Myosin 1e is a component of the glomerular slit diaphragm complex that regulates actin reorganization during cell-cell contact formation in podocytes.

Glomerular visceral epithelial cells, also known as podocytes, are critical to both normal kidney function and the development of kidney disease. Podocyte actin cytoskeleton and their highly specialized cell-cell junctions (also called slit diaphragm complexes) play key roles in controlling glomerular filtration. Myosin 1e (myo1e) is an actin-based molecular motor that is expressed in renal glomeruli. Disruption of the Myo1e gene in mice and humans promotes podocyte injury and results in the loss of the integrity of the glomerular filtration barrier. Here, we have used biochemical and microscopic approaches to determine whether myo1e is associated with the slit diaphragm complexes in glomerular podocytes. Myo1e was consistently enriched in the slit diaphragm fraction during subcellular fractionation of renal glomeruli and colocalized with the slit diaphragm markers in mouse kidney. Live cell imaging studies showed that myo1e was recruited to the newly formed cell-cell junctions in cultured podocytes, where it colocalized with the actin filament cables aligned with the nascent contacts. Myo1e-null podocytes expressing FSGS-associated myo1e mutant (A159P) did not efficiently assemble actin cables along new cell-cell junctions. We have mapped domains in myo1e that were critical for its localization to cell-cell junctions and determined that the SH3 domain of myo1e tail interacts with ZO-1, a component of the slit diaphragm complex and tight junctions. These findings suggest that myo1e represents a component of the slit diaphragm complex and may contribute to regulating junctional integrity in kidney podocytes.

Induction of apoptosis in oral cancer cells by an anti-bcl-2 ribozyme delivered by an adenovirus vector.

Human oral cancer cells may have any of several genetic changes, but the role of the bcl-2 oncogene is relatively unexplored. To find out if this gene plays a significant role and whether it could act as a target for gene therapy of oral cancer, we have examined the effects of an anti-bcl-2 ribozyme on the phenotype of oral cancer cells. A hammer-head ribozyme was designed to cleave the bcl-2 transcript after nucleotide 279 and was confirmed to be effective against a synthetic bcl-2 transcript. A gene encoding the ribozyme was cloned into an adenovirus vector and transferred to the human oral cancer cell lines 686LN, 1483, and Tu183. Over a 6-day period, the growth of each cancer cell line was reduced, whereas growth of the fibroblast cell line FS7 was less inhibited. Inhibition of the oral cancer cells could be attributed to apoptosis, as indicated by the detection of histone-associated DNA fragments in an immunoassay. Northern blots showed no detectable reduction in the level of bcl-2 mRNA of Tu183 cells, but Western blots showed a reduction of Bcl-2 protein at 24 h after infection with the ribozyme-expressing adenovirus vector. The results imply that (a) expression of the bcl-2 oncogene is necessary for the survival of oral cancer cells, (b) the bcl-2 gene transcript presents a target for gene therapy by ribozymes, and (c) an adenovirus vector is a suitable method for transfection of the ribozyme-expressing gene.

Effects of herpes simplex virus on human oral cancer cells, and potential use of mutant viruses in therapy of oral cancer.

The herpes simplex virus type-1 (HSV-1) might be useful in treatment of oral cancer because it is strongly cytolytic, and its natural target tissue is the source of oral squamous cell carcinomas. Use of a wild-type virus would be limited by its spread and neurotoxicity, but it might be possible to develop mutants whose range could be restricted to oral cancers. Thus we have investigated the effects of HSV-1 on human oral cancer cells and have used both wild-type virus and a mutant that lacks UL42--an essential gene of the virus. Growth of the oral cancer cell line 686LN was readily inhibited by wild-type HSV-1, with only 10(2) plaque forming units (pfu) per milliliter required for 50% inhibition. In contrast, the mutant HSV-1 required a titer of 10(6) pfu/ml for 50% inhibition of growth. The mutant virus did, however, inhibit cell growth through the activation of ganciclovir and thus might be able to amplify its cytotoxicity through a bystander effect. When wild-type HSV-1 was injected into 686LN cells which were growing as tumors in nude mice, the virus spread through the tumor. Treated tumors were smaller, of lower weight, and significantly more necrotic than either untreated tumors or tumors which had been treated with the mutant virus. The wild-type virus spread to the skin and nervous system of most animals causing zosteriform skin rash, neurological symptoms and death, while the mutant virus produced none of these side-effects. These results show that HSV-1 might be used to treat oral cancer if its replication could be limited to the tumor cells, and that controlled expression of the UL42 gene would be one way to obtain that limitation.