Harnessing the Regenerative Potential of Purified Bovine Dental Pulp and Dentin Extracellular Matrices in a Chitosan/Alginate Hydrogel.

When a tooth is diseased or damaged through caries, bioactive molecules are liberated from the pulp and dentin as part of the natural response to injury and these are key molecules for stimulating stem cell responses for tissue repair. Incorporation of these extracellular-matrix (ECM)-derived molecules into a hydrogel model can mimic in vivo conditions to enable dentin-pulp complex regeneration. Here, a chitosan/alginate (C/A) hydrogel is developed to sequester bovine ECM extracts. Human dental pulp cells (hDPCs) are cultured with these constructs and proliferation and cytotoxicity assays confirm that these C/A hydrogels are bioactive. Sequential z-axis fluorescent imaging visualizes hDPCs protruding into the hydrogel as it degraded. Alizarin red S staining shows that hDPCs cultured with the hydrogels display increased calcium-ion deposition, with dentin ECM stimulating the highest levels. Alkaline phosphatase activity is increased, as is expression of transforming growth factor-beta as demonstrated using immunocytochemistry. Directional analysis following phase contrast kinetic image capture demonstrates that both dentin and pulp ECM molecules act as chemoattractants for hDPCs. Data from this study demonstrate that purified ECM from dental pulp and dentin when delivered in a C/A hydrogel stimulates dental tissue repair processes in vitro.

Activated CD90/Thy-1 fibroblasts co-express the Δ133p53ß isoform and are associated with highly inflamed rheumatoid arthritis.

BACKGROUND: The p53 isoform Δ133p53ß is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53ß is driving aggressive disease in RA. METHODS: Using RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53ß. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software. RESULTS: Elevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of Δ133TP53 and TP53ß mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of 'activated fibroblasts' together with elevated levels of Δ133p53ß. CONCLUSIONS: Induction of Δ133p53ß in CD90+ synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53ß could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53ß could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6.

Calpains Released from Necrotic Tumor Cells Enhance Antigen Cross-Presentation to Activate CD8+ T Cells In Vitro.

The initiation of CD8+ T cell responses against dead cell-associated Ags is tightly regulated, facilitating adaptive immunity against pathogens and tumors while preventing autoimmunity. It is now well established that dying cells actively regulate the generation of CD8+ T cell responses via the release or exposure of damage-associated molecular patterns. However, it is unclear whether nonproteasomal proteases (activated in stressed and dying cells) can influence the availability of Ags for cross-presentation. Using a mouse model of immunogenic necrosis, we investigated the role of tumor-derived proteases in the priming of CD8+ T cells. We demonstrate that proteases released from necrotic tumor cells can degrade whole-protein Ag, generating proteolytic intermediates that are efficiently cross-presented by dendritic cells and enhance CD8+ T cell cross-priming. We identify a dominant role for calpain proteases, which are activated during necrotic cell death induced by severe heat shock. Mechanistically, proteolytic intermediates generated by tumor-derived proteases associate with necrotic tumor cell debris, which acts as a vehicle for Ag transfer that facilitates highly efficient cross-presentation in dendritic cells. Our results suggest that proteolytic systems activated in Ag donor cells during cell death may influence the availability of antigenic substrates for cross-presentation, thereby regulating the antigenicity of cell death.

Chemical Synthesis of the PAX Protein Inhibitor EG1 and Its Ability to Slow the Growth of Human Colorectal Carcinoma Cells.

Colorectal cancer is primarily a disease of the developed world. The incidence rate has continued to increase over time, reflecting both demographic and lifestyle changes, which have resulted in genomic and epigenomic modifications. Many of the epigenetic modifications occur in genes known to be closely associated with embryonic development and cellular growth. In particular, the paired box (PAX) transcription factors are crucial for correct tissue development during embryogenesis due to their role in regulating genes involved in proliferation and cellular maintenance. In a number of cancers, including colorectal cancer, the PAX transcription factors are aberrantly expressed, driving proliferation and thus increased tumour growth. Here we have synthesized and used a small molecule PAX inhibitor, EG1, to inhibit PAX transcription factors in HCT116 colorectal cell cultures which resulted in reduced proliferation after three days of treatment. These results highlight PAX transcription factors as playing an important role in the proliferation of HCT116 colorectal cancer cells, suggesting there may be a potential therapeutic role for inhibition of PAX in limiting cancer cell growth.

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, ß-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.

