Loss of p38δ mitogen-activated protein kinase expression promotes oesophageal squamous cell carcinoma proliferation, migration and anchorage-independent growth.

Oesophageal cancer is an aggressive tumour which responds poorly to both chemotherapy and radiation therapy and has a poor prognosis. Thus, a greater understanding of the biology of oesophageal cancer is needed in order to identify novel therapeutic targets. Among these targets p38 MAPK isoforms are becoming increasingly important for a variety of cellular functions. The physiological functions of p38α and -β are now well documented in contrast to -γ and -δ which are comparatively under-studied and ill-defined. A major obstacle to deciphering the role(s) of the latter two p38 isoforms is the lack of specific chemical activators and inhibitors. In this study, we analysed p38 MAPK isoform expression in oesophageal cancer cell lines as well as human normal and tumour tissue. We observed specifically differential p38δ expression. The role(s) of p38δ and active (phosphorylated) p38δ (p-p38δ) in oesophageal squamous cell carcinoma (OESCC) was delineated using wild-type p38δ as well as active p-p38δ, generated by fusing p38δ to its upstream activator MKK6b(E) via a decapeptide (Gly-Glu)5 linker. OESCC cell lines which are p38δ-negative (KE-3 and -8) grew more quickly than cell lines (KE-6 and -10) which express endogenous p38δ. Re-introduction of p38δ resulted in a time-dependent decrease in OESCC cell proliferation which was exacerbated with p-p38δ. In addition, we observed that p38δ and p-p38δ negatively regulated OESCC cell migration in vitro. Finally both p38δ and p-p38δ altered OESCC anchorage-independent growth. Our results suggest that p38δ and p-p38δ have a role in the suppression of OESCC. Our research may provide a new potential target for the treatment of oesophageal cancer.

X-linked inhibitor of apoptosis protein mediates tumor cell resistance to antibody-dependent cellular cytotoxicity.

Inflammatory breast cancer (IBC) is the deadliest, distinct subtype of breast cancer. High expression of epidermal growth factor receptors [EGFR or human epidermal growth factor receptor 2 (HER2)] in IBC tumors has prompted trials of anti-EGFR/HER2 monoclonal antibodies to inhibit oncogenic signaling; however, de novo and acquired therapeutic resistance is common. Another critical function of these antibodies is to mediate antibody-dependent cellular cytotoxicity (ADCC), which enables immune effector cells to engage tumors and deliver granzymes, activating executioner caspases. We hypothesized that high expression of anti-apoptotic molecules in tumors would render them resistant to ADCC. Herein, we demonstrate that the most potent caspase inhibitor, X-linked inhibitor of apoptosis protein (XIAP), overexpressed in IBC, drives resistance to ADCC mediated by cetuximab (anti-EGFR) and trastuzumab (anti-HER2). Overexpression of XIAP in parental IBC cell lines enhances resistance to ADCC; conversely, targeted downregulation of XIAP in ADCC-resistant IBC cells renders them sensitive. As hypothesized, this ADCC resistance is in part a result of the ability of XIAP to inhibit caspase activity; however, we also unexpectedly found that resistance was dependent on XIAP-mediated, caspase-independent suppression of reactive oxygen species (ROS) accumulation, which otherwise occurs during ADCC. Transcriptome analysis supported these observations by revealing modulation of genes involved in immunosuppression and oxidative stress response in XIAP-overexpressing, ADCC-resistant cells. We conclude that XIAP is a critical modulator of ADCC responsiveness, operating through both caspase-dependent and -independent mechanisms. These results suggest that strategies targeting the effects of XIAP on caspase activation and ROS suppression have the potential to enhance the activity of monoclonal antibody-based immunotherapy.

