技术资料

Despite many years of intensive effort,there is surprisingly little consensus on the most suitable markers with which to locate and isolate stem cells from adult tissues. By comparison,the study of cancer stem cells is still in its infancy; so,unsurprisingly,there is great uncertainty as to the identity of these cells. Stem cell markers can be broadly categorized into molecular determinants of self-renewal,clonogenicity,multipotentiality,adherence to the niche,and longevity. This review assesses the utility of recognizing cancer stem cells by virtue of high expression of aldehyde dehydrogenases (ALDHs),probably significant determinants of cell survival through their ability to detoxify many potentially cytotoxic molecules,and contributing to drug resistance. Antibodies are available against the ALDH enzyme family,but the vast majority of studies have used cell sorting techniques to enrich for cells expressing these enzymes. Live cells expressing high ALDH activity are usually identified by the ALDEFLUOR kit and sorted by fluorescence activated cell sorting (FACS). For many human tumours,but notably breast cancer,cell selection based upon ALDH activity appears to be a useful marker for enriching for cells with tumour-initiating activity (presumed cancer stem cells) in immunodeficient mice,and indeed the frequency of so-called ALDH(bri) cells in many tumours can be an independent prognostic indicator. View Publication

In models of acute lung injury,CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands,including CXCL1 and CXCL2/3,are chemotactic for PMNs,CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung,aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice,but not in littermate CXCR2-/- mice. Surprisingly,lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium,but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely,CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment,inconsistent with a role of CXCR2 on PMNs alone. Cell culture,immunohistochemistry,flow cytometry,and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury. View Publication

In recent years,the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated,but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs,and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor,Gleevec (imatinib mesylate; Novartis International,Basel,Switzerland,http://www.novartis.com),to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5,sarcomeric proteins beta-MHC and MLC-2v,and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy,the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating,myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters. View Publication

Little is known about the origin of basal-like breast cancers,an aggressive disease that is highly similar to BRCA1-mutant breast cancers. p63 family proteins that are structurally related to the p53 suppressor protein are known to function in stem cell regulation and stratified epithelia development in multiple tissues,and p63 expression may be a marker of basal-like breast cancers. Here we report that ΔNp63 isoforms of p63 are transcriptional targets for positive regulation by BRCA1. Our analyses of breast cancer tissue microarrays and BRCA1-modulated breast cancer cell lines do not support earlier reports that p63 is a marker of basal-like or BRCA1 mutant cancers. Nevertheless,we found that BRCA1 interacts with the specific p63 isoform ΔNp63γ along with transcription factor isoforms AP-2α and AP-2γ. BRCA1 required ΔNp63γ and AP-2γ to localize to an intronic enhancer region within the p63 gene to upregulate transcription of the ΔNp63 isoforms. In mammary stem/progenitor cells,siRNA-mediated knockdown of ΔNp63 expression resulted in genomic instability,increased cell proliferation,loss of DNA damage checkpoint control,and impaired growth control. Together,our findings establish that transcriptional upregulation of ΔNp63 proteins is critical for BRCA1 suppressor function and that defects in BRCA1-ΔNp63 signaling are key events in the pathogenesis of basal-like breast cancer. View Publication

The origins of the epithelial cells participating in the development,tissue homeostasis,and cancer of the human breast are poorly understood. However,emerging evidence suggests a role for adult tissue-specific stem cells in these processes. In a hierarchical manner,these generate the two main mammary cell lineages,producing an increasing number of cells with distinct properties. Understanding the biological characteristics of human breast stem cells and their progeny is crucial in attempts to compare the features of normal stem cells and cancer precursor cells and distinguish these from nonprecursor cells and cells from the bulk of a tumor. A historical overview of research on human breast stem cells in primary tissue and in culture reveals the progress that has been made in this area,whereas a focus on the cell-of-origin and reprogramming that occurs during neoplastic conversion provides insight into the enigmatic way in which human breast cancers are skewed toward the luminal epithelial lineage. View Publication