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 YB1 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 YB1 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 YB1 determines its cellular location.

A novel EGR-1 dependent mechanism for YB-1 modulation of paclitaxel response in a triple negative breast cancer cell line.

Chemotherapy with taxanes such as paclitaxel (PTX) is a key component of triple negative breast cancer (TNBC) treatment. PTX is used in combination with other drugs in both the adjuvant setting and in advanced breast cancer. Because a proportion of patients respond poorly to PTX or relapse after its use, a greater understanding of the mechanisms conferring resistance to PTX is required. One protein shown to be involved in drug resistance is Y-box binding protein 1 (YB-1). High levels of YB-1 have previously been associated with resistance to PTX in TNBCs. In this study, we aimed to determine mechanisms by which YB-1 confers PTX resistance. We generated isogenic TNBC cell lines that differed by YB-1 levels and treated these with PTX. Using microarray analysis, we identified EGR1 as a potential target of YB-1. We found that low EGR1 mRNA levels are associated with poor breast cancer patient prognosis, and that EGR1 and YBX1 mRNA expression was inversely correlated in a TNBC line and in a proportion of TNBC tumours. Reducing the levels of EGR1 caused TNBC cells to become more resistant to PTX. Given that PTX targets cycling cells, we propose a model whereby high YB-1 levels in some TNBC cells can lead to reduced levels of EGR1, which in turn promotes slow cell cycling and resistance to PTX. Therefore YB-1 and EGR1 levels are biologically linked and may provide a biomarker for TNBC response to PTX.

Variable Expression of GLIPR1 Correlates with Invasive Potential in Melanoma Cells.

GLI pathogenesis-related 1 (GLIPR1) was previously identified as an epigenetically regulated tumor suppressor in prostate cancer and, conversely, an oncoprotein in glioma. More recently, GLIPR1 was shown to be differentially expressed in other cancers including ovarian, acute myeloid leukemia, and Wilms' tumor. Here we investigated GLIPR1 expression in metastatic melanoma cell lines and tissue. GLIPR1 was variably expressed in metastatic melanoma cells, and transcript levels correlated with degree of GLIPR1 promoter methylation in vitro. Elevated GLIPR1 levels were correlated with increased invasive potential, and siRNA-mediated knockdown of GLIPR1 expression resulted in reduced cell migration and proliferation in vitro. Immunohistochemical studies of melanoma tissue microarrays showed moderate to high staining for GLIPR1 in 50% of specimens analyzed. GLIPR1 staining was observed in normal skin in merocrine sweat glands, sebaceous glands, and hair follicles within the dermis.

YB-1: oncoprotein, prognostic marker and therapeutic target?

Hanahan and Weinberg have proposed the 'hallmarks of cancer' to cover the biological changes required for the development and persistence of tumours [Hanahan and Weinberg (2011) Cell 144, 646-674]. We have noted that many of these cancer hallmarks are facilitated by the multifunctional protein YB-1 (Y-box-binding protein 1). In the present review we evaluate the literature and show how YB-1 modulates/regulates cellular signalling pathways within each of these hallmarks. For example, we describe how YB-1 regulates multiple proliferation pathways, overrides cell-cycle check points, promotes replicative immortality and genomic instability, may regulate angiogenesis, has a role in invasion and metastasis, and promotes inflammation. We also argue that there is strong and sufficient evidence to suggest that YB-1 is an excellent molecular marker of cancer progression that could be used in the clinic, and that YB-1 could be a useful target for cancer therapy.

YB-1, the E2F pathway, and regulation of tumor cell growth.

