Melinda E. Christensen

Comprehensive transcriptome and immunophenotype analysis of renal and cardiac MSC-like populations supports strong congruence with bone marrow MSC despite maintenance of distinct identities.

Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.

Isolation of clonogenic, long-term self renewing embryonic renal stem cells

A tissue stem cell should exhibit long-term self-renewal, clonogenicity and a capacity to differentiate into the tissue of origin. Such a postnatal renal stem cell has not been formally identified. The metanephric mesenchyme (MM) of the developing kidney gives rise to both the renal interstitium and the nephrons and is regarded as the progenitor population of the developing kidney. However, isolated MM does not self renew and requires immortalization for survival in culture. Here we report the isolation and sustained culture of long-term repopulating, clonal progenitors from the embryonic kidney as free floating nephrospheres. Such cells displayed clonal self renewal for in excess of twenty passages when cultured with bFGF and thrombin, showed broad mesodermal multipotentiality, but retained expression of key renal transcription factors (Wt1, Sall1, Eya1, Six1, Six2, Osr1 and Hoxa11). While these cells did display limited capacity to contribute to developing embryonic kidney explants, nephrospheres did not display in vitro renal epithelial capacity. Nephrospheres could be cultured from both Sall1(+) and Sall1(-) fractions of embryonic kidney, suggesting that they were derived from the MM as a whole and not specifically the MM-derived cap mesenchyme committed to nephron formation. This embryonic renal stem cell population was not able to be isolated from postnatal kidney confirming that while the embryonic MM represents a mulitpotent stem cell population, this does not persist after birth.

Flow cytometry based assays for the measurement of apoptosis-associated mitochondrial membrane depolarisation and cytochrome c release.

Mitochondria play a pivotal role in life and death of the cell because they produce the majority of energy required for survival and also regulate the intrinsic pathway to apoptosis. The involvement of mitochondria in cell death is generally measured by following mitochondrial membrane depolarisation or mitochondrial outer membrane permeabilisation (MOMP). These events can be assayed using cationic dyes that are attracted to the negative charge across the inner membrane of healthy mitochondria or by following translocation of cytochrome c from the mitochondria to the cytoplasm respectively. These events progress rapidly in individual cells but are observed as bi-phasic peaks in flow cytometry assays because cell death generally occurs asynchronously in a population. This allows researchers to use flow cytometry to easily distinguish healthy cells with intact mitochondria healthy from dying cells with permeabilised mitochondria. This article will therefore review methods using flow cytometry to follow mitochondrial membrane depolarisation and cytochrome c release during apoptosis, and will highlight some studies that resulted in development of these assays.

The Role of Palmitoylation in Signalling, Cellular Trafficking and Plasma Membrane Localization of Protease-Activated Receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.

Cellular Settings Mediating Src Substrate Switching between Focal Adhesion Kinase Tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) Tyrosine 734

Background: Focal adhesion kinase (FAK) and CUB-domain-containing protein 1 (CDCP1) are Src family kinase (SFK) substrates. Results: SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 is induced by increased CDCP1 expression and changes in cell attachment. Conclusion: SFK switching between FAK and CDCP1 may be relevant to malignant transformation. Significance: Targeting of this switch may be a rational approach to treat diseases such as cancer. Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.

Granzyme B triggers a prolonged pressure to die in Bcl-2 overexpressing cells, defining a window of opportunity for effective treatment with ABT-737.

Overexpression of Bcl-2 contributes to resistance of cancer cells to human cytotoxic lymphocytes (CL) by blocking granzyme B (GraB)-induced mitochondrial outer membrane permeabilization (MOMP). Drugs that neutralise Bcl-2 (e.g., ABT-737) may therefore be effective adjuvants for immunotherapeutic strategies that use CL to kill cancer cells. Consistent with this we found that ABT-737 effectively restored MOMP in Bcl-2 overexpressing cells treated with GraB or natural killer cells. This effect was observed even if ABT-737 was added up to 16 h after GraB, after which the cells reset their resistant phenotype. Sensitivity to ABT-737 required initial cleavage of Bid by GraB (gctBid) but did not require ongoing GraB activity once Bid had been cleaved. This gctBid remained detectable in cells that were sensitive to ABT-737, but Bax and Bak were only activated if ABT-737 was added to the cells. These studies demonstrate that GraB generates a prolonged pro-apoptotic signal that must remain active for ABT-737 to be effective. The duration of this signal is determined by the longevity of gctBid but not activation of Bax or Bak. This defines a therapeutic window in which ABT-737 and CL synergise to cause maximum death of cancer cells that are resistant to either treatment alone, which will be essential in defining optimum treatment regimens.

