No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Establishment and Characterization of a New Canine B-Cell Leukemia Cell Line
Abstract
A new cell line derived from a spontaneous canine leukemia was established and designated GL-1. The cells have been cultured in a floating fashion and passaged for over two years. They were round with rich cytoplasm containing many rough endoplasmic reticula and mitochondria. Peroxidase staining was negative. The nuclei of many cells were round, but segmented nuclei were seen frequently. The doubling time of the cells was 27.3 hr and they had 78 chromosomes. Surface marker analysis using monoclonal antibodies (MABs) and flowcytometry revealed that GL-1 possessed CD45 and surface IgG. However, the cells did not react with MABs detecting T-cell markers. These results indicate that GL-1 has a lymphocytic lineage and is derived from a B-cell leukemia.
... Xanthohumol (1) was used as a reference compound. The results revealed that CLB70 (B-cell leukemia) [24] and CLBL-1 (B-cell lymphoma) [25] cells were relatively sensitive for the applied compounds, whereas GL-1 (B-cell leukemia) cells [26] were the most resistant among the tested lines. Compounds 1, 3 and 5 displayed the lowest IC 50 values ( Figure S1). ...
... Xanthohumol (1) was used as a reference compound. The results revealed that CLB70 (B-cell leukemia) [24] and CLBL-1 (B-cell lymphoma) [25] cells were relatively sensitive for the applied compounds, whereas GL-1 (B-cell leukemia) cells [26] were the most resistant among the tested lines. Compounds 1, ...
... The following canine cell lines: CLB70 (B-cell leukemia), CLBL-1 (B-cell lymphoma) and GL-1 (B/T-cell leukemia) were used in this study. The CLB70 cell line was obtained by Aleksandra Pawlak and coworkers [24]; CLBL-1 was obtained from Barbara C. Ruetgen, Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria [25]; and GL-1 cells were obtained from Yasuhito Fujino and Hajime Tsujimoto from the University of Tokyo, Department of Veterinary Internal Medicine [26]. ...
Abstract: Xanthohumol is a cancer chemopreventive agent that can interfere with the initiation, promotion, and progression phase of carcinogenesis via a variety of inhibitory mechanisms. Xantho-humol was reported as an effective agent against leukemia/lymphoma cells. In the present study, we investigated the effect of xanthohumol and its natural and semisynthetic derivatives against various canine leukemia/lymphoma cell lines. Xanthohumol, three hops minor prenylflavonoids (xanthohumol C, xanthohumol D, α,β-dihydroxanthohumol) and four derivatives obtained by biotransformation (xanthohumol 4-O-β-D-(4-O-methyl)-glucopyranoside) as well as by chemical modification (1 ,2-dihydroxanthohumol K, 2,3-dehydroisoxanthohumol, (Z)-6,4-dihydroxy-4-methoxy-7-prenylaurone) were tested for their antiproliferative and pro-apoptotic activities against the following canine leukemia/lymphoma cell lines: CLBL-1 (B-cell lymphoma), CLB70 (B-cell leukemia), and GL-1 (B-cell leukemia). The compounds were tested at a final concentration range of 0.1-30 µM for 48 h. All eight of the tested flavonoids exerted concentration-dependent cytotoxicity in the selected canine lymphoma/leukemia cell lines. Three compounds markedly decreased the viability of all cell lines with IC 50 in the range of 0.5 to 8 µM. Double-staining of the treated cells with AnnexinV and propidium iodide revealed that the dying cells were mostly in the late apoptosis stage. ROS production and changes in mitochondrial potential were detected. Western blot analysis showed a decreased expression of Bcl-2. Canine lymphoma and leukemia cell lines are sensitive to xanthohumol derivatives, and the compounds acted through an apoptotic cell-death mechanism. These compounds, either used alone or in combination with other therapies, may be useful for the treatment of canine leukemia/lymphoma.
... CL-1 represents immature T lymphoma cells. GL-1 cell line was previously described as B-cell leukemia GL-1 cell line was previously described as B-cell leukemia (Nakaichi et al., 1996), but currently this cell line has unclear phenotype with reported positive results for TCR < gamma > gene rearrangements and altered cell surface markers (Rutgen et al., 2010). CLBL-1 represents diffuse large Bcell lymphoma (Rutgen et al., 2010). ...
... In the present study, the concentration-dependent cytotoxicity of MTX was assessed for the first time against different types of established canine lymphoma/leukemia cell lines: CLBL-1 (Rutgen et al., 2010), GL-1 (Momoi et al., 1997), and CL-1 (Nakaichi et al., 1996). ...
... The canine cell lines CLBL-1 (B-cell lymphoma cell line), GL-1 (Bcell leukemia), and CL-1 (T-cell lymphoma) were used in this study. CLBL-1 was obtained from Barbara C. Ruetgen, Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria (Rutgen et al., 2010), and GL-1 and CL-1 were obtained from Yasuhito Fujino and Hajime Tsujimoto from the University of Tokyo, Department of Veterinary Internal Medicine (Momoi et al., 1997;Nakaichi et al., 1996). ...
Introduction:
Methotrexate is an antimetabolite used in the treatment of cancer and non-malignant diseases including rheumatoid arthritis, psoriasis and graft vs. host disease. Combination therapy with methotrexate was successful in the treatment of canine lymphoma, mammary tumor and invasive urinary bladder cancer. Lymphoma, the most common hematopoietic cancer in dogs, and leukemia are sensitive to chemotherapy, which is why methotrexate may be an important treatment option for these diseases. Although methotrexate is already used in veterinary oncology its effects on canine cancer cells has not been tested. The aim of the study was to evaluate for the first time methotrexate concentration-dependent cytotoxicity and its capability of inducing apoptosis in selected canine lymphoma/leukemia cell lines: CLBL-1, GL-1 and CL-1 as a first step before the in vitro development of new therapeutic options with the use of methotrexate.
Results:
Methotrexate exhibited concentration-dependent inhibitory effect on proliferation of all the examined cell lines with different degree of apoptosis induction. The most methotrexate sensitive cells belonged to CL-1 cell line derived from T cell neoplasia and previously characterized by high resistance to the majority of anticancer drugs used in the therapy of lymphoma/leukemia in dogs. Canine lymphoma and leukemia cell lines are sensitive to methotrexate, and this drug may be useful in effective treatment of canine neoplasms and especially of T-type leukemia/lymphoma.
... The oldest, 17-71 B-cell lymphoma line was developed by Steplewski et al. 9 The canine cell line GL-1 was derived from peripheral blood of a dog with acute B-cell leukaemia. 10 The CL-1 T-cell lymphoma cell line was derived from pleural fluid of a dog with thymic lymphoma. 11 The T-cell line DCL-01 was established from a lymph node of a dog with cutaneous lymphoma 12 and DLC-02 line was established from peripheral lymphocytes of a dog with large granular lymphocyte leukaemia. ...
... The results of western blot analysis are shown in Figure 4. We also analyzed three other established canine cell lines -CL-1, GL-1, and CLBL-1. 10,11,14 Expression of the investigated proteins in the tested cell lines was variable. We observed an elevated level of Bcl-2, Mcl-1 and Ras, decreased expression of p53, and activation of NF-kB in most of the tested cell lines compared to normal canine peripheral blood mononuclear cells (PBMC). ...
... Several canine lymphoma and acute leukaemia cell lines have been reported 10,11,14 but no cell line derived from a chronic B-cell leukaemia dog has been described so far. We established B-type leukaemia cell line CLB70 from FNA of a dog diagnosed with CLL. ...
We established a new B-cell leukaemia cell line CLB70 from a dog with chronic lymphocytic leukaemia. This cell line is positive for CD20, CD45, CD79a, MHC class II, IgG, IgM; weakly positive for CD21; and negative for CD3, CD4, CD5, CD8, CD14, CD34, CD117. PCR for antigen receptor gene rearrangement (PARR) analysis revealed a biclonal immunoglobulin heavy chain (IgH) gene rearrangement and negative result for TCRγ. Western blot analysis of anti- and pro-apoptotic proteins showed increased expression of Bcl-2, Mcl-1, NF-kB, and Ras, and decreased expression of p53. CLB70 cells grow rapidly in vitro and are tumourigenic in nude mice. The CLB70 line is highly sensitive to doxorubicin, less sensitive to etoposide and imatinib, and resistant to piroxicam, celecoxib and dexamethasone. Our results indicate that CLB70 cells are derived from mature B-cells and they may be a useful tool for the development of new therapeutic strategies for both dogs and humans.
... A panel of canine lymphoma/leukemia cell lines: CLBL-1 (B-cell lymphoma), CLB70 (B-cell chronic lymphocytic leukemia), and GL-1 (B-cell leukemia) was used in this study. The CLBL-1 cell line was a gift from Barbara Rütgen from the Institute of Immunology, Department of Pathobiology from the University of Vienna (29), the GL-1 cell line was received from Yasuhito Fujino and Hajime Tsujimoto from the Department of Veterinary Internal Medicine at the University of Tokyo (30), and the CLB70 cell line (31) was established with the participation of researchers from our laboratory; the studies involving animals participants were reviewed and approved by the New York Academy of Sciences Ad Hoc Committee on Animal Research and were approved by the First Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland (approval no. 24/2014). ...
... In our study, the replication fork speed in the canine cells seemed to be lower, around 1.5 Kb/ min for the GL-1 cell line, and 0.86 Kb/min for the CLBL-1 cell line ( Figure 6; Supplementary Table S1). It can be concluded that GL-1 cells have a higher replication speed than CLBL-1, which is interesting as the GL-1 cell line's doubling time is 27.3 h, and for the CLBL-1 it is 19 h (30,86). The mean values of fork asymmetry were higher than 1 in both cell lines (Supplementary Table S1), indicating a significant number of replication forks terminate asymmetrically in both cell lines (87). ...
Background
Dogs present a significant opportunity for studies in comparative oncology. However, the study of cancer biology phenomena in canine cells is currently limited by restricted availability of validated antibody reagents and techniques. Here, we provide an initial characterization of the expression and activity of key components of the DNA Damage Response (DDR) in a panel of hematopoietic canine cancer cell lines, with the use of commercially available antibody reagents.
Materials and methods
The techniques used for this validation analysis were western blot, qPCR, and DNA combing assay.
