in the mussel Mytilus galloprovincialis hemocytes. Flow cytometric analyses were performed to detect changes in hemocytes DNA content distribution throughout the cell cycle. Organic seawater extracts from sampling sites S2, S3 and S4 induced an increase in the coefficient of variation of the G0/G1 peak and an increase in the number of cells in the G2/M phase reflecting the extent of DNA damage and G2/M arrest, respectively. The G2/M arrest in mussel hemocytes was concentration-dependent upon injection with organic seawater extracts from the S3 site and time dependant for S2, S3 and S4 sampling sites. The time dependence of the induction of the G2/M arrest showed a characteristic pattern for each site due to the different quantitative and qualitative composition of the organic seawater extracts. The G2/M arrest was reversible 24 or 72 hours after treatment with organic seawater extracts from S2 or S3, and S4 sites, respectively. This reversibility was time- and site-specific indicating that such DNA damage is repairable to a certain degree according to the organic seawater extract composition. Thus, the hemocytes cell cycle alterations in the mussel Mytilus galloprovincialis caused by organic seawater extracts reliably reflect the extent of organic contamination effects for selected marine areas

Physiological levels of Pik3ca(H1047R) mutation in the mouse mammary gland results in ductal hyperplasia and formation of ER alpha-positive tumors

PIK3CA, the gene coding for the p110α subunit of phosphoinositide 3-kinase, is frequently mutated in a variety of human tumors including breast cancers. To better understand the role of mutant PIK3CA in the initiation and/or progression of breast cancer, we have generated mice with a conditional knock-in of the common activating mutation, Pik3ca(H1047R), into one allele of the endogenous gene in the mammary gland. These mice developed a ductal anaplasia and hyperplasia by 6 weeks of age characterized by multi-layering of the epithelial lining of the mammary ducts and expansion of the luminal progenitor (Lin(−); CD29(lo); CD24(+); CD61(+)) cell population. The Pik3ca(H1047R) expressing mice eventually develop mammary tumors with 100% penetrance but with a long latency (>12 months). This is significantly longer than has been reported for transgenic models where expression of the mutant Pik3ca is driven by an exogenous promoter. Histological analysis of the tumors formed revealed predominantly ERα-positive fibroadenomas, carcinosarcomas and sarcomas. In vitro induction of Pik3ca(H1047R) in immortalized mammary epithelial cells also resulted in tumor formation when injected into the mammary fat pad of immunodeficient recipient mice. This novel model, which reproduces the scenario of a heterozygous somatic mutation occurring in the endogenous PIK3CA gene, will thus be a valuable tool for investigating the role of Pik3ca(H1047R) mutation in mammary tumorigenesis both in vivo and in vitro

The Type Iα Inositol Polyphosphate 4-Phosphatase Generates and Terminates Phosphoinositide 3-Kinase Signals on Endosomes and the Plasma Membrane

Endosomal trafficking is regulated by the recruitment of effector proteins to phosphatidylinositol 3-phosphate [PtdIns(3)P] on early endosomes. At the plasma membrane, phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P(2)] binds the pleckstrin homology (PH) domain-containing proteins Akt and TAPP1. Type Iα inositol polyphosphate 4-phosphatase (4-phosphatase) dephosphorylates PtdIns(3,4)P(2), forming PtdIns(3)P, but its subcellular localization is unknown. We report here in quiescent cells, the 4-phosphatase colocalized with early and recycling endosomes. On growth factor stimulation, 4-phosphatase endosomal localization persisted, but in addition the 4-phosphatase localized at the plasma membrane. Overexpression of the 4-phosphatase in serum-stimulated cells increased cellular PtdIns(3)P levels and prevented wortmannin-induced endosomal dilatation. Furthermore, mouse embryonic fibroblasts from homozygous Weeble mice, which have a mutation in the type I 4-phosphatase, exhibited dilated early endosomes. 4-Phosphatase translocation to the plasma membrane upon growth factor stimulation inhibited the recruitment of the TAPP1 PH domain. The 4-phosphatase contains C2 domains, which bound PtdIns(3,4)P(2), and C2-domain-deletion mutants lost PtdIns(3,4)P(2) 4-phosphatase activity, did not localize to endosomes or inhibit TAPP1 PH domain membrane recruitment. The 4-phosphatase therefore both generates and terminates phosphoinositide 3-kinase signals at distinct subcellular locations

