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Focal Adhesion Kinase (FAK) is an important regulator of tumor cell proliferation, survival and metastasis. As such it has become a therapeutic target of interest in cancer. Previous studies suggested that use of FAK tyrosine kinase inhibitors (TKIs) blocks osteolysis in in vivo models of bone metastasis. However, from these studies it was not clear whether FAK TKIs blocked bone degradation by osteoclasts or also promoted bone formation by osteoblasts. In this study we evaluated whether use of the FAK TKI PF-562,271 affected the differentiation of pre-osteoblasts, or activity of mature differentiated osteoblasts. MC3T3-E1 pre-osteoblastic cells were treated with various doses of PF-562,271 following 3 or 10 days of differentiation which led to the inhibition of alkaline phosphatase (ALP) expression and reduced viable cell numbers in a dose-dependent manner. MC3T3-E1 cells which had been differentiated for 21 days prior to treatment with PF-562,271 showed a dose dependent decrease in mineralization as assessed by Alizarin Red staining, with concomitant decreased expression of ALP which is known to facilitate the bone mineralization activity of osteoblasts, however mRNA levels of the transcription factors RUNX2 and osterix which are important for osteoblast maturation and mineralization appeared unaffected at this time point. We speculated that this may be due to altered function of RUNX2 protein due to inhibitory phosphorylation by GSK3β. We found treatment with PF-562,271 resulted in increased GSK3β activity as measured by reduced levels of phospho-Ser9-GSK3β which would result in phosphorylation and inhibition of RUNX2. Treatment of 21 day differentiated MC3T3-E1 cells with PF-562,271 in combination with GSK3β inhibitors partially restored mineralization however this was not statistically significant. As we observed that FAK TKI also resulted in suppression of Akt, which is known to alter osterix protein stability downstream of RUNX2, we examined protein levels by western blot and found a dose-dependent decrease in osterix in FAK TKI treated differentiated MC3T3-E1 cells which is likely responsible for the reduced mineralization observed. Taken together our results suggest that use of FAK TKIs as therapeutics in the bone metastatic setting may block new bone formation as an off-target effect and thereby exacerbate the defective bone regulation that is characteristic of the bone metastatic environment., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors. Published by Elsevier GmbH.)

The calcium-activated cysteine protease m-calpain plays a pivotal role during the earlier stages of myogenesis, particularly during fusion. The enzyme is a heterodimer, encoded by the genes capn2, for the large subunit, and capn4, for the small subunit. To study the regulation of m-calpain, the DNA sequence upstream of capn2 was analyzed for promoter elements, revealing the existence of five consensus-binding sites (E-box) for several myogenic regulatory factors and one binding site for myocyte enhancer factor-2 (MEF-2). Transient transfections with reporter gene constructs containing the E-box revealed that MyoD presents a high level of transactivation of reporter constructs containing this region, in particular the sequences including the MEF-2/E4-box. In addition, over-expression of various myogenic factors demonstrated that MyoD and myogenin with much less efficiency, can up-regulate capn2, both singly and synergistically, while Myf5 has no effect on synthesis of the protease. Experiments with antisense oligonucleotides directed against each myogenic factor revealed that MyoD plays a specific and pivotal role during capn2 regulation, and cannot be replaced wholly by myogenin and Myf5. [Copyright &y& Elsevier]

Bax inhibitor-1 (BI-1) protein is proposed to be a conserved programmed cell death suppressor. In this report, we investigate the anti-apoptotic function of plant BI-1 by antisense (AS) down regulation of NtBI-1 in Nicotiana tabacum cv. BY-2 cells. We observed that AS cell lines were more susceptible to autophagy, internucleosomal DNA fragmentation and death than control cells when subjected to sucrose starvation and hypo-osmotic shock, in agreement with a role of BI-1 as a death inhibitor. [Copyright &y& Elsevier]

The present study was designed to elucidate the consequences of antisense oligonucleotide-mediated knockdown of striatal dopamine reuptake transporters on 3,4-methylenedioxymethamphetamine (MDMA)-induced neurotoxicity. Antisense oligonucleotide complementary to the mRNA translational start site of the rat dopamine transporter was delivered by constant (7 days) intranigral infusion with an osmotic minipump. Delivery of the antisense oligonucleotide by this method resulted in a 70% reduction in the density of the dopamine transporter in the ipsilateral striatum, as measured by [3H]mazindol binding. The effect of this transporter knockdown on MDMA-induced serotonergic neurotoxicity was then examined. MDMA (2×20 mg/kg, s.c., given 12 h apart) administered to control rats produced hyperthermia following the first dose and led to a 45–50% reduction in striatal serotonin, 5-hydroxyindoleacetic acid, and serotonin reuptake transporter density 1 week after the second dose. Conversely, in antisense-, but not missense-treated rats, a significant attenuation of MDMA-induced neurotoxicity was observed only in the ipsilateral striatum. The hyperthermic response elicited by MDMA was not altered by prior administration of antisense. In vivo microdialysis revealed that the antisense treatment attenuated MDMA-induced dopamine release in the ipsilateral striatum.These results suggest that the dopamine transporter plays an essential role in the neurodegeneration induced by MDMA, and provides additional support for the hypothesis that extracellular dopamine is involved in the neurotoxic process, at least in the striatum. [Copyright &y& Elsevier]