Results
Substantial variations in both the basal expression (ATR, Claspin, Chk1, and Rad51) and agonist-induced activation (p-Chk1) of DDR components were observed in canine cancer cell lines. The expression was stronger in the CLBL-1 (B-cell lymphoma) and CLB70 (B-cell chronic lymphocytic leukemia) cell lines than in the GL-1 (B-cell leukemia) cell line, but the biological significance of these differences requires further investigation. We also validated methodologies for quantifying DNA replication dynamics in hematopoietic canine cancer cell lines, and found that the GL-1 cell line presented a higher replication fork speed than the CLBL-1 cell line, but that both showed a tendency to replication fork asymmetry.
Conclusion
These findings will inform future studies on cancer biology, which will facilitate progress in developing novel anticancer therapies for canine patients. They can also provide new knowledge in human oncology.
... A structural isomer of lactone 3, namely 2 -methoxy derivative 2, showed lower cytotoxic potency, as illustrated in Table 1 by IC 50 values for each derivative. Both compounds were more effective towards CLBL-1 and CLB70 cell lines (representing B-cell lymphoma and chronic type B leukemia, respectively) [21,22] than towards the rarer and more difficultto-treat types of hematopoietic cancer in dogs-acute leukemia of not clearly defined phenotype (B/T) [22,23], or NK cell lymphoma [24]. The remaining tests confirmed that the difference between these isomers concerns only the strength of their action and not the way in which they kill cancer cells. ...
... The study involved a panel of canine cancer cell lines: CLBL-1 (B-cell lymphoma), GL-1 (B/T-cell leukemia), CLB70 (B-cell chronic lymphocytic leukemia) and CNK-89 (NK-cell lymphoma), and a normal canine cell line (MDCK). CLBL-1 was obtained from Barbara C. Ruetgen from the Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria [22]; GL-1 from Yasuhito Fujino and Hajime Tsujimoto of the University of Tokyo, Department of Veterinary Internal Medicine [23]; while CLB70 [21] and CNK-89 [24] were established in our laboratory. The MDCK cell line was purchased from Sigma Aldrich (Steinheim, Germany). ...
Background:
The study investigated four flavanone-derived γ-oxa-ε-lactones: a parent unsubstituted compound and its three derivatives with the methoxy group in positions 2', 4' and 8. Our objective was to find out if the introduction of the methoxy group into the aromatic ring affects in vitro anti-tumor potency of the investigated lactones.
Methods:
Cytotoxic and pro-apoptotic effects were assessed with cytometric tests with propidium iodide, annexin V, and Western blot techniques. We also investigated potential synergistic potency of the tested lactones and glucocorticoids in canine lymphoma/leukemia cell lines.
Results:
The tested flavanone-derived lactones showed anti-cancer activity in vitro. Depending on its location, the methoxy group either increased or decreased cytotoxicity of the derivatives as compared with the parent compound. The most potent lactone was the one with the methoxy group at position 4' of the B ring (compound 3), and the weakest activity was observed when the group was located at C-8 in the A ring. A combination of the lactones with glucocorticoids confirmed their synergy in anti-tumor activity in vitro.
Conclusions:
Methoxy-substituted flavanone-derived lactones effectively kill canine lymphoma/leukemia cells in vitro and, thanks to their synergistic action with glucocorticoids, may potentially be applied in the treatment of hematopoietic cancers.
... CLBL-1 was obtained from Barbara C.Ruetgen from the Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna [48]. GL-1 from Yasuhito Fujino and Hajime Tsujimoto of the University of Tokyo, Department of Veterinary Internal Medicine [49]. The CF2TH cell line was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) K9NK cell line was generous gifts from Deepika Dhawan (Purdue University College of Veterinary Medicine, USA) and RDSVS-TCC1 was a gift from Maciej Parys (Royal (Dick) School of Veterinary Studies and The Roslin Institute, University of Edinburgh, Scotland), respectively. ...
This study focuses on understanding the structural and molecular changes in lipid membranes under the influence of six halogenated flavonoid derivatives differing in the number and position of substitution of chlorine and bromine atoms (D1-D6). Utilizing various analytical techniques, including fluorometric methods, dynamic light scattering (DLS), attenuated Fourier transform infrared spectroscopy (ATR- FTIR), and Raman spectroscopy, the research seeks to elucidate the mechanisms underlying the interaction of flavonoids with cell membranes. Additionally, the study includes in silico analyses to explore the physicochemical properties of these compounds and their potential pharmaceutical applications, along with toxicity studies to assess their effects on cancer, normal, and red blood cells. Our study showed the ability of halogenated derivatives to interact mostly with the outer part of the membrane, especially in the lipid heads region however, some of them were able to penetrate deeper into the membrane and affect the fluidity of hydrocarbon chains. The potential to reduce cancer cell viability, the lack of toxicity towards erythrocytes, and the favourable physicochemical and pharmacokinetic properties make these halogenated flavonoids potential candidates for exploring their potential for medical use.
... The U2OS human osteosarcoma cell line (obtained from ATCC) was used as a control. The CLBL-1 cell line was kindly provided by Dr. Barbara Rütgen from the Institute of Immunology, Department of Pathobiology at the University of Vienna (29), the GL-1 line was received from Dr. Yasuhito Fujino and Dr. Hajime Tsujimoto of the Department of Veterinary Internal Medicine at the University of Tokyo (22), and CLB70 (25) and CNK-89 (12) lines were established with the participation of researchers from our laboratory. Fig. 1. ...
Introduction
New and more effective therapies for canine cancer patients are urgently required and this necessitates advanced experimental research. Dogs are good models for studies in comparative oncology; however, canine cancer cell biology research is currently limited by low availability of validated antibody reagents and techniques. This study characterises the expression of key components of the unfolded protein response (UPR) in a panel of haematopoietic canine cancer cell lines using commercially available antibodies, and validates the methods used to study this pathway.
Material and Methods
The CLBL-1 canine lymphoma cell line and the GL-1 canine leukaemia cell line sourced externally and two counterparts established in house (CNK-89 and CLB70) were used as models of different lymphoma and leukaemia canine cell lines for the study. The human U2OS cell line served as the control. Antibodies were selected for identifying UPR proteins according to known canine cell reactivity and canine–murine and canine–human homology. Endoplasmic reticulum stress was induced with thapsigargin and MG132 in the cell lines. Etoposide was used to induce DNA damage in the cells. The techniques used for this validation analysis were RNA sequencing to observe the expression of UPR components in canine cell lines, Western blot to observe changes of protein expression levels after inducing ER stress in the cells, and flow cytometry in order to study cell death.
Results
Substantial variations in both the basic expression and agonist-induced activation of the UPR pathway were observed in canine cancer cell lines, although the biological significance of these differences requires further investigation.
Conclusion
These findings will be a starting point for future studies on cancer biology in dogs. They will also contribute to developing novel anticancer therapies for canine patients and may provide new insights into human oncology.
... In the present study, we used eight canine lymphoma and leukemia cell lines derived from dogs with naturally occurring lymphoid malignancies. These included the following B-cell lines: 17-71 (multicentric B-cell lymphoma), CLBL-1 (multicentric B-cell lymphoma), and GL-1 [B-cell acute lymphoblastic leukemia (ALL)] [26][27][28]; and T-cell lines: CLC (gastrointestinal T-cell lymphoma), CLGL-90 (chronic large granular lymphocytic T-cell leukemia), Ema (mediastinal T-cell lymphoma), Nody-1 (alimentary T-cell lymphoma), and UL-1 (renal T-cell lymphoma) [29][30][31][32]. All canine lymphoma and leukemia cell lines were maintained in a complete medium [RPMI-1640 (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) containing 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin (Nacalai Tesque, Kyoto, Japan)]. ...
Cyclin-dependent kinase inhibitor p16 (CDKN2A) primarily functions as a negative regulator of the retinoblastoma protein (pRb) pathway to prevent pRb phosphorylation, thus playing a critical role in cell cycle arrest. In canine lymphoma cells, methylation due to inactivation of the p16 gene has been reported. However, its protein expression has not been examined in previous studies. In our in vitro study, the gene and protein expression of p16 and phosphorylated pRb were examined simultaneously in eight canine lymphoma and leukemia cell lines (17-71, CLBL-1, GL-1, CLC, CLGL-90, Ema, Nody-1, and UL-1). Methylation of the p16 gene was also explored using the demethylation drug 5-Aza-2'-deoxycytidine (5-Aza). After 5-Aza treatment, p16 gene and protein expression increased and pRb phosphorylation decreased, suggesting that both hypermethylation of the p16 gene and pRb hyperphosphorylation occurred in four out of eight cell lines (CLBL-1, CLC, Nody-1, and UL-1). Moreover, the estimation of p16's protein expression was better than that of p16's mRNA expression because the expression of the protein was more stable than those of the gene, and highly related to the phosphorylation of pRb. These results revealed that p16's protein expression could be a promising biomarker for canine lymphoma cells.
... The well-characterized canine lymphoid B-cell line GL-1 and the canine T-cell lymphoma cell line CL-1 were obtained from Prof. Yuko Goto-Koshino (Tokyo University, Japan) and validated elsewhere. [10][11][12] Canine DLBCL cell line CLBL-1 was obtained from Prof. Barbara C Rütgen (University of Veterinary Medicine Vienna, Austria). 13 All cell lines were validated for canine origin by karyotyping. ...
The interleukin‐1 receptor‐related kinase 4 (IRAK4), downstream of myd88, plays an essential role in hyperactive TLR signaling seen in some B‐cell lymphomas. In particular, efficient IRAK4 inhibitors of activated B‐cell subtype of human diffuse large B‐Cell lymphoma (DLBCL) are being developed. However, the anticancer effect of IRAK‐4 inhibitors in veterinary medicine has not been elucidated. It is therefore explored in this study involving the GL‐1 and CL‐1 canine lymphoma cell lines in vitro. MyD88 expression was analysed using polymerase chain reaction. GL‐1 and CL‐1 cells were subjected to concentration‐ and time‐dependent treatment with an IRAK‐4 inhibitor and assessed for viability, TLR signalling association, and apoptosis using a cell counting Kit‐8 assay, Western blotting, and flow cytometry. The GL‐1 and CL‐1 cells exhibited enhanced MyD88 expression, however, canine peripheral blood mononuclear cells (cPBMCs) did not. The IRAK‐4 inhibitor reduced cell viability in a dose‐ and time‐dependent manner, significantly reduced the phosphorylation of molecules associated with TLR signalling at IC50 such as IRAK1, IRAK4, NF‐κB and STAT3, and induced apoptosis in GL‐1 and CL‐1 cells. The anticancer effect of the IRAK‐4 inhibitor on canine lymphoma cells is mediated by apoptosis via downregulation of TLR signalling. This article is protected by copyright. All rights reserved.