The Inositol Polyphosphate 5-Phosphatase, PIPP, Is a Novel Regulator of Phosphoinositide 3-Kinase-dependent Neurite Elongation

The spatial activation of phosphoinositide 3-kinase (PI3-kinase) signaling at the axon growth cone generates phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P(3)), which localizes and facilitates Akt activation and stimulates GSK-3β inactivation, promoting microtubule polymerization and axon elongation. However, the molecular mechanisms that govern the spatial down-regulation of PtdIns(3,4,5)P(3) signaling at the growth cone remain undetermined. The inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P(2)) and/or PtdIns(3,4,5)P(3). We demonstrate here that PIPP, an uncharacterized 5-phosphatase, hydrolyzes PtdIns(3,4,5)P(3) forming PtdIns(3,4)P(2), decreasing Ser473-Akt phosphorylation. PIPP is expressed in PC12 cells, localizing to the plasma membrane of undifferentiated cells and the neurite shaft and growth cone of NGF-differentiated neurites. Overexpression of wild-type, but not catalytically inactive PIPP, in PC12 cells inhibited neurite elongation. Targeted depletion of PIPP using RNA interference (RNAi) resulted in enhanced neurite differentiation, associated with neurite hyperelongation. Inhibition of PI3-kinase activity prevented neurite hyperelongation in PIPP-deficient cells. PIPP targeted-depletion resulted in increased phospho-Ser473-Akt and phospho-Ser9-GSK-3β, specifically at the neurite growth cone, and accumulation of PtdIns(3,4,5)P(3) at this site, associated with enhanced microtubule polymerization in the neurite shaft. PIPP therefore inhibits PI3-kinase-dependent neurite elongation in PC12 cells, via regulation of the spatial distribution of phospho-Ser473-Akt and phospho-Ser9-GSK-3β signaling

Immortalized mammary epithelial and mammary tumor cells expressing <i>Pik3ca</i>^H1047R mutation form tumors when injected into the mammary fat pad of SCID mice.

<p>Immortalized mammary epithelial and mammary tumor cells expressing <i>Pik3ca</i>^H1047R mutation form tumors when injected into the mammary fat pad of SCID mice.</p

Expression of <i>Pik3ca</i>^H1047R in developing mammary gland induces ductal hyperplasia.

<p>(A, B) Formalin-fixed paraffin-embedded sections of mammary glands from control and mutant (<i>Pik3ca</i>^H1047R–expressing) mice were stained with hematoxylin and eosin (H&amp;E). Shown are representative sections showing the presence of a multilayered epithelium (arrow) in the ducts of <i>Pik3ca</i>^H1047R –expressing mammary gland from 6 week old mice (A) and regions of hyperplastic epithelium (arrow) in the <i>Pik3ca</i>^H1047R expressing mammary gland of 12 week old mice (B). Sections from control and mutant mammary glands are pictured at same magnification (scale bar 50 µm). (C, D) Representative sections were also stained for K5 (green), K8 (red) and E-cadherin (pink) using immunofluorescence. DAPI (blue) was used to stain nuclei. Note that, in addition to staining the basal layer of the epithelium, K5 also stains anaplastic cells (arrows) in the terminal end buds (TEB) and ducts of the <i>Pik3ca</i>^H1047R –expressing mammary gland of 6 week (C) or 12 week mice (D). All sections were imaged at the same magnification using appropriate excitation filters. Scale bar, 50 µm.</p

Expression of <i>Pik3ca</i>^H1047R induces pre-neoplastic lesions and tumorigenesis in aged mice.