Mir-190b negatively contributes to the Trypanosoma cruzi -infected cell survival by repressing PTEN protein expression

Chagas disease, which is caused by the intracellular protozoan Trypanosoma cruzi , is a serious health problem in Latin America. The heart is one of the major organs affected by this parasitic infection. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection, and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. Previous studies have reported that the establishment of parasitism is connected to the activation of the phosphatidylinositol- 3 kinase (PI3K), which controls important steps in cellular metabolism by regulating the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Particularly, the tumour suppressor PTEN is a negative regulator of PI3K signalling. However, mechanistic details of the modulatory activity of PTEN on Chagas disease have not been elucidated. To address this question, H9c2 cells were infected with T. cruzi Berenice 62 strain and the expression of a specific set of microRNAs (miRNAs) were investigated. Our cellular model demonstrated that miRNA-190b is correlated to the decrease of cellular viability rates by negatively modulating PTEN protein expression in T. cruzi-infected cells.

Microfluidics-based single-cell functional proteomics microchip for portraying protein signal transduction networks within the framework of physicochemical principles, with applications in fundamental and translational cancer research

Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing unique opportunities. This thesis describes an emerging microfluidics-based toolkit for single cell functional proteomics, focusing on the development of the single cell barcode chips (SCBCs) with applications in fundamental and translational cancer research.

The microchip designed to simultaneously quantify a panel of secreted, cytoplasmic and membrane proteins from single cells will be discussed at the beginning, which is the prototype for subsequent proteomic microchips with more sophisticated design in preclinical cancer research or clinical applications. The SCBCs are a highly versatile and information rich tool for single-cell functional proteomics. They are based upon isolating individual cells, or defined number of cells, within microchambers, each of which is equipped with a large antibody microarray (the barcode), with between a few hundred to ten thousand microchambers included within a single microchip. Functional proteomics assays at single-cell resolution yield unique pieces of information that significantly shape the way of thinking on cancer research. An in-depth discussion about analysis and interpretation of the unique information such as functional protein fluctuations and protein-protein correlative interactions will follow.

The SCBC is a powerful tool to resolve the functional heterogeneity of cancer cells. It has the capacity to extract a comprehensive picture of the signal transduction network from single tumor cells and thus provides insight into the effect of targeted therapies on protein signaling networks. We will demonstrate this point through applying the SCBCs to investigate three isogenic cell lines of glioblastoma multiforme (GBM).

The cancer cell population is highly heterogeneous with high-amplitude fluctuation at the single cell level, which in turn grants the robustness of the entire population. The concept that a stable population existing in the presence of random fluctuations is reminiscent of many physical systems that are successfully understood using statistical physics. Thus, tools derived from that field can probably be applied to using fluctuations to determine the nature of signaling networks. In the second part of the thesis, we will focus on such a case to use thermodynamics-motivated principles to understand cancer cell hypoxia, where single cell proteomics assays coupled with a quantitative version of Le Chatelier's principle derived from statistical mechanics yield detailed and surprising predictions, which were found to be correct in both cell line and primary tumor model.

The third part of the thesis demonstrates the application of this technology in the preclinical cancer research to study the GBM cancer cell resistance to molecular targeted therapy. Physical approaches to anticipate therapy resistance and to identify effective therapy combinations will be discussed in detail. Our approach is based upon elucidating the signaling coordination within the phosphoprotein signaling pathways that are hyperactivated in human GBMs, and interrogating how that coordination responds to the perturbation of targeted inhibitor. Strongly coupled protein-protein interactions constitute most signaling cascades. A physical analogy of such a system is the strongly coupled atom-atom interactions in a crystal lattice. Similar to decomposing the atomic interactions into a series of independent normal vibrational modes, a simplified picture of signaling network coordination can also be achieved by diagonalizing protein-protein correlation or covariance matrices to decompose the pairwise correlative interactions into a set of distinct linear combinations of signaling proteins (i.e. independent signaling modes). By doing so, two independent signaling modes – one associated with mTOR signaling and a second associated with ERK/Src signaling have been resolved, which in turn allow us to anticipate resistance, and to design combination therapies that are effective, as well as identify those therapies and therapy combinations that will be ineffective. We validated our predictions in mouse tumor models and all predictions were borne out.