... The CLBL-1 cell line was isolated from a male Bernese mountain dog with confirmed stage IV diffuse large cell lymphoma [27]. GL-1 was a B-cell leukemia cell line, derived from a German Shepherd [40]. The DLBCL cell line SU-DHL-4 was extracted and cultured from a 38-year-old male patient with diffuse histiocytic lymphoma [41]. ...
Bruton’s tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3β, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL.
... CLBL-1 [59] was a kind gift from Barbara C. Ruetgen from the Institute of Immunology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria. GL-1 [60] was provided by Yasuhito Fujino and Hajime Tsujimoto from the University of Tokyo, Department of Veterinary Internal Medicine. CLB70 [61] and CNK-89 [62] were established in our laboratory. ...
Treatment of neoplastic diseases in companion animals is one of the most important problems of modern veterinary medicine. Given the growing interest in substances of natural origin as potential anti-cancer drugs, our goal was to examine the effectiveness of benzyl isothiocyanate (BITC), a compound found in cruciferous vegetables, against canine lymphoma and leukemia. These are the one of the most common canine cancer types, and chemotherapy is the only treatment option. The study involved established cell lines originating from various hematopoietic malignancies: CLBL-1, GL-1, CLB70 and CNK-89, immortalized noncancerous cell lines: MDCK and NIH-3T3 and canine peripheral blood mononuclear cells (PBMCs). The cytotoxic activity of BITC, apoptosis induction, caspase activity and ROS generation were evaluated by flow cytometry. H2AX phosphorylation was assessed by western blot. The study showed that the compound was especially active against B lymphocyte-derived malignant cells. Their death resulted from caspase-dependent apoptosis. BITC induced ROS accumulation, and glutathione precursor N-acetyl-l-cysteine reversed the effect of the compound, thus proving the role of oxidative stress in BITC activity. In addition, exposure to the compound induced DNA damage in the tested cells. This is the first study that provides information on the activity of BITC in canine hematopoietic malignancies and suggests that the compound may be particularly useful in B-cell neoplasms treatment.
... The antiproliferative tests were performed on three types of cancer cell lines: canine B-cell lymphoma (CLBL-1) [33], canine B-cell chronic leukemia (CLB70) [34] and NK-cell lymphoma (CNK-89) (Grudzień et al. in press). The antiproliferative activity was investigated by MTT test after 72 h of treatment, according to the procedure described previously [16]. ...
The β-aryl-δ-halo-γ-lactones are known for their antiproliferative activity towards numerous cancer cell lines. The aim of this study was to obtain in the biotransformation process new β-aryl-δ-hydroxy-γ-lactones and compare their activity with the antiproliferative activity of parent compounds. The racemic cis-5-(1-iodoethyl)-4-phenyldihydrofuran-2-one as well as separate enantiomers were transformed in fungal cultures. Among ten tested biocatalysts, three (Absidia cylindrospora AM336, Absidia glauca AM254, and Fusarium culmorum AM10) were able to catalyze the hydrolytic dehalogenation process. The biotransformations processes were highly stereoselective and enantiomerically pure hydroxylactones were obtained (ee ≥ 99%). The iodo- and hydroxylactone enantiomers were subjected to cytotoxic activity evaluation on canine leukemia and lymphoma cell lines. The iodolactones exhibited higher biological potential towards tested cell lines than hydroxylactones. Higher cytotoxic potential was also characteristic for (+)-(4S,5S,6R)-enantiomer of iodolactone compared to its antipode.
... The GL-1 (canine B-cell leukemia) was obtained from Yasuhito Fujino and Hajime Tsujimoto from the Department of Veterinary Internal Medicine University of Tokyo [27], the CLB70 (canine B-cell chronic leukemia) was described earlier by Pawlak et al. [28]. The GL-1 cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, 100 U/mL penicillin and 2 mM L-glutamine (Sigma-Aldrich, Stainheim, Germany). ...
Starting from 1-acetyl-1-cyclohexene, three enantiomeric pairs (ee ≥99%) of bicyclic δ-halo-γ-lactones with cyclohexane ring were obtained in five-step synthesis. The key stereochemical steps were lipase-catalyzed kinetic resolution of racemic 1-(cyclohex-1-en-1-yl) ethanol followed by transfer of chirality to ethyl 2-(2-ethylidenecyclohexyl) acetate in the Johnson–Claisen rearrangement. Synthesized halolactones exhibited antiproliferative activity towards canine B-cell leukemia cells (GL-1) and canine B-cell chronic leukemia cells (CLB70) and the most potent (IC50 18.43 ± 1.46 μg/mL against GL-1, IC50 11.40 ± 0.40 μg/mL against CLB70) comparable with the control etoposide, was (1R,6R,1’S)-1-(1’-chloroethyl)-9- oxabicyclo[4.3.0]nonan-8-one (8b). All halolactones did not have a toxic effect on erythrocytes and did not change the fluidity of membranes in the hydrophobic region of the lipid bilayer. Only weak changes in the hydrophilic area were observed, like the degree of lipid packing and associated hydration. The racemic halolactones were also tested for their antimicrobial properties and found to exhibit selectivity towards bacteria, in particular, towards Proteus mirabilis ATCC 35659.
... CLB70 [40] was established by us. GL-1 [41] was kindly provided by Drs Y. Fujino and H.Tsujimoto (University of Tokyo, Tokyo, Japan). All cell lines were cultured in RPMI with 15% FBS. ...
Canine B-cell lymphoma (CBL) is an incurable, spontaneous lymphoid malignancy constituting an accurate animal model for testing novel therapeutic strategies in human medicine. Resources of available species-specific therapeutic monoclonal antibodies (mAbs) targeting CBL are scarce. The aim of the present study was to evaluate the therapeutic potential of mAb B5, specific for the dog leukocyte antigen DR (DLA-DR) and its antibody-drug conjugate with methotrexate (B5-MTX). B5 induced caspase-dependent apoptosis of DLA-DR-expressing canine B cell lymphoma/CLBL1 and CLB70 leukemia lines, but not the GL-1 line not expressing DLA-DR. The cytotoxicity of B5-MTX to sensitive cells was further potentiated by a payload of MTX, but without any substantial off-target effects. The infusion of B5 and B5-MTX in a murine model of disseminated, advanced canine lymphoma, mediated >80% and >90% improvement in survival, respectively, and was well tolerated by the animals. Interestingly, the concentrations of soluble DLA-DR (sDLA-DR) antigens present in the blood serum of tumor-bearing mice were found proportional to the tumor burden. On this basis, sDLA-DR levels were evaluated as a potential biomarker using samples from canine lymphoma patients. In summary, the action of B5 and B5-MTX holds promise for further development as an alternative/complementary option for the diagnosis and treatment of canine lymphoma.
... D10.G4.1 and Jurkat cell lines were obtained from the collection of the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland. GL-1 (B-cell leukemia) and CL-1 (T-cell lymphoma) were obtained from Yasuhito Fujino and Hajime Tsujimoto from the University of Tokyo, Department of Veterinary Internal Medicine, Tokyo, Japan [22,23]. ...
Context: Leaf extracts of plants of the genus Betula have traditionally been used as diuretic, anti-rheumatic and diaphoretic preparations. One of the main active ingredients of Betula bark is betulin, lupane-type triterpene alcohol, with multiple biological activities.
Objectives: The aim of this study was to investigate in vitro and in vivo immunomodulatory effects of a newly synthesized ester of betulin: 28-O-phosphatidylbetulin [28-O-(1,2-diacyl-sn-glycero-3-phospho)-betulin, DAPB] in comparison with betulin in mice.
Materials and methods: Cytotoxic activity of DAPB or betulin was tested against non-cancer (D10.G4.1 and J774E.1) and cancer (GL-1; CL-1 and Jurkat) cell lines. The in vivo part assessed total lymphocyte count, weight ratio and subsets of lymphocytes in the lymphatic organs, and humoral immune response to sheep erythrocytes (SRBC).
Results: In vitro assay showed that DAPB, contrary to betulin, had no antiproliferative activity. Exposure to four doses of DAPB increased the absolute count of immature CD4⁺CD8⁺ thymic cells as well as the percentage and absolute count of mature CD4⁺ and CD8⁺ thymocytes. DAPB enhanced the percentage or absolute count of CD3⁺ cells in spleen and lymph nodes with corresponding decrease in the percentage and/or absolute count of CD19⁺ cells. Both DAPB and betulin enhanced the percentage and absolute count of CD8⁺ lymphocytes in lymph nodes. In SRBC-immunized mice, betulin contrary to DAPB enhanced the number of splenocytes producing anti-SRBC antibodies (PFC). Both DAPB and betulin increased the level of total (IgM + IgG) and IgG titers.
Conclusion: Despite the lack of cytotoxic activity, DAPB shows valuable immunomodulatory properties.
... Four canine lymphoid tumour cell lines (CLBL-1, GL-1, UL-1, and Ema) were used in this study: CLBL-1, a canine B-cell lymphoma cell line [42]; GL-1, a canine B-cell leukaemia cell line [43]; UL-1, a canine T-cell lymphoma cell line [44]; and Ema; a canine T-cell lymphoma cell line [45]. UL-1 and Ema were established from dogs with lymphoma showing drug resistance after chemotherapy, whereas CLBL-1 and GL-1 were established from dogs with leukaemia or lymphoma who were not subjected to chemotherapy. ...
Exosomes are small extracellular vesicles released from almost all cell types, which play roles in cell-cell communication. Recent studies have suggested that microenvironmental crosstalk mediated by exosomes is an important factor in the escape of tumour cells from the anti-tumour immune system in human haematopoietic malignancies. Here, we conducted comprehensive analysis of the miRNA and protein profiles within the exosomes released from four canine lymphoid tumour cell lines as a model of human lymphoid tumours. The results showed that the major miRNAs and proteins extracted from the exosomes were similar among the four cell lines. However, the miRNA profiles differed among the exosomes of each cell line, which corresponded to the expression patterns of the parent cells. In the comparison of the amounts of miRNAs and proteins among the cell lines, those of three miRNAs (miR-151, miR-8908a-3p, and miR-486) and CD82 protein differed between exosomes derived from vincristine-sensitive and resistant cell lines. Further investigations are needed to elucidate the biological functions of the exosomal contents in the microenvironmental crosstalk of lymphoid tumours.