<p>(A). Representative whole mounts of control (MMTV-Cre alone) and mutant (<i>Pik3ca</i>^H1047R:MMTV-Cre) mammary glands from 13 month old nulliparous (i) and biparous (ii) mice. Arrows indicate foci of sclerosis adenosis. Scale bars, 500 µm. (B) Representative sections from control (MMTV-Cre alone) and mutant (<i>Pik3ca</i>^H1047R:MMTV-Cre) mammary glands from 13 month old biparous mice were stained with H&amp;E or anti-pS6 antibody. Scale bars, 100 µm. (C) Representative sclerosing adenosis lesion from <i>Pik3ca</i>^H1047R-expressing mammary glands from aged (&gt;12-month) old mice were stained with H&amp;E, and with anti-pS6 and anti-ERα antibodies. Scale bars, 100 µm. (D) Kaplan-Meier survival curves for aged control and mutant (<i>Pik3ca</i>^H1047R:MMTV-Cre) mice. Shown are nulliparous (green broken line, n = 10) and biparous (blue solid line, n = 11) and respective control mice (nulliparous, gray line, n = 30; biparous, orange line, n = 10). ** The median tumor free survival in biparous mice was significantly (p&lt;0.005) reduced compared to nulliparous mice. (E) Protein lysates from control and mutant mammary glands and tumor tissue from nulliparous aged (&gt;12 month) mice were separated by SDS-PAGE and immunoblotted for analysis of PI3K pathway activation using anti-pAKT, anti-pGSK3β, and anti-pS6, as well as anti-tubulin (loading control). Immunoreactive proteins were detected with horse-radish peroxidase conjugated secondary antibodies and visualized with enhanced chemiluminescence. Samples are identified by their corresponding mouse ID. MG, mammary gland.</p

<i>Pik3ca</i>^H1047R inducible knock-in mouse model.

<p>(A) A schema outlining the generation of mice expressing <i>Pik3ca</i>^H1047R in the mammary gland. Mice heterozygous for the latent mutant allele (<i>Pik3ca</i>^wt/LatH1047R) were crossed with mice heterozygous for the transgenic MMTV–Cre allele (MMTV-Cre^−/+). Cre-mediated recombination results in the excision of everything between the LoxP sites (triangles) allowing the mutant version of exon 20 (dark rectangle) to replace the wild type exon 20, thus generating mice that express the <i>Pik3ca</i>^H1047R mutation in mammary epithelial cells. (B) Cre-mediated knock-in of the mutant exon 20 was confirmed by sequencing RNA extracted from the mammary fat pad (MFP) from heterozygous <i>Pik3ca</i>^H1047R:MMTV-Cre (HET) and <i>Pik3ca^wt</i> (WT) mice. RNA extracted from WT and HET MFP was reverse transcribed into cDNA and sequenced from exon 19 into exon 20 (exon 19-specific primer: 5′-CAAGAGTACACCAAGACCAGAGAGTT-3′) and confirmed by sequencing in the reverse direction from exon 20 (5′- TCCAATCCATTTTTGTCGTCC-3′). Shown are the expected sequences (1, 2) and the actual sequence traces (3, 4) for cDNA from WT MFP (<i>Pik3ca</i> control) (1, 3) and HET-MFP (<i>Pik3ca</i> mutant) (2, 4). Note that the representative cDNA sequence from the HET-MFP is heterozygous at the sites of silent base changes engineered into the mutant exon 20 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036924#pone.0036924-Kinross1" target="_blank">[28]</a> confirming successful knock-in of the <i>Pik3ca</i>^H1047R mutation in HET MFP. (C) The section of the sequencing trace from (B) showing the region spanning position 1047 showing the bases changes responsible for the His→Arg substitution (*). (D) Whole mounts (i) and 10 µm sections (ii) of mammary tissue from (ROSA)26Sor control mice (left) and (ROSA)26Sor:MMTV-Cre mice (right) were stained using X-Gal and counterstained with nuclear fast red. MMTV-Cre-mediated recombination, where present, is identified by blue staining. Scale bar, 50 µm.</p