[PubMed] [Google Scholar]Heby O, Marton LJ, Zardi L, Russell DH, Baserga R

[PubMed] [Google Scholar]Heby O, Marton LJ, Zardi L, Russell DH, Baserga R. polyamines. In addition, intercellular relationships mediated by polyamines can coordinate the translational response to oxidative stress through the formation of stress granules. Some putative in vivo consequences of polyamine-mediated intercellular interactions are also discussed regarding cancer invasiveness and tissue regeneration. INTRODUCTION Natural polyamines, that is, divalent putrescine, trivalent spermidine, and tetravalent spermine, are small cationic organic molecules present in the millimolar range in mammalian cells and are necessary for cell proliferation (Tabor and Tabor, 1984 ; Thomas and Thomas, 2001 ), in line with their higher concentration in malignancy cells compared with normal cells (Heby, 1981 ; Pegg, 2009 ). Polyamines, as major counterions of negatively charged nucleic acids (RNA and DNA to a lesser degree; Watanabe 0.05 by test. n.s., not significant. Polyamines and space junction communications The overall effect of polyamines within the epithelial cell cytoskeleton raised interesting issues regarding the consequences for intercellular relationships. After DFMO/APCHA treatment, fewer space junction plaques were observed compared with control cells (Number 2A). We may then assume that polyamines favor the formation of space junction plaques. However, in apparent contradiction with this assumption, agmatine treatment, although leading also to polyamine depletion, induced the formation of larger space junction plaques in the cell/cell interface than in control cells (Number 2A). This is WJ460 further confirmed by Western blotting, where agmatine but not DFMO/APCHA treatment indeed led to an overexpression of Cx43, this pattern becoming reversed by putrescine supplementation (Number 2B). A closer look at the space junctions created in the presence of agmatine exposed particularly solid plaques, raising doubts about whether such space junction plaques were functional. To answer this question, we performed scrape-loading assays (el-Fouly plaques. * 0.05 by test on the null hypothesis that the two human population means are equal. n.s., not significant. (B) Western blotting indicates that agmatine improved the expression level of Cx43. DFMO/APCHA treatment did not switch the Cx43 manifestation level. GAPDH was used as a loading control. (C) The transfer of Lucifer yellow, a small hydrophilic dye, from cell to cell was observed after indicated treatments using the scrape-loading process. Long-range dye transfer via space junction communications was recognized in both agmatine- and DFMO/APCHA-treated cells, even though slightly reduced after the second option treatment. As control, 50 M of the space junction inhibitor oleamide for 2 h strongly inhibited space junction communication and, in this case, only cells in the vicinity of the wound appeared bright (arrow). Level pub, 80 m. Statistical analysis of dye diffusion through space junctions was performed as explained in Supplemental Number S4. Results are mean SD attained on five different areas. ** 0.005; * 0.05 by test. Impaired microtubule dynamics and maintenance of difference junctions in polyamine-depleted cells To research whether polyamines regulate difference junction company through their actions on microtubule dynamics, we initial analyzed whether disruption of microtubules by itself (using nocodazole) may lead to mislocated difference junctions in charge or DFMO/APCHA-treated cells. We discovered after nocodazole treatment (5 M going back 24 h) the current presence of abundant connexin-rich vesicles in the cytosol of both cells, in contract with impaired transportation (Supplemental Amount S5A). In both depleted and polyamine-supplemented cells, after Taxol treatment (100 nM going back 24 h), which abolishes microtubule dynamics, the current presence of connexin was noticed on the cell/cell user interface, but large difference junction plaques tended to vanish as though microtubule dynamics allowed the higher-order set up of difference junction protein. In agmatine-treated cells (Amount 2A), we hypothesized which the observed thick difference junction plaques may derive from an impaired microtubule-related transportation of connexins. To explore this simple idea, we disrupted difference junction plaques with the reversible inhibitor oleamide (50 M; Guan 0.05 by test. n.s., not really significant (find Supplemental Amount S5B for a good example displaying how this proportion was approximated). No difference within