... The GL-1 (canine B-cell leukemia) was obtained from Yasuhito Fujino and Hajime Tsujimoto from the Department of Veterinary Internal Medicine University of Tokyo [39], the CLB 70 (canine B-cell chronic leukemia) was established by Pawlak et al. [40]. The GL-1 cells were maintained in RPMI 1640 culture medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin and 10% fetal bovine serum (FBS) (Sigma-Aldrich, Stainheim, Germany). ...
Searching for the new anticancer compounds we prepared three new β-cyclocitral-derived hydroxyl-γ-lactones by microbial hydroxylation of tetramethyl-substituted bicyclic γ-lactone. The substrate was transformed by the enzymatic system of filamentous fungi. Three out of fifteen strains were selected as effective biocatalysts (Fusarium culmorum AM10, Armillaria mellea AM296, Trametes versicolor AM536). The hydroxylation processes were not only regioselective but also stereoselective. The hydroxylation products of each secondary carbon atom in the cyclohexane ring were obtained by the application of the selected fungal strains. The Fusarium culmorum AM10 introduced the hydroxy function at C-3 and C-4, Armillaria mellea AM296 incorporated the hydroxy function at C-3 and C-5 and Trametes versicolor AM536 transformed the substrate to the mixture of C-3, C-4 and C-5 hydroxylactones. The hydroxylactones obtained were enantiomericaly enriched (ee values in the range 17–99%). The in vitro antiproliferative activities of the functionalization products were also evaluated. Regardless of the hydroxy substituent location all tested lactones exhibited similar, significant activity towards selected cancer cell lines (IC50 in the range 22.8–33.9 µg/mL).
... Therefore, the majority of canine lymphoma xenograft murine models described until now represent T-cell lymphoid malignancies [20][21][22]. Notably, only four of the available cell lines are reportedly of B-cell origin, including the GL-1 cell line derived from a dog with B-cell acute lymphoblastic leukemia [34]; the 17-71, a B-cell cell line not initially phenotyped and that does not express typical B-cell lymphoma markers [35]; 3132, a cell line that probably is not of B-cell origin despite initial reports of surface immunoglobulin [36] and CLBL-1, the only available cell line that has been well-characterized both in vitro and in vivo [11,23,24,27,37]. As a matter of fact, CLBL-1 appears to be the only exclusive cell line that faithfully represents DLBCL, reproducibly inducing tumors and preserving its phenotype in the xenotransplantation setting [23,24]. ...
Canine diffuse large B-cell lymphoma (DLBCL) is one of the most common cancers in dogs which shares remarkable similarities with its human counterpart, making the dog an excellent model for the investigation of novel therapeutic agents. However, the integration of canine lymphoma in comparative studies has been limited due in part to the lack of suitable xenograft mouse models for preclinical studies. To overcome these limitations, we established and characterized a localized subcutaneous bioluminescent canine DLBCL xenograft mouse model. The canine CLBL-1 cell line stably expressing the luciferase and green fluorescent protein reporters was generated and used to establish the xenograft tumor model. A pilot study was first conducted with three different cell densities (0.1×10 6 , 0.5×10 6 and 1×10 6 cells) in SCID mice. All mice presented homogeneous tumor induction within eight days after subcutaneous injection, with a 100% engraftment efficiency and no significant differences were observed among groups. The tumors were highly aggressive and localized at the site of inoculation and reproduced histological features and immunophenotype consistent with canine DLBCL. Importantly, xenograft tumors were detected and quantified by bio-luminescent imaging. To assess response to therapy, a therapeutic study with a histone deacetylase inhibitor, panobinostat, was performed. The results demonstrated that panobi-nostat (20 mg/kg) efficiently inhibited tumor growth and that bioluminescent imaging allowed the monitorization and quantification of tumor response to therapy. In summary, this study provides a bioluminescence canine DLBCL model that offers high engraftment efficiency, preservation of tumor features, and noninvasive monitoring of tumor progression, validating the model as a promising preclinical tool for both veterinary and human medicine.
... The B cell lymphoma cell line (CLBL-1) was obtained from Barbara C. Rütgen, Institute of Immunology, Department of Pathobiology of the University of Veterinary Medicine, Vienna, Austria [45]. The canine cell line GL-1 (B cell leukemia) was obtained from Yasuhito Fujino and Hajime Tsujimoto from the University of Tokyo, Department of Veterinary Internal Medicine, Tokyo, Japan [46]. CLB70 line (B cell chronic leukemia) was established by one of the coauthors of the manuscript [47]. ...
Three novel enantiomeric pairs of bromolactones possesing a 2,5-dimethylphenyl substituent at the β-position of the lactone ring have been synthesized from corresponding enantiomeric (E)-3-(2′,5′-dimethylphenyl)hex-4-enoic acids (4) by kinetically controlled bromolactonization with N-bromosuccinimide (NBS). γ-Bromo-δ-lactones (5) were isolated as the major products. Absolute configurations of stereogenic centers of γ-bromo-δ-lactones (5) were assigned based on X-ray analysis; configurations of cis δ-bromo-γ-lactones (6) and trans δ-bromo-γ-lactones (7) were determined based on mechanism of bromolactonization. Synthesized compounds exhibited significant antiproliferative activity towards the four canine cancer cell lines (D17, CLBL-1, CLB70, and GL-1) and one human cancer line (Jurkat). Classifying the compounds in terms of activity, the most active were enantiomers of trans δ-bromo-γ-lactones (7) followed by enantiomers of cis isomer (6) and enantiomeric γ-bromo-δ-lactones (5). Higher activity was observed for all stereoisomers with S configuration at C-4 in comparison with their enantiomers with 4R configuration. Synthesized compounds did not induce hemolysis of erythrocytes. The results of the interaction of bromolactones with red blood cell membranes suggest that these compounds incorporate into biological membranes, concentrating mainly in the hydrophilic part of the bilayer but have practically no influence on fluidity in the hydrophobic region. The differences in interactions with the membrane between particular enantiomers were observed only for γ-lactones: stronger interactions were found for enantiomer 4R,5R,6S of cis γ-lactone (6) and for enantiomer 4S,5R,6S of trans γ-lactone (7).
... In searching for tumour-associated antigens expressed by CL cells, we established 2 hybridomas producing mAbs, which were designated as GL1 28 and NK cell lymphoma CH89 (A. Rapak, unpublished) cell lines, where both the latter lacked the expression of DLA-DR molecules. ...
Spontaneous canine lymphoma (CL) has become a promising, nonrodent model for advancing the therapeutic strategies of human hematological malignancies. As new resources for veterinary and comparative studies on CL‐associated antigens, we developed 2 novel mouse monoclonal antibodies, denoted B5 and E11, that recognized the canine major histocompatibility Class II DR antigens (dog leukocyte antigen DR). Using flow cytometry and solid phase immunoenzymatic assays, we showed that the antigens recognized by B5 and E11 were strongly expressed in several CL cell lines and the ex vivo canine neoplastic cells of B and mixed B/T immunophenotypes. Additionally, we evaluated a minimal cross‐reactivity of B5 and E11 with the human B‐cell line, Raji. By the ectopic expression of the hybrid murine/canine I‐E/DR dimers in the HEK293 cells, we demonstrated that the epitope of B5 was localized to the invariant DRα chain, whereas the epitope of E11 was collectively formed by the DRα and DRβ chains. Both epitopes were conformational and conserved in all the tested unrelated individuals of different dog breeds. In vitro treatment of 2 CL B‐cell lines (CLBL1 and CLB70) with B5 and E11 rapidly induced a direct apoptotic cell death. Similarily, both mouse monoclonal antibodies efficiently killed the above cell lines through the mechanisms of complement‐dependent and antibody‐mediated cellular phagocytosis. Collectively, our data support the further development of B5 and E11 as novel tools for dog leukocyte antigen DR‐targeted, preclinical trials involving CL.
... We hypothesized that MDR lymphoma would cause high levels of expression of PDGFRα, VEGFR2 and c-KIT, and that TOC had potential as an effective canine anticancer therapy. Canine lymphoma cell lines (CL-1 and GL-1) [8,10] and DOX-resistant lymphoma cells were used in this study. The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, U.S.A.), supplemented with 10% heat-inactivated fetal bovine serum (Cosmo Bio, Tokyo, Japan) and 1% L-glutamine (BioWhittaker, Walkersville, MD, U.S.A.) with 5% CO 2 at 37°C. ...
We examined whether multidrug resistant (MDR) canine lymphoma increases gene expression for platelet-derived growth factor receptor α (PDGFRα), vascular endothelial growth factor receptor 2 (VEGFR2), and c-KIT, and whether toceranib phosphate (TOC) has potential as a treatment for MDR canine lymphoma. The clinical data showed that PDGFRα expression was higher in canine subjects with MDR T-cell lymphoma than in those with untreated T-cell lymphoma, and that c-KIT expression was greater in subjects with T-cell lymphoma than in those with B-cell lymphoma. TOC monotherapy was well tolerated without serious adverse effects, and two of the five subjects that received TOC exhibited partial responses. The data presented here could contribute to the assessment of TOC-based therapy for dogs with MDR or T-cell lymphoma.
... Canine lymphoma cells (CL-1 and GL-1) [14,15], DOX-resistant lymphoma cells (CL-1DR and GL-1DR) and mononuclear cells were used in this study. The CL-1DR and GL-1DR were generated from the corresponding parental cells (CL-1 and GL-1) with a previously reported procedure [16], the details of which are given in the S1 File and S1 Table. ...
We tested the hypotheses that hypoxic stimulation enhances growth potentials of canine lymphoma cells by activating hypoxia-inducible factor 1α (HIF-1α), and that the hypoxia-activated prodrug (TH-302) inhibits growth potentials in the cells. We investigated how hypoxic culture affects the growth rate, chemoresistance, and invasiveness of canine lymphoma cells and doxorubicin (DOX)-resistant lymphoma cells, and influences of TH-302 on survival rate of the cells under hypoxic conditions. Our results demonstrated that hypoxic culture upregulated the expression of HIF-1α and its target genes, including ATP-binding cassette transporter B1 (ABCB1), ATP-binding cassette transporter G2 (ABCG2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and survivin, and enhanced the growth rate, DOX resistance, and invasiveness of the cells. Additionally, TH-302 decreased the survival rate of the cells under hypoxic condition. Our studies suggest that hypoxic stimulation may advance the tumorigenicity of canine lymphoma cells, favoring malignant transformation. Therefore, the data presented may contribute to the development of TH-302-based hypoxia-targeting therapies for canine lymphoma.
