Therapeutic implications of cancer stem cells

Most cancers comprise a heterogenous population of cells with marked differences in their proliferative potential as well as the ability to reconstitute the tumor upon transplantation. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. In addition, dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. This new model for cancer will have significant ramifications for the way we study and treat cancer. In addition, through targeting the cancer stem cell and its dysregulated self-renewal, our therapies for treating cancer are likely to improve.     

Formation of 4,4'-dimethoxytrityl-C-phosphonate oligonucleotides

Incomplete sulfurization during solid-phase synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry was identified as the cause of formation of two new classes of process-related oligonucleotide impurities containing a DMTr-C-phosphonate (DMTr=4,4'-dimethoxytrityl) moiety. Phosphite triester intermediates that failed to oxidize (sulfurize) to the corresponding phosphorothioate triester react during the subsequent acid-induced (dichloroacetic acid) detritylation with the DMTr cation or its equivalent in an Arbuzov-type reaction. This leads to formation of DMTr-C-phosphonate mono- and diesters resulting in oligonucleotides modified with a DMTr-C-phosphonate moiety located internally or at the 5'terminal hydroxy group. DMTr-C-phosphonate derivatives are not detected when optimized sulfurization conditions are employed.     

Early activation of sphingosine kinase in mast cells and recruitment to FcepsilonRI are mediated by its interaction with Lyn kinase

Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca(2+), released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-gamma and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcepsilonRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcepsilonRI triggering, enhanced sphingosine kinase activity was associated with FcepsilonRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn(-/)(-) mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcepsilonRI proximal event.     

Quantifying structure-function uncertainty: a graph theoretical exploration into the origins and limitations of protein annotation

Since the advent of investigations into structural genomics, research has focused on correctly identifying domain boundaries, as well as domain similarities and differences in the context of their evolutionary relationships. As the science of structural genomics ramps up adding more and more information into the databanks, questions about the accuracy and completeness of our classification and annotation systems appear on the forefront of this research. A central question of paramount importance is how structural similarity relates to functional similarity. Here, we begin to rigorously and quantitatively answer these questions by first exploring the consensus between the most common protein domain structure annotation databases CATH, SCOP and FSSP. Each of these databases explores the evolutionary relationships between protein domains using a combination of automatic and manual, structural and functional, continuous and discrete similarity measures. In order to examine the issue of consensus thoroughly, we build a generalized graph out of each of these databases and hierarchically cluster these graphs at interval thresholds. We then employ a distance measure to find regions of greatest overlap. Using this procedure we were able not only to enumerate the level of consensus between the different annotation systems, but also to define the graph-theoretical origins behind the annotation schema of class, family and superfamily by observing that the same thresholds that define the best consensus regions between FSSP, SCOP and CATH correspond to distinct, non-random phase-transitions in the structure comparison graph itself. To investigate the correspondence in divergence between structure and function further, we introduce a measure of functional entropy that calculates divergence in function space. First, we use this measure to calculate the general correlation between structural homology and functional proximity. We extend this analysis further by quantitatively calculating the average amount of functional information gained from our understanding of structural distance and the corollary inherent uncertainty that represents the theoretical limit of our ability to infer function from structural similarity. Finally we show how our measure of functional "entropy" translates into a more intuitive concept of functional annotation into similarity EC classes.     

Macrophage population dynamics within fetal mouse fibroblast cultures derived from C57BL/6, CD-1, CF-1 mice and interleukin-6 and granulocyte colony stimulating factor knockout mice

