Feline Cell Line Research Articles

Micro-RNA 10a Is Increased in Feline T Regulatory Cells and Increases Foxp3 Protein Expression Following In Vitro Transfection.

CD4+CD25+Foxp3+ T regulatory (Treg) cells are activated during the course of lentiviral infection and exhibit heightened suppressor function when compared to Treg cells from uninfected controls. Foxp3 is essential to Treg cell function and multiple studies have documented that lentivirus-activated Treg cells exhibit heightened Foxp3 expression when compared to Treg cells from uninfected controls. Our hypothesis was that lentivirus-induced micro-RNAs (miRNAs) contribute to heightened Treg cell suppressor function by stabilizing Foxp3 expression. We demonstrated that CD4+CD25+ T cells from both feline immunodeficiency virus infected (FIV+) cats and uninfected control cats exhibit increased miRNA 10a and 21 levels compared to autologous CD4+CD25− T cells but there was no difference in the levels of these miRNAs when Treg cells from FIV+ cats were compared to Treg cells from uninfected controls. Further, there was no increase in Foxp3 mRNA following transfection of miRNA 10a or 21 into a feline cell line. However, transfection with miRNA 10a resulted in increased Foxp3 protein expression.

Mutations in the CXCR4 gene cause a population shift in the feline lymphoma cell line 3201 surviving feline immunodeficiency virus infection

The feline 3201-S cell line was established from original 3201 cell populations that survived productive infection of feline thymic lymphoma 3201 cells with feline immunodeficiency virus (FIV). The 3201-S cells expressing a mutated CXCR4 are unresponsive to the CXCR4 ligand SDF-1α, and are refractory to FIV reinfection. In this mini-review, possible mechanisms of how the 3201-S cells could restrict FIV infection and could not respond to SDF-1α are described and discussed.

Pharmacokinetics and derivation of an anticancer dosing regimen for the novel anti-cancer agent isobutyl-deoxynyboquinone (IB-DNQ), a NQO1 bioactivatable molecule, in the domestic felid species.

Isobutyl-deoxynyboquinone (IB-DNQ) is a selective substrate for NAD(P)H:quinone oxidoreductase (NQO1), an enzyme overexpressed in many solid tumors. Following activation by NQO1, IB-DNQ participates in a catalytic futile reduction/reoxidation cycle with consequent toxic reactive oxygen species generation within the tumor microenvironment. To elucidate the potential of IB-DNQ to serve as a novel anticancer agent, in vitro studies coupled with in vivo pharmacokinetic and toxicologic investigations in the domestic felid species were conducted to investigate the tractability of IB-DNQ as a translationally applicable anticancer agent. First, using feline oral squamous cell carcinoma (OSCC) as a comparative cancer model, expressions of NQO1 were characterized in not only human, but also feline OSCC tissue microarrays. Second, IB-DNQ mediated cytotoxicity in three immortalized feline OSCC cell lines were studied under dose-dependent and sequential exposure conditions. Third, the feasibility of administering IB-DNQ at doses predicted to achieve cytotoxic plasma concentrations and biologically relevant durations of exposure were investigated through pharmacokinetic and tolerability studies in healthy research felines. Intravenous administration of IB-DNQ at 1.0-2.0mg/kg achieved peak plasma concentrations and durations of exposure reaching or exceeding predicted in vitro cytotoxic concentrations. Clinical adverse side effects including ptyalism and tachypnea exhibited during and post-IV infusion of IB-DNQ were transient and tolerable. Additionally, IB-DNQ administration did not produce acute or delayed-onset unacceptable hematologic, non-hematologic, or off-target oxidative toxicities. Collectively, the findings reported here within provide important safety and pharmacokinetic data to support the continued development of IB-DNQ as a novel anticancer strategy for NQO1 expressing cancers.