BACKGROUND: Y-box binding factor 1 (YB-1) has been associated with prognosis in many tumor types. Reduced YB-1 expression inhibits tumor cell growth, but the mechanism is unclear. METHODS: YB-1 mRNA levels were compared with tumor grade and histology using microarray data from 771 breast cancer patients and with disease-free survival and distant metastasis-free survival using data from 375 of those patients who did not receive adjuvant therapy. Microarrays were further searched for genes that had correlated expression with YB-1 mRNA. Small interfering RNA (siRNA) was used to study the effects of reduced YB-1 expression on growth of three tumor cell lines (MCF-7 breast, HCT116 colon, and A549 lung cancer cells), on tumorigenesis by A549 cells in nude mice, and on global transcription in the three cancer cell lines. Reporter gene assays were used to determine whether YB-1 siRNAs affected the expression of E2F1, and chromatin immunoprecipitation was used to determine whether YB-1 bound to various E2F promoters as well as E2F1-regulated promoters. All P values were from two-sided tests. RESULTS: YB-1 levels were elevated in more aggressive tumors and were strongly associated with poor disease-free survival and distant metastasis-free survival. YB-1 expression was often associated with the expression of genes with E2F sites in their promoters. Cells expressing YB-1 siRNA grew substantially more slowly than control cells and formed tumors less readily in nude mice. Transcripts that were altered in cancer cell lines with YB-1 siRNA included 32 genes that are components of prognostic gene expression signatures. YB-1 regulated expression of an E2F1 promoter-reporter construct in A549 cells (eg, relative E2F1 promoter activity with control siRNA = 4.04; with YB-1 siRNA = 1.40, difference= -2.64, 95% confidence interval = -3.57 to -1.71, P < .001) and bound to the promoters of several well-defined E2F1 target genes. CONCLUSION: YB-1 expression is associated with the activity of E2F transcription factors and may control tumor cell growth by this mechanism.

Prognostic association of YB-1 expression in breast cancers: a matter of antibody.

The literature concerning the subcellular location of Y-box binding protein 1 (YB-1), its abundance in normal and cancer tissues, and its prognostic significance is replete with inconsistencies. An explanation for this could be due in part to the use of different antibodies in immunohistochemical and immunofluorescent labeling of cells and tissues. The inconsistencies could also be due to poor resolution of immunohistochemical data. We analyzed two cohorts of breast tumours for both abundance and subcellular location of YB-1 using three different antibodies; two targeting N-terminal epitopes (AB-a and AB-b) and another (AB-c) targeting a C-terminal epitope. We also investigated stress-induced nuclear translocation of YB-1 in cell culture. We report that both AB-a and AB-c detected increased YB-1 in the cytoplasm of high-grade breast cancers, and in those lacking estrogen and progesterone receptors; however the amount of YB-1 detected by AB-a in these cancers is significantly greater than that detected by AB-c. We confirm our previously published findings that AB-b is also detecting hnRNP A1, and cannot therefore be used to reliably detect YB-1 by immunohistochemistry. We also report that AB-a detected nuclear YB-1 in some tumour tissues and stress treated cells, whereas AB-c did not. To understand this, cancer cell lines were analyzed using native gel electrophoresis, which revealed that the antibodies detect different complexes in which YB-1 is a component. Our data suggest that different YB-1 antibodies show different staining patterns that are determined by the accessibility of epitopes, and this depends on the nature of the YB-1 complexes. It is important therefore to standardize the protocols if YB-1 is to be used reproducibly as a prognostic guide for different cancers.

Investigation of neuromuscular abnormalities in neurotrophin-3-deficient mice.

Neurotrophin-3 (NT-3) is a trophic factor that is essential for the normal development and maintenance of proprioceptive sensory neurons and is widely implicated as an important modulator of synaptic function and development. We have previously found that animals lacking NT-3 have a number of structural abnormalities in peripheral nerves and skeletal muscles. Here we investigated whether haploinsufficiency-induced reduction in NT-3 resulted in impaired neuromuscular performance and synaptic function. Motor nerve terminal function was tested by monitoring the uptake/release of the fluorescent membrane dye FM1-43 by the electrophysiological examination of synaptic transmission and electron microscopic determination of synaptic vesicle density at the presynaptic active zone. We investigated skeletal muscle form and function by measuring force in response to both nerve-mediated and direct muscle stimulation and by quantification of fiber number and area from transverse sections. Synaptic transmission was not markedly different between the two groups, although the uptake and release of FM1-43 were impaired in mature NT-3-deficient mice but not in immature mice. The electron microscopic examination of mature nerve terminals showed no genotype-dependent variation in the number of synaptic vesicles near the active zone. NT-3(+/-) mice had normal soleus muscle fiber numbers but their fibers had smaller cross-sectional areas and were more densely-packed than wild-type littermates. Moreover, the muscles of adult NT-3-deficient animals were weaker than those of wild-type animals to both nerve and direct muscle stimulation. The results indicate that a reduction in NT-3 availability during development impairs motor nerve terminal maturation and synaptic vesicle recycling and leads to a reduction in muscle fiber diameter.

A gene expression signature of invasive potential in metastatic melanoma cells.

