G12D and perturbations in p53, we developed a novel genetically engineered mouse model (GEMM) for RMS. The anatomic sites of primary RMS development recapitulated human disease, including tumors in the head, neck, extremities and abdomen. We confirmed RMS histology and diagnosis through Hematoxylin and Eosin staining, and positive immunohistochemical staining for desmin, myogenin, and phosphotungstic acid–Hematoxylin. Cell lines from GEMM tumors were established with the ability to engraft in immunocompetent mice with comparable histological and staining features as the primary tumors. Tail vein injection of cell lines had high metastatic potential to the lungs. Transcriptomic analyses of p53R172H/K-RasG12D GEMM-derived tumors showed evidence of high molecular homology with human RMS. Finally, pre-clinical use of these murine RMS lines showed similar therapeutic responsiveness to chemotherapy and targeted therapies as human RMS cell lines.Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, with overall long-term survival rates of ∼65-70%. Thus, additional molecular insights and representative models are critical for identifying and evaluating new treatment modalities. Using MyoD-Cre-mediated introduction of mutant K-Ras Editors' choice: We developed a new conditional genetically engineered mouse model of rhabdomyosarcoma (RMS) with homologous molecular signature to human RMS that provides valuable pre-clinical models for evaluating novel therapies. Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Despite improvements in patient survival rates over the past few decades, over one-third of RMS patients still succumb to the disease. Therefore, identification of additional effective therapeutic regimens is crucial towards improving the outcome for these patients. RMS can be categorized histologically into alveolar RMS (ARMS) and embryonal RMS (ERMS) . The alvKRAS oncogene has been implicated in various cancers, with up to one-third of RMS cases involving the activation of one of the three RAS isoforms, including K-RAS , rendering ARMS to be grouped as fusion positive and ERMS as fusion negative. Although ARMS rarely involves the inactivation or mutations of p53 (TP53), ERMS has a relatively high frequency of mutations or aberrations in the p53/MDM2 axis . In adding K-RAS .Although rapid progress has been made in the field of RMS, most research has been based on xenotransplantation using human cell lines into immunodeficient models. These xenograft models do not recapitulate the complete tumor biology, including the tumor immune microenvironment. Thus, there is a significant need to develop murine RMS models that can develop and progress in immunocompetent model systems.In this study, we developed and characterized a conditional p53 and/or K-Ras fusion-negative RMS (FN-RMS) genetically engineered mouse model (GEMM). We also performed histologic and genetic analysis of GEMM tumors, and found that these tumors had similar phenotypic and molecular features to human RMS. Based on the homologous histological and molecular traits to RMS, we believe that this mouse model, and the derived cell lines, could significantly contribute to our understanding of the molecular pathogenesis of RMS and provide valuable immunocompetent models for investigating novel therapeutic strategies for treating RMS.R172H or K-RasG12D alleles. Four distinct MyoD-Cre transgenic genotypes were monitored for tumor incidence: K-Ras-wild type (WT) mice with floxed p53 allele/WT p53 allele or LSL-p53R172H allele/WT p53 allele (K-RasWTp53F/+ and K-RasWTp53R172H/+) and K-Ras-mutated mice with floxed p53 allele/WT p53 allele or LSL-p53R172H allele/WT p53 allele (K-RasG12Dp53F/+ and K-RasG12Dp53R172H/+) -Cre mice with germline floxed p53, Lox-Stop-Lox (LSL) p53R172H/+) A.Fig. 1During necropsy, we assessed for metastatic lesions in all of the major organs, noting lungs to be the most frequent site of metastasis, which we noted ranged between 10% and 20% of the animals, while there seemed to be no significant difference in incidence of tumor generation between each group B. The siWTp53F/+ and K-RasWTp53R172H/+) lived significantly longer than K-Ras-mutated mice (K-RasG12Dp53F/+ and K-RasG12Dp53R172H/+) (P<0.0001). In addition, there was a significant survival difference between K-RasWTp53F/+ and K-RasWTp53R172H/+ (P=0.0269) mice, but there were no statistically significant differences between the K-RasG12Dp53F/+ and K-RasG12Dp53R172H/+ cohorts (P=0.58).We examined the overall survival of these genetically engineered mice, comparing the survival rates of K-Ras-mutated and K-Ras-WT mice D. We notFig. S1). Most tumors had identifiable elongated ‘strap’ cells or cells with visible cross striations that are found in RMS.Following the evaluation of tumor incidence, primary tumor development and metastatic potential in this murine model, we next sought to characterize the tumors histologically. Tumor morphology was evaluated by a veterinary pathologist with experience in mouse sarcoma models, and skeletal muscle differentiation was evaluated using phosphotungstic acid–Hematoxylin (PTAH) histochemical staining and immunohistochemistry for desmin and myogenin, markers of muscle and skeletal muscle differentiation A-D. PrimG12D tumors (p53R172H/+), three K-RasWT tumors (two p53R172H/+and one p53F/+) and normal gastrocnemius muscle tissue from three non-tumor bearing mice as controls. We generated gene signatures of at least 1.5-fold change and a false discovery rate (FDR)≤0.05 for K-RasG12D or K-RasWT compared to normal murine gastrocnemius muscle tissue, as well as human RMS (either fusion negative or fusion positive) to normal muscle tissue. To determine the extent of overlap between the differentially expressed genes (DEGs) from the K-RasG12D/+ and K-RasWT tumors with human RMS, we compared the murine DEGs to the human fusion-positive RMS (FP-RMS) and FN-RMS gene signatures. Of the 7900 FN-RMS DEGs, 30% (2391 genes) and 45% (3585 genes) overlapped with our K-RasWT and K-RasG12D, respectively (WT and 43% (3547 genes) overlap with K-RasG12D (We performed RNA sequencing (RNA-seq) from primary GEMM tumors to examine whether gene expression in tumors from our model overlapped with gene expression changes seen in human RMS. For our studies, we used three K-Rasectively A. Simila-RasG12D A.Fig. 3GSE108022 and murine Kras gene signatures in other human RMS cohorts. Although both the K-RasG12D and K-RasWT signatures were significantly correlated with our human RMS signature in all four cohorts analyzed, the K-RasG12D signature consistently had much higher Pearson correlation coefficients, similar to those of FN-RMS and FP-RMS, compared to K-RasWT (R172H/+ resulted in the rapid development of UPS in the setting of an activating K-Ras mutation (WT signature had a higher correlation coefficient (0.97) with the UPS gene signature compared to K-RasG12D (0.89), which had comparable correlation coefficients to UPS with human FN-RMS and FP-RMS. Taken together, these data indicate that the K-RasG12D GEMM more closely mimics the gene expression changes seen in human RMS across multiple datasets compared to the K-RasWT model, which has gene expression changes more closely related to those seen in human UPS.We further asked whether the overlapping gene changes were consistent across multiple human RMS datasets. We performed signature correlation between our human RMS gene signature derived from the Gene Expression Omnibus dataset  K-RasWT B. A prev K-RasWT , and Doymutation . We therG12D mouse model captures human RMS pathway changes, we ran gene set enrichment analysis (GSEA) using the mouse and human DEG signatures. Interestingly, based on unbiased hierarchical clustering using the resulting normalized enrichment scores (NESs), the K-RasG12D clustered closer to human RMS than the K-RasWT . Not unexpectedly, immune-related pathways were not consistent between our K-RasG12D tumors and cell lines. We further assessed for the ability of these cell lines to generate tumors in vivo by injecting 1 million cells for each K-RasG12D cell line, KMR19 (p53R172H/+), KMR46 (p53F/+) and KMR72 (p53R172H/+), into the gastrocnemius of C57BL/6J mice  and p53F/+ (MF21) in C57BL/6 mice (Table S1), but, after several months (2-4 months), no tumors were evident upon necropsy of the animals.After isolation of GEMM tumors, as outlined in our methods, we developed primary cell lines from several GEMM tumors. We performed RNA-seq analysis on four cell lines  and subsequently performed GSEA analysis on the DEGs between our cell lines and normal gastrocnemius muscle tissue. RNA splicing, mRNA processing and signaling pathways were consistent with the results from our K-Ras/6J mice A, with pM tumors B,C. In aM tumors B,C. The G12D cell lines were injected via the tail vein into three C57BL/6J mice. One to two weeks following injection, mice were sacrificed and examined for metastatic disease. We observed that all mice showed evidence of macroscopic lung metastasis, which was further analyzed by Hematoxylin and Eosin (H&E) staining  values between our murine RMS lines and the human RMS cell lines  were purchased from American Type Culture Collection . The cells were grown and maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (Thermo Fisher Scientific), and cultured in a humidified 5% COR172H mice or K-RasG12D mice. Both male and female mice were used. Genotyping was performed using PCR. All research was performed in compliance with the Baylor College of Medicine IACUC  and AAALAC recommendations as published in The Guide for the Care and Use of Laboratory Animals (NRC1996). All animals were monitored closely and underwent comprehensive necropsies following humane euthanasia at an endpoint. All major organs and tumors were evaluated, and tissues suspected of metastatic disease were submitted for histological analysis.Transgenic mouse lines were generated through MyoD promoter-driven Cre recombinase with core enhancer, which directs the early activation of MyoD, as reported by 2 incubator at 37°C. All murine RMS cell lines are available upon request from the corresponding author (J.T.Y.).Murine RMS cell lines  were isolated from primary K-Ras-mutated RMS tumors. Resected tumors were diced and dissociated using a Tumor Dissociation Kit, mouse  and a gentleMACS Dissociator (Miltenyi Biotec). The dissociated cells were resuspended and filtered through a 70 μm cell strainer (Corning), then seeded, established