YB-1 oncoprotein in cancer and drug resistance

Y-box-binding protein 1 (YB-1) is a biomarker that is predictive of poor prognosis in cancer. Various molecular functions of YB-1 in cancer have been proposed, including the transcriptional regulation of gene expression. YB-1 also binds to RNA transcripts to influence gene expression. In the present study, the status of YB-1 as a biomarker was confirmed by immunohistochemistry using two antibodies against YB-1. However, the prognostic sensitivity of these two antibodies differed. The observed difference in antibody affinity was most likely due to the tertiary structure or protein-protein interactions (PPI) associated with various functions of YB-1 in situ. To gain further insights into the molecular functions and potential mechanisms of YB-1 in cancer biology the state of phosphorylation of YB-1 and the PPI were investigated in the cytoplasm and nucleus of two cancer cell lines. The YB-1 from the cytoplasm and nucleus of the cell lines was extensively phosphorylated. These experiments identified >250 proteins. These binding partners confirmed the multifunctionality of YB-1 as the proteins that co-purify with YB-1 participate in glycolysis, RNA splicing, RNA stabilization, translation, mitochondrial localisation, and chromosomal association. These data suggest that the bulk of YB-1 function may be explained by non-transcriptional mechanisms. Mechanisms of drug resistance were also investigated. Depleting YB-1, using siRNA duplexes, reduced MDA-MB231 cell growth and increased cell death. The loss of YB-1 sensitised MDA-MB231 cells to cisplatin exposure by increasing cell death. Cisplatin exposure altered the distribution of YB-1 protein to perinuclear spots and to foci in the nucleus of many cells. The molecular basis of YB-1 mediated cisplatin resistance was analysed by examining the alterations of YB-1 PPI during cisplatin exposure using co-immunoprecipitation of YB-1 binding partners and mass spectrometry-based protein identification. Quantitative analyses of the co-immunoprecipitated proteins from MDA-MB231 cells indicated that a subset of the proteins, such as TRIM28 and FAM120A, increased markedly after 48 and 96 hours of cisplatin exposure. The chromosomal proteins that interacted with YB-1 were disproportionately affected by cisplatin exposure. The importance of FAM120A, TRIM28, and C1QBP, three YB-1 binding partners identified here, during cisplatin exposure was studied. The subcellular distribution of FAM120A was most similar that of YB-1 in MDA-MB231 cells. Depleting YB-1 or FAM120A, but not TRIM28 or C1QBP, sensitised MDA-MB231 cells to cisplatin exposure. Depleting YB-1 alongside either FAM120A or C1QBP partially restored the growth of MDA-MB231 cells. YB-1 does not appear to participate in the repair of double-strand DNA breaks during cisplatin exposure as depleting YB-1 had no effect on the number of γH2AX foci that formed during cisplatin exposure. This is the first report that integrates findings of protein-binding partners, state of phosphorylation, and subcellular localisation of endogenous YB-1 to understand the complex functions of YB-1. These results confirm the importance of RNA binding to the molecular function of YB-1. The interaction of YB-1 with FAM120A, a novel finding, increases during cisplatin exposure and both proteins together, via an unknown molecular pathway, confer cisplatin resistance to breast cancer cells

Site Occupancy and Lattice Parameters in Sigma-Phase Co-Cr alloys

Neutron diffraction technique was used to study distribution of Co and Cr atoms over different lattice sites as well as lattice paramaters in sigma-phase Co100-xCrx compounds with x = 57.0, 62.7 and 65.8. From the diffractograms recorded in the temperature range of 4.2 - 300 K it was found that all five sites A, B, C, D and E are populated by both kinds of atoms. Sites A and D are predominantly occupied by Co atoms while sites B, C and E by Cr atoms. The unit cell parameters a and c, hence the unit cell volume, increase with x, the increase being characteristic of the lattice paramater and temperature. Both a and c show a non-linear increase with temperature.Comment: 5 figure

Dephosphorylation of YB-1 is Required for Nuclear Localisation During G2 Phase of the Cell Cycle