Genetic and epigenetic aberrations of p16 in feline primary neoplastic diseases and tumor cell lines of lymphoid and non-lymphoid origins

The p16 gene acts as a tumor suppressor by regulating the cell cycle and is frequently inactivated in human and canine cancers. The aim of this study was to characterize genetic and epigenetic alterations of the p16 in feline lymphoid and non-lymphoid malignancies, using 74 primary tumors and 11 tumor cell lines. Cloning of feline p16 and subsequent sequence analysis revealed 11 germline sequence polymorphisms in control cats. Bisulfite sequencing analysis of the p16 promoter region in a feline lymphoma cell line revealed that promoter methylation was associated with decreased mRNA expression. Treatment with a demethylating agent restored mRNA expression of the silenced p16. PCR amplification and sequencing analysis detected homozygous loss (five tumors, 6.7%) and a missense mutation (one tumor, 1.4%) in the 74 primary tumors analyzed. Methylation-specific PCR analysis revealed promoter methylation in 10 primary tumors (14%). Promoter methylation was frequent in B cell lymphoid tumors (7/21 tumors, 33%). These genetic and epigenetic alterations were also observed in lymphoma and mammary gland carcinoma cell lines, but not detected in non-neoplastic control specimens. These data indicate that molecular alterations of the p16 locus may be involved in the development of specific types of feline cancer, and warrant further studies to evaluate the clinical value of this evolutionarily-conserved molecular alteration in feline cancers.

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk-3).

Reduced expression in immortalized cells (REIC/Dkk-3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk-3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk-3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk-3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk-3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk-3 represents a potential molecular target for the development of therapies against feline mammary cancers.

Transduction of hematopoietic stem cells to stimulate RNA interference against feline infectious peritonitis.

Objectives The goals of the study were: (1) to develop and evaluate non-replicating lentivirus vectors coding for feline coronavirus (FCoV)-specific micro (mi)RNA as a potential antiviral therapy for feline infectious peritonitis (FIP); (2) to assess the feasibility of transducing hematopoietic stem cells (HSCs) with ex vivo introduction of the miRNA-expressing lentivirus vector; and (3) to assess the ability of the expressed miRNA to inhibit FCoV replication in HSCs in vitro. Methods HSCs were obtained from feline bone marrow and replicated in vitro. Three lentiviruses were constructed, each expressing a different anti-FCoV miRNA. HSCs were stably transduced with the miRNA-expressing lentivirus vector that produced the most effective viral inhibition in a feline cell line. The effectiveness of the transduction and the expression of anti-FCoV miRNA were tested by infecting the HSCs with two different strains of FCoV. The inhibition of coronavirus replication was determined by relative quantification of the inhibition of intracellular viral genomic RNA synthesis using real-time, reverse-transcription PCR. The assessment of virus replication inhibition was determined via titration of extracellular virus using the TCID50 assay. Results Inhibition of FCoV was most significant in feline cells expressing miRNA-L2 that targeted the viral leader sequence, 48 h postinfection. miRNA-L2 expression in stably transduced HSCs resulted in 90% and 92% reductions in FIPV WSU 79-1146 genomic RNA synthesis and extracellular virus production, respectively, as well as 74% and 80% reduction in FECV WSU 79-1683 genomic RNA synthesis and extracellular virus production, respectively, as compared with an infected negative control sample producing non-targeting miRNA. Conclusions and relevance These preliminary results show that genetic modification of HSCs for constitutive production of anti-coronavirus miRNA will reduce FCoV replication.

Establishment and Characterization of New Canine and Feline Osteosarcoma Primary Cell Lines.

Osteosarcomas are the most abundant form of bone malignancies in multiple species. Canine osteosarcomas are considered a valuable model for human osteosarcomas because of their similar features. Feline osteosarcomas, on the other hand, are rarely studied but have interesting characteristics, such as a better survival prognosis than dogs or humans, and less likelihood of metastasis. To enable experimental approaches to study these differences we have established five new canine osteosarcoma cell lines out of three tumors, COS_1186h, COS_1186w, COS_1189, and COS_1220, one osteosarcoma-derived lung metastasis, COS_1033, and two new feline osteosarcoma cell lines, FOS_1077 and FOS_1140. Their osteogenic and neoplastic origin, as well as their potential to produce calcified structures, was determined by the markers osteocalcin, osteonectin, tissue unspecific alkaline phosphatase, p53, cytokeratin, vimentin, and alizarin red. The newly developed cell lines retained most of their markers in vitro but only spontaneously formed spheroids produced by COS_1189 showed calcification in vitro.