BACKGROUND: We are investigating the molecular basis of melanoma by defining genomic characteristics that correlate with tumour phenotype in a novel panel of metastatic melanoma cell lines. The aim of this study is to identify new prognostic markers and therapeutic targets that might aid clinical cancer diagnosis and management. PRINCIPAL FINDINGS: Global transcript profiling identified a signature featuring decreased expression of developmental and lineage specification genes including MITF, EDNRB, DCT, and TYR, and increased expression of genes involved in interaction with the extracellular environment, such as PLAUR, VCAN, and HIF1a. Migration assays showed that the gene signature correlated with the invasive potential of the cell lines, and external validation by using publicly available data indicated that tumours with the invasive gene signature were less melanocytic and may be more aggressive. The invasion signature could be detected in both primary and metastatic tumours suggesting that gene expression conferring increased invasive potential in melanoma may occur independently of tumour stage. CONCLUSIONS: Our data supports the hypothesis that differential developmental gene expression may drive invasive potential in metastatic melanoma, and that melanoma heterogeneity may be explained by the differing capacity of melanoma cells to both withstand decreased expression of lineage specification genes and to respond to the tumour microenvironment. The invasion signature may provide new possibilities for predicting which primary tumours are more likely to metastasize, and which metastatic tumours might show a more aggressive clinical course.

Developmental loss of NT-3 in vivo results in reduced levels of myelin-specific proteins, a reduced extent of myelination and increased apoptosis of Schwann cells.

This work investigates the role of NT-3 in peripheral myelination. Recent articles, based in vitro, propose that NT-3 acting through its high-affinity receptor TrkC may act to inhibit myelin formation by enhancing Schwann cell motility and/or migration. Here, we investigate this hypothesis in vivo by examining myelination formation in NT-3 mutant mice. On the day of birth, soon after the onset of myelination, axons showed normal ensheathment by Schwann cells, no change in the proportion of axons which had begun to myelinate, and no change in either myelin thickness or number of myelin lamellae. However in postnatal day 21 mice, when myelination is substantially complete, we observed an unexpected reduction in mRNA and protein levels for MAG and P(0), and in myelin thickness. This is the opposite result to that predicted from previous in vitro studies, where removal of an inhibitory NT-3 signal would have been expected to enhance myelination. These results suggest that, in vivo, the importance of NT-3 as a major support factor for Schwann cells (Meier et al., (1999) J Neurosci 19:3847-3859) over-rides its potential role as an myelin inhibitor, with the net effect that loss of NT-3 results in degradation of Schwann cell functions, including myelination. In support of this idea, Schwann cells of NT-3 null mutants showed increased expression of activated caspase-3. Finally, we observed significant reduction in width of the Schwann cell periaxonal collar in NT-3 mutant animals suggesting that loss of NT-3 and resulting reduction in MAG levels may alter signaling at the axon-glial interface.

Neurotrophin-3 null mutant mice display a postnatal motor neuropathy.

This paper examines early postnatal development of the neuromuscular system in mice with a null mutation in the gene for neurotrophin-3. We report that alpha-motoneurons at first develop substantially normally, despite a known 15% deficit in their somal size [Woolley et al. (1999)Neurosci. Lett., 272, 107-110.] and the absence of proprioceptive input [Ernfors et al. (1994)Cell, 77, 503-512]. At birth, motor axons have extended into the muscle, forming normal-looking neuromuscular junctions with focal accumulations of acetylcholine receptors. Detailed ultrastructural analysis does however, reveal subtle abnormalities at this time, particularly a decrease in the extent of occupancy of the postsynaptic site by nerve terminals, and a small but significant deficit in myofibre number. After the relative normality of this early neuromuscular development, there then occurs a catastrophic postnatal loss of motor nerve terminals, resulting in complete denervation of hindlimb muscles by P7. In systematic semi-serial samples through the entire muscle endplate zones, no neuromuscular junctions can be found. Intramuscular axons are fragmented, as shown by both electron microscopic observations and neurofilament immunohistochemistry, and alpha-bungarotoxin detection of acetylcholine receptors indicates dispersal of the junctional accumulation. At earlier times (postnatal days three and four) the terminal Schwann cells show ultrastructural abnormalities, and preliminary observations suggest marked disturbance of myelination. Based on comparison with other literature, the peripheral nerve degeneration seems unlikely to have arisen as a secondary effect of de-afferentation. We discuss whether the neural degeneration is secondary to the disturbance of Schwann cell function, or due directly to a loss of neurotrophin-3 based support of the motoneuron.