Intracellular Fas ligand in normal and malignant breast epithelium does not induce apoptosis in Fas-sensitive cells

Fas ligand (FasL) is expressed on some cancers and may play a role in the immune evasion of the tumour. We used immuno-histochemistry to study the expression of Fas and FasL in tissue samples from breast cancer patients, as well as normal breast tissue. Our results show that Fas and FasL are co-expressed both in normal tissue and in breast tumours. Fas and FasL mRNA were expressed in fresh normal and malignant breast tissue, as well as cultured breast epithelium and breast cancer cell lines. Flow cytometry analysis of live cells failed to detect FasL on the surface of normal or malignant breast cells; however, both stained positive for FasL after permeabilization. Fas was detected on the surface of normal breast cells and T47D and MCF-10A cell lines but only intracellularly in other breast cell lines tested. Neither normal breast epithelium nor breast cell lines induced Fas-dependent apoptosis in Jurkat cells. Finally, 20 tumour samples were stained for apoptosis. Few apoptotic cells were detected and there was no increase in apoptotic cells on the borders between tumour cells and lymphocytes. We conclude that FasL is expressed intracellularly in both normal and malignant breast epithelium and unlikely to be important for the immune evasion of breast tumours. © 2000 Cancer Research Campaign http://www.bjcancer.co

A short isoform of ATG7 fails to lipidate LC3/GABARAP

Publisher's version (útgefin grein)Autophagy is a degradation pathway important for cellular homeostasis. The E1-like enzyme ATG7 is a key component of the autophagy machinery, with the main function of mediating the lipidation of LC3/GABARAP during autophagosome formation. By analysing mRNA-sequencing data we found that in addition to the full-length ATG7 isoform, various tissues express a shorter isoform lacking an exon of 27 amino acids in the C-terminal part of the protein, termed ATG7(2). We further show that ATG7(2) does not bind LC3B and fails to mediate the lipidation of members of the LC3/GABARAP family. We have thus identified an isoform of ATG7 that is unable to carry out the best characterized function of the protein during the autophagic response. This short isoform will have to be taken into consideration when further studying the role of ATG7.This work was supported by a START Marie Curie/Icelandic Research Fund grant (M.H.O.; grant number 120457-041), Icelandic Research Fund grant (M.H.O.; grant number 184727-051), an Icelandic Cancer Society Research Fund grant (M.H.O.), Icelandic Research Fund grant (E.S.; grant number 152715) and by an Erwin Schrödinger fellowship grant from the Austrian Science Fund (V.F.; grant number: J 3864-B26).Peer Reviewe

Proton-Assisted Amino Acid Transporter PAT1 Complexes with Rag GTPases and Activates TORC1 on Late Endosomal and Lysosomal Membranes

Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H+-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments

PAT1 modulates the AA-dependent relocalisation of mTOR to LELs.

<p>(<b>A</b>, <b>B</b>) Knockdown of PAT1 (PAT1 kd) in HEK-293 cells reduces the AA-stimulated accumulation of mTOR (green) to LAMP2-positive (red) LELs (B), when compared to cells treated with a scrambled siRNA (scr; A). (<b>C</b>, <b>D</b>) Importantly, <i>PAT1</i> knockdown does not eliminate all PAT1 protein from cells (compare D with control cells in C), so residual mTOR relocalisation in B may result from the presence of low levels of PAT1. PAT1 antibody staining is shown in green, LAMP2 in red. Scale bar in A is 10 µm and applies to all panels.</p

PAT1 co-localises and can physically interact with Rag GTPases.

<p>(<b>A</b>, <b>B</b>) Flag-PAT1 co-localises with a subset of the compartments containing endogenous RagC under both AA-starved (A) and AA-stimulated (B) conditions in a stable HEK-293 cell line overexpressing Flag-PAT1. (<b>C</b>) Under steady state conditions, immunoprecipitation of Flag-PAT1 leads to co-immunoprecipitation of endogenous RagC, but not tubulin. (<b>D</b>) Conversely, immunoprecipitation of Flag-RagD, but not Flag-Rap2A, both transiently expressed in HEK-293 cells, leads to co-immunoprecipitation of endogenous PAT1, but not tubulin, suggesting that the Rag GTPases complex with PAT1 in cells. Scale bar in A is 10 µm and applies to all panels in A and B.</p

PI3K/Akt/Rheb signalling promotes shuttling of the PATs to endosomal compartments.

