Canine Lymphoid Cell Lines Research Articles

Expression of asparagine synthetase predicts in vitro response to l-asparaginase in canine lymphoid cell lines

l-asparaginase (L-asp), a bacterial enzyme that depletes extracellular asparagine, is used to treat acute lymphoblastic leukemia in humans and a variety of aggressive lymphoid malignancies in dogs. Resistance to this drug is an important cause of treatment failure in both species. Using canine lymphoid cell lines, we found that L-asp sensitivity is strongly negatively correlated with the level of methylation of the asparagine synthetase (ASNS) promoter. Selection for in vitro resistance was accompanied by increased ASNS promoter methylation and decreased ASNS mRNA expression. In addition, treatment with the hypomethylating agent 5-azacytidine increased resistance to L-asp. ASNS methylation and expression is not predictive of overall survival or progression-free survival in canine lymphoma patients treated with L-asp. Our data suggest that ASNS is an important factor in mediating the in vitro response of canine lymphoid cells to L-asp; however, resistance mechanisms may be more complex in dogs treated clinically with L-asp, potentially due to concurrent treatments.

Masitinib reverses doxorubicin resistance in canine lymphoid cells by inhibiting the function of P‐glycoprotein

Overexpression of ABC-transporters including Pgp, MRP1, and BCRP has been associated with multidrug resistance (MDR) in both human and canine oncology. Therapeutic interventions to reverse MDR are limited, but include multidrug protocols and the temporary concomitant use of inhibitors of ABC-transporters. Recently, the use of tyrosine kinase inhibitors has been proposed to overcome MDR in human oncology. One of the tyrosine kinase inhibitors, masitinib, is licensed for veterinary use in the treatment of canine mast cell tumors. Therefore, this study aimed to assess the potential of masitinib to revert MDR in canine malignant lymphoma using an in vitro model with canine lymphoid cell lines. Masitinib had a mild antiproliferative effect on lymphoid cells, inhibited Pgp function at concentrations equal to or exceeding 1μm and was able to reverse doxorubicin resistance. The current findings provide the rationale for a combined use of masitinib with doxorubicin in the treatment of dogs with doxorubicin-resistant malignant lymphoma but await confirmation in clinical trials.

Evaluation of the Aurora Kinase Inhibitor, ZM447439, in Canine Malignant Lymphoid Cells <i>in Vitro</i>

Aurora kinases play an important role in the cell cycle. These enzymes help establish mitotic spindles by directing centrosome duplication and separation and by regulating the spindle assembly checkpoint thereby helping control cytokinesis. An over-expression of aurora kinases has been reported in a variety of human tumors. In this study, we identified the expression of aurora-A and aurora-B kinases in canine malignant lymphoid cells. We also evaluated the effects of the aurora kinase inhibitor (ZM447439), and found that this inhibitor decreases cell viability, increases DNA content change, and leads to apoptosis in canine B- and T-cell lymphoid cell lines. The lymphotoxicity induced by ZM447439 in these canine lymphoid cell lines suggests that further in vivo evaluation of aurora kinase inhibitors as a potential treatment for canine malignant lymphoid tumors is warranted.

Identification of altered MicroRNA expression in canine lymphoid cell lines and cases of B‐ and T‐Cell lymphomas

Canine lymphoma is a common spontaneous tumor with many similarities to human lymphoma, and thus has potential to be an important animal model of lymphomagenesis. This study determined that microRNA (miRNA) expression in canine tumors can be assessed using a commercially available human cancer miRNA qPCR array. miRNA expression in six different canine lymphoid cell lines and in naturally occurring canine B- and T-cell lymphomas was compared using RNA harvested from normal canine peripheral blood mononuclear cells (PBMC) and normal lymph nodes (LN) as controls. We found that false discovery rate (FDR) correction for multiple testing after quantile normalization controlled for variation across arrays and that they were the best methods for normalization and statistical analysis. Increases in miRNAs known to upregulate oncogenes (miR19a+b, miR17-5p) and decreased expression of miRNAs with tumor suppressor functions (miR-203, miR-218, and miR-181a) also seen in human lymphoid malignancies were observed. However, there were few similarities between canine groups. The results of this study indicate that the use of both PBMC and LN cells as controls provides different, but potentially equally important targets for further analysis. Our findings of miRNA dysregulation in canine lymphoid cell lines and clinical cases of lymphoma emphasize the potential of canine lymphoma as an important spontaneous, large animal model of human B- and T-cell lymphomas.