... All three cell lines were obtained from American Type Culture Collection e ATCC, Rockville, MD, USA and kindly provided from the collection of the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroc?aw, Poland. The non-adherent canine cell lines CL-1 (T-lymphoblastoid cell line), GL-1 (B cell lymphoma cell line), both obtained from Yasuhito Fujino and Hajime Tsujimoto from the University of Tokyo, Department of Veterinary Internal Medicine [21,22] and D-17 (canine osteosarcoma, obtained from American Type Culture Collection e ATCC, Rockville, MD, USA) were also used. ...
... Also in humans, the success rates for leukaemia/lymphoma cell line establishment are poor and depend on sampling site and treatment status (Drexler 2001). A few years ago, the only three canine lymphoma/leukaemia cell lines were the CL-1 (Momoi et al. 1996), GL-1 (Nakaichi et al. 1996) and OSW respect of radiosensitivity together with gene expression profiling by quantitative real-time PCR (qPCR) assays for tumorigenic important genes playing key roles in cell proliferation, apoptosis, lymphopoiesis, lymphocyte differentiation, cell survival, proliferation, differentiation and tumorigenicity. ...
This issue of the Graue Reihe is based on the international summer school “Does size matter? Ethical, societal, legal and biological aspects of large animals as biomedical models”, which took place in Freising and Munich from October 10–14, 2011. The summer school was a co-operation of the Europäische Akademie GmbH with the Chair of Livestock Biotechnology at the Technische Universität München. The 15 junior participants representing various relevant disciplines at PhD and Postdoc level came from seven European countries and Canada to present and discuss their work. Leading researchers in veterinary science, biomedical research, philosophy and law brought diverse perspectives to a complex topic. Here, we present papers by eight junior and three senior participants.
... We used 4 canine lymphoid tumor cell lines, i.e., CLBL-1 22) , GL-1 20) , UL-1 27) , and CL-1 19) , which were established from canine patients with multicentric B-cell lymphoma, B-cell acute lymphoblastic leukemia, renal T-cell lymphoma, and mediastinal T-cell lymphoma, respectively. All cell lines were maintained in an atmosphere of 5% CO 2 and cultivated under normal conditions 4) in the presence or absence of 25 ng/mL (82.7 nM) trichostatin A (TSA) (Upstate Biotechnology, US) for 24 hours. ...
In order to investigate whether suppression of the p16 gene is mediated by histone H3 acetylation in 4 canine lymphoid tumor cell lines, the gene's acetylation status was examined. In 2 canine lymphoid tumor cell lines with low p16 mRNA expression (GL-1 and UL-1), the acetylation level was lower than that in CL-1 cells with high p16 mRNA expression. The expression of the p16 gene in these 2 cell lines was markedly restored after culture in the presence of a histone deacetylase inhibitors trichostatin A, indicating that p16 was inactivated by hypoacetylation. Findings obtained this study will add new insights and lead to the better understanding of the disease pathogenesis and future development of epigenetic therapeutic strategies.
... The lymphoma/leukemia cell lines included CLBL-1 (B-cell lymphoma cell line), GL-1 (B-cell leukemia), CL-1 (T-cell lymphoma) and CLB70 (B-cell chronic leukemia). CLBL-1 cells were obtained from Barbara C. Ruetgen, Institute of Immunology, Department of Pathobiology of the University of Veterinary Medicine in Vienna [13], GL-1 and CL-1 cells were provided by Yasuhito Fujino and Hajime Tsujimoto from the University of Tokyo, Department of Veterinary Internal Medicine [14,15]. CLB70 line was established in our laboratory [16]. ...
Four stereoisomers of δ-iodo-γ-lactones with p-isopropylphenyl substituent at β-position: cis-(4R,5R,6S)-1, cis-(4S,5S,6R)-2, trans-(4R,5S,6R)-3, trans-(4S,5R,6S)-4 with proved antiproliferative activity were subjected to in vitro tests for a better understanding of their anticancer activity. The subject of our interest was a possible relationship between a configuration of chiral centers of the studied lactones and their anticancer potency against a panel of canine cell lines representing hematopoietic (CLBL-1, GL-1, CL-1, CLB70) and mammary gland cancers (P114, CMT-U27, CMT-U309). To determine the anticancer activity of the tested compounds, cell viability and cell metabolic activity were checked using propidium iodide staining and the MTT test. To determine whether the studied compounds cause necrotic or apoptotic cell death, two assays for apoptosis evaluation were performed, annexin V staining and detection of caspase 3/7 activation. Simultaneously, the effects of the compounds on the cell cycle were also examined. The conducted research confirmed the anticancer potential of the tested lactones against canine cancers. The investigated isomers exerted higher activity against canine lymphoma/leukemia cell lines than against mammary tumors, whereas the configuration of stereogenic centers of the examined compounds affected their activity. It has been shown that stereoisomers with 4S configuration (2,4) were more active, and the most potent one was the cis-(4S,5S,6R)-2 isomer. The investigated lactones seemed to initiate the process of apoptosis rather than acting as typical cytostatic agents, as cell death via apoptosis, and no increase in G2-M population in the cell cycle analysis were observed. The presented study demonstrated that all four stereoisomers of δ-iodo-γ-lactones with p-isopropylphenyl substituent at β-position induced apoptosis via a mitochondrial-mediated, caspase-dependent pathway.
... The canine lymphoma cell lines 17-71 [23,24] and GL-1 [25] and PBMC obtained from lymphoma dogs were used for this assay. ...
The nitrosourea drug lomustine is used clinically for treating a wide variety of malignancies, most commonly brain tumors and lymphoma. Lomustine undergoes hydrolysis in vivo to form isomeric metabolites, primarily trans-4-hydroxylomustine (trans-4) and cis-4-hydroxylomustine (cis-4) in various animal species including humans. Despite its widespread usage to treat canine lymphoma, the metabolism of lomustine has not been studied in dogs. It is reported that 4'-hydroxylation products of lomustine (trans-4 and cis-4) have enhanced alkylating activity and reduced toxic effects relative to lomustine, resulting in a better therapeutic index of each of the metabolites relative to the parent compound. Our results show that the metabolic profile of lomustine in dogs is similar to that in humans with trans-4 being the major metabolite and cis-4 as the minor metabolite. Comparative cytotoxicity studies of lomustine and its trans-4 and cis-4 metabolites in canine lymphoma cell lines 17–71 and GL-1 show that there is no difference in the cytotoxicity of the three compounds. In addition, a concentration and time-dependent cell killing was seen in both of these cell lines. Also, primary canine cells like peripheral blood mononuclear cells (PBMC) from lymphoma dogs did not show any sensitivity towards lomustine and its metabolites.
... All three cell lines were obtained from American Type Culture Collection e ATCC, Rockville, MD, USA and kindly provided from the collection of the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland. The non-adherent canine cell lines CL-1 (T-lymphoblastoid cell line), GL-1 (B cell lymphoma cell line), both obtained from Yasuhito Fujino and Hajime Tsujimoto from the University of Tokyo, Department of Veterinary Internal Medicine [21,22] and D-17 (canine osteosarcoma, obtained from American Type Culture Collection e ATCC, Rockville, MD, USA) were also used. ...
New butyltin complexes with 2-sulfobenzoic acid: [Sn(C4H9)2{O3SC6H4COO-2}(H2O)]·(C2H5OH) (DBTsbz), [Sn(C4H9)3{O3SC6H4COOH-2}] (TBTsbz) and [Sn2(C4H9)6{μ-O3SC6H4COO-2}] (DTBTsbz) are very effective cytotoxic agents against tumor cells. The molecular interaction of these complexes with lipid membranes and DNA has been investigated.
The IR spectra and changes of ¹H, ¹³C chemical shifts suggest that SO3 and COO groups of 2-sulfobenzoato ligand interact with O atom of glycerin fragment of DPPC. Moreover, the compounds form Sn–OP bonds with phosphate groups of DPPC, which was shown by the lower frequency shift of the νs(PO2⁻) and νas(PO2⁻) band, by change of ³¹P NMR signals and by DFT calculation. Another possibility is the interaction of the phosphate group of DPPC owing to formation of hydrogen bond O–H…O–P between water molecule coordinated to Sn and oxygen atom from the phosphate group.
Using TCSPC-FCS we characterized DNA supramolecular assemblies' formation upon increasing TBTsbz, DTBTsbz and DBTsbz concentration. Diffusion time, lifetime and particle number changes are altered systematically with increasing Ccomp/CDNAbp ratio in following effectiveness order DBTsbz > TBTsbz > DTBTsbz. From those parameters we can conclude that all these compounds lead to a change of DNA winding, strand but not to DNA compaction.
Investigated compounds show very high cytotoxic activity against cancer cell lines. All compounds exhibit efficient in vitro antitumor activity toward Jurkat (T-cell leukemia), CL-1 (T-lymphoblastoid cell line), GL-1 (B cell lymphoma cell line) and D-17 (canine osteosarcoma). The DBTsbz is more effective then carboplatin against canine osteosarcoma.
... To date, twelve still available canine cell lines have been described and characterized according to their morphology, immunophenotype, pattern of TCR genes status and ability to induce tumours in mice (Kisseberth et al., 2007;Momoi et al., 1997;Nakaichi et al., 1996;Rutgen et al., 2010;Steplewski et al., 1987;Suter et al., 2005;Umeki et al., 2013;Yamazaki et al., 2008). Importantly, cell phenotype may change either from the primary material to the establishment of the cell line (Rutgen et al., 2010) or during in vitro maintenance (Umeki et al., 2013), leading to differences in the pattern of expression of specific markers. ...
Dogs with lymphoma are established as good model for human non-Hodgkin lymphoma studies. Canine cell lines derived from lymphomas may be valuable tools for testing new therapeutic drugs. In this context, we established a canine T-cell line, PER-VAS, from a primary aggressive T-cell lymphoma with large granular morphology. Flow cytometric analysis revealed a stable immunophenotype: PER-VAS cells were positively labelled for CD5, CD45, MHC II and TLR3, and were negative for CD3, CD4 and CD8 expression. Although unstable along the culture process, IL-17 and MMP12 proteins were detectable as late as at passages 280 and 325i.e. respectively 24 and 29 months post isolation. At passage 325, PER-VAS cells maintained the expression of IL-17, CD3, CD56, IFNγ and TNFα mRNAs as shown by RT-PCR analysis. Stable rearrangement of the TCRγ gene has been evidenced by PCR. PER-VAS cells have a high proliferation index with a doubling time of 16.5h and were tumorigenic in Nude mice. Compared to the canine cell lines already reported, PER-VAS cells display an original expression pattern, close to NKT cells, which makes them valuable tools for in vitro comparative research on lymphomas.
