Improved Long-term Culture of Epidermal Stem Cells Utilizing CD200R-expressing Feeder Cells

Tissue stem cells have great potential as a source of tissue for regenerative medicine. Epidermal stem cells (EpSCs) are the most accessible population of tissue stem cells that can regenerate the specialized cell types of tissues including the epidermis, smooth muscle and the sciatic nerve. However, the difficulties in isolation of the high numbers of EpSCs and their long-term culture have hampered the development of wider clinical applications of EpSCs. Here, we present a novel approach to EpSC culture that utilizes a feeder layer of Swiss 3T3 cells expressing the putative EpSC niche-specific molecule, CD200R. The colony forming efficiency of CD34+, α6-integrin+ EpSCs was increased on CD200R-expressing Swiss 3T3 feeder cells compared with normal Swiss 3T3 feeder cells. Furthermore, treatment with glycogen synthase kinase (GSK)-3 inhibitor, an activator of Wnt signaling, synergistically enhanced the proliferation of EpSCs. These results raise the possibility that an artificial microenvironment equivalent to in vivo niches will enable the persistent culture of EpSCs, thereby increasing the utility of EpSCs for tissue engineering and regeneration

PKCα mediates TGFβ-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11

Growth regulation of epithelial cells is of major concern because most human cancers arise from them. We demonstrated previously a novel signal pathway involving S100C/A11 for high Ca2+-induced growth inhibition of normal human keratinocytes (Sakaguchi, M., M. Miyazaki, M. Takaishi, Y. Sakaguchi, E. Makino, N. Kataoka, H. Yamada, M. Namba, and N.H. Huh. 2003. J. Cell Biol. 163:825–835). This paper addresses a question whether transforming growth factor β (TGFβ) shares the pathway with high Ca2+. On exposure of the cells to TGFβ1, S100C/A11 was phosphorylated, bound to nucleolin, and transferred to the nucleus, resulting in induction of p21WAF1/CIP1 and p15INK4B through activation of Sp1. Protein kinase C α (PKCα) was shown to phosphorylate 10Thr of S100C/A11, which is a critical event for the signal transduction. The TGFβ1-induced growth inhibition was almost completely mitigated when PKCα activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCα–S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFβ1

Molecular-Targeted Therapies for Epidermal Growth Factor Receptor and Its Resistance Mechanisms

Cancer therapies targeting epidermal growth factor receptor (EGFR), such as small-molecule kinase inhibitors and monoclonal antibodies, have been developed as standard therapies for several cancers, such as non-small cell lung cancer, colorectal cancer, pancreatic cancer, breast cancer, and squamous cell carcinoma of the head and neck. Although these therapies can significantly prolong progression-free survival, curative effects are not often achieved because of intrinsic and/or acquired resistance. The resistance mechanisms to EGFR-targeted therapies can be categorized as resistant gene mutations, activation of alternative pathways, phenotypic transformation, and resistance to apoptotic cell death. Analysis of the processes that modulate EGFR signal transduction by EGFR-targeted inhibitors, such as tyrosine kinase inhibitors and monoclonal antibodies, has revealed new therapeutic opportunities and has elucidated novel mechanisms contributing to the discovery of more effective anticancer treatments. In this review, we discuss the roles of EGFR in cancer development, therapeutic strategies for targeting EGFR, and resistance mechanisms to EGFR-targeted therapies, with a focus on cancer therapies for individual patients

Up-regulation of Syndecan-4 contributes to TGF-β1-induced epithelial to mesenchymal transition in lung adenocarcinoma A549 cells

Syndecan-4 (SDC4) is a cell-surface proteoglycan associated with cell adhesion, motility, and intracellular signaling. Here, we present that SDC4 functions as a positive regulator of the transforming growth factor (TGF)-β1-induced epithelial to mesenchymal transition (EMT) via Snail in lung adenocarcinoma, A549 cells. TGF-β1 up-regulated the expression of SDC4, accompanied by the induction of EMT. Wound-healing and transwell chemotaxis assay revealed that SDC4 promoted cell migration and invasion. SDC4 knockdown recovered the E-cadherin and decreased vimentin and Snail expression in EMT-induced A549 cells. However, depletion of SDC4 resulted in little change of the Slug protein expression and mesenchymal cell morphology induced by TGF-β1. The double knockdown of SDC-4 and Slug was required for reversal of epithelial morphology; it did not occur from the SDC4 single knockdown. These findings suggest that Snail is a transcriptional factor downstream of SDC4, and SDC4 regulates TGF-β1-induced EMT by cooperating with Slug. Our data provide a novel insight into cellular mechanisms, whereby the cell-surface proteoglycan modulated TGF-β1-induced EMT in lung adenocarcinoma, A549 cells

Receptor Tyrosine Kinase-Targeted Cancer Therapy

In the past two decades, several molecular targeted inhibitors have been developed and evaluated clinically to improve the survival of patients with cancer. Molecular targeted inhibitors inhibit the activities of pathogenic tyrosine kinases. Particularly, aberrant receptor tyrosine kinase (RTK) activation is a potential therapeutic target. An increased understanding of genetics, cellular biology and structural biology has led to the development of numerous important therapeutics. Pathogenic RTK mutations, deletions, translocations and amplification/over-expressions have been identified and are currently being examined for their roles in cancers. Therapies targeting RTKs are categorized as small-molecule inhibitors and monoclonal antibodies. Studies are underway to explore abnormalities in 20 types of RTK subfamilies in patients with cancer or other diseases. In this review, we describe representative RTKs important for developing cancer therapeutics and predicting or evaluated resistance mechanisms

Novel protein kinase C isoforms regulate human keratinocyte differentiation by activating a p38 delta mitogen-activated protein kinase cascade that targets CCAAT/enhancer-binding protein alpha

The novel protein kinase C (nPKC) isoforms are important regulators of human involucrin (hINV) gene expression during keratinocyte differentiation (Efimova, T., and Eckert, R. L. (2000) J. Biol. Chem. 275, 1601-1607). Although the regulatory mechanism involves mitogen-activated protein kinase (MAPK) activation, the role of individual MAPK isoforms has not been elucidated. We therefore examined the effects of individual nPKCs on MAPK activation. We observe unique changes whereby nPKC expression simultaneously increases p38 activity and decreases ERK1 and ERK2 activity. Although p38 alpha, p38 beta, and p38 delta are expressed in keratinocytes, only a single isoform, p38 delta, accounts for the increased p38 activity. Parallel studies indicate that this isoform is also activated by treatment with the keratinocyte regulatory agents, 12-O-tetradecanoylphorbol-13-acetate, calcium, and okadaic acid. These changes in MAPK activity are associated with increased C/EBP alpha transcription factor expression and DNA binding to the hINV promoter and increased hINV gene expression. Expression of PKC delta, PKC epsilon, or PKC eta causes a 10-fold increase in hINV promoter activity, whereas C/EBP alpha expression produces a 25-fold increase. However, simultaneous expression of both proteins causes a synergistic 100-fold increase in promoter activity. These responses are eliminated by the dominant-negative C/EBP isoform, GADD153, and are also inhibited by dominant-negative forms of Ras, MEKK1, MEK3, and p38. These results suggest that the nPKC isoforms produce a unique shift in MAPK activity via a Ras, MEKK1, MEK3 pathway, to increase p38 delta and inhibit ERK1/2 and ultimately increase C/EBP alpha binding to the hINV promoter and hINV gene expression