Premature expression of cyclin B sensitizes human HT1080 cells to caffeine-induced premature mitosis

Eukaryotic cells do not normally initiate mitosis when DNA replication is blocked. This cell cycle checkpoint can be bypassed in some cells, however, by treatment with caffeine and certain other chemicals. Although S-phase arrested hamster cells undergo mitosis-specific events such as premature chromosome condensation (PCC) and nuclear envelope disassembly when exposed to caffeine, human cells show little response under the same conditions. To further investigate the molecular basis of this cell type specificity, a panel of hamster/human whole cell hybrids was created. The frequency of caffeine-induced PCC and the level of cyclin B-associated H1 kinase activity in the various hybrids were directly correlated with the extent of cyclin B synthesis during S-phase arrest. To determine whether expression of cyclin B alone could sensitize human cells to caffeine, cyclin B1 was transiently overexpressed in S-phase arrested HT1080 cells. The transfected cell population displayed a 5-fold increase in the frequency of caffeine-induced PCC when compared with normal HT1080 cells, roughly equivalent to the frequency of cells expressing exogenous epitope-tagged cyclin B1. In addition, immunofluorescent microscopy showed that individual cells overexpressing cyclin B1 during S phase arrest underwent PCC when exposed to caffeine. These results provide direct evidence that premature expression of cyclin B1 can make cells more vulnerable to chemically-induced uncoupling of mitosis from the completion of DNA replication. © 1995 Wiley-Liss, Inc.     

The anti-genomic (negative) strand of Hepatitis C Virus is not targetable by shRNA

Hepatitis C Virus (HCV) and other plus-strand RNA viruses typically require the generation of a small number of negative genomes (20–100× lower than the positive genomes) for replication, making the less-abundant antigenome an attractive target for RNA interference(RNAi)-based therapy. Because of the complementarity of duplex short hairpin RNA/small interfering RNA (shRNA/siRNAs) with both genomic and anti-genomic viral RNA strands, and the potential of both shRNA strands to become part of the targeting complexes, preclinical RNAi studies cannot distinguish which viral strand is actually targeted in infected cells. Here, we addressed the question whether the negative HCV genome was bioaccessible to RNAi. We first screened for the most active shRNA molecules against the most conserved regions in the HCV genome, which were then used to generate asymmetric anti-HCV shRNAs that produce biologically active RNAi specifically directed against the genomic or antigenomic HCV sequences. Using this simple but powerful and effective method to screen for shRNA strand selectivity, we demonstrate that the antigenomic strand of HCV is not a viable RNAi target during HCV replication. These findings provide new insights into HCV biology and have important implications for the design of more effective and safer antiviral RNAi strategies seeking to target HCV and other viruses with similar replicative strategies.     

Tissue interaction in chick liver development: A reevaluation: II. Parenchymal differentiation: Mesenchymal influence and morphogenetic independence

Previous investigators of liver differentiation have described its dependence upon an epithelial-mesenchymal interaction of partial specificity, based upon the embryonic origin of the mesodermal component. Derivatives of the ventral mesoderm encourage epithelial differentiation (PAS-detectable glycogen synthesis), whereas those of the dorsal mesoderm do not. The possibility of a causal relationship between normal histogenesis and functional differentiation was not explored, but a suggestive correlation was established.The present study reinvestigates the development of hepatic function, reexamining the nature of the mesenchymal role and the apparent interdependence of differentiation and morphogenesis. Differentiation was assessed by the criterion of UDP-glucuronyltransferase activity. The glucuronide product was measured by a highly sensitive spectrofluorometric technique following its isolation by thin layer chromatography.It is demonstrated, in agreement with previous studies, that differentiated levels of specialized function are achieved only in the presence of mesenchyme, but that this need not be the homologous mesenchyme. Not all ventrally-derived mesenchymes are permissive in this regard, however. Neither is there any reason to believe that favorable mesenchymes play an instructional, as opposed to a nutritional role. Augmentation, rather than acquisition, of function is the product of this interaction. Functional differentiation of hepatic endoderm appears to require its cytological maturation, but its histological architecture is probably irrelevant.     

A mechanical dissociation method for preparation of muscle cell cultures

A cell preparation method by which large numbers of embryonic chick skeletal muscle cells may be obtained is described. The procedure requires fewer manipulations and much less time than standard trypsinization. By the criteria used, both methods are comparable with respect to percent viable cells and survival of plated cells. However, in addition to the ease of preparation, the mechanical dissociation method offers the significant advantage that the cell suspension is greatly enriched for myoblasts without the necessity of an additional preplating step.