this proportion was noticed between agmatine-treated cells and putrescine-supplemented cells (check not really significant) prior to the treatment with oleamide. These outcomes prompted us to help expand investigate the function of polyamines over the elongation price of dynamical microtubule plus ends. Videomicroscopy of EB1 uncovered which the elongation price of microtubules and the amount of Rabbit polyclonal to FUS dynamical plus ends on the cell cortex had been considerably reduced due to polyamine depletion after both DFMO/APCHA and agmatine remedies (Amount 4, A and B, and Supplemental Movies S1CS3). Once again, polyamine-depleted cells could recover dynamical microtubules on the cell cortex after putrescine supplementation. An identical pattern was attained.Cancer Res. of polyamine-mediated intercellular interactions are discussed relating to cancer invasiveness and tissues regeneration also. INTRODUCTION Organic polyamines, that’s, divalent putrescine, trivalent spermidine, and tetravalent spermine, are little cationic organic substances within the millimolar range in mammalian cells and so are essential for cell proliferation (Tabor and Tabor, 1984 ; Thomas and Thomas, 2001 ), consistent with their higher focus in cancers cells weighed against regular cells (Heby, 1981 ; Pegg, 2009 ). Polyamines, as main counterions of adversely billed nucleic acids (RNA and DNA to a smaller level; Watanabe 0.05 by test. n.s., not really significant. Polyamines and difference junction communications The entire aftereffect of polyamines over the epithelial cell cytoskeleton elevated interesting problems with respect to the results for intercellular connections. After DFMO/APCHA treatment, fewer difference junction plaques had been observed weighed against control cells (Amount 2A). We might after that assume that polyamines favour the forming of difference junction plaques. Nevertheless, in obvious contradiction with this assumption, agmatine treatment, although leading also to polyamine depletion, induced the forming of bigger difference junction plaques on the cell/cell user interface than in charge cells (Amount 2A). That is additional verified by Traditional western blotting, where agmatine however, not DFMO/APCHA treatment certainly resulted in an overexpression of Cx43, this design getting reversed by putrescine supplementation (Amount 2B). A nearer go through the difference junctions produced in the current presence of agmatine uncovered particularly dense plaques, raising uncertainties about whether such difference junction plaques had been functional. To reply this issue, we performed scrape-loading assays (el-Fouly plaques. * 0.05 by test within the null hypothesis that both people means are equal. n.s., not really significant. (B) Traditional western blotting indicates that agmatine elevated the expression degree of Cx43. DFMO/APCHA treatment didn’t transformation the Cx43 appearance level. GAPDH was utilized as a launching control. (C) The transfer of Lucifer yellowish, a little hydrophilic dye, from cell to cell was noticed after indicated remedies using the scrape-loading method. Long-range dye transfer via difference junction marketing communications was discovered in both agmatine- and DFMO/APCHA-treated cells, despite the fact that slightly reduced following the last mentioned treatment. As control, 50 M from the difference junction inhibitor oleamide for 2 h highly inhibited difference junction conversation and, in cases like this, only cells on the vicinity from the wound made an appearance bright (arrow). Range club, 80 m. Statistical evaluation of dye diffusion through difference junctions was performed as described in Supplemental Physique S4. Results are mean SD obtained on five different areas. ** 0.005; * 0.05 by test. Impaired microtubule dynamics and maintenance of gap junctions in WJ460 polyamine-depleted cells To investigate whether polyamines regulate gap junction business through their action on microtubule dynamics, we first examined whether disruption of microtubules per se (using nocodazole) could lead to mislocated gap junctions in control or DFMO/APCHA-treated cells. We found after nocodazole treatment (5 M for the last 24 h) the presence of abundant connexin-rich vesicles in the cytosol of both cells, in agreement with impaired transport (Supplemental Physique S5A). In both polyamine-supplemented and depleted cells, after Taxol treatment (100 nM for the last 24 h), which abolishes microtubule dynamics, the presence of connexin was observed at the cell/cell interface, but large gap junction plaques tended to disappear