Dual targeting of EGFR and ERBB2 pathways produces a synergistic effect on cancer cell proliferation and migration in vitro

Members of the epidermal growth factor receptor (EGFR/ERBB) gene family are frequently dysregulated in a range of human cancers, and therapeutics targeting these proteins are in clinical use. We hypothesized that similar pathways are involved in feline and canine tumours and that the same drugs may be of clinical use in veterinary patients. We investigated EGFR and ERBB2 targeting using a panel of feline and canine cell lines. EGFR and ERBB2 were targeted with siRNAs or tyrosine kinase inhibitors (TKIs) and their effect on cellular proliferation, colony formation and migration was investigated in vitro. Here we report that EGFR and ERBB2 combined siRNA targeting produced synergistic effects in feline and canine cell lines similar to that reported in human cell lines. We conclude that dual EGFR and ERBB2 targeting using TKIs should be further evaluated as a potential new therapeutic strategy in feline head and neck and mammary tumours and canine mammary tumours.

Establishment and characterization of a new feline mammary cancer cell line, FkMTp.

Studies on tumours in domestic animals are believed to greatly contribute to a better understanding of similar diseases in humans. Comparative studies have shown that feline mammary carcinomas share important features with human breast cancers, including a similar biological behaviour and histological appearance. In the present study we have established and characterized at different cellular levels one feline mammary cancer cell line, FkMTp, derived from a cat mammary carcinoma. The FkMTp cell line revealed to be a promising resource and tool to study tumour microevolution and all the mechanisms and processes involved in carcinogenesis from the tumour (primary culture) to the immortalized cell line. Several assays were conducted to assess the growth behaviour, differentiated morphology, anchorage independent growth in soft agar, wound-healing invasion and migration of the cell line across time (from the primary culture until the 160th passage). FkMTp revealed increased levels of anchorage independence, migration and invasion according to the course of time as well as different numbers of ploidy. These results demonstrate and validate the in vitro tumorigenicity of the FkMTp cell line. During the cell line establishment, it was cryopreserved approximately every six passages, including the tumour primary culture, allowing now the possibility to access almost any specific momento of the tumour progression.

An in vitro and in vivo characterization of the cadherin-catenin adhesion complex in a feline mammary carcinoma cell line

An in vitro and in vivo characterization of the cadherin-catenin adhesion complex in a feline mammary carcinoma cell line

Detection of Immunodominant Proteins of Felis catus Gammaherpesvirus 1

We recently identified and sequenced a novel herpesvirus of domestic cats, Felis catus gammaherpesvirus 1 (FcaGHV1). FcaGHV1 is a member of the gammaherpesvirus subfamily which also includes the human cancer-associated herpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). As a first step toward developing a serologic assay to detect exposure to FcaGHV1, we are seeking to determine which viral proteins elicit an antibody response in naturally occurring domestic cat infections. We cloned selected FcaGHV1 genes into a mammalian expression vector and performed transfections of a feline cell line for expression of recombinant FcaGHV1 proteins. We fixed cells with paraformaldehyde and methanol-acetone and tested reactivity to serum from cats naturally infected with FcaGHV1 using immunofluorescent antibody staining. An FIV immunoflouresence test was developed as a positive control for transfection and assay function. Serum from specific pathogen-free laboratory cats served as negative controls. Preliminary data from 9 cats with FcaGHV1 infection indicates that capsid protein ORF 65 and tegument protein ORF38 may elicit antibodies during naturally occurring FcaGHV1 infection. Results of this study will suggest which FcaGHV1 proteins are immunodominant during natural infection. With this information we plan to develop a serologic assay and further evaluate FcaGHV1 as a model for EBV and KSHV.

Characterization of STAT3 expression, signaling and inhibition in feline oral squamous cell carcinoma.