In vitro models of macrophage growth, differentiation, and function are needed to facilitate the study of their biology as important immune facilitator cells and as frequent targets of bacterial and viral infection. A simple method for the selective expansion and continuous culture of mouse macrophages from primary explant cultures of mouse embryonic tissue is described. Culture in Dulbecco modified Eagle medium (DMEM) low-glucose (1 g/L) formulation (DMEM/L) inhibited fibroblast growth. In contrast, macrophages continued to proliferate in the presence of DMEM/L when in contact with the fibroblasts. Alternating growth in high-glucose DMEM with DMEM/L produced a 1.16- to 2.1-fold increase (depending on mouse strain) in the percentage of macrophages within the cell culture in comparison with culturing in DMEM with high glucose exclusively. Macrophage yields of over 1 million cells/T12.5 flask were achieved by passages 3-4, and, thereafter, declined over the next 5-10 passages. The peak percentage of macrophages within a culture varied depending on the strain of mouse (C57BL/6, CD-1, and CF-1 and two knockout C57BL/6 strains deficient in either interleukin-6 [IL-6] or granulocyte colony stimulating factor [GCSF]). The GCSF (-/-)-derived cultures had the lowest peak macrophage content (30%) and CD-1 the highest content (64.9%). The IL-6 (-/-) and CD-1 cultures appeared to spontaneously transform to create cell lines (IL6MAC and CD1MAC, respectively) that were composed of 50-75% macrophages. The macrophages were phagocytic and were positive for CD14, acetylated low-density lipoprotein receptors, and F4-80 antigen. Light and electron microscopy showed that the cultured macrophages had in vivo-like morphological features, and they could be plated to high purity by differential attachment to petri dishes in serum-free medium.     

The peripheral-type benzodiazepine receptor and tumorigenicity: isoquinoline binding protein (IBP) antisense knockdown in the C6 glioma cell line

Peripheral-type benzodiazepine receptors (PBR) are constituted by three protein components, the isoquinoline binding protein (IBP), the voltage-dependent anion channel (VDAC), and the adenine nucleotide transporter (ANT). Recently, we found that high levels of PBR ligand binding in glioma cell lines correlate with in vitro tumorigenicity. To study whether enhanced PBR expression is causative or in response to cancer, we genetically modified C6 glioma cells. Antisense knockdown of the IBP resulted in more than 50% reductions in PBR ligand binding both in the mitochondrial and whole cell fractions, accompanied by similar reductions in IBP levels in these respective fractions. The IBP knockdown was accompanied by a 25% increase in cell number in confluent cultures. This correlated with an 8-fold increase in in vitro tumorigenicity, as assessed by anchorage independent growth. Cell cycle analysis indicated that knockdown of the IBP resulted in a 60% reduction in the number of cells in the pre-G1 apoptosis phase. This paralleled the reduction seen in apoptosis and cell death shown by DNA fragmentation and Trypan blue assays, respectively. Furthermore, knockdown of the IBP appeared to prevent induction of apoptosis by the antineoplastic agent, erucylphosphocholine. In addition, IBP knockdown prevented processing of the caspase 3 component of the apoptosis cascade by the erucylphosphocholine congener, erucylphospho-N,N,N-trimethylammonium. In conclusion, our results suggest that enhanced IBP expression, including enhanced PBR ligand binding, such as occurring in untreated C6 glioma cells, may provide a mechanism to increase apoptotic rates of cancer cells.     

Scale-dependent feedback and regular spatial patterns in young mussel beds

In the past decade, theoretical ecologists have emphasized that local interactions between predators and prey may invoke emergent spatial patterning at larger spatial scales. However, empirical evidence for the occurrence of emergent spatial patterning is scarce, which questions the relevance of the proposed mechanisms to ecological theory. We report on regular spatial patterns in young mussel beds on soft sediments in the Wadden Sea. We propose that scale-dependent feedback, resulting from short-range facilitation by mutual protection from waves and currents and long-range competition for algae, induces spatial self-organization, thereby providing a possible explanation for the observed patterning. The emergent self-organization affects the functioning of mussel bed ecosystems by enhancing productivity and resilience against disturbance. Moreover, self-organization allows mussels to persist at algal concentrations that would not permit survival of mussels in a homogeneous bed. Our results emphasize the importance of self-organization in affecting the emergent properties of natural systems at larger spatial scales.     