... Although multiple canine lymphoid cell lines have been reported including a T-cell leukemia cell line (CL-1) [132] and B-cell lymphoma cell line (CLBL-1) [133], most studies have been performed in the canine B-cell leukemia cell line GL-1 [134]. In two separate studies DR to doxorubicin was established through exposure of GL-1 cells to increasing concentrations of doxorubicin [62,86]. ...
Drug resistance (DR) is the major limiting factor in the successful treatment of systemic neoplasia with cytotoxic chemotherapy. DR can be either intrinsic or acquired, and although the development and clinical implications are different, the underlying mechanisms are likely to be similar. Most causes for DR are pharmacodynamic in nature, result from adaptations within the tumor cell and include reduced drug uptake, increased drug efflux, changes in drug metabolism or drug target, increased capacity to repair drugǦinduced DNA damage or increased resistance to apoptosis. The role of active drug efflux transporters, and those of the ABCͲtransporter family in particular, have been studied extensively in human oncology and to a lesser extent in veterinary medicine. Methods reported to assess ABCͲtransporter status include detection of the actual protein (Western blot, immunohistochemistry), mRNA or ABCͲtransporter function. The three major ABCͲtransporters associated with DR in human oncology are ABCB1 or PͲgp, ABCC1 or MRP1, and ABCG2 or BCRP, and have been demonstrated in canine cell lines, healthy dogs and dogs with cancer. Although this supports a causative role for these ABCͲtransporters in DR cytotoxic agents in the dog, the relative contribution to the clinical phenotype of DR in canine cancer remains an area of debate and requires further prospective studies.
... Cells: Seven canine lymphoma cell lines; the CLBL-1 [24], GL-1 [20] and 17-71 [23] B cell lines; the CL-1 [19], CLC [29], CLGL90 [28] and Nody-1 [29] T cell lines; and a Raji human lymphoma cell line were used. CLBL-1, GL-1, CL-1, CLC, Nody-1 and Raji cell lines were cultured in RPMI1640 medium, and CLGL90 and 17-71 cell lines were cultured in Dulbecco's Modified Eagle medium, which were supplemented 10% fetal bovine serum, penicillin (100 U/ ml), streptomycin (100 µg/ml) and 2-mercaptoethanol (55 µM). ...
Hypoxic conditions in various cancers are believed to relate with their
malignancy, and hypoxia inducible factor-1α (HIF-1α) has been shown to be a major
regulator of the response to low oxygen. In this study, we examined HIF-1α expression in
canine lymphoma using cell lines and clinical samples and found that these cells expressed
HIF-1α. Moreover, the HIF-1α inhibitors, echinomycin, YC-1 and 2-methoxyestradiol,
suppressed the proliferation of canine lymphoma cell lines. In a xenograft model using
NOD/scid mice, echinomycin treatment resulted in a dose-dependent regression of the tumor.
Our results suggest that HIF-1α contributes to the proliferation and/or survival of canine
lymphoma cells. Therefore, HIF-1α inhibitors may be potential agents to treat canine
lymphoma.
... The O 6 hydroxyethyldeoxyguanosine and N 7 hydroxyethylguanine adducts were measured in canine lymphoid cell lines 17-71 and GL-1 [26,27] and primary canine hepatocytes (isolated from six dogs that were euthanized at the Champaign county animal control facility) after exposure to lomustine, trans-4 and cis-4 by the LC/MS/MS method as mentioned below. The cell lines were grown and maintained at 37 °C in 5% CO2, and cultures were passaged as necessary to maintain high cell viability (above 90%) in culture media Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. ...
DNA Alkylation is thought to be the reason for the efficacy of lomustine while carbamylation has been implicated as the cause for the side effects seen with lomustine treatment such as hepatotoxicity. In the alkylation study we show that lomustine and its metabolites form similar levels of the DNA adducts N7 hydroxyethylguanine and O6 hydroxyethyldeoxyguanosine. In terms of carbamylation, lomustine showed greater extent of carbamylation in the canine hepatocytes and lymphoma cell lines. The DNA repair enzyme O6 methylguanine DNA methyltransferase (MGMT) causes resistance of tumor cells to bifunctional nitrosourea, like lomustine. There is no data available regarding MGMT expression/activity in canine cells or tissues. Our study shows that there is low MGMT activity in the canine lymphoid cell line 17–71 while the GL-1 cells did not show any detectable enzyme activity or mRNA expression. The MGMT enzyme activity measured in canine hepatocytes is about 250–350 fmol/mg protein as compared to about 90 fmol/mg protein in 17–71 cells. We also show that MGMT mRNA expression in 17–71 cells and canine hepatocytes positively correlates with its enzyme activity in these cells.
... Cell culture: Canine neoplastic lymphoid cell lines of GL-1 [15], UL-1 [20], CLBL-1 [17], CL-1 [14], Nody-1 [9] and Ema [9] were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, U.S.A.) supplemented with penicillin-streptomycin and 10% fetal bovine serum. ...
Lymphoid malignancies, such as leukemia, and many types of lymphoma are common and severe disorders in dogs. Since shortening remission duration caused by resistance to chemotherapy often becomes clinically critical problems, development of novel and effective therapy should be required. The present study investigated the status of NF-kappa B and effect of its inhibitor, bortezomib, in six canine neoplastic lymphoid cell lines. NF-kappa B p65 and p50 were detected in the nuclear fraction of GL-1, CLBL-1 and CL-1, suggesting that NF-kappa B was constitutively activated in the cells. NE-kappa B p65 was detected in the cytoplasmic fraction of UL-1 and Ema. After incubation with bortezomib, NF-kappa B p50 and p65 became undetectable in the nuclear fraction of GL-1, CLBL-1 and CL-1, and CLBL-1, respectively, and p65 was clearly degraded in the cytoplasmic fraction of CLBL-1 and CL-1. Bortezomib inhibited the proliferation of all cell lines except Nody-1 in a concentration-dependent manner. The results indicated that constitutive activation of NF-kappa B could contribute to the proliferation of canine neoplastic lymphoid cells, and bortezomib would have suppressive effects on the NF-kappa B activation and the proliferation of neoplastic lymphoid cells in dogs.
This study focuses on understanding the structural and molecular changes in lipid membranes under the influence of six halogenated flavonoid derivatives differing in the number and position of substitution of chlorine and bromine atoms (D1–D6). Utilizing various analytical techniques, including fluorometric methods, dynamic light scattering (DLS), attenuated Fourier transform infrared spectroscopy (ATR- FTIR), and FT-Raman spectroscopy, the research aims to elucidate the mechanisms underlying the interaction of flavonoids with cell membranes. Additionally, the study includes in silico analyses to explore the physicochemical properties of these compounds and their potential pharmaceutical applications, along with toxicity studies to assess their effects on cancer, normal, and red blood cells. Our study showed the ability of halogenated derivatives to interact mostly with the outer part of the membrane, especially in the lipid heads region however, some of them were able to penetrate deeper into the membrane and affect the fluidity of hydrocarbon chains. The potential to reduce cancer cell viability, the lack of toxicity towards erythrocytes, and the favourable physicochemical and pharmacokinetic properties suggest these halogenated flavonoids potential candidates for exploring their potential for medical use.
Canine lymphoma/leukemia cell lines with p16 protein expressions: high (17-71 and GL-1) and low (CLBL-1, CLC, Nody-1, and UL-1) were treated in vitro with cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, palbociclib or abemaciclib. Cell proliferation decreased as a result, with higher IC50 levels observed in the high p16 (17-71 and GL-1) and one low p16 (UL-1) cell lines compared with the low p16 cells (CLBL-1, CLC, and Nody-1). As expected, palbociclib and abemaciclib treatment reduced pRb phosphorylation in a dose-dependent manner, especially in cells with low p16. These results suggest that CDK4/6 inhibitors have potential as new chemotherapeutic agents for canine lymphoma and high p16 protein expression may be used as a biomarker for resistance to CDK4/6 inhibitor therapy.
Canine lymphoma is the most common haematological malignancy in dogs and is typically treated with multidrug chemotherapy. Most cases are at risk of relapse after several courses of chemotherapy and the oncogenic mechanism remains unknown. This study was aimed at identifying genes expressed in canine lymphoma by cDNA microarray. We found elevated expression of Dishevelled, EGL-10 and pleckstrin (DEP) domain-containing 1B (DEPDC1B) in canine lymphoma cells compared with cells and tissues from healthy dogs. Canine DEPDC1B protein was detected in 13 of 41 lymphoma specimens by immunohistochemistry, but was not detected in lymph nodes from normal dogs. Immunoreactive DEPDC1B protein was also detected in several other types of canine tumour. This is the first report documenting the association of DEPDC1B with canine cancer and the results suggest that DEPDC1B might serve as a potential marker or therapeutic target for canine malignancies.
We established a canine NK‐type cell line called CNK‐89 derived from a dog with NK cell neoplasia. Immunophenotyping analysis showed positive staining for CD5, CD8, CD45, CD56, CD79a and NKp46, while negative for CD3, CD4, CD14, CD20, CD21, CD34, Thy1, IgG, IgM and MHCII. PCR analysis revealed the presence of CD56, NKG2D, NKp30, NKp44, NKp46 and perforin, but the absence of CD16, Ly49 and granzyme B mRNA. Treating CNK‐89 cells with IL‐2 did not change the expression of activating receptors, TNFα and IFNγ secretion and cytotoxic activity, however, treatment with IL‐12 alone or in combinations with IL‐15, IL‐18 and IL‐21 caused an increase in granzyme B and CD16 mRNA, IFNγ secretion and cytotoxic properties of the CNK‐89 cell line.
This article is protected by copyright. All rights reserved.
Background:
Malignant lymphoma is the most common hematopoietic malignancy in dogs, and relapse is frequently seen despite aggressive initial treatment. In order for the treatment of these recurrent lymphomas in dogs to be effective, it is important to choose a personalized and sensitive anticancer agent. To provide a reliable tool for drug development and for personalized cancer therapy, it is critical to maintain key characteristics of the original tumor.
Objectives:
In this study, we established a model of hybrid tumor/stromal spheroids and investigated the association between canine lymphoma cell line (GL-1) and canine lymph node (LN)-derived stromal cells (SCs).