BackgroundSignal transducer and activator of transcription 3 (STAT3) plays a critical role in tumor development by regulating signaling pathways involved in cell proliferation, survival, metastasis and angiogenesis. STAT3 is activated in many cancers, including head and neck squamous cell carcinoma (HNSCC) in people. Feline oral squamous cell carcinoma (OSCC) is similar to advanced or recurrent HNSCC as it is poorly responsive to traditional therapies and carries a poor long-term prognosis. The purpose of this study was to characterize expression and activation of STAT3 in feline OSCC cell lines and tumor samples and to investigate the biologic activity of a novel, allosteric STAT3 inhibitor, LLL12, in feline OSCC cell lines.ResultsWe evaluated 3 feline OSCC cell lines and one of these (SCCF2) exhibited high levels of constitutive STAT3 phosphorylation and high sensitivity to LLL12 treatment. Exposure of SCCF2 cells to LLL12 resulted in decreased expression of pSTAT3 and total STAT3, apoptosis as assessed by caspase 3/7 activation, inhibition of colony formation and reduced expression of the STAT3 transcriptional target survivin. In contrast, the STAT3 transcriptional targets VEGF and MCL-1 increased after LLL12 treatment. This was, in part, likely due to LLL12 mediated upregulation of HIF-1α, which is known to drive VEGF and MCL-1 expression. The OSCC cell lines with low basal STAT3 phosphorylation did not exhibit these effects, suggesting that STAT3 inhibition was responsible for the observed results. Lastly, immunohistochemistry for pSTAT3 was performed using a feline OSCC tissue microarray, demonstrating expression in 48 % of samples tested.ConclusionsThese data demonstrate that LLL12 has biologic activity against a feline OSCC cell line expressing pSTAT3 and that STAT3 represents a target for therapeutic intervention in this disease. However, given the up-regulation of several STAT3 transcriptional targets following treatment, further investigation of STAT3 and its related signaling pathways in OSCC is warranted.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0505-7) contains supplementary material, which is available to authorized users.

Expression of platelet-derived growth factor BB, erythropoietin and erythropoietin receptor in canine and feline osteosarcoma

The discovery of expression of the erythropoietin receptor (EPO-R) on neoplastic cells has led to concerns about the safety of treating anaemic cancer patients with EPO. In addition to its endocrine function, the receptor may play a role in tumour progression through an autocrine mechanism. In this study, the expression of EPO, EPO-R and platelet-derived growth factor BB (PDGF-BB) was analysed in five feline and 13 canine osteosarcomas using immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR).EPO expression was positive in all tumours by IHC, but EPO mRNA was only detected in 38% of the canine and 40% of the feline samples. EPO-R was expressed in all samples by quantitative RT-PCR (RT-qPCR) and IHC. EPO-R mRNA was expressed at higher levels in all feline tumours, tumour cell lines, and kidney when compared to canine tissues. PDGF-BB expression was variable by IHC, but mRNA was detected in all samples. To assess the functionality of the EPO-R on tumour cells, the proliferation of canine and feline osteosarcoma cell lines was evaluated after EPO administration using an alamarBlue assay and Ki67 immunostaining. All primary cell lines responded to EPO treatment in at least one of the performed assays, but the effect on proliferation was very low indicating only a weak responsiveness of EPO-R. In conclusion, since EPO and its receptor are expressed by canine and feline osteosarcomas, an autocrine or paracrine tumour progression mechanism cannot be excluded, although in vitro data suggest a minimal role of EPO-R in osteosarcoma cell proliferation.

Enhancing anti-tumor efficacy of Doxorubicin by non-covalent conjugation to gold nanoparticles - in vitro studies on feline fibrosarcoma cell lines.