and subcultured. The established cell lines were cultured in RPMI-1640 medium  supplemented with 1% penicillin/streptomycin and 10% FBS, and cultured in a humidified 5% CO6 cells per 25 μl PBS and then mixed with 25 μl Matrigel matrix . The cell–Matrigel mixture was subsequently injected into the gastrocnemius of C57BL/6J mice (n=3/cell line), and the tumors were resected once palpable.KMR19, KMR46, KMR72, KMR78, RD, Rh30 and PDX40 cells were harvested and resuspended at a concentration of 1×106 cells per 200 μl PBS. The cells were injected into the tail vein of the mice (n=3/cell line), which were subsequently sacrificed a week after injection. Cell lines were authenticated using short tandem repeat analysis and frequently checked for mycoplasma contamination.KMR19, KMR46, KMR72 and KMR78 cell lines were harvested and resuspended at a concentration of 1×103 cells per 96-well plate (Corning) for 24 h and then incubated with various concentrations of vincristine, irinotecan, SN38, MK1775 and trametinib  for 72 h. Subsequently, the degree of cell viability was evaluated using the Cell Counting Kit-8  according to the manufacturer's protocol.To assess cell viability, cells were seeded at 5×10GEMM and orthotopic model-derived xenograft tumors, as well as clinical specimens, were embedded in paraffin, fixed, and analyzed by Hematoxylin, Eosin, myogenin  and desmin  staining. For IHC analysis, a VECTASTAIN ABC Kit, Rabbit IgG  was used, and the staining was visualized using a Vector NovaRED Peroxidase Substrate Kit (Vector Laboratories).For PTAH staining, we used a Rapid PTAH Stain Kit . Briefly, tumor sections slides were placed in Langeron's iodine after deparaffinization and rehydration, and then transferred to 5% sodium thiosulfate. After rinsing in water, slides were stained in pre-heated PTAH.Images of H&E and IHC staining were obtained using a BX53 Biological Microscope . The whole pictures of lung sections were obtained by tile scanning, which merged 20× images and allowed the acquisition of overview images of the specimen, and Leica DMi8 and LAS X software .RNA samples underwent quality control assessment using the RNA tape on Tapestation 4200 (Agilent Technologies) and were quantified using a Qubit Fluorometer (Thermo Fisher Scientific). The RNA libraries were prepared and sequenced at the University of Houston Seq-N-Edit Core per standard protocols. RNA libraries were prepared with QIAseq Stranded Total RNA Library Kit (Qiagen) using 500 ng input RNA. mRNA was enriched with Oligo-dT probes attached to Pure mRNA beads (Qiagen). RNA was fragmented, reverse transcribed into cDNA, and ligated with Illumina sequencing adaptors. The size selection for libraries was analyzed using the DNA 1000 tape Tapestation 4200 (Agilent Technologies). The prepared libraries were pooled and sequenced using NextSeq 500 (Illumina), generating ∼17million 2×76 bp paired-end reads per samples.GSE108022, which includes 101 RMS (35 FP-RMS and 66 FN-RMS) samples and five normal muscle samples. Differential expression analysis was performed using DESeq2 R package (1.28.1)  software package; significance was achieved for adjusted q-value<0.25.Paired-end sequencing reads were trimmed using trimGalore software. The trimmed reads were mapped using STAR against the murine genome build UCSC mm10 and quantified using featureCounts  against (1.28.1) . P-valuen=30; n=147; n=58; GSE66533) datasets in R2:Genomics Analysis and Visualization Platform (http://r2.amc.nl). The UPS cohort RNA-seq counts data were kindly provided by Dr Nischalan Pillay .


Wolbachia is an intracellular bacterium that occurs in arthropods and in filarial worms. First described nearly a century ago in the reproductive tissues of Culex pipiens mosquitoes, Wolbachia is now known to occur in roughly 50% of insect species, and has been considered the most abundant intracellular bacterium on earth. In insect hosts, Wolbachia modifies reproduction in ways that facilitate spread of the microbe within the host population, but otherwise is relatively benign. In this “gene drive” capacity, Wolbachia provides a tool for manipulating mosquito populations. In mosquitoes, Wolbachia causes cytoplasmic incompatibility, in which the fusion of egg and sperm nuclei is disrupted, and eggs fail to hatch, depending on the presence/absence of Wolbachia in the parent insects. Recent findings demonstrate that Wolbachia from infected insects can be transferred into mosquito species that do not host a natural infection. When transinfected into Aedes aegypti, an important vector of dengue and Zika viruses, Wolbachia causes cytoplasmic incompatibility and, in addition, decreases the mosquito’s ability to transmit viruses to humans. This review addresses the maintenance of Wolbachia in insect cell lines, which provide a tool for high-level production of infectious bacteria. In vitro technologies will improve use of Wolbachia for pest control, and provide the microbiological framework for genetic engineering of this promising biocontrol agent.Wolbachia pipientis , is a Gram-negative member of the alpha proteobacteria that infects arthropods and filarial worms. Although closely related to the genera Anaplasma and Ehrlichia, which include pathogens of humans, Wolbachia is uniquely associated with invertebrate hosts in the clade Ecdysozoa. Originally described in Culex pipiens mosquitoes, Wolbachia is currently represented by 17 supergroups and is believed to occur in half of all insect species. In mosquitoes, Wolbachia acts as a gene drive agent, with the potential to modify vector populations; in filarial worms, Wolbachia functions as a symbiont, and is a target for drug therapy. A small number of Wolbachia strains from supergroups A, B, and F have been maintained in insect cell lines, which are thought to provide a more permissive environment than the natural host. When transferred back to an insect host, Wolbachia produced in cultured cells are infectious and retain reproductive phenotypes. Here, I review applications of insect cell lines in Wolbachia research and describe conditions that facilitate Wolbachia infection and replication in naive host cells. Progress in manipulation of Wolbachia in vitro will enable genetic and biochemical advances that will facilitate eventual genetic engineering of this important biological control agent.The obligate intracellular microbe,  Wolbachia is an obligate intracellular microbe first described in reproductive tissues of Culex pipiens mosquitoes nearly a century ago [Escherichia coli, Wolbachia is a Gram-negative bacterium in the phylum Proteobacteria: the purple bacteria and their relatives. Proteobacteria include nine monophyletic classes representing tremendous biodiversity. Among these, the genera Ehrlichia and Anaplasma, which can cause disease in humans, are classified with Wolbachia as members of the alpha-proteobacteria, in the order Rickettsiales, family Anaplasmataceae. Wolbachia is uniquely associated with invertebrates, does not infect vertebrate hosts, and replicates only within a eukaryotic host cell. In contrast, E. coli and many familiar Gram-negative pathogens of humans classified as gamma-proteobacteria can be cultured in liquid medium and plated on solid media as free-living microbes.tury ago ,2. Like Wolbachia, now known to infect a high proportion of insect species, in addition to other arthropods and filarial worms, all members of the Ecdysozoa. Because of its widespread distribution among insects [Wolbachia provides a model system for exploring biological interactions between an intracellular microbe, the invertebrate host cells in which it resides, and the diversity of reproductive phenotypes with which it is associated [Wolbachia, the bacterium is transmitted vertically, from mother to offspring, which retain the infection. In most arthropods, Wolbachia alters reproduction in diverse ways that favor its invasion of naive populations, and is sometimes considered a reproductive parasite. In contrast, Wolbachia is an essential symbiont in filarial worms [Wolbachia causes a reproductive distortion called cytoplasmic incompatibility (CI), which has important applications in vector control [Knowledge of well-studied free-living bacteria provides an important framework for investigating the genetics and physiology of  insects ,4, Wolbasociated ,6. In spal worms ,8,9. In  control .Wolbachia, Wolbachia pipientis, reflects its discovery in reproductive tissues of the mosquito Cx. pipiens [Wolbachia was described during the historical period when arthropod-borne intracellular bacteria were first appreciated as pathogens that cause disease in humans. Notable discoveries during that time included those of Howard Ricketts, who observed that a tick-borne bacterium, now known as Rickettsia rickettsii, was the causative agent of Rocky Mountain Spotted Fever [Rickettsia prowazekii as the cause of epidemic typhus [Wolbachia. Within the Rickettsiales, members of the genus Rickettsia are now assigned to the family Rickettsiaceae (short rods or coccobacilli), and Wolbachia, to the family Anaplasmataceae ; these families have been distinguished based on genetic analyses [Wolbachia was thought to be unique to Cx. pipiens mosquitoes because it appeared to be restricted to reproductive tissues, whereas other microbial symbionts in arthropods were more widely distributed among host tissues [The species name for  pipiens ,2. Wolbaed Fever , and Henc typhus . Simeon c typhus , and it analyses . For man tissues ,2. In reWolbachia is the reproductive distortion known as cytoplasmic incompatibility (CI). Discovery of CI was a fortuitous result of the fact that Cx. pipiens mosquitoes mate in small cages and establish breeding populations under laboratory conditions. In studies unrelated to the microbiology of Wolbachia, crosses between Cx. pipiens from independent laboratory colonies representing diverse geographic regions sometimes exhibited the peculiar, maternally inherited mating distortion called CI. CI was manifested when eggs from crosses between male and female mosquitoes originating from different regions failed to hatch, in a pattern eventually defined by 17 distinct cytotypes [Wolbachia was established, maternal inheritance of CI was recognized as a gene drive mechanism potentially useful for population replacement of vector mosquitoes [Wolbachia and CI was established when Yen and Barr demonstrated reversal of the CI phenotype in Cx. pipiens after treatment