Studies on the replication of adenovirus-2 DNA

Monolayer cultures of KB cells infected with adenovirus type 2 (Ad2) were subjected to short pulses of tritiated thymidine, at the time of maximal viral DNA synthesis. Nascent viral DNA was selectively extracted from the cells, purified, and fractionated into partly single-stranded (or replicating) molecules and double-stranded (or completed) molecules. Completed molecules were cleaved by the restriction endonucleases EcoRI, HindIII, Hpal, or by the latter two enzymes in succession, and the resulting fragments were separated by electrophoresis through agarose-acrylamide slab gels. In the case of replicating molecules, the double-stranded fragments generated after treatment with the same enzymes were first separated from the partly single-stranded fragments by chromatography on benzoylated-naphthoylated DEAE (BND) cellulose before being subjected to electrophoresis. The relative yields of tritium of the various fragments resolved by the gels were then determined. The analysis of the completed molecules obtained after the shorter pulses revealed a strong preferential labelling of the fragments derived from the molecular ends, as expected if both of these ends contained termini for replication. The analysis of double-stranded fragments from replicating molecules revealed a predominant labelling of the central portion of the genome which could be reconciled with initiation occurring either centrally or at both molecular ends. In addition, two gradients of labelling, ascending toward each molecular end and encompassing the outer quarters of the molecule, were noted which slowly receded when the pulse length was increased. The latter finding could reflect an overrepresentation of molecules in the late stages of replication in the pool of nascent DNA, such as one would expect if replication slows down when it nears the termination site(s). This interpretation is consistent with the kinetics of labelling of the various portions of the genome observed in the pool of completed molecules. Our present work deals also with the characterization of three different fractions of replicating Ad2 DNA, separated by MAP chromatography. Two of these fractions, eluted with 140 or 180 mM phosphate respectively, display properties distinct from the third fraction, eluted with 240 mM phosphate or the mature DNA. The progressive disappearance of all three fractions upon chases of sufficient durations, suggest that they are all eventually converted into mature DNA. Based on the density in CsC1 or CS2SO4, before or after treatment by various enzymes (N. crassa nuclease, RNAase, DNA polymerases, alone or in various combinations), it is concluded that the first two fractions contain a substantial amount of ss DNA, or RNA in the form of RNA-DNA hybrid. These fractions may thus be composed of molecules at rather late stages of displacement of incipient stages of complementary strand synthesis. The presence of RNA in these molecules accounts for the high buoyant density difference found in several laboratories (up to 10 mg/mL) between replicating and completed Adenovirus DNA molecules, which cannot be explained on the sole basis of partial single-strandedness

Differential effects of c-Ras upon transformation, adipocytic differentiation, and apoptosis mediated by the simian virus 40 large tumor antigen

To investigate the functional relationship between the ability of the simian virus 40 large tumor antigen (TAg) to transform and its ability to block adipocytic differentiation and induce apoptosis, we expressed TAg in C3H10T1/2 (10T1/2)-derived preadipocytes. The results demonstrated that differentiation could be suppressed at lower TAg levels than at the levels required for full neoplastic conversion. Progressively higher TAg levels were accompanied by apoptosis induction in this system. To further examine the role of the cellular Ras protooncogene product (Ras) in TAg function, TAg was expressed in 10T1/2-derived preadipocytes rendered deficient in Ras activity by transfection with inducible or constitutive antisense ras gene constructs. The results indicated that Ras is required for TAg-mediated transformation and for suppression of adipocytic differentiation, while TAg-mediated apoptosis following serum starvation was independent from Ras action. Unexpectedly, our results further demonstrated a dramatic reduction in the levels of the TAg protein itself as differentiation progressed in Ras-knockdown cells, with a concomitant reduction in TAg’s ability to induce apoptosis as a result. These findings suggest that Ras, although cytoplasmic, is an integral component of the pathway whereby TAg, an oncoprotein believed to have primarily nuclear targets, suppresses differentiation or induces neoplastic conversion of murine preadipocytes

Examination of gap junctional, intercellular communication by in situ electroporation on two co-planar indium-tin oxide electrodes

Gap junctions are plasma membrane channels between neighboring cells. We previously described a powerful technique where gap junctional, intercellular communication (GJIC) of adherent cells can be examined by in situ electroporation on a slide, part of which is coated with electrically conductive and transparent indium-tin oxide. An electric pulse is applied through an electrode placed on the cells in the presence of the tracking dye, Lucifer yellow (LY). The pulse causes LY's penetration into the cells growing on the conductive part of the slide, and the subsequent migration of the dye to the non-electroporated cells growing on the non-conductive area is microscopically observed under fluorescence illumination. Although this technique is adequate for a number of cell lines, the turbulence generated as the electrode is removed can cause cell detachment, which makes GJIC examination problematic. In this communication, we describe a slide configuration where junctional communication can be examined in the absence of an upper electrode: Cells are grown on two co-planar electrodes separated by a barrier which diverts the electric field, rendering it vertical to the cell layer. The elimination of an upper electrode is especially valuable for the electroporation of sensitive cells, such as terminally differentiated adipocytes. This technique can also be used for the introduction of other non-permeant molecules such as peptides or siRNA, followed by examination of the cellular phenotype or gene expression levels in situ

Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (RacV12), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and RacV12 with cell density, which was due to inhibition of proteasomal degradation. In addition, RacV12-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that RacV12 is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that RacV12 expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by RacV12. The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by RacV12, as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions

Stat3 and Gap Junctions in Normal and Lung Cancer Cells

Gap junctions are channels linking the interiors of neighboring cells. A reduction in gap junctional intercellular communication (GJIC) correlates with high cell proliferation, while oncogene products such as Src suppress GJIC, through the Ras/Raf/Erk and other effector pathways. High Src activity was found to correlate with high levels of the Src effector, Signal Transducer and Activator of Transcription-3 (Stat3) in its tyrosine-705 phosphorylated, i.e., transcriptionally activated form, in the majority of Non-Small Cell Lung Cancer lines examined. However, Stat3 inhibition did not restore GJIC in lines with high Src activity. In the contrary, Stat3 inhibition in normal cells or in lines with low Src activity and high GJIC eliminated gap junctional communication. Therefore, despite the fact that Stat3 is growth promoting and in an activated form acts like an oncogene, it is actually required for junctional permeability

Synthesis, Characterization and Stat3 Inhibitory Properties of the Prototypical Platinum(IV) Anticancer Drug, [PtCl3(NO2)(NH3)2] (CPA-7)

This paper describes a reinvestigation of the literature concerning the synthesis and structural characterization of the platinum(IV)-based anticancer drug known as CPA-7 and believed to be the compound fac-[PtCl3(NO2)(NH3)2]. CPA-7 has previously been extensively investigated for its ability to control tumor cell growth by inhibition of Stat3 signaling, but very little information is available concerning its synthesis or spectroscopic properties. A reproducible synthetic route is shown to produce an active material which is characterized by IR and 1H, 14N, 15N, and 195Pt NMR spectroscopy, and single crystal X-ray crystallography. The freshly prepared drug is obtained as a single isomer which may in fact be fac- or mer-[PtCl3(NO2)(NH3)2], but recrystallization resulted in a disordered crystal containing approximately equal amounts of the two geometric isomers

The R(H)Oads To Stat3: Stat3 Activation By The Rho Gtpases

The signal transducer and activator of transcription-3 (Stat3) is a member of the STAT family of cytoplasmic transcription factors. Overactivation of Stat3 is detected with high frequency in human cancer and is considered a molecular abnormality that supports the tumor phenotype. Despite concerted investigative efforts, the molecular mechanisms leading to the aberrant Stat3 activation and Stat3-mediated transformation and tumorigenesis are still not clearly defined. Recent evidence reveals a crosstalk close relationship between Stat3 signaling and members of the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Specifically, Rac1, acting in a complex with the MgcRacGAP (male germ cell RacGAP), promotes tyrosine phosphorylation of Stat3 by the IL6-receptor family/Jak kinase complex, as well as its translocation to the nucleus. Studies have further revealed that the mutational activation of Rac1 and Cdc42 results in Stat3 activation, which occurs in part through the upregulation of IL6 family cytokines that in turn stimulates Stat3 through the Jak kinases. Interestingly, evidence also shows that the engagement of cadherins, cell to cell adhesion molecules, specifically induces a striking increase in Rac1 and Cdc42 protein levels and activity, which in turn results in Stat3 activation. In this review we integrate recent findings clarifying the role of the Rho family GTPases in Stat3 activation in the context of malignant progression. © 2011 Elsevier Inc

Stat3 and Gap Junctions in Normal and Lung Cancer Cells

Gap junctions are channels linking the interiors of neighboring cells. A reduction in gap junctional intercellular communication (GJIC) correlates with high cell proliferation, while oncogene products such as <i>Src</i> suppress GJIC, through the Ras/Raf/Erk and other effector pathways. High <i>Src</i> activity was found to correlate with high levels of the <i>Src</i> effector, Signal Transducer and Activator of Transcription-3 (Stat3) in its tyrosine-705 phosphorylated, <i>i.e.</i>, transcriptionally activated form, in the majority of Non-Small Cell Lung Cancer lines examined. However, Stat3 inhibition did not restore GJIC in lines with high <i>Src</i> activity. In the contrary, Stat3 inhibition in normal cells or in lines with low <i>Src</i> activity and high GJIC eliminated gap junctional communication. Therefore, despite the fact that Stat3 is growth promoting and in an activated form acts like an oncogene, it is actually required for junctional permeability