In the last part, some preliminary results about the clinical translation of single-cell proteomics chips will be presented. The successful demonstration of our work on human-derived xenografts provides the rationale to extend our current work into the clinic. It will enable us to interrogate GBM tumor samples in a way that could potentially yield a straightforward, rapid interpretation so that we can give therapeutic guidance to the attending physicians within a clinical relevant time scale. The technical challenges of the clinical translation will be presented and our solutions to address the challenges will be discussed as well. A clinical case study will then follow, where some preliminary data collected from a pediatric GBM patient bearing an EGFR amplified tumor will be presented to demonstrate the general protocol and the workflow of the proposed clinical studies.

Purification, biochemical characterization and localization at single cell resolution of Matrix Gla Protein (MGP) and Bone Gla Protein (BGP) in the teleost fish Argyrosomus regius

Tese de dout. em Química, Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2002

Analysis of contributions of herpes simplex virus type 1 UL43 protein to induction of cell-cell fusion

Purpose: To investigate whether UL43 protein, which is highly conserved in alpha- and gamma herpes viruses, and a non-glycosylated transmembrane protein, is involved in virus entry and virus-induced cell fusion. Methods: Mutagenesis was accomplished by a markerless two-step Red recombination mutagenesis system implemented on the Herpes simplex virus 1 (HSV-1) bacterial artificial chromosome (BAC). Growth properties of HSV-1 UL43 mutants were analyzed using plaque morphology and one-step growth kinetics. SDS-PAGE and Western blot was employed to assay the synthesis of the viral glycoproteins. Virus-penetration was assayed to determine if UL43 protein is required for efficient virus entry. Results: Lack of UL43 expression resulted in significantly reduced plaque sizes of syncytial mutant viruses and inhibited cell fusion induced by gBΔ28 or gKsyn20 (p < 0.05). Deletion of UL43 did not affect overall expression levels of viral glycoproteins gB, gC, gD, and gH on HSV-1(F) BAC infected cell surfaces. Moreover, mutant viruses lacking UL43 gene exhibited slower kinetics of entry into Vero cells than the parental HSV-1(F) BAC. Conclusion: Thus, these results suggest an important role for UL43 protein in mediating virus-induced membrane fusion and efficient entry of virion into target cells.

Deciphering proteome dynamics using cell-type selective metabolic protein labeling in the fruit fly Drosophila melanogaster

Otto-von-Guericke-Universität Magdeburg, Fakultät für Naturwissenschaften, Dissertation, 2016

Regulation of electrical transmission by protein kinase C in Aplysia bag cell neurons

Electrical synapses are composed of gap junctions, made from paired hemi-channels that allow for the transfer of current from one neuron to another. Gap junctions mediate electrical transmission in neurons, where they synchronize spiking and promote rapid transmission, thereby influencing the coordination, pattern, and frequency of firing. In the marine snail, Aplysia calfornica, two clusters of neuroendocrine bag cell neurons use electrical synapses to synchronize a 30-min burst of action potentials, known as the afterdischarge, which releases egg-laying hormone and induces reproduction. In culture, paired bag cell neurons present a junctional conductance that is non-rectifying and largely voltage-independent. During the afterdischarge, PKC is activated, which is known to increase voltage-gated Ca2+ current; yet, little is understood as to how this pathway impacts electrical transmission. The transfer of presynaptic spike-like waveforms (generated in voltage-clamp) to the postsynaptic cell (measured in current-clamp) was monitored with or without PKC activation. It was found that pretreatment with the PKC activator, phorbol-12-myristate-13-acetate (PMA), enhanced junctional conductance between bag cell neurons. Furthermore, in control, presynaptic action potential waveforms mainly evoked postsynaptic electrotonic potentials at both -60 and -40 mV. However, with PKC activation the presynaptic stimulus consistently elicited postsynaptic action potentials from resting potentials of -40 mV, and would occasionally result in firing from repetitive input at -60 mV. Moreover, to assess whether this enhanced electrical transmission genuinely reflects a greater junctional conductance or a change in postsynaptic responsiveness, a fast-phase junctional-like current was applied to single bag cell neurons. Neurons in PMA always fired action potentials in response to current injection as opposed to control, which were less likely to spike. This outcome did not change when the junctional-like current was artificially enhanced in control conditions. Also, in response to fast- and slow-phase electrotonic potential (ETP) waveforms, Ca2+ current was markedly larger in single PMA-treated neurons. These findings suggest that PKC activation may contribute to afterdischarge fidelity by recruiting postsynaptic Ca2+ current to promote synchronous network firing. Finally, Aplysia gap junction genes (innexins) were transfected into mouse N2A cells and characterized. This revealed a biophysical and pharmacological profile similar to native gap junctions.