FLT3 mutations in canine acute lymphocytic leukemia

BackgroundFMS-like tyrosine kinase 3 (FLT3) is a commonly mutated protein in a variety of human acute leukemias. Mutations leading to constitutively active FLT3, including internal tandem duplications of the juxtamembrane domain (ITD), result in continuous cellular proliferation, resistance to apoptotic cell death, and a poorer prognosis. A better understanding of the molecular consequences of FLT3 activation would allow improved therapeutic strategies in these patients. Canine lymphoproliferative diseases, including lymphoma and acute leukemias, share evolutionarily conserved chromosomal aberrations and exhibit conserved mutations within key oncogenes when compared to their human counterparts. A small percentage of canine acute lymphocytic leukemias (ALL) also exhibit FLT3 ITD mutations.MethodsWe molecularly characterized FLT3 mutations in two dogs and one cell line, by DNA sequencing, gene expression analysis via quantitative real-time PCR, and sensitivity to the FLT3 inhibitor lestaurtinib via in vitro proliferation assays. FLT 3 and downstream mediators of FLT3 activation were assessed by Western blotting.ResultsThe canine B-cell leukemia cell line, GL-1, and neoplastic cells from 2/7 dogs diagnosed cytologically with ALL were found to have FLT3 ITD mutations and FLT3 mRNA up-regulation. Lestaurtinib, a small molecule FLT3 inhibitor, significantly inhibited the growth of GL-1 cells, while not affecting the growth of two other canine lymphoid cell lines without the FLT3 mutation. Finally, western blots were used to confirm the conserved downstream mediators of FLT3 activating mutations.ConclusionsThese results show that ALL and FLT3 biology is conserved between canine and human patients, supporting the notion that canine ALL, in conjunction with the GL-1 cell line, will be useful in the development of a relevant large animal model to aid in the study of human FLT3 mutant leukemias.

GCP, a genistein-rich compound, inhibits proliferation and induces apoptosis in lymphoma cell lines

Genistein combined polysaccharide (GCP), derived from soy bean extract, is comprised of deglycosylated isoflavones, such as genistein and daidzein. The goal of this work was to determine if GCP is effective in lymphoid cancers. In three human and four canine lymphoid cell lines, GCP inhibited proliferation and induced G2/M arrest. Additionally, increased apoptosis was observed in Ramos and Jurkat lines. These results demonstrate that GCP is effective in lymphoid cell lines of both human and canine origin. Due to its minimal toxicities, increased bioavailability, and in vitro efficacy, GCP may have clinical utility in the treatment of patients with lymphoma.

MiRNA Profiles of Canine Lymphoid Cell Lines

miRNAs are very highly conserved across species, however their role in the regulation of cell proliferation and differentiation in normal and neoplastic canine tissues is unknown. Lymphoma is one of the most common malignant tumors of the dog, accounting for about 25% of all cancers. The purpose of this study was to determine whether miRNA arrays developed for human samples can be used to characterize canine tumors. Human cancer microRNA qPCR arrays were used to establish the miRNA profiles of 4 canine lymphoid cell lines: 2 of B cell origin and 2 of T cell origin. Over ninety percent of the human miRNA probes detected canine miRNAs. miRNA analysis of canine lymphoid cell lines identified 12‐15 miRNAs that are either 10‐fold over or 10‐fold under expressed compared to normal canine peripheral blood mononuclear cells. In addition, the relative expression of six miRNAs known to be upregulated in human chronic lymphoid leukemia was determined in the four canine lymphoid cell lines, and expression of miR‐20a and miR‐92 were found to be markedly increased in the cell lines. The results of this study indicate that miRNAs are highly conserved between humans and dogs, and that miRNA profiles have potential predictive, diagnostic and prognostic potential in canine lymphoma.

