Antiviral Activity of Cyclosaligenyl Prodrugs of Acyclovir, Carbovir and Abacavir

The cyclosaligenyl ( cycloSal) derivatives of the monophosphates of three acyclic or carbocyclic guanosine analogues, for example, acyclovir (ACV), carbovir (CBV) and abacavir (ABC), were investigated for their activity against retrovirus (HIV, Moloney sarcoma virus) and herpes simplex virus (HSV) activity in cell culture. The extent of the antiviral potency of the prodrugs depended on the nature of the nucleoside, the substituent on the cycloSal moiety and the virus investigated. Most notably, and unlike the parent compound ACV, cycloSal-ACV monophosphate (MP) prodrugs retained pronounced activity against ACV-resistant (thymidine kinase-deficient) HSV-1 and also gained anti-HIV activity. While the cycloSal-CBVMP prodrugs did not show enhanced activity compared with the parent compound CBV, the cycloSal-ABCMP prodrugs afforded markedly increased potency against both HSV and HIV. Our data indicate that the cycloSal prodrug approach can be useful to deliver directly the MP derivatives of nucleoside analogues into the intact, virus-infected cells, thus improving and extending the antiviral potency and spectrum of the drugs against retro- and herpesvirus strains.

Na,K-ATPase β-Subunit Is Required for Epithelial Polarization, Suppression of Invasion, and Cell Motility

The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an α- and β-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin–mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and β1-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase β1-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin–mediated cell-cell adhesion requires the Na,K-ATPase β-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the β1-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells.

The MN1-TEL Fusion Protein, Encoded by the Translocation (12;22)(p13;q11) in Myeloid Leukemia, Is a Transcription Factor with Transforming Activity

ABSTRACT The Tel gene (or ETV6) is the target of the translocation (12;22)(p13;q11) in myeloid leukemia. TEL is a member of the ETS family of transcription factors and contains the pointed protein interaction (PNT) domain and an ETS DNA binding domain (DBD). By contrast to other chimeric proteins that contain TEL's PNT domain, such as TEL–platelet-derived growth factor β receptor in t(5;12)(q33;p13), MN1-TEL contains the DBD of TEL. The N-terminal MN1 moiety is rich in proline residues and contains two polyglutamine stretches, suggesting that MN1-TEL may act as a deregulated transcription factor. We now show that MN1-TEL type I, unlike TEL and MN1, transforms NIH 3T3 cells. The transforming potential depends on both N-terminal MN1 sequences and a functional TEL DBD. Furthermore, we demonstrate that MN1 has transcription activity and that MN1-TEL acts as a chimeric transcription factor on the Moloney sarcoma virus long terminal repeat and a synthetic promoter containing TEL binding sites. The transactivating capacity of MN1-TEL depended on both the DBD of TEL and sequences in MN1. MN1-TEL contributes to leukemogenesis by a mechanism distinct from that of other chimeric proteins containing TEL.

AMF-R Tubules Concentrate in a Pericentriolar Microtubule Domain After MSV Transformation of Epithelial MDCK Cells

Autocrine motility factor receptor (AMF-R) is localized to an intracellular microtubule-associated membranous organelle, the AMF-R tubule. In well-spread untrans-formed MDCK epithelial cells, the microtubules originate from a broad perinuclear region and AMF-R tubules extend throughout the cytoplasm of the cells. In Moloney sarcoma virus (mos)-transformed MDCK (MSV-MDCK) cells, microtubules accumulate around the centrosome, forming a microtubule domain rich in stabilized detyrosinated microtubules. AMF-R tubules are quantitatively associated with this pericentriolar microtubule domain and the rough endoplasmic reticulum and lysosomes also co-distribute with the pericentriolar mass of microtubules. The Golgi apparatus is closely associated with the microtubule organizing center (MTOC) within the juxtanuclear mass of AMF-R tubules, and no co-localization of AMF-R tubules with the Golgi marker β-COP could be detected by confocal microscopy. After nocodazole treatment and washout, microtubule nucleation occurs exclusively at the centrosome of MSV-MDCK cells, and only after microtubule extension to the cell periphery does the microtubule cytoskeleton reorganize to generate the pericentriolar microtubule domain after 30–60 min. AMF-R tubules dispersed by nocodazole treatment concentrate in the pericentriolar region in parallel with the reorganization of the microtubule cytoskeleton. MSV transformation of epithelial MDCK cells results in the stabilization of a pericentriolar microtubule domain responsible for the concentration and polarized distribution of AMF-R tubules.

Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions.

We characterized the role of the E-cadherin adhesion system in the formation of epithelial tight junctions using the calcium switch model. In MDCK cells cultured in low (micromolar) calcium levels, the tight junctional protein Zonula Occludens-1 (ZO-1) is distributed intracellularly in granular clusters, the larger of which codistribute with E-cadherin. Two hours after activation of E-cadherin adhesion by transfer to normal (1.8 mM) calcium levels, ZO-1 dramatically redistributed to the cell surface, where it localized in regions rich in E-cadherin. Immunoprecipitation with ZO-1 antibodies of extracts from cells kept in low calcium and 2 h after shifting to 1.8 mM Ca2+ demonstrated the association of ZO-1 with alpha-, beta-, and gamma-catenins. E-cadherin was not detected in the ZO-1 immunoprecipitates but it was found in beta-catenin immunoprecipitates that excluded ZO-1, suggesting that the binding of ZO-1 to catenins may weaken the interaction of these proteins with E-cadherin. Immunofluorescence and immunoelectron microscopy confirmed a close association of beta-catenin and ZO-1 at 0 and 2 h after Ca2+ switch. 48 h after Ca2+ switch, upon complete polarization of the epithelium, most of the ZO-1 had segregated from lateral E-cadherin and formed a distinct, separate apical ring. The ZO-1-catenin complex was not detected in fully polarized monolayers. MDCK cells permanently transformed with Moloney sarcoma virus, which expresses low levels of E-cadherin, displayed clusters of cytoplasmic ZO-1 granules and very little of this protein at the cell surface. Upon transfection with E-cadherin into Moloney sarcoma virus-MDCK cells, ZO-1 redistributed to E-cadherin-rich lateral plasma membrane but later failed to segregate into mature tight junctions. Our experiments suggest that catenins participate in the mobilization of ZO-1 from the cytosol to the cell surface early in the development of tight junctions and that neoplastic transformation may block the formation of tight junctions, either by decreasing the levels of E-cadherin or by preventing a late event: the segregation of tight junction from the zonula adherens.