Elevated levels of nuclear Y-box binding protein 1 (YB-1) are linked to poor prognosis in cancer. It has been proposed that entry into the nucleus requires specific proteasomal cleavage. However, evidence for cleavage is contradictory and high YB-1 levels are prognostic regardless of cellular location. Here, using confocal microscopy and mass spectrometry, we find no evidence of specific proteolytic cleavage. Doxorubicin treatment, and the resultant G2 arrest, leads to a significant increase in the number of cells where YB-1 is not found in the cytoplasm, suggesting that its cellular localisation is variable during the cell cycle. Live cell imaging reveals that the location of YB-1 is linked to progression through the cell cycle. Primarily perinuclear during G1 and S phases, YB-1 enters the nucleus as cells transition through late G2/M and exits at the completion of mitosis. Atomistic modelling and molecular dynamics simulations show that dephosphorylation of YB-1 at serine residues 102, 165 and 176 increases the accessibility of the nuclear localisation signal (NLS). We propose that this conformational change facilitates nuclear entry during late G2/M. Thus, the phosphorylation status of YB-1 determines its cellular location

Critical Role for Cold Shock Protein YB-1 in Cytokinesis

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, β-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division

Critical Role for Cold Shock Protein YB-1 in Cytokinesis

High levels of the cold shock protein Y-box-binding protein-1, YB-1, are tightly correlated with increased cell proliferation and progression. However, the precise mechanism by which YB-1 regulates proliferation is unknown. Here, we found that YB-1 depletion in several cancer cell lines and in immortalized fibroblasts resulted in cytokinesis failure and consequent multinucleation. Rescue experiments indicated that YB-1 was required for completion of cytokinesis. Using confocal imaging we found that YB-1 was essential for orchestrating the spatio-temporal distribution of the microtubules, β-actin and the chromosome passenger complex (CPC) to define the cleavage plane. We show that phosphorylation at six serine residues was essential for cytokinesis, of which novel sites were identified using mass spectrometry. Using atomistic modelling we show how phosphorylation at multiple sites alters YB-1 conformation, allowing it to interact with protein partners. Our results establish phosphorylated YB-1 as a critical regulator of cytokinesis, defining precisely how YB-1 regulates cell division

YB-1 oncoprotein in cancer and drug resistance

Y-box-binding protein 1 (YB-1) is a biomarker that is predictive of poor prognosis in cancer. Various molecular functions of YB-1 in cancer have been proposed, including the transcriptional regulation of gene expression. YB-1 also binds to RNA transcripts to influence gene expression. In the present study, the status of YB-1 as a biomarker was confirmed by immunohistochemistry using two antibodies against YB-1. However, the prognostic sensitivity of these two antibodies differed. The observed difference in antibody affinity was most likely due to the tertiary structure or protein-protein interactions (PPI) associated with various functions of YB-1 in situ. To gain further insights into the molecular functions and potential mechanisms of YB-1 in cancer biology the state of phosphorylation of YB-1 and the PPI were investigated in the cytoplasm and nucleus of two cancer cell lines. The YB-1 from the cytoplasm and nucleus of the cell lines was extensively phosphorylated. These experiments identified >250 proteins. These binding partners confirmed the multifunctionality of YB-1 as the proteins that co-purify with YB-1 participate in glycolysis, RNA splicing, RNA stabilization, translation, mitochondrial localisation, and chromosomal association. These data suggest that the bulk of YB-1 function may be explained by non-transcriptional mechanisms. Mechanisms of drug resistance were also investigated. Depleting YB-1, using siRNA duplexes, reduced MDA-MB231 cell growth and increased cell death. The loss of YB-1 sensitised MDA-MB231 cells to cisplatin exposure by increasing cell death. Cisplatin exposure altered the distribution of YB-1 protein to perinuclear spots and to foci in the nucleus of many cells. The molecular basis of YB-1 mediated cisplatin resistance was analysed by examining the alterations of YB-1 PPI during cisplatin exposure using co-immunoprecipitation of YB-1 binding partners and mass spectrometry-based protein identification. Quantitative analyses of the co-immunoprecipitated proteins from MDA-MB231 cells indicated that a subset of the proteins, such as TRIM28 and FAM120A, increased markedly after 48 and 96 hours of cisplatin exposure. The chromosomal proteins that interacted with YB-1 were disproportionately affected by cisplatin exposure. The importance of FAM120A, TRIM28, and C1QBP, three YB-1 binding partners identified here, during cisplatin exposure was studied. The subcellular distribution of FAM120A was most similar that of YB-1 in MDA-MB231 cells. Depleting YB-1 or FAM120A, but not TRIM28 or C1QBP, sensitised MDA-MB231 cells to cisplatin exposure. Depleting YB-1 alongside either FAM120A or C1QBP partially restored the growth of MDA-MB231 cells. YB-1 does not appear to participate in the repair of double-strand DNA breaks during cisplatin exposure as depleting YB-1 had no effect on the number of γH2AX foci that formed during cisplatin exposure. This is the first report that integrates findings of protein-binding partners, state of phosphorylation, and subcellular localisation of endogenous YB-1 to understand the complex functions of YB-1. These results confirm the importance of RNA binding to the molecular function of YB-1. The interaction of YB-1 with FAM120A, a novel finding, increases during cisplatin exposure and both proteins together, via an unknown molecular pathway, confer cisplatin resistance to breast cancer cells

