Nischarin is a novel protein that regulates cell migration by inhibiting p21-activated kinase (PAK). LIM kinase (LIMK) is a downstream effector of PAK, and it is known to play an important role in cell invasion.Here we show that nischarin also associates with LIMK to inhibit LIMK activation, cofilin phosphorylation, and LIMK-mediated invasion of breast cancer cells, suggesting that nischarin regulates cell invasion by negative modulation of the LIMK/cofilin pathway. The amino terminus of nischarin binds to the PDZ and kinase domains of LIMK. Although LIMK activation enhances the interaction with nischarin, only phosphorylation of threonine 508 of LIMK is crucial for the interaction. Inhibition of endogenous nischarin expression by RNA interference stimulates breast cancer cell invasion. Also, nischarin small interfering RNA (siRNA) enhances cofilin phosphorylation. In addition, knock-down of nischarin showed branched projection actin structures. Collectively these data indicate that nischarin siRNA may enhance random migration, resulting in stimulation of invasion.Tumor cell migration/invasion is an important factor in solid tumor formation and is necessary for the spread to different organs (47), and thus cellular invasion is a hallmark of metastasis (25). Cellular invasion is a complicated process that involves cytoskeletal reorganization, formation of lamellipodia, membrane ruffling, and altered cell morphology (47). For instance, cell invasion requires partial detachment from intercellular adhesions and from cell-extracellular matrix interactions, reorganization of the actin cytoskeleton, and movement through the extracellular matrix (47, 57). Thus, the actin cytoskeleton is an important determinant of cell motility and cell invasion (5). The actin cytoskeleton drives formation and extension of lamellae at the leading edge of the cells, while the actin-based molecular motor myosin provides the force necessary for cell movement (36).Members of Rho family GTPases are crucial regulators of several biological events, and they are particularly important in the organization of the actin cytoskeleton, as well as in cell migration and invasion (26,27,29,58). Several effectors of Rho GTPases have been identified, but signal transduction pathways that link these to the actin cytoskeleton are not completely understood. A number of actin-associated proteins that regulate actin polymerization and depolymerization are potential downstream mediators. For example, the biological effects of Rac are exerted through the activation of several downstream effectors (11). One important family of Rac effectors is the p21-activated kinases (PAKs), which play a role in cytoskeletal reorganization (9) and cell migration (30, 35).Actin cytoskeletal reorganization is initiated when PAK1 is activated by GTP-bound Rac or Cdc42. PAK then transphosphorylates and activates LIM kinase 1 (LIMK1). Active LIMK1 in turn catalyzes phosphorylation of an N-terminal serine residue of cofilin, thereby inactivating its F actin-depolymerizing activity a...

Turpentine-induced acute-phase (AP) response in rats is followed by transcriptional activation of the alpha2-macroglobulin (MG) gene mediated by cytokine interleukin-6 (IL-6) and glucocorticoids. Based on nucleotide sequence analysis of the alpha2-MG gene promoter regions responsive to IL-6, we postulated that binding of members of the liver-enriched CCAAT-enhancer-binding proteins (C/EBP) family of transcription factors to the type I IL-6 responsive element (IL-6RE) may participate in the transcriptional activation of this gene during AP response. Results of Western immunoblot and Northern-blot assays revealed coordinate changes in the pool levels of C/EBPalpha, -beta. and -delta protein isoforms and their genes expression in liver in response to turpentine. By means of an in vitro phosphorylation assay, South-Western blot, and selective proteolysis we have also found that only abilities of 35-kD C/EBPbeta and 27-kD C/EBPdelta to bind to the alpha2-MG gene promoter were affected by phosphorylation. Based on these data we concluded that transcriptional induction of the rat alpha2-MG gene during AP response correlates with both increased synthesis and phosphorylation-induced binding of 35-kD C/EBPbeta and 27-kD C/EBPdelta.

Atomic force microscopy (AFM), a unique tool to investigate drug treatment of cancer cells, was used to analyze the anti-neoplastic activity of adriamycin by comparing DNA structures of non-treated and adriamycin-treated Ehrlich tumor cells. The non-treated cells exhibited a highly branched intact chromatin structure, related to the intensive DNA replication in cancer cells. Images from adriamycin-treated tumor cells showed that the DNA chains were broken and the chromatin structure had been destroyed. Possible explanations for these effects of adriamycin are considered: breakage of hydrogen bonding, oxidation and intercalation effects, as well as the poisoning of topoisomerase enzyme. DNA fractal and multifractal analyses performed in order to evaluate the degree of bond scission, showed that the treated DNA had become more fractal compared to non-treated DNA.

Transcription of the rat gene encoding haptoglobin (Hp) is highly induced during acute phase (AP) response which has been previously shown to be mediated by inducible STAT3 member of the Signal Transducer and Activators of Transcription (STATs) family proteins. In this study, we observed that under normal but not in the turpentine induced AP conditions, another member of the STAT family proteins, STAT5b is expressed and binds to the hormone regulatory element (HRE) of the rat Hp gene. We found that the nuclear amounts of constitutively active STAT5b in rat liver decreased significantly with time of turpentine treatment as opposed to that of cytosol STAT5b, suggesting possible export of constitutive STAT5b from the nucleus. Nuclear accumulation and binding of inducible STAT3 proteins to the rat Hp gene HRE following turpentine treatment implicated that STAT5b negatively regulates Hp gene expression during normal conditions.

Turpentine-induced acute-phase (AP) response in rats is followed by transcriptional activation of the haptoglobin (Hp) gene in liver. Analysing the promoter sequence of the rat Hp gene we postulated an involvement of Signal Transducer and Activator of Transcription 3 (STAT3) in the regulation of this process. Results obtained by using a combination of Western immunoblot and DNA-binding assays revealed AP-induced binding of constitutive 86kD-and inducible 91kD-STAT3 isoforms to the rat Hp gene inducible promoter element. On the basis of these data we assumed that AP-related interactions of these two STAT3 isoforms correlates with an activated transcriptional status of the rat Hp gene.