Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.
Cardiac development arises from two sources of mesoderm progenitors, the first heart field (FHF) and the second (SHF). Mesp1 has been proposed to mark the most primitive multipotent cardiac progenitors common for both heart fields. Here, using clonal analysis of the earliest prospective cardiovascular progenitors in a temporally controlled manner during early gastrulation, we found that Mesp1 progenitors consist of two temporally distinct pools of progenitors restricted to either the FHF or the SHF. FHF progenitors were unipotent, whereas SHF progenitors were either unipotent or bipotent. Microarray and single-cell PCR with reverse transcription analysis of Mesp1 progenitors revealed the existence of molecularly distinct populations of Mesp1 progenitors, consistent with their lineage and regional contribution. Together, these results provide evidence that heart development arises from distinct populations of unipotent and bipotent cardiac progenitors that independently express Mesp1 at different time points during their specification, revealing that the regional segregation and lineage restriction of cardiac progenitors occur very early during gastrulation.
Surveillance for alveolar echinococcosis in central Europe was initiated in 1998. On a voluntary basis, 559 patients were reported to the registry. Most cases originated from rural communities in regions from eastern France to western Austria; single cases were reported far away from the disease-“endemic” zone throughout central Europe. Of 210 patients, 61.4% were involved in vocational or part-time farming, gardening, forestry, or hunting. Patients were diagnosed at a mean age of 52.5 years; 78% had symptoms. Alveolar echinococcosis primarily manifested as a liver disease. Of the 559 patients, 190 (34%) were already affected by spread of the parasitic larval tissue. Of 408 (73%) patients alive in 2000, 4.9% were cured. The increasing prevalence of Echinococcus multilocularis in foxes in rural and urban areas of central Europe and the occurrence of cases outside the alveolar echinococcosis–endemic regions suggest that this disease deserves increased attention.
Papillary thyroid carcinomas (PTCs) that invade into local structures are associated with a poor prognosis, but the mechanisms for PTC invasion are incompletely defined, limiting the development of new therapies. To characterize biological processes involved in PTC invasion, we analyzed the gene expression profiles of microscopically dissected intratumoral samples from central and invasive regions of seven widely invasive PTCs and normal thyroid tissue by oligonucleotide microarray and performed confirmatory expression and functional studies. In comparison with the central regions of primary PTCs, the invasive fronts overexpressed TGF , NFB and integrin pathway members, and regulators of small G proteins and CDC42. Moreover, reduced levels of mRNAs encoding proteins involved in cell-cell adhesion and communication were identified, consistent with epithelial-to-mesenchymal transition (EMT). To confirm that aggressive PTCs were characterized by EMT, 34 additional PTCs were examined for expression of vimentin, a hallmark of EMT. Overexpression of vimentin was associated with PTC invasion and nodal metastasis. Functional, in vitro studies demonstrated that vimentin was required both for the development and maintenance of a mesenchymal morphology and invasiveness in thyroid cancer cells. We conclude that EMT is common in PTC invasion and that vimentin regulates thyroid cancer EMT in vitro.cdc42 ͉ runx2 ͉ thyroid cancer ͉ vimentin T hyroid carcinoma is the most common classical endocrine malignancy, and its incidence is rising rapidly, due almost entirely to an increase in papillary thyroid carcinoma (PTC) diagnoses (1). Patients diagnosed with PTC at an early stage have an excellent prognosis; however, individuals with large, invasive tumors and/or distant metastases have a 5-year survival rate of Ϸ40% (2, 3). Thus, there is a need to better understand the molecular causes of thyroid cancer progression to develop new treatment options.The genetic defects believed to be responsible for PTC initiation have been identified in the majority of cases; these include genetic rearrangements involving the tyrosine kinase domain of RET and activating mutations of BRAF and RAS (3-5). Although some correlation studies support an association between specific genetic alterations and aggressive cancer behavior (6-9), there are a number of events that are found nearly exclusively in aggressive PTCs, including mutations of P53 (10, 11), dysregulated -catenin signaling (12), up-regulation of cyclin D1 (13), and overexpression of metastasis-promoting, angiogenic, and/or cell adhesion-related genes (14-20). We have determined that invasive regions of primary PTCs are frequently characterized by enhanced Akt activity and cytosolic p27 localization (21, 22). We, and others, have also demonstrated functional roles for PI3 kinase, Akt, and p27 in PTC cell invasion in vitro (16,23,24). However, the correlation between increased Akt activity and invasion was not found for PTCs with activating BRAF mutations. Most importantly, these focused s...