tRNA anticodon loop modifications ensure protein homeostasis and cell morphogenesis in yeast

Using budding yeast, we investigated a negative interaction network among genes for tRNA modifications previously implicated in anticodon-codon interaction: 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm5s2U34: ELP3, URM1), pseudouridine (Ψ38/39: DEG1) and cyclic N6-threonyl-carbamoyl-adenosine (ct6A37: TCD1). In line with functional cross talk between these modifications, we find that combined removal of either ct6A37 or Ψ38/39 and mcm5U34 or s2U34 results in morphologically altered cells with synthetic growth defects. Phenotypic suppression by tRNA overexpression suggests that these defects are caused by malfunction of tRNALysUUU or tRNAGlnUUG, respectively. Indeed, mRNA translation and synthesis of the Gln-rich prion Rnq1 are severely impaired in the absence of Ψ38/39 and mcm5U34 or s2U34, and this defect can be rescued by overexpression of tRNAGlnUUG. Surprisingly, we find that combined modification defects in the anticodon loops of different tRNAs induce similar cell polarity- and nuclear segregation defects that are accompanied by increased aggregation of cellular proteins. Since conditional expression of an artificial aggregation-prone protein triggered similar cytological aberrancies, protein aggregation is likely responsible for loss of morphogenesis and cytokinesis control in mutants with inappropriate tRNA anticodon loop modifications.

