Rab3D is critical for secretory granule maturation in PC12 cells.

Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs

Zoledronic acid renders human M1 and M2 macrophages susceptible to Vδ2(+) γδ T cell cytotoxicity in a perforin-dependent manner.

Vδ2(+) T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2(+) T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2(+) T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C-C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2(+) T cell cytotoxicity. Vδ2(+) T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive-at least in part-to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2(+) T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy

Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: a mechanism of action for cancer immunotherapy

Attenuated and heat-killed mycobacteria display demonstrable activity against cancer in the clinic; however, the induced immune response is poorly characterised and potential biomarkers of response ill-defined. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour effector responses in human γδ T-cells. γδ T-cell responses were characterised by measuring cytokine production, expression of granzyme B and cytotoxicity against tumour target cells. Results show that γδ T-cells are activated by these mycobacterial preparations, as indicated by upregulation of activation marker expression and proliferation. Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B expression, production of the TH1 cytokines IFN-γ and TNF-α, and enhanced degranulation in response to susceptible and zoledronic acid-treated resistant tumour cells. Moreover, γδ T-cell activation is induced by IL-12, IL-1β and TNF-α from circulating type 1 myeloid dendritic cells (DCs), but not from type 2 myeloid DCs or plasmacytoid DCs. Taken together, we show that BCG, M. vaccae and M. obuense induce γδ T-cell anti-tumour effector responses indirectly via a specific subset of circulating DCs and suggest a mechanism for the potential immunotherapeutic effects of BCG, M. vaccae and M. obuense in cancer

Functional Analysis of the Transcription Repressor PLU-1/JARID1B▿ †

The PLU-1/JARID1B nuclear protein, which is upregulated in breast cancers, belongs to the ARID family of DNA binding proteins and has strong transcriptional repression activity. To identify the target genes regulated by PLU-1/JARID1B, we overexpressed or silenced the human PLU-1/JARID1B gene in human mammary epithelial cells by using adenovirus and RNA interference systems, respectively, and then applied microarray analysis to identify candidate genes. A total of 100 genes showed inversely correlated differential expression in the two systems. Most of the candidate genes were downregulated by the overexpression of PLU-1/JARID1B, including the MT genes, the tumor suppressor gene BRCA1, and genes involved in the regulation of the M phase of the mitotic cell cycle. Chromatin immunoprecipitation assays confirmed that the metallothionein 1H (MT1H), -1F, and -1X genes are direct transcriptional targets of PLU-1/JARID1B in vivo. Furthermore, the level of trimethyl H3K4 of the MT1H promoter was increased following silencing of PLU-1/JARID1B. Both the PLU-1/JARID1B protein and the ARID domain selectively bound CG-rich DNA. The GCACA/C motif, which is abundant in metallothionein promoters, was identified as a consensus binding sequence of the PLU-1/JARID1B ARID domain. As expected from the microarray data, cells overexpressing PLU-1/JARID1B have an impaired G2/M checkpoint. Our study provides insight into the molecular function of the breast cancer-associated transcriptional repressor PLU-1/JARID1B

Proteomic Footprinting of Drug-Treated Cancer Cells as a Measure of Cellular Vaccine Efficacy for the Prevention of Cancer Recurrence*

The comparative proteomic study of cell surfaces of native and drug-treated cancer cells was performed. To this end, cell proteomic footprinting, which reflects the mass spectrometry profiling of cell surface proteins, was applied to breast adenocarcinoma cells (MCF-7), which were untreated or treated with doxorubicin, tamoxifen, or etoposide. The footprints of drug-treated cells were compared with the footprints of untreated cells and the footprint of a randomly selected control cancer cell culture. It was found that drug-treated cells have reproducible, pronounced, and drug-specific changes in cell surface protein expression. Cytotoxicity assays, which are an in vitro model of human antitumor vaccination, revealed that the degree of these changes correlates directly with the ability of the cancer cells to escape cell death induced by a cytotoxic T-cell-mediated immune response. Moreover, cancer cells escape from the immune response was linearly approximated (R2 equal to 0.99) with the degree by which their proteomic footprints diverged from the footprint of the targeted (native) cancer cells. From these findings, it was concluded that the design of anticancer vaccines intended to prevent cancer recurrence after primary treatment should consider the drug-specific changes in cancer cell-surface antigens. Such changes can be easily identified by cell proteomic footprinting, renewing hopes for development of efficient cellular cancer vaccines

myc-Rab3D is recruited to ISGs.

<p>PC12 cells were cotransfected with hCgB-GFP(S65T) and myc-Rab3D, myc-Rab3A or control vector. Cells were cultured for 2 days including sodium butyrate induction and then subjected to the long pulse/chase-like protocol. After 12 min of chase, SGs were isolated, spun down on coverslips, fixed and stained against the myc-tag (see Experimental). (<b>A</b>) Maximum projections of processed confocal image stacks, which were used to count the percent of colocalization of spots of hCgB-GFP(S65T) signals (top) with spots of myc signals (bottom). Red circles, non-colocalizing spots, green circles, colocalizing spots; scalebars, 10 µm. (<b>B</b>) Amount of fluorescent ISGs colocalizing with myc signal in corresponding frames (left) and non-corresponding frames (right) as a control. Bars, mean ± SEM; students two-tailed t-test confidence interval: *<0,05; for each condition, ≤143 hCgB-GFP(S65T) puncta on ≤7 frames for each condition and each of 3 independent experiments. For non-corresponding frames, the green channel of all frames was paired with the red channel of the following frame.</p

Myc-Rab3D(N135I) but not myc-Rab3A(N135I) inhibits the removal of bfurin from maturing SGs to the same extent as FLAG-MyoVa-tail.