A comparison of factual/low context versus emotional/high context message appeals: persuading Chinese smokers to ring the Chinese quitline

This study does not support the notion developed from cross cultural advertising literature that members of a particular cultural group will prefer a particular message advertising appeal if it reflects values that are congruent to its culture. An experimental design is applied to examine whether emotional/ high context appeals (n=120) are more persuasive than factual/ low context appeals (n=124) among target groups from collectivist cultural backgrounds. Analysis of Chinese smokers’ pre-and-post intentions to contact the Chinese Quitline after viewing print advertisements determined that there was no difference between the two ad groups’ shift scores. The common use of direct translations of Australian mainstream campaigns for this culturally and linguistically diverse (CALD) target group could be viewed as an acceptable practice

Nano-sunscreens - a double-edged sword in protecting consumers from harm: viewing Australian regulatory policies through the lenses of the European Union

Nanotechnology has the potential to bring about revolutionary changes in manufacturing products, including sunscreens. However, a knowledge gap between benefits and detriments of engineered nano-materials used in sunscreens exists, which gives rise to safety concerns. This article is concerned with the protection of consumers without impairing the embellishment of this promising technology. It is widely argued that the harm associated with nano-sunscreens may only occur under certain conditions related mainly to users skin vulnerability, which can be avoided by informed and careful use of such a product. We thus recognize the need for fostering the growth of nanotech simultaneously with preventing potential harm. We revisit the Australian sunscreens regulatory policies, which embrace a wait and see approach, through the lens of regulatory policies in the European Union (EU) that are influenced by a precautionary principle. We highlight the importance of informing consumers about the sunscreen they are using and recommend that product labels should disclose the presence of nano-ingredients in line with the EU disclosure requirements. This will allow users to carefully apply the product in order to avoid any potential harm and to protect manufacturers from possible costly litigation in future. This can be achieved through a combined collaborative effort of regulators, supply chain entities, and end users

Nano-sunscreens - a double-edged sword in protecting consumers from harm: viewing Australian regulatory policies through the lenses of the European Union

Nanotechnology has the potential to bring about revolutionary changes in manufacturing products, including sunscreens. However, a knowledge gap between benefits and detriments of engineered nano-materials used in sunscreens exists, which gives rise to safety concerns. This article is concerned with the protection of consumers without impairing the embellishment of this promising technology. It is widely argued that the harm associated with nano-sunscreens may only occur under certain conditions related mainly to users skin vulnerability, which can be avoided by informed and careful use of such a product. We thus recognize the need for fostering the growth of nanotech simultaneously with preventing potential harm. We revisit the Australian sunscreens regulatory policies, which embrace a wait and see approach, through the lens of regulatory policies in the European Union (EU) that are influenced by a precautionary principle. We highlight the importance of informing consumers about the sunscreen they are using and recommend that product labels should disclose the presence of nano-ingredients in line with the EU disclosure requirements. This will allow users to carefully apply the product in order to avoid any potential harm and to protect manufacturers from possible costly litigation in future. This can be achieved through a combined collaborative effort of regulators, supply chain entities, and end users