with antibiotics, which eliminated the bacterium [Drosophila and mosquitoes have been reviewed, and will not be discussed in detail here [The best-studied effect of ytotypes ,16,17,18squitoes ,20. The acterium ,22. The Wolbachia sequences in DNA samples from Drosophila melanogaster and other insects, contributed substantially to our understanding of the biology, distribution, and diversity of Wolbachia. Advancement of Wolbachia research through the use of D. melanogaster mutants is only beginning and, among mosquitoes, the presence of Wolbachia in Aedes albopictus, and its absence in important vectors such as Aedes aegypti and Anopheles gambiae, has provided an important incentive for Wolbachia research. Wolbachia also occurs in agricultural pests, including planthoppers that feed on rice, and lepidopteran pests of food crops.Development of genomic sequencing and related molecular technologies, and the discovery of Wolbachia genome from strain wMel infecting D. melanogaster revealed a streamlined, AT-rich genome of 1.27 Mbp encoding 1144 proteins (NC_002978.6), compared to the E. coli genome of 4.64 Mbp, 4242 proteins for K-12 sub-strain MG1655 (NC_000913.3). The wMel genome contains a putative prophage/s and an unusual abundance of mobile elements, relative to genomes from other intracellular bacteria [Wolbachia genomes has provided important insights into the correlations between gene loss, symbiosis, Wolbachia’s dependence on its host for essential nutrients, and its genetic capabilities; complete or nearly-complete sequence annotation for several representative genomes is available on the NCBI website (ncbi.nih.nlm.gov).Publication of the first bacteria . ComparaWolbachia isolate is typically called a strain, and is usually named for its host species; for example, Wolbachia from D. melanogaster is called wMel; that from Cx. pipiens is known as wPip. Individual strains are further subdivided into 17 monophyletic clusters of diversity, or supergroups, designated by letters [wCtub could refer to a supergroup J Wolbachia from a filarial worm [Wolbachia from a termite [As noted above, a  letters ,25. As mial worm  or a sup termite .Wolbachia infections are associated with phenotypes other than CI, including parthenogenesis, male killing, and feminization of genetic males, which are not necessarily uniform among members of a supergroup. Like CI, these phenotypes have a net effect of increasing the abundance of Wolbachia in host populations [Wolbachia infection, with loss of associated phenotypes, by treatment with rifampicin and/or tetracycline [Cimex lectularius (Hemiptera), the wasp Asobara tabida (Hymenoptera), and the springtail Folsomia candida (Collembola), in which Wolbachia has become a symbiont, are refractory to antibiotic cure. In humans, antibiotics have therapeutic value for reducing filarial infection [Consistent with the wide diversity of its hosts, ulations ,6,28 andacycline . Filarianfection .Wolbachia’s more general effects on the biology and ecology of its hosts [Cx. pipiens adults in the author’s laboratory have thus far yielded inconsistent results that may reflect, at least in part, variability in larval rearing conditions. Wolbachia’s individual associations with diverse hosts may preclude broad generalizations, and the complexity of Wolbachia’s interactions with host tissues provides a rich field for further investigation. As described below, fitness issues have become particularly important in efforts to establish novel Wolbachia associations for control of mosquito vectors.Although reproductive phenotypes can be validated by comparing infected relative to antibiotic-cured populations, it has been difficult to document ts hosts . Evaluatts hosts , and altts hosts ,32,33. SWolbachia as an environmentally-friendly approach to vector control builds upon early successful demonstrations that its associated gene drive can be harnessed for vector replacement [Wolbachia infections; successful introduction (transinfection) into non-host species such as the dengue vector, Ae aegypti, and the malaria vector, Anopheles gambiae [Wolbachia density, and tissue distribution, and virus-blocking capacity, are similarly important for successful Wolbachia-based control applications. Several ongoing studies address optimization of Wolbachia to control disease transmission, including combination with sterile insect techniques [Wolbachia, and the potential for its genetic modification using technologies that have become routine for E. coli.Present day interest in lacement ,20,34, c gambiae ; retenti gambiae ,35,36,37chniques . AlthougWolbachia’s obligate intracellular lifestyle complicates the biochemical and genetic analyses that could advance pest control and anti-filarial applications. Even with hosts amenable to laboratory rearing, maintenance of colonies, dissection of infected tissues, and embryonic microinjection are labor-intensive and time-consuming. Moreover, many existing laboratory colonies are highly inbred, complicating cage studies that address fitness. The utility of Wolbachia in control applications would be enhanced if the microbe could be experimentally manipulated by genetic engineering to express selectable markers, which in turn will be advanced by improving manipulation of Wolbachia in cell lines and expanding the diversity of Wolbachia strains that can be investigated in culture. A modest advance would be adaptation of a filarial strain of Wolbachia to a cell line; at present, Wolbachia-infected insect cell lines are used as a surrogate to identify new drugs that target Wolbachia for treatment of filarial diseases [diseases ,40,41.Wolbachia propagation in cultured cells as a substitute for the differentiated host tissues, such as ovaries and testes, in which Wolbachia is most abundant. Cell lines used to propagate Wolbachia are listed in wPip_B indicates that wPip is classified in supergroup B. With the exception of a single member of supergroup F from the cat flea [Wolbachia by different investigators, and given a different name. An important incentive for employing cell lines was the possibility that preadaption to cultured cells might improve the likelihood that Wolbachia would establish in novel hosts infected by embryonic microinjection, and towards this end, a few lines have been maintained for several years [Wolbachia on viral replication in efforts that generally validate the anti-pathogen responses seen in transinfected mosquitoes. Finally, as with Wolbachia itself, a uniform descriptive label for infected cell lines remains to be developed.The author’s research focuses on systematic exploration of cat flea , only meal years . In otheWolbachia-infected cell line are noted: those established directly from infected tissues, which retain Wolbachia from the host, and lines established from uninfected cells into which Wolbachia has been artificially introduced, either from infected tissues or from a previously infected cell line. O’Neill and coworkers were the first to describe a line with a host-derived Wolbachia infection, which they named Aa23 [wAlbB, one of two strains that co-infected the host Ae. albopictus mosquitoes. Curiously, at least in some geographic areas, when a single Wolbachia strain is found in Ae. albopictus, it types to strain wAlbA [Aedes albopictus hosts a natural infection, one might wonder why Wolbachia is absent from older Ae. albopictus lines dating back to the late 1960s, before Wolbachia was known to be widespread in insects [Wolbachia over decades of use in multiple laboratories.In med Aa23 . Aa23 cein wAlbA . Given t insects . The poswMelPop from D. melanogaster [Wolbachia strain in a primary culture called Dm2008Wb1, from which the Wolbachia was subsequently transferred to Drosophila S2 cells, has been developed in Russia [Drosophila mutants will provide insights into Wolbachia’s interactions with host cells.Other cell lines developed directly from infected hosts include JW-18, which carries nogaster . In addin Russia . AlthougWolbachia’s behavior in host tissues to cultured cells is not straightforward; similarly, it is not known how cultured cells should be manipulated to favor successful establishment of a persistent infection. For example, studies with Ae. albopictus eggs suggest that the highest Wolbachia densities occur during embryogenesis, and that replication is dependent on host cell division [wMelPop replicates best in postmitotic cells of adult flies [Wolbachia from metabolically active and/or infectious Wolbachia are not yet available, and will be needed to determine whether maintenance of Wolbachia in one cell line facilitates its establishment in a second cell line, or whether the initial cell line simply provides a higher multiplicity of infectious particles. When examined by fluorescence microscopy, a single Cx. pipiens ovary appears to contain innumerable Wolbachia, but it is not known whether all are equally infectious, whether the proportion of microbes capable of initiating infection changes during the mosquito reproductive cycle, and what factors define the small proportion of Wolbachia that establish in the embryonic germline. Finally, it will be of interest to learn whether Wolbachia (or other endogenous infectious agents such as insect-specific flaviviruses) influence how easily a permanent cell line can be established from a host species. Compared to those from Ae. albopictus and Ae. aegypti mosquitoes, relatively few established cell lines are available from Cx pipiens [Extrapolation of division , whereaslt flies . How est pipiens ,73.wAlbB can be removed from Aa23 cells by treatment with tetracycline, and these cured Aa23(T) cells have been a popular host for establishment of new infections [Wolbachia has been introduced derive from Ae. albopictus, and the possibility that these successes are influenced by host factors that coevolved with the natural Wolbachia infection in ancestral mosquitoes has not been addressed. Note, for example, that the RML-12 cell line, originally used to “preadapt” Wolbachia for transfer to Ae. aegypti, is now known to have originated from Ae. albopictus [wPip in culture [wPip as one of the best-understood models for CI [wPip in culture, and is not aware of a cell line that maintains a long-term persistent infection with wPip.As with infected insects, fections ,45,46. Ibopictus ,45. Aa23s for CI ,75,76, tWolbachia strains introduced into Aa23(T) cells is wMelPop, an unusually virulent strain