<p>(A) PC12 cells were cotransfected with hCgB-EGFP, bfurin and FLAG, FLAG-MyoVa-tail, myc-Rab3D or myc-Rab3D(N135I) or with hCgB-EGFP, ECFP-bfurin, myc-Rab3A or myc-Rab3A(N135I). Subsequently, cells were subjected to the shorter pulse/chase-like protocol with chase times of 2, 12, 30 or 180 min, respectively, and fixed. Cells were stained against bfurin, except for cotransfections with myc-Rab3A and myc-Rab3A(N135I), imaged by confocal microscopy and analyzed for colocalization. The graphs show the percentage of hCgB-EGFP positive SGs colocalizing with bfurin signal (n = 6 cells per experiment, 2 independent experiments for myc-Rab3A and myc-Rab3A(N135I), and n≥4 cells per experiment, ≥3 independent experiments, for all other conditions); bars: mean ± SEM). Results of unpaired two-tailed student' t-tests are shown. (B) Myc-Rab3D and myc-Rab3D(N135I) do not induce clustering of SGs. PC12 cells were cotransfected with hCgB-GFP(S65T) and FLAG-MyoVa-tail, myc-Rab3D or myc-Rab3D(N135I). Cells were subjected to the long pulse/chase like protocol using a chase time of 90 min. Then, cells were fixed and imaged by confocal microscopy. The images show 3D reconstructions (Imaris) of fluorescence signals of hCgB-GFP(S65T). Scalebar: 10 µm.</p

Effects of myc-Rab3D and myc-Rab3D(N135I) on buoyant density of SGs and processing of SgII.

<p>PC12 cells were cotransfected with hCgB-EGFP and FLAG, myc-Rab3D, myc-Rab3D(N135I), FLAG or FLAG-MyoVa-tail. (<b>A and B</b>) Cells were cultured for two days including sodium butyrate induction. Cell fractions enriched in SGs were analyzed by sucrose gradient centrifugation followed by Western blotting. (<b>A</b>) Western blots of one representative experiment. (<b>A′</b>) Quantification of the hCgB-EGFP signal as percent of the maximum value upon co-expression of FLAG (black squares on black line), myc-Rab3D (grey circles on grey line) or myc-Rab3D(N135I) (light grey triangles on light grey line). (<b>A″</b>) Sucrose concentrations of the respective fractions in (<b>A′</b>) are shown. (<b>A′</b>, <b>A″</b>) The published density of ISGs and MSGs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057321#pone.0057321-Urbe2" target="_blank">[7]</a> is indicated by unfilled and filled arrowheads, respectively. Graphs, mean ± SEM (n = 4 independent experiments) (<b>B</b>): FLAG-MyoVa-tail does not impede the maturation-dependent increase in buoyant density of SGs compared to FLAG expression only. (<b>B</b>) Representative Western blots of hCgB-EGFP upon co-expression of FLAG or FLAG-MyoVa-tail, repectively. (<b>B′</b>) Quantification of the hCgB-EGFP signals as for (<b>A′</b>) with FLAG (black squares on black line) or FLAG-MyoVa-tail (light grey line). (<b>B, B′</b>) Graphs, mean ± SEM (N = 4 independent experiments). (<b>C</b>) Expression of myc-Rab3D(N135I) impairs the processing of SgII during SG maturation. PC12 cells were cotransfected with PC2 and FLAG, myc-Rab3D or myc-Rab3D(N135I). Cells were cultured for one day including sodium butyrate induction. Then, cells were pulse-labeled with [³⁵S]-sulphate for 1 hour followed by a chase of 3 hours (see Experimental). Thereafter cells were lysed, the processing product p18 (<b>C</b>, lower panel, <b>C′</b>, right panel) was immunoprecipitated and analyzed by SDS-PAGE and radiofluorography. Aliquots of the cell lysates were analyzed for endogenous rSgII (loading control C, upper panel, C′, left panel). One respresentative radiofluorography (<b>C, top</b>) for each condition and the quantitation (C′) (mean ± SEM, n = 3 independent experiments for p18, mean ± stdev, n = 2 independent experiments for rSgII) is shown.</p

Myc-Rab3A(N135I) and myc-Rab3D(N135I) impede localization of SGs in the F-actin rich cell cortex.

<p>PC12 cells were cotransfected with hCgB-GFP(S65T) and FLAG or FLAG-MyoVa-tail, myc-Rab3A, B, C or D or their (N135I) mutants. Subsequently, cells were cultured for 2 days at 37 °C including sodium butyrate induction, and then subjected to the longer pulse/chase-like protocol with a chase time of 1 h. Cells were then fixed, stained with TRITC-phalloidin and imaged by confocal microscopy. (<b>A</b>) Representative single optical sections of cells cotransfected with hCgB-GFP(S65T) and FLAG (left), myc-Rab3D (middle) or myc-Rab3D(N135I) (right). Green, hCgB- GFP(S65T); magenta, TRITC-phalloidin; arrowheads, SGs colocalizing with F-actin; arrows, SGs not colocalizing with F-actin; scalebar, 5 µm. (<b>B</b>) Quantification of colocalization between TRITC-phalloidin and GFP. Bars, percent of colocalization; error bars, SEM (n>6 cells from at least 2 independent experiments). Unpaired two-tailed student' t-tests are indicated.</p