Global gene expression analysis reveals a link between NDRG1 and vesicle transport

-N-myc downstream-regulated gene 1 (NDRG1) is induced by cellular stress such as hypoxia and DNA damage, and in humans, germ line mutations cause Charcot-Marie-Tooth disease. However, the cellular roles of NDRG1 are not fully understood. Previously, NDRG1 was shown to mediate doxorubicin resistance under hypoxia, suggesting a role for NDRG1 in cell survival under these conditions. We found decreased apoptosis in doxorubicin-treated cells expressing NDRG1 shRNAs under normoxia, demonstrating a requirement for NDRG1 in apoptosis in breast epithelial cells under normal oxygen pressure. Also, different cellular stress regimens, such as hypoxia and doxorubicin treatment, induced NDRG1 through different stress signalling pathways. We further compared expression profiles in human breast epithelial cells ectopically over-expressing NDRG1 with cells expressing NDRG1 shRNAs in order to identify biological pathways where NDRG1 is involved. The results suggest that NDRG1 may have roles connected to vesicle transport

Knock down of NDRG1 expression by shRNAs.

<p>Western blots detect reduced NDRG1 expression in the breast cell lines ME16C2 and SUM102 expressing the shRNAs NDRG1si4 (si4) and NDRG1si7 (si7), transduced in parallel experiments (A and B) compared to control cells (C) transduced with the empty vector pSiRPG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087268#pone.0087268-Storvold1" target="_blank">[27]</a>. α-tubulin was used as loading control.</p

Increased NDRG1 expression due to hypoxia or ectopic expression.

<p>(<b>A</b>) Increased NDRG1 expression detected by Western blotting in the breast cell lines MCF-7 and ZR-75-1 grown at 1% O₂ for 24, 48 and 72 hours as indicated, compared to control cells grown at 20% O₂. (<b>B</b>) Western blots document increased NDRG1 expression in cells grown at 20% O₂. The ZR-75-1 cell populations transduced with the NDRG1-cDNA (six biological parallels indicated A–F), are compared to ZR-75-1 transduced with the vector pSiRPG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087268#pone.0087268-Storvold1" target="_blank">[27]</a> without insert (C2 and C3). α-tubulin was used as loading control (<b>A</b> and <b>B</b>). Note that a reduced level of total protein is loaded in the lane containing extract from MCF-7 grown at 1% O₂ for 24 hours. (<b>C</b>) Treeview presentation of the expression of 18 genes in a hypoxia signature gene list <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087268#pone.0087268-Chi1" target="_blank">[24]</a> in MCF-7 and ZR-75-1 cells grown at 1% O₂ for 24 or 48 hours as indicated. (<b>D</b>) Treeview presentation of the hypoxia signature genes in six ZR-75-1 cell populations ectopically over-expressing NDRG1 (indicated A–F). The red and green colours show increased and reduced expression levels, respectively (<b>C</b> and <b>D</b>). The colour intensity is from −3 to 3 indicating the magnitude of the fold change in gene expression between the cell population and its control.</p

Immunofluorescence staining of SUM102 cell cultures.

<p>Colors were applied artificially for graphical purposes. (<b>A</b>) NDRG1 (green) and calnexin (red) in untreated, hypoxia and doxorubicin treated cells. Bar: 5 µm. (<b>B</b>) NDRG1 (green) and EEA1 (red) in hypoxia treated cells. Bar: 10 µm.</p

Gypsies/Travellers and health: risk categorisation versus being ‘at risk’

Risk categorisation provides a routine and necessary contribution to the way people make sense of and impute predictability in a complex human world of which their knowledge is limited. It is a precursor to the development of risk management strategies. Risk categorisation schemes can vary depending on the underlying perspective and knowledge used in their construction. There are estimated to be between 120,000 and 300,000 Gypsies and Travellers in the United Kingdom. They have been categorised on the one hand as a group that is ‘at risk’ suffering wide-ranging inequalities and on the other they can be seen as the archetypical ‘other’ posing risks to normative stability. Public policies to manage their health risks have been limited in contrast to policies to address their ‘otherness’ status which have aimed to exclude, relocate and forcibly remove them from public space. Little is understood about the way in which Gypsies and Travellers categorise and manage the risks to their health within the context of adverse public policies. In-depth qualitative interviews were undertaken during 2010 and 2011 with 39 Gypsies and Travellers aged between 18 and 66 years comprising 20 females and 19 males living in an area of South East England. Respondents framed risk in terms of threats to their health, culture and traditional way of life and issues of trust were central to this. They sought to devise risk management strategies that would maintain boundaries between their community and outsiders who were perceived to be the source of risks to their health. A consequence of their risk management strategies was the potential perpetuation of threats to their health and well-being