Methods:
A hybrid spheroid model consisting of GL-1 cells and LN-derived SC was created using ultra low attachment plate. The relationship between SCs and tumor cells (TCs) was investigated using a coculture system.
Results:
TCs cocultured with SCs were found to have significantly upregulated multidrug resistance genes, such as P-qp, MRP1, and BCRP, compared with TC monocultures. Additionally, it was revealed that coculture with SCs reduced doxorubicin-induced apoptosis and G2/M cell cycle arrest of GL-1 cells.
Conclusions:
SCs upregulated multidrug resistance genes in TCs and influenced apoptosis and the cell cycle of TCs in the presence of anticancer drugs. This study revealed that understanding the interaction between the tumor microenvironment and TCs is essential in designing experimental approaches to personalized medicine and to predict the effect of drugs.
Background
Ubiquitin‐specific protease 7 (USP7) belongs to the group of deubiquitinating enzymes (DUBs), which remove ubiquitin which controls various cellular processes such as chromosome segregation, DNA repair, gene expression, protein localization, kinase activity, protein degradation, cell cycle progression, and apoptosis. It is critical for several important functions in the cell, and therefore dysregulation of USP7 can contribute to tumorigenesis.
Objectives
Alterations in the USP7 protein have been identified in various malignancies of humans. Our aim was to examine whether USP7 could be a potential therapeutic target in hematopoietic cancers of dogs.
Methods
The expression level of USP7 in lymphocytes from healthy dogs and canine lymphoma cells was determined, and the effect of USP7 inhibition on the vital functions of canine cancer cells was examined.
Results
We showed that USP7 was overexpressed in lymphomas in dogs. The USP7 inhibitor P5091 has selective cytotoxic activity in canine lymphoma and leukemia cell lines. Our results indicate that inhibition of USP7 leads to a disruption of cell cycle progression, and triggers DNA damage and apoptosis. The observed proapoptotic effect of the USP7 inhibitor most likely is not dependent on the p53 pathway.
Conclusions and Clinical Importance
Our results suggest that USP7 could be explored as a potential therapeutic target in dogs with lymphoma. The effectiveness of USP7 inhibition in malignant cells is predicted to be independent of their p53 status.
Traditional laboratory model organisms are indispensable for cancer research and have provided insight into numerous mechanisms that contribute to cancer development and progression in humans. However, these models do have some limitations, most notably related to successful drug translation, because traditional model organisms are often short-lived, small-bodied, genetically homogeneous, often immunocompromised, are not exposed to natural environments shared with humans, and usually do not develop cancer spontaneously. We propose that assimilating information from a variety of long-lived, large, genetically diverse, and immunocompetent species that live in natural environments and do develop cancer spontaneously (or do not develop cancer at all) will lead to a more comprehensive understanding of human cancers. These non-traditional model organisms can also serve as sentinels for environmental risk factors that contribute to human cancers. Ultimately, expanding the range of animal models that can be used to study cancer will lead to improved insights into cancer development, progression and metastasis, tumor microenvironment, as well as improved therapies and diagnostics, and will consequently reduce the negative impacts of the wide variety of cancers afflicting humans overall.
Chalcones are interesting candidates for anti-cancer drugs due to the ease of their synthesis and their extensive biological activity. The study presents antitumor activity of newly synthesized chalcone analogues with a methoxy group on a panel of canine lymphoma and leukemia cell lines. The antiproliferative effect of the 2-hydroxychalcone and its methoxylated derivatives was evaluated in MTT assay after 48 h of treatment in different concentrations. The proapoptotic activity was studied by cytometric analysis of cells stained with Annexin V/FITC and propidium iodide and by measure caspases 3/7 and 8 activation. The DNA damage was evaluated by Western blot analysis of phosphorylated histone H2AX. The new compounds had selective antiproliferative activity against the studied cell lines, the most effective were the 2-hydroxy-2 ,5-dimethoxychalcone and 2-hydroxy-4 ,6-dimethoxychalcone. 2-Hydroxychalcone and the two most active derivatives induced apoptosis and caspases participation, but some percentage of necrotic cells was also observed. Comparing phosphatidylserine externalization after treatment with the different compounds it was noted that the addition of two methoxy groups increased the proapoptotic potential. The most active compounds triggered DNA damage even in the cell lines resistant to chalcone-induced apoptosis. The results confirmed that the analogues could have anticancer potential in the treatment of canine lymphoma or leukemia.
Recombinant canine interferon-γ (rc-IFNγ; InterdogⓇ) was exclusively approved as a therapeutic for canine atopic dermatitis. However, it has been used off-label for the treatment of canine cancer. We examined the inhibitory effect of rc-IFNγ on the growth of canine tumor cell lines and analyzed its mechanism of action. Three (CTB-p, CTB-m, and CNM-m) out of seven mammary gland tumor cell lines and two (VIMC and CoMS) out of four mast cell tumor cell lines showed remarkable growth inhibition after treatment with rc-IFNγ. However, one (CLBL-1) out of nine lymphoma cell lines showed a significant amount of cell death. Using CTB-p and CTB-m cell lines, we showed that STAT1 was essential for inducing the growth inhibitory effect of rc-IFNγ. Although rc-IFNγ induced G1 growth arrest in CTB-p cell line, treatment with rc-IFNγ did not alter the expression of cell cycle regulatory proteins. In this study, we observed direct cytotoxicity or cytostatic effects of rc-IFNγ in canine tumor cell lines. However, the detailed mechanisms responsible for these effects need to be elucidated in the future.
Lymphoma is the most common hematological cancer in dogs. Canine diffuse large B cell lymphoma shows a relatively good response to treatment with multi-agent cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy; however, the 2-year survival rate is as low as 20%. For human B cell type lymphoma, the anti-CD20 chimeric antibody, rituximab, was developed two decades ago. The combination of rituximab and CHOP chemotherapy was highly successful in improving patient prognosis. However, no anti-canine CD20 antibody is available for the treatment of canine lymphoma. During this study, a rat anti-canine CD20 monoclonal antibody was established. We also generated a rat-canine chimeric antibody against canine CD20 designed for clinical application. This chimeric antibody (4E1-7-B) showed in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against the canine B cell lymphoma cell line CLBL-1. Moreover, to obtain stronger ADCC activity, a defucosylated 4E1-7-B antibody (4E1-7-B_f) was also generated, and it showed tenfold stronger ADCC activity compared with 4E1-7-B. 4E1-7-B_f as well as 4E1-7-B suppressed the growth of CLBL-1 tumors in an immunodeficient xenotransplant mouse model. Finally, a single administration of 4E1-7-B_f induced considerable peripheral B cell depletion in healthy beagles. Thus, 4E1-7-B_f is a good antibody drug candidate for canine B cell type lymphoma.
Exosomes are small extracellular vesicles released from almost all cell types, which play roles in cell-cell communication. Recent studies have suggested that microenvironmental crosstalk mediated by exosomes is an important factor in the escape of tumour cells from the anti-tumour immune system in human haematopoietic malignancies. Here, we conducted comprehensive analysis of the miRNA and protein profiles within the exosomes released from four canine lymphoid tumour cell lines as a model of human lymphoid tumours. The results showed that the miRNAs and proteins abundantly contained in exosomes were similar among the four cell lines. However, the profiles of miRNA within exosomes differed among the cell lines and reflected the expression pattern of miRNAs of the parent cells. In the comparison of the amounts of miRNAs and proteins among the cell lines, those of three miRNAs (miR-151, miR-8908a-3p, and miR-486) and CD82 protein differed between exosomes derived from vincristine-sensitive and resistant cell lines. Further investigations are needed to elucidate the biological functions of the exosomal contents in the microenvironmental crosstalk of lymphoid tumours.
In dogs as well as humans, lymphoma is one of the most common hematopoietic malignancies. Furthermore, due to its characteristics, canine lymphoma is recognized as a clinically relevant in vivo model to study the corresponding human disease. Immortalized cell lines are widely used as in vitro models to evaluate novel therapeutic agents and characterize their molecular mechanisms. However, it is known that long-term cultivation leads to clonal selection, genetic instability, and loss of the initial heterogenic character, limiting the usefulness of cell lines as preclinical models. Herein, we present a systematic characterization and comparison of the transcriptomic landscape of canine primary B- and T-cell lymphomas, five lymphoid cell lines (CLBL-1, CLBL-1M, GL-1, CL-1, and OSW) and four non-neoplastic control samples. We found that lymphomas and cell lines exhibit a common "differentiation and proliferation signature". However, our analysis also showed that, independently of the cell of origin, the transcriptional signatures of lymphomas are more similar to each other than they are to those of cell lines. In particular, we observed that not all common therapeutic targets are similarly expressed between lymphomas and lymphoid cell lines, and provide evidence that different lymphoid cell-lines should be used to model distinct aspects of lymphoma dysregulation.
For many years, studies focused on developing new natural or synthetic compounds with antineoplastic activity have attracted the attention of researchers. An interesting group of such compounds seem to be those with both lactone moiety and an aromatic ring which, in addition to antimicrobial or antiviral activity, also exhibit antitumor properties. The study shows antitumor activity of two enantiomeric trans isomers of 5-(1-iodoethyl)-4-(2',5'-dimethylphenyl)dihydrofuran-2-one. Our aim was to determine their antitumor activity manifested as an ability to induce apoptosis in selected canine cancer cell lines as well as to evaluate differences in their strength depending on the configuration of their stereogenic centers. The enantiomers (+)-(4R,5S,6R)-1 and (-)-(4S,5R,6S)-2 were found to induce classical caspase-dependent apoptosis through downregulation of the expression of anti-apoptotic proteins Bcl-xL and Bcl-2. Although the mechanism of apoptosis induction was the same for both enantiomers, they differed in their strength, as stronger antineoplastic activity in vitro was exhibited by isomer (+)-(4R,5S,6R)-1.