as if microtubule dynamics allowed the higher-order assembly of gap junction proteins. WJ460 In agmatine-treated cells (Physique 2A), we hypothesized that this observed thick gap junction plaques may result from an impaired microtubule-related transport of connexins. To explore this idea, we disrupted gap junction plaques by the reversible inhibitor oleamide (50 M; Guan 0.05 by test. n.s., not significant (see Supplemental Physique S5B for an example showing how this ratio was estimated). No difference in this ratio was observed between agmatine-treated cells and putrescine-supplemented cells (test not significant) before the treatment with oleamide. These results prompted us to further investigate the role of polyamines around the elongation rate of dynamical microtubule plus ends. Videomicroscopy of EB1 revealed that this elongation rate of microtubules and the number.GFP emission was detected with a 63/1.4 numerical aperture objective. discussed regarding malignancy invasiveness and tissue regeneration. INTRODUCTION Natural polyamines, that is, divalent putrescine, trivalent spermidine, and tetravalent spermine, are small cationic organic molecules present in the millimolar range in mammalian cells and are necessary for cell proliferation (Tabor and Tabor, 1984 ; Thomas and Thomas, 2001 ), in line with their higher concentration in cancer cells compared with normal cells (Heby, 1981 ; Pegg, 2009 ). Polyamines, as major counterions of negatively charged nucleic acids (RNA and DNA to a lesser extent; Watanabe 0.05 by test. n.s., not significant. Polyamines and gap junction communications The overall effect of polyamines around the epithelial cell cytoskeleton raised interesting issues regarding the consequences for intercellular interactions. After DFMO/APCHA treatment, fewer gap junction plaques were observed compared with control cells (Physique 2A). We may then assume that polyamines favor the formation of gap junction plaques. However, in apparent contradiction with this assumption, agmatine treatment, although leading also to polyamine depletion, induced the formation of larger gap junction plaques at the cell/cell interface than in control cells (Physique 2A). This is further confirmed by Western blotting, where agmatine but not DFMO/APCHA treatment indeed led to an overexpression of Cx43, this pattern being reversed by putrescine supplementation (Physique 2B). A closer look at the gap junctions formed in the presence of agmatine revealed particularly thick plaques, raising doubts about whether such gap junction plaques were functional. To answer this question, we performed scrape-loading assays (el-Fouly plaques. * 0.05 by test over the null hypothesis that the two populace means are equal. n.s., not significant. (B) Western blotting indicates that agmatine increased the expression level of Cx43. DFMO/APCHA treatment did not change the Cx43 expression level. GAPDH was used as a loading control. (C) The transfer of Lucifer yellow, a small hydrophilic dye, from cell to cell was observed after indicated treatments using the scrape-loading procedure. Long-range dye transfer via gap junction communications was detected in both agmatine- and DFMO/APCHA-treated cells, even though slightly reduced after the latter treatment. As control, 50 M of the gap junction inhibitor oleamide for 2 h strongly inhibited gap junction communication and, in this case, only cells at the vicinity of the wound appeared bright (arrow). Scale bar, 80 m. Statistical analysis of dye diffusion through gap junctions was performed as described in Supplemental Figure S4. Results are mean SD obtained on five different areas. ** 0.005; * 0.05 by test. Impaired microtubule dynamics and maintenance of gap junctions in polyamine-depleted cells To investigate whether polyamines regulate gap junction organization through their action on microtubule dynamics, we first examined whether disruption of microtubules per se (using nocodazole) could lead to mislocated gap junctions in control or DFMO/APCHA-treated cells. We found after nocodazole treatment (5 M for the last 24 h) the presence of abundant connexin-rich vesicles in the cytosol of both cells, in agreement with impaired transport (Supplemental Figure S5A). In both polyamine-supplemented and depleted cells, after Taxol treatment (100 nM for the last 24 h), which abolishes microtubule dynamics, the presence of connexin was observed at the cell/cell interface, but large gap junction plaques tended to disappear as if microtubule dynamics allowed the higher-order assembly of gap junction proteins. In agmatine-treated cells (Figure 2A), we hypothesized that the observed thick gap junction plaques may result from an impaired microtubule-related transport of connexins. To explore this idea, we disrupted gap junction plaques by the reversible inhibitor oleamide (50 M; Guan 0.05 by test. n.s., not significant (see Supplemental Figure S5B for an example showing how this ratio was estimated). No difference in this ratio was observed between agmatine-treated cells and putrescine-supplemented cells (test not significant) before the treatment with oleamide. These results prompted us to further investigate the role of polyamines on the elongation rate of dynamical.See Supplemental Videos S1CS3. Supplementary Material Supplemental Materials: Click here to view. Acknowledgments This work was supported by funds from the Institut National de la Sant et de la Recherche Mdicale, the Genopole Evry, and an ANR grant (NAFLD-Citrulline). Abbreviation used: Puputrescine Footnotes This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E12-10-0729) on March 20, 2013. REFERENCES Arimoto K, Fukuda H, Imajoh-Ohmi S, Saito H, Takekawa M. oxidative stress through the formation of stress granules. Some putative in vivo consequences of polyamine-mediated intercellular interactions are also discussed regarding cancer invasiveness and tissue regeneration. INTRODUCTION Natural polyamines, that is, divalent putrescine, trivalent spermidine, and tetravalent spermine, are small cationic organic molecules present in the millimolar range in mammalian cells and are necessary for cell proliferation (Tabor and Tabor, 1984 ; Thomas and Thomas, 2001 ), in line with their higher concentration in cancer cells compared with normal cells (Heby, 1981 ; Pegg, 2009 ). Polyamines, as major counterions of negatively charged nucleic acids (RNA and DNA to a lesser extent; Watanabe 0.05 by test. n.s., not significant. Polyamines and gap junction communications The overall effect of polyamines on the epithelial cell cytoskeleton raised interesting issues regarding the consequences for intercellular relationships. After DFMO/APCHA treatment, fewer space junction plaques were observed compared with control cells (Number 2A). We may then assume that polyamines favor the formation of space junction plaques. However, in apparent contradiction with this assumption, agmatine treatment, although leading also to polyamine depletion, induced the formation of larger space junction plaques in the cell/cell interface than in control cells (Number 2A). This is further confirmed by Western blotting, where agmatine but not DFMO/APCHA treatment indeed led to an overexpression of Cx43, this pattern becoming reversed by putrescine supplementation (Number 2B). A closer look at the space junctions created in the presence of agmatine exposed particularly solid plaques, raising doubts about whether such space junction plaques were functional. To solution this query, we performed scrape-loading assays (el-Fouly plaques. * 0.05 by test on the null hypothesis that the two human population means are equal. n.s., not significant. (B) Western blotting indicates that agmatine improved the expression level of Cx43. DFMO/APCHA treatment did not switch the Cx43 manifestation level. GAPDH was used as a loading control. (C) The transfer of Lucifer yellow, a small hydrophilic dye, from cell to cell was observed after indicated treatments using the scrape-loading process. Long-range dye transfer via space junction communications was recognized in both agmatine- and DFMO/APCHA-treated cells, even though slightly reduced after the second option treatment. As control, 50 M of the space junction inhibitor oleamide for 2 h strongly inhibited space junction communication and, in this case, only cells in the vicinity of the wound appeared bright (arrow). Level pub, 80 m. Statistical analysis of dye diffusion through space junctions was performed as explained in Supplemental Number S4. Results are mean SD acquired on five different areas. ** 0.005; * 0.05 by test. Impaired microtubule dynamics and maintenance of space junctions in polyamine-depleted cells To investigate whether polyamines regulate space junction corporation through their action on microtubule dynamics, we 1st examined whether disruption of microtubules per se (using nocodazole) could lead to mislocated space junctions in control or DFMO/APCHA-treated cells. We found after nocodazole treatment (5 M for the last 24 h) the presence of abundant connexin-rich vesicles in the cytosol of both cells, in agreement with impaired transport (Supplemental Number S5A). In both polyamine-supplemented and depleted