Temporal expression profile of lef4, the gene encoding late gene expression factor 4 (LEF4) from the baculovirus, Bombyx mori nucleopolyhedrovirus (BmNPV), has been analysed. lef4 behaved like an early gene and the transcripts were detectable from 6 h post infection (hpi) which reached maximal levels by 18–24 hpi, and declined considerably at later times. The LEF4 open reading frame was bacterially expressed as a glutathione S-transferase (GST) fusion protein which was solubilized from the inclusion bodies and purified by adsorption to the affinity matrix, GST–Sepharose. Using polyclonal antibodies raised against the bacterially expressed protein, the temporal profile of LEF4 synthesis in BmNPV-infected BmN cells was analysed. The LEF4 protein levels were also higher at 24 hpi compared to 12 or 36 hpi, correlating with the RNA patterns. The protein was predominantly localized to the nucleus of the infected BmN cell and only a small portion was present in the cytosolic fraction. Preliminary studies with antisense lef4 expression revealed substantial reduction in expression from the viral polyhedrin promoter without significantly affecting the viral DNA replication. [Copyright &y& Elsevier]

p94 belongs to the calpain family of enzymes, also called calcium-activated neutral proteases and is mainly expressed in the skeletal muscle. Mutations affecting the gene coding for p94 are responsible for a myopathy syndrome called Limb Girdle Muscular Dystrophy type 2A (LGMD2A). Although the activity of p94 seems necessary for muscle function, the biological role of the enzyme is still unknown. The goal of this study was to develop a muscle cell line in which the expression level of p94 can be regulated, by an inducible way. In this study, a biological system was developed which allowed mimicking, in vitro, of part of the events occurring in patients (i.e. a decrease of p94 activity). The first results indicate that the decrease in p94 activity results in a significant increase of myogenin level, a high specific transcription factor involved in myoblast fusion. This muscle specific inducible system is an interesting biological tool to assess specifically p94 function(s) in cultured muscle cells. According to the present results, p94 seems at least to be involved in a myogenesis regulation pathway via its action on certain proteins belonging to the myogenic regulator factor family. [Copyright &y& Elsevier]

The objectives of this study were to investigate the effect of antisense (AS) oligodeoxynucleotides (ODNs) directed against gastrin releasing peptide (GRP) receptor mRNA on proliferation of human small cell lung cancer (SCLC) NCI-H345 cells which express the autocrine system for GRP. The methods used were to expose human SCLC cell lines to antisense ODNs or sense ODNs and to measure their proliferation by spectrophotometric assay or viable cell counts. Our results demonstrated that the single or combined AS ODNs against GRP receptor inhibited proliferation of human SCLC NCI-H345 cells significantly by 37% (P<0.01), but did not inhibit proliferation of either human bronchial epithelial BEAS 2B cells or human SCLC NCI-N417 cells, neither of which express the GRP autocrine system. The sense controls did not significantly inhibit proliferation compared with no treatment controls. Specificity was also demonstrated by the observation that cells exposed to AS ODNs had a decrease in GRP receptor expression as measured by specific binding of 34% (P<0.01), and when all three AS ODNs were used, binding was decreased by 60% (P<0.03). Furthermore, AS ODNs decreased by 75% the maximum percentage of cells responding to GRP in an intracellular calcium release assay. Our conclusions are that antisense ODNs directed against a GRP receptor which is involved in an autocrine loop in human SCLC cells inhibited proliferation of these cells by their impact on reducing GRP receptor expression. Further development of means of increasing AS ODN specificity and effectiveness in human SCLC cell is warranted. [Copyright &y& Elsevier]

Hepatocellular lineage cell lines (two hepatoma and Chang liver cell lines) were found to produce interleukin-6 (IL-6). As the human hepatoma cell line, HCC-M expresses mRNA for both IL-6 and IL-6 receptor, we examined the possibility that IL-6 acted as an autocrine growth factor for HCC-M cells using two IL-6 antisense oligonucleotides (AS-1 and AS-2 oligomers) which were synthesized from different regions of an IL-6 cDNA clone. Both IL-6 antisense oligonucleotides inhibited the growth of HCC-M cells within 48 h (% inhibition by AS-1 and AS-2 oligomers was 53 and 21%, respectively). Although inhibition of HCC-M cell growth induced by AS-2 oligomer was restored by addition of exogenous recombinant IL-6 (rIL-6), the inhibition of growth induced by AS-1 oligomer was not fully restored by exogenous rIL-6, implicating the involvement of a nonantisense mechanism associated with four contiguous guanosine residues in this sequence. The inhibitory effect of AS-2 oligomer was attenuated after 72 h (% inhibition was 8%), whereas the AS-1 oligomer-induced inhibition of growth was sustained beyond 72 h (% inhibition was 38–39%). Therefore, these dual-function oligonucleotides that act via both an antisense and nonantisense mechanism may be of potential therapeutic value against hepatoma. [ABSTRACT FROM AUTHOR]