Abstract. Relatively little is known about the mechanisms used by somatic cells to regulate the replication of the centrosome complex. Centrosome doubling was studied in CHO cells by electron microscopy and immunofluorescence microscopy using human autoimmune anticentrosome antiserum, and by Northern blotting using the cDNA encoding portion of the centrosome autoantigen pericentriolar material (PCM)-I. Centrosome doubling could be dissociated from cycles of DNA synthesis and mitotic division by arresting cells at the G1/S boundary of the cell cycle using either hydroxyurea or aphidicolin. Immunofluorescence microscopy using SPJ human autoimmune anticentrosome antiserum demonstrated that arrested cells were able to undergo numerous rounds of centrosome replication in the absence of cycles of DNA synthesis and mitosis. Northern blot analysis demonstrated that the synthesis and degradation of the mRNA encoding PCM-1 occurred in a cell cycle--dependent fashion in CHO cells with peak levels of PCM-1 mRNA being present in G1 and S phase cells before mRNA amounts dropped to undetectable levels in G2 and M phases. Conversely, cells arrested at the G1/S boundary of the cell cycle maintained PCM-1 mRNA at artificially elevated levels, providing a possible molecular mechanism for explaining the multiple rounds of centrosome replication that occurred in CHO cells during prolonged hydroxyurea-induced arrest. The capacity to replicate centrosomes could be abolished in hydroxyurea-arrested CHO cells by culturing the cells in dialyzed serum. However, the ability to replicate centrosomes and to synthesize PCM-1 mRNA could be re-initiated by adding EGF to the dialyzed serum. This experimental system should be useful for investigating the positive and negative molecular mechanisms used by somatic cells to regulate the replication of centrosomes and for studying and the methods used by somatic cells for coordinating centrosome duplication with other cell cycle progression events.

Nonviral gene delivery is limited to a large extent by multiple extracellular and intracellular barriers. One of the major barriers, especially in nondividing cells, is the nuclear envelope. Once in the cytoplasm, plasmids must make their way into the nucleus in order to be expressed. Numerous studies have demonstrated that transfections work best in dividing populations of cells in which the nuclear envelope disassembles during mitosis, thus largely eliminating the barrier. However, since many of the cells that are targets for gene therapy do not actively undergo cell division during the gene transfer process, the mechanisms of nuclear transport of plasmids in nondividing cells are of critical importance. In this review, we summarize recent studies designed to elucidate the mechanisms of plasmid nuclear import in nondividing cells and discuss approaches to either exploit or circumvent these processes to increase the efficiency of gene transfer and therapy. Gene Therapy (2005) 12, 881-890.

Genetic disruption of the pancreatic mesenchyme reveals that it is critical for the expansion of epithelial progenitors and for the proliferation of insulin-producing beta cells.

No abstract

One factor limiting the success of non-viral gene therapy smooth muscle cells, we have created a series of reporter vectors is the relative inability to target genes specifically plasmids that are expressed selectively in smooth muscle to a desired cell type. To address this limitation, we have cells. Moreover, when injected into the cytoplasm, plasbegun to develop cell-specific vectors whose specificity is mids containing portions of the SMGA promoter localize to at the level of the nuclear import of the plasmid DNA. We the nucleus of smooth muscle cells, but remain cytoplashave recently shown that nuclear import of plasmid DNA mic in fibroblasts and CV1 cells. In contrast, a similar plasis a sequence-specific event, requiring the SV40 enhancer, mid carrying the SV40 enhancer is transported into the a region known to bind to a number of general transcription nuclei of all cell types tested. Nuclear import of the SMGA factors (Dean DA, Exp Cell Res 1997; 230: 293). From promoter-containing plasmids could be achieved when the these studies we developed a model whereby transcription smooth muscle specific transcription factor SRF was factor(s) bind to the DNA in the cytoplasm to create a proexpressed in stably transfected CV1 cells, supporting our tein-DNA complex that can enter the nucleus using the model for the nuclear import of plasmids. Finally, these protein import machinery. Our model predicts that by using nuclear targeting sequences were also able to promote DNA elements containing binding sites for transcription facincreased gene expression in liposome-and polycationtors expressed in unique cell types, we should be able to transfected non-dividing cells in a cell-specific manner, create plasmids that target to the nucleus in a cell-specific similar to their nuclear import activity. These results promanner. Using the promoter from the smooth muscle vide proof of principle for the development of cell-specific gamma actin (SMGA) gene whose expression is limited to non-viral vectors for any desired cell type.