Distribution and status of the medicinal leech (Hirudo medicinalis L.) in Turkey

A survey of all the major potential habitats in western Turkey showed that medicinal leeches, Hirudo medicinalis L., are widely distributed over the country and are not rare. They occur in practically all suitable habitats and the only region where they were found to be absent is that of the large river deltas in the south of the country (Çukurova deltas, Göksu delta). There may be zoogeographic reasons for this (Taurus mountains barrier). The application of a semi-quantitative survey method using collecting efficiency (number of leeches collected per hour by a single person) allowed a rapid assessment to be made of its status in a large number of wetlands. Leech density varied considerably from wetland to wetland, and the results enabled a ranking of the Turkish wetlands to be made according to their importance for medicinal leeches. Taking both the leech density and the size of leech habitats into account, the largest populations were identified on the Black Sea coast (Kizilirmak delta, Yeilirmak delta and Karagöl Marshes near Sinop) and in inner and south-west Anatolia (Eber Gölü, Karamik and Sultan Marshes). Commercial exploitation for the pharmaceutical industry and for other purposes takes place at only a few places and does not appear to affect the population seriously. However, many populations are threatened by the draining of their habitats.     

Adult corneal epithelium basal cells possess the capacity to activate epidermal, pilosebaceous and sweat gland genetic programs in response to embryonic dermal stimuli

Recent work has shown remarkable plasticity between neural and hematopoeitic, as well as between hematopoeitic and muscle stem cells, depending on environmental stimuli (Fuchs, E. and Segre, J. A. (2000) Cell 100, 143-155). Stem cells give rise to a proliferative transient amplifying population (TA), which is generally considered to be irreversibly committed. Corneal epithelium provides a particularly useful system for studying the ability of TA cells to activate different genetic programs in response to a change in their fibroblast environment. Indeed, corneal stem and TA cells occupy different localities - stem cells at the periphery, and TA cells more central (Lehrer, M. S., Sun, T. T. and Lavker, R. M. (1998) J. Cell Sci. 111, 2867-2875) - and thus can be discretely dissected from each other. It is well known that pluristratified epithelia of cornea and skin display distinct programs of differentiation: corneal keratinocytes express keratin pair K3/K12 and epidermal keratinocytes keratin pair K1-2/K10; moreover, the epidermis forms cutaneous appendages, which express their own set of keratins. In our experiments, central adult rabbit corneal epithelium was thus associated either with a mouse embryonic dorsal, upper-lip or plantar dermis before grafting onto nude mice. Complementary experiments were performed using adult mouse corneal epithelium from the Rosa 26 strain. The origin of the differentiated structures were identified in the first case by Hoechst staining and in the second by the detection of beta-galactosidase activity. The results show that adult central corneal cells are able to respond to specific information originating from embryonic dermis. They give rise first to a new basal stratum, which does not express anymore corneal-type keratins, then to pilosebaceous units, or sweat glands, depending of the dermis, and finally to upper layers expressing epidermal-type keratins. Our results provide the first evidence that a distinct TA cell population can be reprogrammed.     

Putative autocleavage of reovirus mu1 protein in concert with outer-capsid disassembly and activation for membrane permeabilization

Capsid proteins of several different families of non-enveloped animal viruses with single-stranded RNA genomes undergo autocatalytic cleavage (autocleavage) as a maturation step in assembly. Similarly, the 76 kDa major outer-capsid protein mu1 of mammalian orthoreoviruses (reoviruses), which are non-enveloped and have double-stranded RNA genomes, undergoes putative autocleavage between residues 42 and 43, yielding N-terminal N-myristoylated fragment mu1N and C-terminal fragment mu1C. Cleavage at this site allows release of mu1N, which is thought to be critical for penetration of the host-cell membrane during cell entry. Most previous studies have suggested that cleavage at the mu1N/mu1C junction precedes addition to the outer capsid during virion assembly, such that only a small number of the mu1 subunits in mature virions remain uncleaved at that site (approximately 5%). In this study, we varied the conditions for disruption of virions before running the proteins on denaturing gels and in several circumstances recovered much higher levels of uncleaved mu1 (up to approximately 60%). Elements of the disruption conditions that allowed greater recovery of uncleaved protein were increased pH, absence of reducing agent, and decreased temperature. These same elements allowed comparably higher levels of the mu1delta protein, in which cleavage at the mu1N/delta junction has not occurred, to be recovered from particle uncoating intermediates in which mu1 had been previously cleaved by chymotrypsin in a distinct protease-sensitive region near residue 580. The capacity to recover higher levels of mu1delta following disruption of these particles for electrophoresis was lost, however, in concert with a series of structural changes that activate the particles for membrane permeabilization, suggesting that the putative autocleavage is itself one of these changes.