isolated from D. melanogaster. Briefly, to investigate brain degeneration in Drosophila, Min and Benzer crossed an X-chromosome deficiency strain with reduced life span into the white mutant 1118,w which had a normal life span [popcorn” strain of Wolbachia. Popcorn proliferates primarily in postmitotic tissues of the adult, causing death of the host fly. Despite its ovarian transmission, popcorn does not cause CI in the original Drosophila strain described by Min and Benzer [Drosophila [wMelPop is caused by copy number variation of a 21 kb “octomom” region encoding eight genes of unknown function [One of the most interesting ife span . Resultid Benzer , but theosophila  and in tosophila . Genome function ,78.wMelPop-CLA designates a “cell-line adapted” version of wMelPop, created by infecting Aa23(T) cells with wMelPop from 1118w eggs [wMelPop to a cell line related to difficulties in using Wolbachia from infected tissues directly to establish a stable transovarial infection in a novel host, transfer of Wolbachia into an insect cell line can also be problematic. For example, establishment of wMelPop in Aa23(T) cells was successful in only two of 68 attempts [wMelPop-CLA was reintroduced into Drosophila embryos, in which it exhibited a diminution, but not complete loss, of properties related to life shortening, bacterial density, and CI, relative to the parental strain [wMelPop was of interest because reduced longevity of vector species would be expected to reduce pathogen transmission [wMelPop-CLA, and to a lesser extent wMel, have deleterious effects on Ae. aegypti eggs and larval development [wMelPop-CLA include RML-12  and Mos-55 (An. gambiae).The strain called 118 eggs . Althougattempts . After al strain . The lifsmission , but wMeelopment ,80, whicelopment . Other mD. melanogaster, wMelCS, was isolated from Canton S flies [wMelCS arose prior to wMel, and is ancestral to wMelPop. In nature, wMel appears to be replacing wMelCS [Wolbachia genome undergoes changes during growth in cultured cells has been evaluated in a genetic comparison of wMel strains, in which five genetic differences were detected after introduction into cell lines: an IS5 insertion, a multi-gene deletion, two point mutations, and a 10 bp deletion [Wolbachia transinfected into mosquitoes, it is possible that these genetic changes were triggered by cross-species transfer. Nevertheless, the changes are not known to affect essential genes or cause major genomic rearrangements, suggesting to a first approximation that use of genetically manipulated Wolbachia produced in cell lines will remain suitable for control applications. Although genome evolution of tissue-derived Wolbachia in cross-species transfers has not yet been investigated, recent in-depth comparisons suggest that relative to wRi (from Drosophila simulans) and wPip (from Cx. pipiens mosquitoes), wMelCS appears to be the best candidate for transinfection into Ae. aegypti for field release [A third variant from  S flies . Genomicg wMelCS . The extdeletion . Because release .Ae. albopictus cell line NIAS-AeAl-2 supports the Hemipteran Wolbachia strain, wStri, which was transferred from ovaries of the planthopper Laodelphax striatellus; this line also supports lepidopteran strains wKue, wCauA, and wCauB [wStri establishes a particularly robust infection in clonal C7-10 Aedes albopictus cells [wStri1 line to develop flow cytometric quantitation of Wolbachia infections [wStri strain also replicates in tick cell lines [wCauA provides an interesting model for investigating a single Wolbachia strain that causes two distinct phenotypes, CI and male killing, in different hosts [Drosophila subquinaria [The nd wCauB ,48. Moreus cells , and usefections . The wStll lines . Strain nt hosts ,85, a phquinaria Ae. aegypti Aa20 (Mos-20) cells [Wolbachia studies, including many that focus on Wolbachia’s antiviral effects. Aag2 cells are a subpopulation of cells isolated in the Peleg laboratory  that we adapted to Eagle’s medium [Ae. albopictus cells by electrophoretic mobility of small heat shock proteins. Aag2 cells host an inapparent infection with the insect-specific flavivirus called Cell Fusing Agent (CFA), which was discovered when Ae. aegypti cells were co-cultured with Ae. albopictus cells [Ae. aegypti cells less desirable, relative to Ae. albopictus cells, for classical studies with mosquito-borne viruses [Aside from a single transfer into 0) cells ,63, the s medium  and desius cells . For vir viruses ,90. Alth viruses , more re viruses .Wolbachia inhibits CFA replication [Wolbachia, possibly reflecting its negative-sense RNA genome [Aedes cell lines and mosquitoes is not inhibited by Wolbachia [In Aag2 cells, lication , in addilication , but thelication . More reA genome ,94. Simiolbachia .Wolbachia include those derived from the sandfly, Lutzomyia longipalpis, the biting midge, Culicoides sonorensis, and the horn fly, Hematobia irritans. Two lepidopteran lines, from the moths Heliothis zea and Spodoptera frugiperda, support wStri, wRi, and wCauB, whereas cell lines from the ticks Ixodes scapularis, Ixodes ricinus, and Riphicephalus microplus support wAlbB and wStri. Uniquely, establishment of wCfe_F in Ixodes scapularis lines raises the expectation that members of supergroups other than A and B will eventually be cultured in vitro [Wolbachia have not yet been cultured; by default, infected mosquito cell lines have been used as surrogates to screen for potential anti-filarial drugs [Newly established dipteran cell lines that support in vitro . Filariaal drugs ,40,41.Aedes albopictus C7-10 cell line [Wolbachia strains wAlbB [wStri [2 atmosphere. The persistently-infected line called C/wStri1 was produced by inoculating wStri from NIAS-AeAl-2 cells [My own lab has worked extensively with the ell line ,96,97 anns wAlbB ,55 and wB [wStri ,53,56. CB [wStri ,96,97. T-2 cells  into a gwStri1 cells are primarily used in current studies, Aa23 cells infected with wAlbB provided the starting point for our investigations [wAlbB in individual cells and between populations of cells [Wolbachia with tetracycline [Wolbachia-infected line, and whether the desirability of cell lines that grow as attached, relatively uniform monolayers may have contributed to inadvertent loss of infection with continued passage of long-established Ae. albopictus cell lines.Although C/igations . Aa23 ceof cells . In our acycline . Becauseacycline . It remaWolbachia replication does not improve the doubling time [Aa23 cells grow relatively slowly with a doubling time of 4–5 days, compared to 20 h with C7-10 cells. Counting Aa23 cells using a Coulter electronic cell counter can be facilitated by adding NP-40 to the culture medium to release cell nuclei, rather than attempting to disrupt clusters of tightly-associated cells . Alternaing time .wAlbB in Aa23 cells led us to transfer wAlbB from Aa23 cells into a TK-6 thymidine kinase-deficient derivative of C7-10 cells [wAlbB infection was lost after five months in TK-6 cells. Nevertheless, analysis of radiolabeled protein profiles in wAlbB-infected TK-6 cells led to the observation that the ubiquitin/proteasome pathway may play an important role in the interaction between Wolbachia and its host cell [Difficulties in obtaining accurate cell counts and enumerating 10 cells . Because10 cells , we reasost cell , and conost cell ,75,76.Laodelphax striatellus, wStri causes CI, as does wPip in Cx. pipiens mosquitoes. Although we have had little success with wPip, transfer of wStri from NIAS-AeAl-2 cells to C7-10 cells was highly successful [wStri1 cells lost the infection after approximately 130 subcultures, suggesting that with continued passage, the cells suppress Wolbachia. Factors that affect stability, such as changes in the ratio of Wolbachia to host cells over time, remain to be systematically examined. These observations support the possibility that there is an unknown fitness cost to maintaining Wolbachia in cell lines, consistent with the absence of Wolbachia in long-established Ae. albopictus lines. As new Wolbachia-infected lines are developed, investigators should bear in mind the importance of periodically storing cells in liquid nitrogen to recover early-passage cultures, against which changes over time can be evaluated.In its native host, the planthopper ccessful , and estWolbachia in cultured cells is flow cytometry, which allows simultaneous evaluation of cell and bacterial particles (A major advance in our ability to work with articles .Wolbachia are those newly-released from fragile host cells, which constitute about 1% of the total Wolbachia population in C/wStri1 cells. Establishment of a robust level of Wolbachia in a naive cell line requires about 6 days, with an infection ratio of 80 to 100 bacteria per cell. Reminiscent of Aa23 cells, mild aggregation of recipient cells is a typical response of C7-10 cells to infection [wStri replicates better in stationary, as opposed to growing, cells, and we have recently found that high yields of infectious Wolbachia can be recovered from mitotically inactivated feeder layers generated by treatment with mitomycin C [Wolbachia infection itself confers a selective advantage will constitute an important resource for recovery of genetically modified Wolbachia.Using this approach, we have determined that the most infectious nfection . Currentomycin C . In addiWolbachia phenotype and/or virulence that accompany long-term maintenance outside the wildtype host, much remains to be learned about optimal conditions for maintaining Wolbachia in vitro. The relationships between cell cycle parameters, doubling time, and ploidy of cultured cells, relative to conditions that support Wolbachia in natural host tissues, have not been systematically explored, nor have effects of nutrient requirements and medium composition. In future studies it will be important to undertake transcriptomic and proteomic analyses to identify genes and gene products that regulate infection and replication of Wolbachia in cell lines. Insect cell culture itself remains a developing field, relative to accomplishments that have been achieved with vertebrate cell lines and embryonic stem cells.In addition to changes in the Wolbachia genome can be genetically engineered in the future, propagation of the altered genome will require efficient reintroduction into a host cell to allow replication and expansion of transformant populations. Use of cell lines offers a practical means of producing the large quantities of Wolbachia that will be needed to develop transformation protocols that are sufficiently robust for use in basic research and pest control applications. Although isolated examples of successful transformation of intracellular microorganisms such as Coxiella burnetti, the pathogen that causes Q fever, have been achieved, these remain labor intensive and have low frequencies of success [Coxiella has been achieved, despite its streamlined 2 Mb genome [Wolbachia lacks pathogenicity to humans, and its genome is more extensively streamlined, relative to that of Coxiella. Nevertheless, the long evolutionary history of Wolbachia’s interaction with invertebrate hosts and its adaptations for germline transmission contribute to the value of Wolbachia as a model system for understanding the biology of obligate intracellular bacteria in invertebrate cells and manipulating their biology for control of insect pests.If the streamlined  success . Neverthb genome ,104. The