Genistein combined polysaccharide (GCP) is highly active in both human and canine lymphoma models

14126 Background: The soy isoflavones genistein and daidzein have demonstrated anticancer activity in vitro in multiple tumor types including leukemia/lymphoma, with evidence for a role in chemoprevention. GCP (Amino Up Chemical Co.) is a fermentation product of soy extract and the mycelium of Basidiomycetes, a process which converts soy isoflavones to their aglycone form, greatly increasing intestinal absorption. In humans, GCP has negligible toxicity at potentially active concentrations. We hypothesized that GCP would have significant anticancer activity in hematologic malignancies. Methods: Three human lymphoma cell lines (Jurkat, Raji, and Ramos), a leukemia line (HL60), and four canine lymphoid cell lines (GL-1, CLGL-90, CL1, and CLL-1390) were treated with GCP at doses ranging from 25 to 200 μg/ml. Growth effects were assessed by trypan blue exclusion 5–7 days following a single treatment. Flow cytometry and Westerns were used to measure changes in cell cycling, protein expression and apoptosis. We are conducting a single-agent dose escalation clinical trial in chemo-naïve canines with lymphoma. Results: GCP had striking cytotoxic effects in both canine and human lymphoma cell lines. In all human lines, the 50% inhibitory concentrations (IC50s) were less than 25μg/ml. At 100μg/ml, a concentration achievable in patient plasma, virtually no viable cells remained. Three of four canine lines were sensitive with IC50s in the range of 15–50μg/ml. Substantial G2 cell cycle accumulation was observed, apparent within two hours and maintained for at least 48h. GCP treatment induced apoptosis, as shown by an increase in sub-G1 DNA content and cleavage of PARP and caspase 3. At higher doses, elevated Bcl-2 levels were observed, suggesting a potential mechanism of resistance. These results provided the rationale for a clinical trial in canine patients at UC Davis. One patient with aggressive diffuse large B-cell lymphoma treated at 93 mg/kg/day (dose level 2) had a confirmed partial response. Conclusions: GCP has potent anticancer activity in vitro against lymphoid cell lines, with almost complete cell kill at doses achievable in patient plasma with minimal toxicity. A phase I clinical trial in canine lymphoma will determine whether testing in human lymphoma patients is warranted. No significant financial relationships to disclose.

In vitroCanine Distemper Virus Infection of Canine Lymphoid Cells: A Prelude to Oncolytic Therapy for Lymphoma

Measles virus (MV) causes the regression of human lymphoma xenografts. The purpose of this study was to determine if canine lymphoid cells could be infected in vitro with MV or canine distemper virus (CDV, the canine Morbillivirus equivalent of MV) and determine if in vitro viral infection leads to apoptotic cell death. Reverse transcriptase-PCR was used to examine the expression of both signal lymphocyte activation molecule (CD150) and membrane cofactor molecule (CD46) mRNA. An attenuated CDV expressing enhanced green fluorescent protein was used to infect canine cells in vitro. Both flow cytometry and reverse transcriptase-PCR was used to document CDV infection. Cell death was examined using a propidium iodide staining assay and Annexin V binding. Canine lymphoid cell lines and neoplastic B and T lymphocytes collected from dogs with spontaneous lymphoma expressed the Morbillivirus receptor CD150 mRNA. In contrast, only neoplastic lymphocytes expressed detectable levels of CD46 mRNA. Although MV did not infect canine cells, CDV efficiently infected between 40% and 70% of all three canine lymphoid lines tested. More importantly, CDV infected 50% to 90% of neoplastic lymphocytes isolated from dogs with both B and T cell lymphoma. Apoptosis of CDV-infected cell lines was documented. Attenuated CDV may be a useful treatment for canine lymphoma. As such, dogs with lymphoma may represent a biologically relevant large animal model to investigate the feasibility, safety, and efficacy of Morbillivirus therapy in a clinical setting with findings that may have direct applicability in the treatment of human non-Hodgkin's lymphoma.