SUMMARYThe events regulating human preimplantation development are still largely unknown owing to a scarcity of material, ethical and legal limitations and a lack of reliable techniques to faithfully amplify the transcriptome of a single cell. Nonetheless, human embryology is gathering renewed interest due to its close relationship with both stem cell biology and epigenetic reprogramming to pluripotency and their importance in regenerative medicine. Carefully timed genome-wide transcript analyses of single oocytes and embryos uncovered a series of successive waves of embryonic transcriptional initiation that start as early as the 2-cell stage. In addition, we identified the hierarchical activation of genes involved in the regulation of pluripotency. Finally, we developed HumER, a database of human preimplantation gene expression, to serve the scientific community. Importantly, our work links early transcription in the human embryo with the correct execution of the pluripotency program later in development and paves the way for the identification of factors to improve epigenetic reprogramming.
Most genes involved in DNA replication in the yeast Saccharomyces cerevisiae are transcribed transiently during late G1 as cells become committed to a new cell cycle at Start. Their promoters all contain one or more versions of an 8-base-pair motif (ACGCGTNA) containing an MluI restriction enzyme site and called the MluI cell-cycle box (MCB). MCBs are both necessary and sufficient for the late G1-specific transcription of the TMP1 thymidylate synthase and POL1 DNA polymerase genes. A different late G1-specific 8-base-pair transcription element called the SCB (CACGAAAA; ref. 5) is bound by a factor containing the Swi4 and Swi6 proteins. We describe here the formation in vitro of complexes on TMP1 MCBs that contain the Swi6 protein and, we suggest, a protein of relative molecular mass 120,000 (p120) that is distinct from Swi4. Transcription due to SCBs and MCBs occurs in the absence of Swi6 but it is no longer correctly regulated in the cell cycle. We suggest that Swi6 is an essential regulatory subunit of two different Start-dependent transcription factors. One factor (SBF) contains Swi4 and binds to SCBs, whereas the other (MBF) contains the protein p120 and binds MCBs.
The cancer microenvironment and interaction between cancer and stromal cells play critical roles in tumor development and progression. The molecular features of cancer stroma are less well understood than those of cancer cells. Cancer-associated stromal fibroblasts are the predominant component of stroma associated with colon cancer and its functions remain unclear. Fibroblast cell cultures were established from metastatic colon cancer in liver, liver away from the metastatic lesions, and skin from three patients with metastatic colorectal cancer. We generated expression profiles of cancer-associated fibroblasts using oligochip arrays and compared them to those of uninvolved fibroblasts. The conditioned media from the cancer-associated fibroblast cultures enhanced proliferation of colon cancer cell line HCT116 to a greater extent than cultures from uninvolved fibroblasts. In microarray expression analysis, cancer-associated fibroblasts clustered tightly into one group and skin fibroblasts into another. Approximately 170 of 22 000 genes were upregulated in cancer-associated fibroblasts (fold change42, Po0.05) as compared to skin fibroblasts, including many genes encoding cell adhesion molecules, growth factors, and COX2. By immunohistochemistry in-vivo, we confirmed COX2 and TGFB2 expression in cancer-associated fibroblasts in metastatic colon cancer. The distinct molecular expression profiles of cancerassociated fibroblasts in colon cancer metastasis support the notion that these fibroblasts form a favorable microenvironment for cancer cells.
Genomic instability has been observed in essentially all sporadic carcinomas. Here we use Drosophila epithelial cells to address the role of chromosomal instability in cancer development as they have proved useful for elucidating the molecular mechanisms underlying tumorigenic growth. We first show that chromosomal instability leads to an apoptotic response. Interestingly, this response is p53 independent, as opposed to mammalian cells, and depends on the activation of the c-Jun N-terminal kinase (JNK) signaling cascade. When prevented from undergoing programmed cell death (PCD), chromosomal instability induces neoplasic overgrowth. These tumor-like tissues are able to grow extensively and metastasize when transplanted into the abdomen of adult hosts. Detailed analysis of the tumors allows us to identify a delaminating cell population as the critical one in driving tumorigenesis. Cells loose their apicalbasal polarity, mislocalize DE-cadherin, and delaminate from the main epithelium. A JNK-dependent transcriptional program is activated specifically in delaminating cells and drives nonautonomous tissue overgrowth, basement membrane degradation, and invasiveness. These findings unravel a general and rapid tumorigenic potential of genomic instability, as opposed to its proposed role as a source of mutability to select specific tumor-prone aneuploid cells, and open unique avenues toward the understanding of the role of genomic instability in human cancer.
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