Rickettsia raoultii is one of the causative agents of tick-borne lymphadenopathy in humans. This bacterium was previously isolated and propagated in tick cell lines; however, the growth characteristics have not been investigated. Here, we present the replication kinetics of R. raoultii in cell lines derived from different tick genera . Tick cell cultures were infected in duplicate with cryopreserved R. raoultii prepared from homologous cell lines. By 12–14 days post infection, 100% of the cells were infected, as visualized in Giemsa-stained cytocentrifuge smears. R. raoultii growth curves, determined by rickettsiae-specific gltA qPCR, exhibited lag, exponential, stationary and death phases. Exponential phases of 4–12 days and generation times of 0.9–2.6 days were observed. R. raoultii in BME/CTVM23 and RSE/PILS35 cultures showed, respectively, 39.5- and 37.1-fold increases compared to the inoculum. In contrast, multiplication of R. raoultii in the IDE8 cultures was 110.1-fold greater than the inoculum with a 7-day stationary phase. These findings suggest variation in the growth kinetics of R. raoultii in the different tick cell lines tested, amongst which IDE8 cells could tolerate the highest levels of R. raoultii replication. Further studies of R. raoultii are needed for a better understanding of its persistence within tick populations. Rickettsia raoultii is a spotted fever group rickettsial species, which was first described in Dermacentor nuttalli (reported then as genotypes DnS14 and DnS28) and Rhipicephalus pumilio (genotype RpA4) ticks from Siberia [Dermacentor spp. ticks, mainly D. nuttalli, Dermacentor reticulatus, Dermacentor marginatus and Dermacentor silvarum, from Russia [R. raoultii has also been reported in Haemaphysalis erinacei [Ixodes ricinus [Melophagus ovinus [Dermacentor spp. ticks. Rickettsia spp., genetically related to R. raoultii (>98% identity in target genes), were also described from Dermacentor spp. ticks from Southeast Asian countries [m Russia ,4,5, andm Russia ,7,8,9. Rerinacei  and Ixod ricinus  ticks, as ovinus , suggestountries ,14. R. raoultii, together with Rickettsia slovaca, is a causative agent of tick-borne lymphadenopathy (TIBOLA), also known as Dermacentor-borne necrosis erythema and lymphadenopathy (DEBONEL) [R. raoultii infections with erythematous rash and fever, but without lymphadenopathy, were also reported in patients from China [R. raoultii infections include meningeal syndrome [R. raoultii. Although TIBOLA/DEBONEL is attributed to both R. slovaca and R. raoultii, studies have suggested that R. raoultii is more highly prevalent among Dermacentor spp. ticks [R. slovaca infection, implying that R. raoultii is less pathogenic [R. raoultii is still scarce; more thorough characterization is necessary to understand these aspects of the bacterium.DEBONEL) . The synDEBONEL) . R. raouom China . Other rsyndrome , and neusyndrome . Althougsyndrome , or suscp. ticks . Howeverthogenic  or less R. raoultii was first isolated from Dermacentor spp. ticks into L929 and Vero cells by Mediannikov and co-workers [R. raoultii was also isolated into embryo-derived tick cell lines originated from Rhipicephalus microplus [Rhipicephalus sanguineus [Dermacentor albipictus and Dermacentor nitens [Rickettsia spp., tick cell lines are a useful system for the isolation and propagation of R. raoultii from ticks or clinical samples for further investigation of its virulence and pathogenicity [icroplus ,21,22,23nguineus . The bacr nitens . Since tgenicity ,24. R. raoultii in three tick cell lines—BME/CTVM23 derived from R. microplus [R. sanguineus [Ixodes scapularis [R. raoultii in tick cell lines and provide the basis for further studies into its invasiveness for host cells.The purpose of this study was to establish the infection rates and replication kinetics of icroplus , RSE/PILnguineus , and IDEapularis . The aimR. microplus-derived cell line BME/CTVM23 at passage 77 [R. sanguineus-derived cell line RSE/PILS35 at passage 16 [I. scapularis-derived cell line IDE8 at passage 98 [The tick cell lines and bacterial culture were obtained from the Tick Cell Biobank, University of Liverpool, UK and maintained in the Tick Cell Biobank Asia Outpost at TIDREC, Universiti Malaya, Malaysia. The ssage 77  and R. sssage 16  were grossage 98  was mainssage 98  supplemeR. raoultii (strain Białystok1) was provided in a culture maintained in the BME/CTVM23 cell line [R. raoultii was maintained by passaging the bacteria onto fresh BME/CTVM23 cells once every three weeks. ell line . The bacg for 5 min at 4 °C, to remove intact cells and cell debris. Next, equal volumes of the suspension were used to inoculate IDE8 and RSE/PILS35 cell cultures. The inoculated cells were maintained at 28 °C and were observed daily for signs of cytopathic effects using a BMI-100 inverted microscope . Once severe cytopathic effects were observed , aliquots of the infected cells were immediately cryopreserved in their respective culture medium with the addition of 10% dimethyl sulfoxide as described previously [Heavily infected BME/CTVM23 cells were resuspended by pipetting and forcibly passed through a 25 G needle 5 times to release the bacteria from the cells. The resulting suspension was filtered through a 2.0 µm membrane filter and centrifuged at 1000× eviously , to be u6 cells/mL, RSE/PILS35 cells were seeded at 2 × 106 cells/mL and IDE8 cells were seeded at 5 × 106 cells/mL. To each culture, 200 µL of thawed, cryopreserved bacterial stock prepared from the homologous cell line was added. Post infection, the cell cultures were monitored daily for signs of cytopathic effects. Cells were collected at the indicated intervals for preparation of Giemsa-stained cytocentrifuge smears and for bacterial quantification by quantitative PCR (qPCR) as described below. Fresh medium was used to replace the volume that was removed when the cell suspension was taken from the culture tube.One day prior to infection, duplicate cultures were set up in 2.2 mL culture medium with the following cell densities for each cell line to be tested: the BME/CTVM23 cells were seeded at 6 × 10Cytocentrifuge smears were prepared from 50 µL of cell suspension centrifuged for 5 min at 1000 rpm. in a Cytospin 3 cytocentrifuge . The resultant smears were air-dried, fixed in methanol for 3 min, stained with Giemsa  and rinsed 3 times with water buffered to pH 7. The Giemsa-stained smears were examined under a compound microscope  at 1000× magnification for the presence of bacteria. The GXCAM digital camera and GXCapture software were used to capture images of the cells. The percentage of infected cells was determined by calculating the number of infected cells × 100, divided by the total number of cells (at least 200 cells examined for each sample).® tissue kit  following the manufacturer’s protocol. DNA was also extracted from a 200 µL aliquot of cryopreserved bacterial stock prepared from the homologous cell line as the representation of the number of bacterial DNA copies present at time point 0 dpi in the infection time course. A previously published qPCR protocol targeting a 74 base-pair fragment of the Rickettsia citrate synthase (gltA) gene [R. raoultii in the infected cultures. The qPCR was performed using a CFX96 touch real-time PCR detection system  with a 6-carboxyfluorescein (FAM) and black-hole quencher (BHQ1-) labelled TaqMan probe . All reactions were prepared in 25 µL reaction volumes, comprising final concentrations of 1 × TaqMan fast advanced master mix , 200 nM of each primer and probe, and 1 µL DNA template. The PCR cycling conditions were as follows: initial holding temperature at 50 °C for 3 min, followed by 95 °C for 5 min and 40 cycles of 95 °C for 20 s and 60 °C for 40 s. To determine the bacterial DNA copy number, a pIDTSmart (Amp) vector  containing the gene target was used to construct a standard curve with serial dilution in the range of 1 × 105 to 1 × 1013 copies. DNA was extracted from 200 µL of cell suspension collected from each time point of the experiment using a NucleoSpintA) gene  was usedrpl6) was performed as described previously [−1 to 5 × 106 copies. The generation times for R. raoultii in each tick cell culture were calculated using the following equation [t is the interval between To determine the numbers of tick cells in the cultures, a qPCR assay targeting a 77 base-pair fragment of a tick single-copy nuclear gene, ribosomal protein L6 . This observation suggests that R. raoultii were able to multiply to higher numbers in the IDE8 cells. Furthermore, a stationary phase lasting up to seven days could be observed in the infected IDE8 cultures, and there was only a marginal decline in the tick gene copy numbers during this phase. This suggests that IDE8 cells may be more tolerant than the other two cell lines of heavy bacterial burden before the onset of cell death in the culture conditions used. The increase in R. raoultii can infect and propagate in BME/CTVM23, RSE/PILS35 and IDE8 cell lines, representing three species of metastriate and prostriate ticks not known to harbor this bacterium in nature. We have further demonstrated the growth kinetics of the bacteria in these cell lines. Our observations include a longer exponential phase and generation times, as well as higher bacterial multiplication levels in tick cell lines as compared to the observations in mammalian cells in previous studies. Further experiments, however, are necessary to examine the influence of incubation temperatures on bacterial growth. When the infected tick cell cultures were maintained under the same culture conditions, the IDE8 cells also appeared to be able to tolerate higher R. raoultii burdens and multiplication levels than the two metastriate tick cell lines. Further investigations will be necessary to determine if similar observations are made under different culture conditions, and to examine if the genotypic and phenotypic differences between the tick cell lines influence R. raoultii growth rates.In conclusion, we have demonstrated that R. raoultii and, therefore, do not represent the natural host cells of the bacteria. Nevertheless, these cell lines will still be useful for investigating arthropod–pathogen interactions, especially in the absence of continuous cell lines from any of the natural arthropod vectors. It is also important to note that the tick cell lines used in this study did not originate from the natural vector of