Regulation of prostate cancer cell function by activators of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is a key regulator of cell energy homeostasis. More recently, it has become apparent that AMPK regulates cell proliferation, migration and inflammation. Previous evidence has suggested that AMPK may influence proliferation and invasion by regulating the pro-proliferative mitogen-activated protein kinases (MAPKs). However, the mechanisms underlying this crosstalk between AMPK and MAPK signalling are not fully understood. As AMPK activation has been reported to have anti-proliferative effects, there has been increasing interest in AMPK activation as a therapeutic target for tumourigenesis. The aim of this study was to investigate whether AMPK activation influenced prostate cancer (PC) cell line proliferation, migration and signalling. Therefore, different PC cell lines were incubated with two structurally-unrelated molecules that activate AMPK by different mechanisms, AICAR and A769662. Both chemicals activated AMPK in a concentration- and time-dependent manner in PC3, DU145 and LNCaP cell lines. AMPK activity as assessed by AMPK activating phosphorylation as well as phosphorylation of the AMPK substrate ACC increased along with tumour severity in PC biopsies. Furthermore, both activators of AMPK decreased cell proliferation and migration in the androgen-independent PC cell lines PC3 and DU145. Inhibition of proliferation by A769662 was attenuated in AMPK α1-/- AMPK α2-/- knockout (KO) mouse embryonic fibroblasts (MEFs) compared to wild type (WT) MEFs, and the inhibitory effect on migration of AICAR lost significance in PC3 cells infected with adenoviruses expressing a dominant negative AMPK α mutant, indicating these effects are partially mediated by AMPK. Furthermore, long-term activation of AMPK was associated with inhibition of both the phosphatidylinositol 3’-kinase/protein kinase B (PI3K/Akt) signalling pathway in addition to the extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathway. Indeed, the actions of AMPK activators on PC cell line viability were mimicked by selective inhibitors of Akt and ERK1/2 pathways. In contrast to the effects of prolonged incubation with AMPK activators, short-term incubation with AMPK activators had no effect on epidermal growth factor (EGF)-stimulated ERK1/2 phosphorylation in PC cell lines. In addition, AMPK activation did not influence phosphorylation of the other MAPK family members p38 and JNK. Interestingly, both AICAR and A769662 decreased EGF-stimulated ERK5 phosphorylation in PC3, DU145 and LNCaP cells as assessed with an anti-phospho-ERK5 antibody. Further characterisation of this effect indicated that prior stimulation with the AMPK activators had no effect on ERK5 phosphorylation stimulated by transient transfection with a constitutively active ERK5 kinase (MEK5DD), which represents the only known canonical kinase for ERK5. Intriguingly, the pattern of EGF-stimulated ERK5 phosphorylation was distinct from that mediated by MEK5DD activation of ERK5. This finding indicates that AMPK activation inhibits EGF-stimulated ERK5 phosphorylation at a point at or above the level of MEK5, although why EGF and constitutively active MEK5 stimulate markedly different immunoreactive species recognised by the anti-phospho-ERK5 antibody requires further study. A769662 had a tendency to reduce EGF-stimulated ERK5 phosphorylation in WT MEFs, yet was without effect in MEFs lacking AMPK. These data indicate that AMPK may underlie the effect of A769662 to reduce EGF-stimulated ERK5 phosphorylation. Prolonged stimulation of PC cell lines with AICAR or A769662 inhibited EGF-stimulated Akt Ser473 phosphorylation, whereas only incubation with A769662 rapidly inhibited Akt phosphorylation. This difference in the actions of the different AMPK activators may suggest an AMPK-independent effect of A769662. Furthermore, AICAR increased phosphorylation of Akt in WT MEFs, an effect that was absent in MEFs lacking AMPK, indicating that this effect of AICAR may be AMPK-dependent. Taken together, the data presented in this study suggest that AMPK activators markedly inhibit proliferation and migration of PC cell lines, reduce EGF-stimulated ERK1/2 and Akt phosphorylation after prolonged incubation and rapidly inhibit ERK5 phosphorylation. Both AMPK activators exhibit a number of effects that are likely to be independent of AMPK in PC cell lines, although inhibition of ERK1/2, ERK5 and Akt may underlie the effects of AMPK activators on proliferation, viability and migration. Further studies are required to understand the crosstalk between those signalling pathways and their underlying significance in PC progression.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

Stem cell regulatory gene expression in human adult dental pulp and periodontal ligament cells undergoing odontogenic/osteogenic differentiation

Introduction During development and regeneration, odontogenesis and osteogenesis are initiated by a cascade of signals driven by several master regulatory genes. Methods In this study, we investigated the differential expression of 84 stem cell–related genes in dental pulp cells (DPCs) and periodontal ligament cells (PDLCs) undergoing odontogenic/osteogenic differentiation. Results Our results showed that, although there was considerable overlap, certain genes had more differential expression in PDLCs than in DPCs. CCND2, DLL1, and MME were the major upregulated genes in both PDLCs and DPCs, whereas KRT15 was the only gene significantly downregulated in PDLCs and DPCs in both odontogenic and osteogenic differentiation. Interestingly, a large number of regulatory genes in odontogenic and osteogenic differentiation interact or crosstalk via Notch, Wnt, transforming growth factor β (TGF-β)/bone morphogenic protein (BMP), and cadherin signaling pathways, such as the regulation of APC, DLL1, CCND2, BMP2, and CDH1. Using a rat dental pulp and periodontal defect model, the expression and distribution of both BMP2 and CDH1 have been verified for their spatial localization in dental pulp and periodontal tissue regeneration. Conclusions This study has generated an overview of stem cell–related gene expression in DPCs and PDLCs during odontogenic/osteogenic differentiation and revealed that these genes may interact through the Notch, Wnt, TGF-β/BMP, and cadherin signalling pathways to play a crucial role in determining the fate of dental derived cell and dental tissue regeneration. These findings provided a new insight into the molecular mechanisms of the dental tissue mineralization and regeneration

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (l-lactide)-block-poly (l-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.