BackgroundFeline injection-site sarcomas are malignant skin tumors of mesenchymal origin, the treatment of which is a challenge for veterinary practitioners. Methods of treatment include radical surgery, radiotherapy and chemotherapy. The most commonly used cytostatic drugs are cyclophosphamide, doxorubicin and vincristine. However, the use of cytostatics as adjunctive treatment is limited due to their adverse side-effects, low biodistribution after intravenous administration and multidrug resistance. Colloid gold nanoparticles are promising drug delivery systems to overcome multidrug resistance, which is a main cause of ineffective chemotherapy treatment. The use of colloid gold nanoparticles as building blocks for drug delivery systems is preferred due to ease of surface functionalization with various molecules, chemical stability and their low toxicity.MethodsStability and structure of the glutathione-stabilized gold nanoparticles non-covalently modified with doxorubicin (Au-GSH-Dox) was confirmed using XPS, TEM, FT-IR, SAXRD and SAXS analyses. MTT assay, Annexin V and Propidium Iodide Apoptosis assay and Rhodamine 123 and Verapamil assay were performed on 4 feline fibrosarcoma cell lines (FFS1WAW, FFS1, FFS3, FFS5). Statistical analyses were performed using Graph Pad Prism 5.0 (USA).ResultsA novel approach, glutathione-stabilized gold nanoparticles (4.3 +/- 1.1 nm in diameter) non-covalently modified with doxorubicin (Au-GSH-Dox) was designed and synthesized. A higher cytotoxic effect (p<0.01) of Au-GSH-Dox than that of free doxorubicin has been observed in 3 (FFS1, FFS3, FFS1WAW) out of 4 feline fibrosarcoma cell lines. The effect has been correlated to the activity of glycoprotein P (main efflux pump responsible for multidrug resistance).ConclusionsThe results indicate that Au-GSH-Dox may be a potent new therapeutic agent to increase the efficacy of the drug by overcoming the resistance to doxorubicin in feline fibrosarcoma cell lines. Moreover, as doxorubicin is non-covalently attached to glutathione coated nanoparticles the synthesized system is potentially suitable to a wealth of different drug molecules.

Multiple invasions of an infectious retrovirus in cat genomes.

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of host germ-line cells. While most ERVs are defective, some are active and express viral proteins. The RD-114 virus is a replication-competent feline ERV, and several feline cell lines produce infectious RD-114 viral particles. All domestic cats are considered to have an ERV locus encoding a replication-competent RD-114 virus in their genomes; however, the locus has not been identified. In this study, we investigated RD-114 virus-related proviral loci in genomes of domestic cats, and found that none were capable of producing infectious viruses. We also found that all domestic cats have an RD-114 virus-related sequence on chromosome C2, termed RDRS C2a, but populations of the other RDRSs are different depending on the regions where cats live or breed. Our results indicate that RDRS C2a, the oldest RD-114-related provirus, entered the host genome before an ancestor of domestic cats started diverging and the other new RDRSs might have integrated into migrating cats in Europe. We also show that infectious RD-114 virus can be resurrected by the recombination between two non-infectious RDRSs. From these data, we conclude that cats do not harbor infectious RD-114 viral loci in their genomes and RD-114-related viruses invaded cat genomes multiple times.

In vitro host range of feline morbillivirus.

Feline morbillivirus (FmoPV) is an emerging virus in cats, which is associated with tubulointerstitial nephritis. To study the in vitro host range of FmoPV, we inoculated FmoPV strain SS1 to 32 cell lines originated from 13 species and cultured for 2 weeks, followed by RNA extraction and reverse-transcription-polymerase chain reaction for FmoPV detection. As a result, only cell lines derived from cats and African green monkeys were susceptible to FmoPV. FmoPV infects diverse feline cell lines: epithelial, fibroblastic, lymphoid and glial cells. These results indicate that the receptor (s) for FmoPV are ubiquitously expressed in cats. No infectivity of FmoPV was observed in human cell lines, which suggests least threatening of cross-species transmission of FmoPV from cats to humans.