Cisplatin is one of the chemotherapeutic drugs used for the management of oral carcinoma, in which combined therapies are estimated to exert superior therapeutic efficacy compared with monotherapy. It is known that poly(ADP-ribosyl)ation is implicated in a multiplicity of cellular activities, such as DNA repair and cell death. Based on these, PARP inhibitors are used for the treatment of cancers; however, the capacity of PARP inhibitors associated to anti-cancer drugs have not been completely assessed in oral carcinoma. Here, we evaluated the effects of PARPi veliparib (ABT888) in combination with cisplatin on the survival of three human oral cancer cell lines HSC-2, Ca9-22 and CAL27 and we observed the effects of ABT888 alone or in combination with cisplatin on apoptosis and DNA damage repair mechanism. The results obtained showed that ABT888 induces a cytotoxicity effect on cell viability increasing the apoptotic pathway as well as DNA strand break; moreover, our results displayed the effects with cisplatin in a dose-dependent manner. Therefore, our results indicate PARP inhibitors as adjuvants for therapeutic strategy of oral cancer. Oral cancer represents one of the most widespread types of tumors that shows high morbidity, mortality  and metaAlthough many drugs have been established as treatments for cancer, they commonly display cytotoxicity and side effects .Specifically, in recent years, new treatments for oral cancer have been advanced; however, overall survival has not meaningfully diminished due to the alterations in tumor suppressor genes and variations in signaling pathways that lead to therapeutic resistance . For thiPoly (ADP-ribose) polymerases (PARPs) are a family of enzymes involved in several cellular processes such as DNA repair for cell survival, inflammatory responses and preservation of genome stability . The PARPARPs are able to catalyze reactions of Poly-ADP-ribosylation or mono-ADP-ribosylation . Among PThus, given the key roles of PARPs, especially PARP1 and PARP2, in repairing cytotoxic chemotherapy-induced DNA damage, they have attracted attention as a new therapeutic strategy for cancer. The potential benefit of targeting the enzymatic activity of PARPs for therapeutic purposes is very high, supported also by its recent approval for clinical usage in the treatment of BRCA DNA repair-associated-mutated ovarian cancer ,14. MoreAmong PARP inhibitors, more attention was given to Veliparib (ABT888), which, as well as other clinically relevant PARPi, targets DNA damage repair PARPs, mainly PARP1 and PARP2 .An important study conducted by Yasukawa revealed the importance of the anti-tumor effects of PARP inhibitors used in combination with chemotherapeutic agents such as cisplatin thanks to their abilities to arrest cell cycle and decrease DNA repair activity through a G2 cell-cycle arrest-like effect in a dose and p53-dependent manner ,22.P53 is a tumor suppressor that responds to various stress signals by modulating specific cellular responses, including transient cell cycle arrest, cellular senescence and apoptosis, all of which are associated with tumor suppression . HoweverP53 deficiency as well as apoptosis alteration can enhance the initiation or progression of cancer , suggestThus, the combination of PARPi with anticancer treatments, including chemotherapy, molecular targeted agents, or immunotherapy, is certainly an encouraging method to maximize the effectiveness of tumor treatment, though they have been proven challenging due to overlapping toxicities with chemotherapy agents .Therefore, on the basis of PARP functions and the key role of DNA damage and the apoptosis pathway on oral cancer ,25,26, hSuch a combination is expected to exert a combinatorial effect, consequently resulting in superior therapeutic effects compared to drug administration alone, thus decreasing the side-effects and preventing drug resistance.Three cell lines, HSC-2 , Ca9-22  and CAL27 (human tongue adenosquamous carcinoma cell line), were obtained from ATCC  and were used in this study. The authors chose to use these three cell lines because they are the most studied cell lines in vitro for these tumors.®, Campinas, Brazil), 100 U/mL penicillin and 100 μg/mL streptomycin  in minimum essential Eagle’s medium (Sigma-Aldrich) for HSC-2 and Ca9-22 cells, and in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich) for CAL27 cells. The cells were kept in an incubator at 37 °C with 5% CO2, and the change of growth medium was performed every three days.The growth medium for cells contained 10% fetal bovine serum (FBS) , as previously described . The firg at 4 °C for 15 min, and the supernatants were collected. Then, cell lysates were electrophoretically separated on an SDS-PAGE gel using a standard protocol. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The blots were then incubated with the following primary antibodies at 4 °C overnight (w/v nonfat dried milk and 0.1% Tween-20 (PMT) and incubated in the membranes at 4 °C, overnight. Subsequently, a secondary antibody was added to the membrane  for 1 h at room temperature. Moreover, to ascertain that the blot was loaded with equal amounts of protein lysate, β-actin antibody  or lamin A/C  were used as an endogenous control for protein expression of blots. Blots were analyzed as previously described [Western blot analysis was performed as previously described . The celvernight : anti-VEescribed .AN immunofluorescence assay was performed on CAL27 cells, as previously described by Donaldson . Cells wThe images were taken with a confocal laser scanning microscope (CLSM) (Zeiss LSM700). The experiments were repeated at least three times to confirm results.6 CAL27 cells per tumor in 0.2 mL of Phosphate Buffered Saline (PBS) and 0.1 mL of Matrigel  into BALB/c nude mice, as previously described by Yasukawa et al. [A Xenograft tumor model was established by subcutaneously inoculating 5 × 10a et al. . Once tha et al. ; (c) ABTa et al. .2 × L/2, where W and L represented the minor and major length. Three days after the last administration, all surviving mice were sacrificed, and tumors samples were processed for several analyses.The tumor size was monitored daily using a caliper and calculated as follows: V = WAnimal experiments were in compliance with Italian regulations on the protection of animals used for experimental and other scientific purposes (DM 116192) as well as EU regulations (OJ of EC L 358/1 18 December 1986). The project identification code was 399/2019-PR released on May 24, 2019.Haematoxylin and eosin (HE) staining was performed as previously described by Casili et al. . Tumor sBAX Inhibiting Peptide V5  and Pifitrin-α  were obtained from Sigma-Aldrich .n” observations. Every analysis was executed three times, as an independent experiment. The results were examined using a one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant.All values are exposed as mean ± standard error of the mean (SEM) of “To evaluate the cytotoxic effects of ABT888 and cisplatin on the three cell lines derived from oral squamous cell carcinoma, we incubated HSC-2, CAL27 and Ca9-22 with different growing concentrations of ABT888  and cisplatin  for 24 h. We performed MTT assays, and we observed weak toxicity effects at the lowest concentration of ABT888  were 60 μM for ABT888 A–C and 1Apoptosis is a crucial pathway involved in the development of tumors such as oral carcinoma. To verify if ABT888 improved the efficacy of chemotherapeutic agents to induce apoptosis in oral squamous carcinoma, we assessed RT-qPCR of pro- and anti-apoptotic proteins in the CAL27 cell line. We observed that a combinatory treatment of ABT888 and cisplatin considerably increased the expression of pro-apoptotic Bax, caspase-3, caspase-9 and p53 in the CAL27 cell line A–D, compTo confirm the ability of ABT888 and cisplatin to modulate the apoptosis pathway, we decided to investigate Bax, Bcl2, p53 and caspase-3 expressions using Western blot analysis in the CAL27, HSC-2 and Ca9-22 cell lines. The results obtained in all three cell lines showed that the protein levels of BAX, p53 and caspase-3 were considerably increased in the combination group of ABT888 and cisplatin  Glycohydrolase (PARG), which is involved in DNA repair , vasculaWe observed that the protein levels of PARP-1 and PARG were considerably increased in the combination group of ABT888 and cisplatin , at a concentration of 50 µM  and an iOn the basis of the obtained results, increasing PARP expression may indicate an increase in cell necrosis . TherefoThe histological analysis revealed a marked subcutaneous mass in the control group, as well as an increase in the neutrophilic permeation, whereas the association between ABT888 and cisplatin showed a significant reduction in tumor sections much more than single components A–D. MoreTargeting DNA damage repair (DDR) signaling is a fast-expanding field for cancer therapy. DNA damage causes genomic instability in cells that require DDR for rescue. DDR signaling triggers the activation of repair protein transcription, hence, leading to overexpression of the related repair protein and activation of mechanisms for DNA repair . PARP isIt has been recognized that PARP inhibition for the management of breast or ovarian cancers with BRAC1/2 mutations (deficiencies in the HR) is now a well-established approach, in fact over the past decade, four PARPi have been FDA-approved for clinical use as single