cells, after Taxol treatment (100 nM for the last 24 h), which abolishes microtubule dynamics, the presence of connexin was observed in the cell/cell interface, but large space junction plaques tended to WJ460 disappear as if microtubule dynamics allowed the higher-order assembly of space junction proteins. In agmatine-treated cells (Number 2A), we hypothesized the observed thick space junction plaques may result from an impaired microtubule-related transport of connexins. To explore this idea, we disrupted space junction plaques from the reversible inhibitor.Proc Natl Acad Sci USA. organic molecules present in the millimolar range in mammalian cells and are necessary for cell proliferation (Tabor and Tabor, 1984 ; Thomas and Thomas, 2001 ), in line with their higher concentration in malignancy cells compared with normal cells (Heby, 1981 ; Pegg, 2009 ). Polyamines, as major counterions of negatively charged nucleic acids (RNA and DNA to a lesser degree; Watanabe 0.05 by test. n.s., not significant. Polyamines and space junction communications The overall effect of polyamines within the epithelial cell cytoskeleton raised interesting issues regarding the consequences for intercellular relationships. After DFMO/APCHA treatment, fewer space junction plaques were observed compared with control cells (Number 2A). We may then assume that polyamines favor the formation of space junction plaques. However, in apparent contradiction with this assumption, agmatine treatment, although leading also to polyamine depletion, induced the formation of larger space junction plaques in the cell/cell interface than in control cells (Number 2A). This is further confirmed by Western blotting, where agmatine but not DFMO/APCHA treatment indeed led to an overexpression of Cx43, this pattern becoming reversed by putrescine supplementation (Number 2B). A closer look at the difference junctions produced in the current presence of agmatine uncovered particularly dense WJ460 plaques, raising uncertainties about whether such difference junction plaques had been functional. To reply this issue, we performed scrape-loading assays (el-Fouly plaques. * 0.05 by test within the null hypothesis that both inhabitants means are equal. n.s., not really significant. (B) Traditional western blotting indicates that agmatine elevated the expression degree of Cx43. DFMO/APCHA treatment didn’t transformation the Cx43 appearance level. GAPDH was utilized as a launching control. (C) The transfer of Lucifer yellowish, a little hydrophilic dye, from cell to cell was noticed after indicated remedies using the scrape-loading method. Long-range dye transfer via difference junction marketing communications was discovered in both agmatine- and DFMO/APCHA-treated cells, despite the fact that slightly reduced following the last mentioned treatment. As control, 50 M from the difference junction inhibitor oleamide for 2 h highly inhibited difference junction conversation and, in cases like this, only cells on the vicinity from the wound made an appearance bright (arrow). Range club, 80 m. Statistical evaluation of dye diffusion through difference junctions was performed as defined in Supplemental Body S4. Email address details are mean SD attained on five different areas. ** 0.005; * 0.05 by test. Impaired microtubule dynamics and maintenance of difference junctions in polyamine-depleted cells To research whether polyamines regulate difference junction firm through their actions on microtubule dynamics, we initial analyzed whether disruption of microtubules by itself (using nocodazole) may lead to mislocated difference junctions in charge or DFMO/APCHA-treated cells. We discovered after nocodazole treatment (5 M going back 24 h) the current presence of abundant connexin-rich vesicles in the cytosol of both cells, in contract with impaired transportation (Supplemental Body S5A). In both polyamine-supplemented and depleted cells, after Taxol treatment (100 nM going back 24 h), which abolishes microtubule dynamics, the current presence of connexin was noticed on the cell/cell user interface, but large difference junction plaques tended to vanish as though microtubule dynamics allowed the higher-order set up of difference junction protein. In agmatine-treated cells (Body 2A), we hypothesized the fact that observed thick difference junction plaques may derive from an impaired microtubule-related transportation of connexins. To explore this notion, we disrupted difference junction plaques with the reversible inhibitor oleamide (50 M; Guan 0.05 by test. n.s., not really significant (find Supplemental Figure.