Bioassay-guided fractionation of extracts from Easter lily (Lilium longiflorum) flowers reveals unprecedented structural variability of steroidal glycoalkaloids

Several Lilium species are nephrotoxic in cats (Felis silvestris catus), among them Easter lilies (Lilium longiflorum). Although clinical trials have been carried out, the causative toxic phytochemicals have not yet been identified. We thus aimed to determine the toxic constituents of Easter lily flowers applying a bioassay-guided approach based on a feline kidney cell line model. The bioassay-guided fractionation traced the observed cytotoxicity to a complex mixture of compounds that were tentatively identified as steroidal glycoalkaloids of the solasodine-type, based on multiple-fragmentation ion trap and high-resolution mass spectrometry. The glycoalkaloids in the active fraction possessed trisaccharide chains, and at least 16 different congeners could be separated using liquid chromatography-mass spectrometry. The two principal compounds were solasodine trisaccharides containing two hexose and one deoxy-hexose unit. In the remaining 14 analogues, one or two of the hydroxyl groups of the second hexose from the aglycone were acetylated. In addition, some of the analogues appeared to be carbonate esters. Esterification of steroidal glycoalkaloids in plants has only been reported once and was in accordance with higher antifungal activity of the acetylated versus the parent congener. Our pilot study shows that esterification of steroidal glycoalkaloids in Lilium species might be common resulting in an array of different analogues with largely unexplored structural variability and bioactivity.

In vitro efficacy of doxorubicin and etoposide against a feline injection site sarcoma cell line

Feline injection site sarcoma (ISS) is a locally invasive tumor, in which surgical treatment is frequently combined with radiation or chemotherapy to improve tumor control. The focus of this study was to evaluate the cytotoxic effects of doxorubicin or etoposide on a feline injection site sarcoma cell line (JB) and to assess the impact of combining these drugs on cell death and cell cycle. Both single agent and combination drug administration increased cell death and significantly reduced the number of viable cells. Cells in G0/G1 were significantly reduced while the G2/M fraction was significantly increased following treatment. Collectively, combining doxorubicin and etoposide at the lower EC yielded comparable results to the EC50 of either drug alone in degree of cytotoxicity, level of apoptosis, and % of cells in G2/M. The results of this study indicate that doxorubicin and etoposide alone and in combination differentially alter ISS cell viability and cycle.

Identification of T cell receptor signaling pathway proteins in a feline large granular lymphoma cell line by liquid chromatography tandem mass spectrometry

Tryptic peptides of a feline large granular lymphoma cell line were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Seventeen proteins of the T cell receptor signaling pathway could be identified by this approach. So far the existence of these proteins has only been postulated in the protein databases while experimental proof of their expression is predominantly pending. This article suggests where these proteins are located within the T cell receptor signaling pathway, thereby giving a short overview of the structure and function of this cascade.

Development of a multiplex amplification refractory mutation system reverse transcription polymerase chain reaction assay for the differential diagnosis of Feline leukemia virus vaccine and wild strains.

A multiplex amplification refractory mutation system reverse transcription polymerase chain reaction (ARMS RT-PCR) was developed for the differential diagnosis of Feline leukemia virus (FeLV) vaccine and wild-type strains based on a point mutation between the vaccine strain (S) and the wild-type strain (T) located in the p27 gene. This system was further upgraded to obtain a real-time ARMS RT-PCR (ARMS qRT-PCR) with a high-resolution melt analysis (HRMA) platform. The genotyping of various strains of FeLV was determined by comparing the HRMA curves with the defined wild-type FeLV (strain TW1), and the results were expressed as a percentage confidence. The detection limits of ARMS RT-PCR and ARMS qRT-PCR combined with HRMA were 100 and 1 copies of transcribed FeLV RNA per 0.5 ml of sample, respectively. No false-positive results were obtained with 6 unrelated pathogens and 1 feline cell line. Twelve FeLV Taiwan strains were correctly identified using ARMS qRT-PCR combined with HRMA. The genotypes of the strains matched the defined FeLV wild-type strain genotype with at least 91.17% confidence. A higher degree of sequence polymorphism was found throughout the p27 gene compared with the long terminal repeat region. In conclusion, the current study describes the phylogenetic relationship of the FeLV Taiwan strains and demonstrates that the developed ARMS RT-PCR assay is able to be used to detect the replication of a vaccine strain that has not been properly inactivated, thus acting as a safety check for the quality of FeLV vaccines.