agents.In particular, previous studies reported that PARP inhibitors enhanced the anti-tumor effects of cisplatin, especially in breast cancer ,44, as wThus, based on this evidence, in this study we evaluate if treatment with specific PARPi such as Veliparib (ABT888) could enhance the anti-tumor effect of cisplatin, when given in combination, for the treatment of oral carcinoma, representing a new therapeutic approach for this type of cancer.The results obtained demonstrated that ABT888 revealed in vitro combinatorial effects with cisplatin in three cell lines derived from oral carcinoma, HSC-2, Ca9-22 and CAL27. In particular, we identified not only a selective anti-proliferative effect of ABT888 in a concentration dependent-manner, but we also observed that ABT888 potentiated the cytotoxic effect of cisplatin. Moreover, we observed that ABT888 exposure shows apoptosis-inducing effects on oral cancer cells, indicating its probable mechanism of action for its specific anti-proliferation effect.Programmed cell death occurs thanks to two main mechanisms, specifically through two apoptotic signaling pathways, one defined as intrinsic and the other extrinsic . The intPrevious papers reported a synergetic effect of a PARPi AZD2281) with cisplatin to cause arrest of the cellular cycle in G2/M and to decrease DNA repair activity in a dose and p53-dependent manner 81 with c.P53 is a tumor suppressor protein with a crucial anti-cancer role promoting cell cycle arrest, senescence and apoptosis in response to stress signals . CurrentTherefore, in the present study, we proved that treatment with ABT888, and much more in combination with cisplatin, inhibited cell apoptosis in the CAL27-treated cell line, resulting in an increase in the mRNA expression of Bax and Bcl-2 downregulation via the p53 dependent pathway. Moreover, we observed that the levels of caspase-3 and 9 were significantly increased in ABT88 and the cisplatin-treated CAL27 cell line.Moreover, to confirm the effect of ABT888 and cisplatin on the apoptosis pathway, we investigated Bax, Bcl2, caspase-3 and p53 protein expression in all three cell lines using Western blot analysis, showing that ABT888, and much more in combination with cisplatin, was able to increase the expression of pro-apoptotic Bax, p53 and caspase-3, while the expression of anti-apoptotic protein Bcl2 was significantly reduced.Thus, in the present study we observed that ABT888, and much more in combination with cisplatin, was able to inhibit CAL27, HSC-2 and Ca9-22 tumor cell growth through the induction of apoptosis inducing p-53 and the caspase-dependent pathway, suggesting that ABT888 can activate the mitochondrial pathway of apoptotic cell death.Moreover, since PARP is an SSB DNA repair enzyme, continuous exposure to PARP inhibitors increases the basal levels of SSBs, which may cause replication fork collapse and the formation of DNA double strand breaks (DSBs) ,53. DSBsHere, we observed that the level of γH2AX was increased in the combination group, although its expression was highest in the CAL-27 cell line treated with ABT888 alone, showing that the PARPi treatment could result in higher levels of DNA damages.Moreover, here, we determined PARG, a glycohydrolase involved in DNA-damage repair , and PARFurther analyses on the molecular basis of DNA breakdown and cytotoxicity were performed by examining the expression of the well-known DNA repair marker, DSB Rad51. Rad51 is a recombinase and an important molecule in the DNA repair pathway of homologous recombination (HR), which is recruited to the DNA break sites and forms separate nuclear foci. We found that the CAL-27 cell lines used in this study expressed high levels of Rad51, suggesting a recombinant functional repair capacity, which was also confirmed using an immunofluorescence assay. However, treatment with ABT888 alone, and much more with cisplatin, reduced the expression of Rad51, indicating a deficiency of human repair that could lead to more DSBs observed through the resulting increase in the expression levels of γ -H2AX. Moreover, to prove that ABT888 in association with cisplatin acts with an apoptosis-dependent and p53-dependent mechanism, we conducted a cell viability assay on a CAL27 cell treated with an inhibitor of apoptosis and an inhibitor of the p53 pathway. Our results showed that the apoptosis inhibitor BIPV5 and the p53 inhibitor PFT-α did not reduce CAL27 cell viability in the control group as well as alone or in association with ABT888 and cisplatin, confirming the hypotheses that ABT888 in association with cisplatin modulates apoptosis and the p53-dependent pathway. Furthermore, it cannot be excluded that in other cellular contexts, PARP inhibitors may act by involving other apoptotic mediators as well. Moreover, an increasing PARP expression may indicate cell necrosis; therefore, we demonstrated that the association of ABT888 and cisplatin significantly increased the expression of necrosis marker as RIP1 and MLKL on the CAL27 cell line, suggesting a promoting of the necrosis phenomenon thanks to the abilities of ABT888 and cisplatin.Additionally, to confirm the data obtained in an in vitro model, we decided to conduct an in vivo xenograft model using CAL27 cells. The results obtained revealed that the association of ABT888 and cisplatin significantly reduced the tumor weight, tumor burden as well as the neutrophil infiltration much more the single treatments.Thus, taken together, our results showed the therapeutic potential of PARPi in the treatment of oral cancer, suggesting that the combination of cisplatin and ABT888 could be a promising candidate for a treatment strategy. However, further investigations into the mechanism responsible for suppressing the DNA repair pathway of homologous recombination will be required. Meanwhile, this study would provide new insights into anticancer therapies, thus representing a new starting point.


Combined NMR and MS analysis revealed that roseabol A (1) contained amino acid residues characteristic of the peptaibol family of peptides such as isovaline, α-aminoisobutyric acid, hydroxyproline, leucinol, and an N-terminal isovaleric acid moiety. The amino acid sequence was established by a combination of NMR studies and tandem MS fragmentation analyses, and the absolute configurations of the constituent amino acids of 1 were determined by the advanced Marfey’s method. Compound 2 showed inhibitory activity against Merkel cell carcinoma, a rare and difficult-to-treat type of skin cancer, with an IC50 value of 16.5 μM.A new 11 amino acid linear peptide named roseabol A ( N-terminus is usually acetylated, whereas the C-terminal amino acid is amide-linked to a 1,2-amino alcohol. While the majority (>50%) of peptaibols have been isolated from different species of Trichoderma 25D +7.7 ; UV (MeOH) λmax (log ε) 195 (3.57) ; CD  λmax (Dε) 194 (+30.79), 206 (−40.01), 224 (−13.69) nm; IR (film) νmax 3286, 2935, 1650, 1540, 1440, 1384, 1201, 1058 cm−1; 1H- and 13C-NMR, m/z 1225.7774 [M + H]+ .1 were individually dissolved in degassed 6 N HCl (0.6 mL) and heated in sealed glass vials at 110 °C for 17 h. The hydrolysates were evaporated to dryness and dissolved in H2O (50 μL); to this solution was added 20 μL of 1 N NaHCO3 and 100 μL of a 1% solution in acetone of either 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA) or a racemic mixture of D/L-FDLA +; L-Iva (26.1), D-Iva (27.7), m/z 412 [M + H]+; L-Leuol (27.2), D-Leuol (31.5), m/z 412 [M + H]+; trans-L-Hyp (18.8), trans-D-Hyp (19.1), m/z 426 [M + H]+; L-Leu (26.7), D-Leu (31.3), m/z 426 [M + H]+; L-Ser (20.8), D-Ser (21.3), m/z 400 [M + H]+; L-Gln (21.8), D-Gln (22.7), m/z 440 [M − H]−.50 values were estimated from dose–response curves using 4-parameter logistic analysis .The purified compounds were assessed for growth inhibition/cytotoxicity against two Merkel cell carcinoma cell lines, MCC26 (MCPyV−) and MKL-1 (MCPyV+), as well as an immortalized human keratinocyte cell line (HaCaT). Briefly, cells were plated in 384-well clear tissue culture plates with 2500 cells/well  or 15,000 cells/well (MKL1) in DME/10% FBS  or RPMI/10% FBS (HaCaT). The plated cells were allowed to grow overnight, followed by addition of the test compound. DMSO solutions of the compounds were diluted in growth medium prior to addition and assessed in a 10-point (1:2) dilution series, top concentration 40 µM. After 3 days, relative cell numbers were assessed using the XTT metabolic growth assay . The res1) was isolated and characterized from the fungus Clonostachys rosea. Studies of secondary metabolite production by various Clonostachys isolates have only been sparsely reported in the chemical literature. There is one prior report of a peptaibol [N-methylated cyclic peptides [1) is only the second member of the peptaibol family of peptides to be discovered from a Clonostachys isolate. The oxidized linoleic acid derivative 13-oxo-trans-9,10-epoxy-11(E)-octadecenoic acid (2) was also obtained from the C. rosea extract. Compound 2 is an epoxyketooctadecenoic acid derivative that is known to stimulate corticosterone production [2 showed selective cytotoxic activity towards the Merkel cell carcinoma cell line MKL-1, which is positive for the Merkel cell polyomavirus (MCPyV+), and was less effective against the MCPyV− MCC26 cell line. Roseabol A (1) was inactive against both Merkel cell carcinoma cell lines. The differential response seen between the virus-positive and virus-negative cell lines suggests that 2, or a related structural analog, could have value as a biological probe to investigate virus-associated aspects of Merkel cell carcinoma.A new 11-residue peptide named roseabol A (eptaibol , as wellpeptides  and a sepeptides  that havoduction  and actioduction . It has oduction . Compoun