<p>(<b>A, C</b>) CG1139-GFP expressed in the larval fat body using Lsp2-GAL4 is localised to both the plasma membrane (white arrow) and also to intracellular LELs throughout the cytoplasm (yellow arrow; A). Overexpression of Rheb leads to an increase in the relative proportion of intracellular, including perinuclear, protein compared to cell surface CG1139-GFP (C). (<b>B</b>, <b>D</b>) When CG1139-GFP is co-expressed with a dominant negative version of Shibire, Shi^K44A, which blocks endocytosis, in either the presence (D) or absence (B) of Rheb, the transporter is mostly located at the plasma membrane (white arrow), strongly suggesting that PATs are normally shuttled to LELs from the cell surface. (<b>E</b>, <b>F</b>) The ratio of the GFP signal intensity in a 2.25 µm perinuclear region and a 2.25 µm region at the plasma membrane (see E) was measured for genotypes in A–D (grey bars; error bars = s.d. in F). Average cell size for each genotype is also shown in F (blue bars; error bars = s.d.). n = 25; *P<0.001 (increased) and _■ P<0.001 (decreased) relative to non-Rheb/non-Shi^K44A-expressing control; _{■ ■} P<0.001, decreased relative to Rheb-overexpressing control. Rheb-induced changes in intracellular PATs and PAT-induced growth are entirely dependent on endocytosis. (<b>G</b>, <b>H</b>) In the larval eye imaginal disc, CG1139-GFP, expressed under GMR-GAL4 control (G), is mostly located at or near the plasma membrane, for example at the surfaces of flattened non-photoreceptor cells that surround each ommatidium (white arrows). Co-overexpression of Rheb (H) results in much larger ommatidia, with reduced staining around the ommatidial border (white arrow) and more CG1139-GFP cytoplasmic expression, including intense intracellular punctae of staining (yellow arrows), consistent with Rheb promoting endocytosis of PATs in this tissue. An outline of an individual ommatidium in G and H is marked with a dashed line. Scale bar in A is 20 µm and applies to panels A–D, scale bar in G is 5 µm and applies to panels G and H.</p

mTOR localises to LAMP2/PAT1-positive compartments upon AA stimulation.

<p>(<b>A</b>, <b>B</b>) Flag-PAT1 (red; B) overexpressed in a stably transfected HEK-293 cell line has a similar intracellular localisation pattern to endogenous PAT1 (green; A). Cells under steady state conditions are shown. (<b>C</b>) Endogenous PAT1 strongly co-localises with LAMP2, which marks LEL compartments. Cells were stained after 50 min AA starvation followed by 10 min AA stimulation. Merge shows LAMP2 (red), PAT1 (green) and DAPI (blue). (<b>D</b>, <b>E</b>) Subcellular localisation of LAMP2, Flag-PAT1 and mTOR after 50 min AA starvation (D) and 50 min AA starvation followed by 10 min AA stimulation (E). Note that Flag-PAT1 and LAMP2 co-localise under both conditions, but mTOR is only recruited to a limited number of LELs upon AA stimulation. Merge shows Flag-PAT1 (red), mTOR (green) and DAPI (blue). Scale bars in B and E are 10 µm, A; scale bar in B also applies to A and C, scale bar in E also applies to D.</p

The growth-promoting activity of PAT transporters is synergistically enhanced by hyperactivation of PI3K/Akt signalling.

<p>(<b>A</b>–<b>C</b>) GAL4-UAS-induced overexpression of the fly PAT transporter genes, <i>path</i> (B) and <i>CG1139</i> (C) in the differentiating eye with GMR-GAL4 promotes increased growth compared to normal animals (A). (<b>D</b>–<b>F</b>) When overexpressed in a <i>PTEN</i> mutant background (D), the effect of <i>path</i> (E) and <i>CG1139</i> (F) on growth is synergistically enhanced, resulting in a highly overgrown, bulging eye phenotype. Ommatidial size measurements are given for eyes where the ommatidial array is regularly arranged (n = 6; bottom of panels A–C, mean ± s.d. relative to control (A); *P<0.001, increased relative to control). Fly genotypes are <i>w</i>; <i>GMR-GAL4</i> (A), <i>w</i>; <i>GMR-GAL4</i>/<i>path^GS13857</i> (B), <i>w</i>; <i>GMR-GAL4</i>/<i>CG1139^GS10666</i> (C), <i>y w</i>; <i>PTEN¹ FRT40A</i>/<i>P[w⁺]l(2)3.1 FRT 40A</i>; <i>GMR-GAL4</i> (D), <i>y w</i>; <i>PTEN¹ FRT40A</i>/<i>P[w⁺]l(2)3.1 FRT 40A</i>; <i>GMR-GAL4</i>/<i>path^GS13857</i> (E) and <i>y w</i>; <i>PTEN¹ FRT40A</i>/<i>P[w⁺]l(2)3.1 FRT 40A</i>; <i>GMR-GAL4</i>/<i>CG1139^GS10666</i> (F).</p