Measuring Cell Death by Trypan Blue Uptake and Light Microscopy

Trypan blue is a colorimetric dye that stains dead cells with a blue color easily observed using light microscopy at low resolution. The staining procedure is rapid and cells can be analyzed within minutes. The number of live (unstained) and dead (blue) cells can be counted using a hemocytometer on a basic upright microscope. Trypan blue staining is therefore a convenient assay for rapidly determining the overall viability of cells in a culture before commencing scientific experimentation, or for quantitating cell death following treatment with any cytotoxic stimuli.

Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer

Human papillomavirus (HPV) is the causative agent in cervical cancer. HPV oncogenes are major drivers of the transformed phenotype, and the cancers remain addicted to these oncogenes. A screen of the human kinome has identified inhibition of Aurora kinase A (AURKA) as being synthetically lethal on the background of HPV E7 expression. The investigational AURKA inhibitor MLN8237/Alisertib selectively promoted apoptosis in the HPV cancers. The apoptosis was driven by an extended mitotic delay in the Alisertib-treated HPV E7–expressing cells. This had the effect of reducing Mcl-1 levels, which is destabilized in mitosis, and increasing BIM levels, normally destabilized by Aurora A in mitosis. Overexpression of Mcl-1 reduced sensitivity to the drug. The level of HPV E7 expression influenced the extent of Alisertib-induced mitotic delay and Mcl-1 reduction. Xenograft experiments with three cervical cancer cell lines showed Alisertib inhibited growth of HPV and non-HPV xenografts during treatment. Growth of non-HPV tumors was delayed, but in two separate HPV cancer cell lines, regression with no resumption of growth was detected, even at 50 days after treatment. A transgenic model of premalignant disease driven solely by HPV E7 also demonstrated sensitivity to drug treatment. Here, we show for the first time that targeting of the Aurora A kinase in mice using drugs such as Alisertib results in a curative sterilizing therapy that may be useful in treating HPV-driven cancers. Mol Cancer Ther; 14(12); 2753–61. ©2015 AACR.

Measuring Survival of Adherent Cells with the Colony-Forming Assay

Measuring cell death with colorimetric or fluorimetric dyes such as trypan blue and propidium iodide (PI) can provide an accurate measure of the number of dead cells in a population at a specific time; however, these assays cannot be used to distinguish cells that are dying or marked for future death. In many cases it is essential to measure the proliferative capacity of treated cells to provide an indirect measurement of cell death. This can be achieved using the colony-forming assay described here. This protocol specifically applies to measurement of HeLa cells but can be used for most adherent cell lines with limited motility.

Measuring Mitochondrial Transmembrane Potential by TMRE Staining

Adenosine triphosphate (ATP) is the main source of energy for metabolism. Mitochondria provide the majority of this ATP by a process known as oxidative phosphorylation. This process involves active transfer of positively charged protons across the mitochondrial inner membrane resulting in a net internal negative charge, known as the mitochondrial transmembrane potential (ΔΨm). The proton gradient is then used by ATP synthase to produce ATP by fusing adenosine diphosphate and free phosphate. The net negative charge across a healthy mitochondrion is maintained at approximately -180 mV, which can be detected by staining cells with positively charged dyes such as tetramethylrhodamine ethyl ester (TMRE). TMRE emits a red fluorescence that can be detected by flow cytometry or fluorescence microscopy and the level of TMRE fluorescence in stained cells can be used to determine whether mitochondria in a cell have high or low ΔΨm. Cytochrome c is essential for producing ΔΨm because it promotes the pumping the protons into the mitochondrial intermembrane space as it shuttles electrons from Complex III to Complex IV along the electron transport chain. Cytochrome c is released from the mitochondrial intermembrane space into the cytosol during apoptosis. This impairs its ability to shuttle electrons between Complex III and Complex IV and results in rapid dissipation of ΔΨm. Loss of ΔΨm is therefore closely associated with cytochrome c release during apoptosis and is often used as a surrogate marker for cytochrome c release in cells.