T-cell lymphoma is a cancer of the immune system. One specific sub-type of T-cell lymphoma is a malignancy called anaplastic large cell lymphoma (ALCL), which is distinct from the other forms, as in general, it has a better prognosis. Research conducted to understand why ALCL develops has shown that a specific genetic event occurs, whereby a new protein is created that drives tumour growth. This protein is called nucleophosmin–anaplastic lymphoma kinase (NPM-ALK). Our research, described here, shows that NPM-ALK regulates another protein, called BRG1, to drive proliferation of tumour cells. In turn, when the gene that leads to expression of BRG1 is inactivated, the tumour cells die. These data suggest that therapeutic targeting of BRG1 might be a novel therapy for this form of cancer.Anaplastic large-cell lymphoma (ALCL) is a T-cell malignancy driven in many cases by the product of a chromosomal translocation, nucleophosmin–anaplastic lymphoma kinase (NPM-ALK). NPM-ALK activates a plethora of pathways that drive the hallmarks of cancer, largely signalling pathways normally associated with cytokine and/or T-cell receptor-induced signalling. However, NPM-ALK is also located in the nucleus and its functions in this cellular compartment for the most part remain to be determined. We show that ALCL cell lines and primary patient tumours express the transcriptional activator BRG1 in a NPM-ALK-dependent manner. NPM-ALK regulates expression of BRG1 by post-translational mechanisms dependent on its kinase activity, protecting it from proteasomal degradation. Furthermore, we show that BRG1 drives a transcriptional programme associated with cell cycle progression. In turn, inhibition of BRG1 expression with specific shRNA decreases cell viability, suggesting that it may represent a key therapeutic target for the treatment of ALCL. The oncactivity activity ,10,11,12 [+ ALCL . It has  [+ ALCL ,14. Howe [+ ALCL ,16.+ ALCL cell line KARPAS-299, suggesting that it is part of a complex with NPM-ALK  [BRM-Related Gene 1 , is a central ATPase subunit of the human SWI/SNF chromatin remodelling complex ,18. BRG1ocation) . Whilst ocation) ,22,23,242. The DHL-T5 A5 (a derivative SUDHL1 cell line expressing an shRNA targeting NPM-ALK in a doxycycline-inducible manner) and MEFNA (murine embryonic fibroblast cell line expressing NPM-ALK in a doxycycline-inducible manner) cell lines were provided by Roberto Chiarle, University of Torino, Torino, Italy. For induction of shRNA expression, the DHL TA A5 cell line was grown in the presence of 1 µg/mL doxycycline . To inhibit expression of NPM-ALK, the MEFNA cell line was grown in the presence of 1 µg/mL doxycycline . A tumour cell line derived from the CD4/NPM-ALK transgenic mouse model [Cell lines Jurkat, SUDHL-1, Karpas-299, DEL and SUP-M2 were purchased from DSMZ . The ALCL, ALK- cell line, FEPD was a kind gift provided by Prof Annarosa Del Mistro, University of Padua, Italy. All of the cell lines were cultured in RPMI-1640/10% FBS  supplemented with 1% Penicillin-Streptomycin , in an incubator at 37 °C/5% COse model   and was resuspended in DMSO  to a stock concentration of 1 mM, before dilution in DMSO (Sigma-Aldrich) or sterile Dulbecco’s PBS  to the required concentration for each assay as indicated. Brigatinib and lorlatinib were purchased from MedChem Express . The BRG1 antibody for IHC was purchased from Abcam . Western blot antibodies were purchased from Cell Signaling Technologies  , Life Technologies  (ALK), Abcam  (Brg1) and Sigma  (Tubulin).Immunohistochemistry was conducted as described previously . In brieMice expressing NPM-ALK from the CD4 promoter  were kin6) were lysed in RIPA buffer, mixed with 2 × Laemmli’s loading buffer before boiling and loading into the wells of an 8% SDS-PAGE. Proteins were transferred to Immobilon PVDF membranes  by wet transfer before incubation in 5% (w/v) BSA  for 1 h at RT followed by primary antibody for 1 h at RT or overnight at 4 °C (phospho-specific antibodies), washed twice in TBS/T and then incubated with horse radish-peroxidase (HRP)-conjugated secondary antibody at 1:10,000 for 1 h at RT. The membrane was subsequently washed twice in TBS/T, and then exposed to Immobilon Western chemiluminescent HRP substrate  and imaged using a Fujifilm LAS-4000 Biomolecular Imager . Analysis and densitometry were performed using Aida software .SDS-PAGE and Western blot were conducted as described previously . Brieflyhttp://colorbrewer2.org, accessed on 31 July 2021) [p-value ≤ 0.05, p-value ≤ 0.005, or adj. p-value ≤ 0.05 are outlined and specified where appropriate) and differentially expressed genes using DESeq2’s negative binomial Wald test, and post-statistical QC steps in R.RNAseq was conducted by Novogene, Cambridge, UK. Data were mapped with HISAT2 , quantifly 2021) . This pi6 cells were lysed in buffer RLT plus and passed through an RNeasy spin column. Subsequently, RNA was eluted in DEPC-treated water and the concentration was measured by NanoDrop . Sufficient RNA purity was indicated by A260/280 ratios of ~2.0 and A260/230 ratios of ~2.0–2.2. To completely remove contaminating DNA, 10 µg purified RNA was diluted in 50 µL 1× Turbo DNase Buffer  supplemented with 2U Turbo DNase  and incubated at 37 °C for 30 min. The DNase was inactivated by the addition of 7.5 µL 100 mM EDTA, pH 8.0.RNA was isolated using the RNeasy Plus mini kit  according to the manufacturer’s instructions. Briefly, 0.5–4.0 × 10Synthesis of cDNA was performed using the SuperScript II Reverse Transcriptase kit  according to the manufacturer’s protocol. Briefly, 500 ng DNase-treated RNA was diluted in DEPC-treated water supplemented with 500 ng oligo(dT) primers  and 10 nmol dNTP mix  up to 12 µL total volume and incubated at 65 °C for 5 min. To this, 4 µL 5× First Strand Buffer and 2 µL 0.1 M DTT were added and then incubated at 42 °C for 2 min. Subsequently, 1 µL SuperScript II Reverse Transcriptase was added and incubated at 42 °C for 50 min to synthesise the cDNA. The reaction was inactivated by incubation at 70 °C for 15 min. The cDNA concentration was measured by NanoDrop analysis . Sufficient cDNA purity was indicated by A260/280 ratios of ~1.8 and A260/ratios of ~2.0–2.2.v/v) SsoFast EvaGreen Supermix  and 25 ng of each primer. The primers and conditions used are detailed in t-tests  on GraphPad Prism 5 with a significance level set at p < 0.05.Samples of cDNA were analysed by quantitative, real-time (qRT)-PCR using the QuantStudio 6 Flex Real-Time PCR system . Reactions were performed in 10 µL volumes with 40 ng cDNA, 50%  in OptiMEM  into the HEK293FT cells. Viral particles were harvested 60 h following transfection and filtered through a 0.45 μm PVDF membrane and either used immediately or stored at −80 °C. Cells were seeded at a concentration of 1 million cells/mL in 1 mL of medium in the wells of a 6-well plate. Dropwise, 500 µL of room temperature-thawed viral particle-containing medium was added to the cells and, 24 h later, transduced cells were selected in puromycin at 1 µg/mL. Lysates were prepared from SUDH1 and FEPD cells 48 and 72 h post-puromycin selection following scrambled and BRG1 shRNA transfection  and anal4) were seeded in 96-well white polystyrene microplates  and transfected with BRG1-targeting siRNAs (10 nM). After 6 days, 20 µL CellTiter-Blue cell viability reagent  was added to each well and cells were incubated at 37 °C for 4 h, according to the manufacturer’s protocol. Fluorescence was read on a SpectraMax i3 microplate reader . For assessment of caspase 3/7 activity, the cells were instead incubated with 100 µL of Caspase 3/7-Glo apoptosis reagent  for 1.5 h. Luminescence was read on a SpectraMax i3 microplate reader . Data were normalised to the scrambled non-coding siRNA.CD4-NA cells (10+ ALCL cell lines) express BRG1 at varying levels, whereas the acute T-cell leukaemia cell line JURKAT expresses BRG1 at higher levels  , PTCL-NOS  and AITL , with only one ALK- ALCL patient showing negative staining for BRG1.Furthermore, Western blot analysis of protein lysates prepared from thymic tumours obtained from the CD4/NPM-ALK transgenic mouse line ,15 also ll lines a. Immunotaining) b–d. Inde+ ALCL cell lines, SU-DHL-1 cells were treated with the ALK inhibitor Crizotinib for 6 h direct regulation of BRG1 expression by NPM-ALK, we sought to uncover the mechanism of this activity. Transcript levels of BRG1 were assessed following inhibition of NPM-ALK activity with the ALK tyrosine kinase inhibitors lorlatinib, brigatinib and crizotinib. Following 6 h of incubation, no significant change in transcript levels were observed, suggesting a post-translational mechanism of regulation a. In supp ≤ 0.05) and upregulation  of genes (p ≤ 0.05). To establish which physiological processes are most affected by the activity of BRG1, pathway enrichment analyses were undertaken. The input comprised of the significant (adj. p ≤ 0.05) protein-coding targets, where the absolute log2-fold change in expression was ±1.5. Transcripts whose expression was either induced (n = 33) or repressed (n = 243) by BRG1 were investigated. Reactome analysis suggests that BRG1 plays roles in upregulating genes associated with the cell cycle  express Brg1. Indeed, recent evidence indicates that BRG1 can fulfil pro-oncogenic roles in some cancers [Loss of BRG1 has been associated with various cancers ,24,26,27 cancers . It is t cancers  and NPM1 cancers .+ ALCL cell lines at a post-translational level. Specifically, NPM-ALK, in a tyrosine kinase-dependent manner protects Brg1 from proteasomal degradation although the exact mechanism remains to be determined. Previously, others have shown that the ALK receptor, post-activation, recruits the Cbl ubiquitin ligase [We also show that Brg1 expression is positively regulated by NPM-ALK tyrosine kinase activity in ALKn ligase . ALK actn ligase . Furthern ligase , and carn ligase . CollectFinally, we demonstrate that BRG1 regulates the transcriptome of ALCL to promote the expression of genes involved in the cell cycle and to maintain cell viability. BRG1 knockdown led to reduced viability; however, the mechanism involved remains to be further substantiated. Notably, a reduction in cell viability was not significant until 14 days following knockdown of BRG1, suggesting that this is a mild effect that might not be therapeutically applicable unless combined with other agents. This clearly requires further investigation should effective and specific pharmacological inhibitors of BRG1 activity be developed. Naturally, the therapeutic efficacy of BRG1 inhibitors would also be dependent on the potential side effects of inhibiting this ubiquitously expressed protein.+ ALCL cell lines. Furthermore, we show that BRG1 drives transcription of genes associated with cell cycle progression and in evidence of this, inhibition of BRG1 expression results in a decrease in cell viability. Overall, these data suggest that NPM-ALK is a key mediator of BRG1 activity in ALCL.In conclusion, we have demonstrated that BRG1 is expressed in a range of T-cell lymphomas and its protein levels are maintained by NPM-ALK activity in ALK