Six enantiomeric pairs of β-aryl-δ-iodo-γ-lactones 8a–c, 9a–c derived from cuminaldehyde, 2,5-dimethylbenzaldehyde and piperonal were synthesized with high enantiomeric purities (ee 93–99%) from enantiomerically enriched allyl alcohols 3a–c. The key step in the synthesis of lactones 8a and 9a was the kinetic resolution of racemic (E)-4-(4′-isopropylphenyl)but-3-en-2-ol 3a by a lipase-catalysed transesterification. Among the five tested enzymes, the most effective and enantioselective was lipase B from Candida antarctica and after 2 h (−)-(S)-alcohol 3a and (+)-(R)-propionate 5 were obtained with ee’s ⩾99%. The transfer of chirality from alcohols (S)-3a–c and (R)-3a–c to γ,δ-unsaturated esters (S)-6a–c and (R)-6a–c via a stereoselective Johnson–Claisen rearrangement followed by hydrolysis and iodolactonization afforded the final lactones 8a–c and 9a–c. The configurations of their stereogenic centres were assigned based on crystallographic analysis and/or the iodolactonization mechanism. In 42 of 48 tests, the synthesized lactones showed antiproliferative activity against four selected cancer lines (Jurkat, D17, GL-1, CLBL-1). The trans-stereoisomers were more active than the cis-stereoisomers and the highest activity was found for lactone (−)-trans-(4S,5R,6S)-9c with a 1,3-benzodioxole substituent and both enantiomers of the trans-lactone with a 2,5-dimethylphenyl substituent: (+)-9b and (−)-9b. Among the trans-lactones, those with a (4S,5R,6S)-configuration exhibited higher activity than their enantiomers and the most significant difference was observed for the enantiomers of the trans-lactone with a 1,3-benzodioxole substituent 9c (IC50 = 5.29 and 5.08 vs 36.47 and 33.77 for Jurkat and GL-1 cancer lines respectively).
The use of animals for the preclinical study of cancer therapeutics has a long history, and important information has been gained regarding new and innovative therapies. Most of this work has been performed on inbred rodent models and laboratory-derived canine populations. Working with inbred populations in controlled, artificial laboratory environments raises some degree of concern over the applicability of information as it relates to naturally occurring tumors in people. Many of these concerns may be allayed through the study of naturally occurring tumors in our companion animal population, (i.e., dog and cat pet population). Companion animals with naturally occurring tumors, although presently underutilized, have and should continue to provide an excellent opportunity to investigate many aspects of malignancy from etiology to treatment.
The DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT) causes resistance to nitrosoureas in various human cancers. In this study, we analyzed the correlation between canine lymphomas and MGMT in vitro. Two of five canine lymphoma cell lines required higher concentrations of lomustine to inhibit cell growth by 50%, but their sensitivity to the drug increased when they were cultured with an MGMT inhibitor. Fluorometric oligonucleotide assay and real-time polymerase chain reaction of these cell lines revealed MGMT activity and high MGMT mRNA expression, respectively. We analyzed the methylation status of the CpG islands of the canine MGMT gene by the bisulfite-sequencing method. Unlike human cells, the canine lymphoma cell lines did not show significant correlation between methylation status and MGMT suppression levels. Our results suggest that in canine lymphoma MGMT activity may influence sensitivity to nitrosoureas; thus, inhibition of MGMT activity would benefit nitrosourea-resistant patients. Additional studies are necessary to elucidate the mechanism of regulation of MGMT expression.
Polymerase chain reaction (PCR) amplification to detect immunoglobulin heavy chain (IgH) and T cell receptor γ-chain (TCRγ) gene rearrangements has recently become widely used as part of the diagnostic strategy for lymphoid tumors in dogs. In this study, we constructed a multicolor GeneScan analytical system to improve the sensitivity and resolution of the clonality analysis of antigen receptor gene rearrangements in dogs. We used 7 reactions per sample, with 2 PCR conditions, to amplify IgH/TCRγ and control genes. By using multicolor-labeled primers, these 7 PCR products could be combined into 3 tubes before capillary electrophoresis. Clonal rearrangement of the IgH/TCRγ genes was detected in 93.3% of dogs with multicentric lymphoma and 84.6% of dogs with gastrointestinal lymphoma. Detection sensitivity of the clonally expanded cells in the background of normal peripheral blood mononuclear cells was 1-10%. The multicolor GeneScan analytical system developed here may prove to be helpful for the diagnosis of lymphoid tumors in dogs.
Copyright © 2015 Elsevier B.V. All rights reserved.
THYMUS-DERIVED (T) lymphocytes play a critical part in the induction of B lymphocytes to antibody production1, especially for conversion of IgM to IgG response2. In humans, the presence of T lymphocytes is also essential for the induction of IgM or IgG production by pokeweed mitogen (PWM) stimulated B lymphocytes3,4. In many experiments it has been shown that antigen-specific5 or nonspecific6-8 soluble factors from T cells, together with antigen or other inducers, for example, anti-immunoglobulin antibody, acting on B-cell surface receptors9, can also provide the stimulus for Ig production in B cells. However, the chemical nature of a B-cell acceptor for the T-effector molecule, the biochemical events responsible for the differentiation of B cells to Ig-producing cells, and the mechanism of the switch of transcription from mu chain to gamma chain genes under the influence of T cells remain largely unknown. Heterogeneity of B-cell population in spleen, lymph node and blood has hindered the molecular analysis of immune phenomena. In this situation, B lymphoblastoid cell lines may be useful models for such analysis, since they may be arrested at certain phases of their differentiative history and if influenced by T cells or T-cell factors might permit incisive analysis of induction and switching of gene action at the molecular level. We report here the induction of IgG production or enhancement of IgM secretion in several human B lymphoblastoid cell lines with the help of normal T cells.
Reports of negative tartrate-resistant acid phosphatase reactions in a few cases of leukemic reticuloendotheliosis prompted the authors to re-evaluate the diagnostic specificity of this test. As a result, they modified the test by (1) incorporating a dual-control system for excluding a false-negative test due to technical errors, and (2) instituting an objective grading system for assuring consistent interpretation of the test on blood smears. When these modifications were applied to materials of patients suspected to have leukemic reticuloendotheliosis, there was an excellent, although not specific, correlation between the positive test and the diagnosis of leukemic reticuloendotheliosis. Tartrate-resistant acid phosphatase reactions were positive, intermediate, and negative for 76, 21, and 3% of 29 patients who had leukemic reticuloendotheliosis, whereas the figures were 3, 32, and 65%, respectively, for 37 patients who had chronic lymphocytic leukemia and other hematologic disorders.
Human interferon-alpha (IFN-alpha) has been shown to be effective in the treatment of Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) in the benign stable phase. The present study indicates that IFN-alpha may have a suppressive effect on Ph1-positive clones not only in the early stable phase but also in the accelerated phase with additional chromosomal abnormalities in some patients. In this study, in addition to 5 benign-phase patients, 3 patients with CML in the accelerated phase who had additional chromosomal abnormalities were treated with IFN-alpha. The presence of the Ph1-positive clone was estimated by chromosomal analysis and by Southern analysis at the DNA level using a 3' breakpoint cluster region (bcr) probe. Hematological remission and the suppression of proliferation of Ph1-positive clone to various extents were achieved by IFN-alpha treatment in 2 benign-phase patients and 3 patients with additional chromosomal abnormalities. Interestingly, in one of the latter three patients, Ph1-positive clones with or without additional chromosomal abnormalities were completely suppressed judging from chromosomal analysis and from the disappearance of bcr gene rearrangements.
Cells acquired from lymph node biopsy specimens obtained from 58 dogs scheduled to undergo chemotherapy for lymphoma were immunophenotyped, using a microlymphocytotoxicity (MLCT) assay comprising a panel of well-characterized monoclonal antibodies (MAB) specific for canine cell surface antigens. Cells from 54 of the dogs concurrently were tested cytofluorometrically, using surface immunoglobulin (SIg) as a marker for B cells and the MAB DT2 specific for peripheral blood T cells. The MLCT results indicated frequent coexpression of antigens identified by DT2 antibody and, to a lesser extent, by 1A1 antibody on SIg-positive cells, suggesting that these antigens may be associated with other types of less-differentiated lymphoid cells, in addition to being associated with mature T cells. Class-II major histocompatibility antigens, as recognized by MAB H81.98.71, HB10a, and H40.315.7, were detected on most SIg-positive cells, but generally were lacking on SIg-negative, DT2-negative cells. The MAB Wig4, reactive with canine monocytes, recognized relatively few cells (11 of 58). Response to chemotherapy was not correlated with reactivity to MAB DLy6 specific for resting lymphocytes or to MAB W3G10 specific for a polymorphic antigen associated with the canine major histocompatibility complex. The MLCT assay appears to be efficient, rapid, and inexpensive for immunophenotyping cells from lymphoma biopsy specimens.
BALB/c mice were immunized with canine T lymphocytes from lymph node and used in cell fusion experiments to derive monoclonal antibodies to the T lymphocyte surface. The cloned hybrid line F3-20-7 described in this paper was shown to be secreting antibodies directed at canine Thy-1 since (1) the tissue distribution of the antigen corresponded precisely to the tissue distribution expected of canine Thy-1 from previous studies and (2) the antigen purified from F3-20-7 monoclonal antibody affinity columns (a) had the same mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as pure rat Thy-1 and (b) could inhibit and assay shown previously to be directed at canine Thy-1 on the basis of cross-reactivity with rat Thy-1. Studies with the fluorescence-activated cell sorter showed that all thymocytes and lymph node T lymphocytes of the dog were Thy-1 positive, establishing that Thy-1 is a T cell marker in the dog, as in the mouse, although the presence of a small percentage of Thy-1-positive B cells could not be entirely excluded. In addition, 15% of the nucleated bone marrow cells were Thy-1 positive, which is similar to the finding in rat bone marrow. Localization studies on frozen sections of skin demonstrated large amounts of Thy-1 organized in a highly structured manner around the basal parts of the hair follicles. This covering of Thy-1 was lost as the follicles approached the surface. Epidermal cells were Thy-1 negative. In the kidney, Thy-1 was shown to be strongly associated with all of the basement membranes of the medulla. The kidney cortex, including glomeruli was essentially negative, except for the occasional staining of basement membranes, possibly of collecting ducts.
A murine monoclonal antibody (DT-2) is described which has been raised against canine thymocytes. DT-2 activates complement and is reactive with most canine thymocytes, peripheral blood T cells, thoracic duct lymphocytes, bronchoalveolar lymphocytes, and T chronic lymphatic leukemia cells. The antibody is nonreactive with surface immunoglobulin-positive blood lymphocytes, monocytes, bronchoalveolar macrophages, null acute lymphatic leukemia cells, granulocytes, erythrocytes, and platelets. In mixed lymphocyte cultures DT-2 and complement eliminate the responding but not the stimulating cell population. Mitogen stimulation (phytohemagglutinin, concanavalin A) experiments revealed that the responding cell affected by DT-2 is of lymphoid and not of monocyte/macrophage origin. All our data suggest that DT-2 is an antibody reacting specifically with canine T cells.
Tumor cells from 15 canine lymphoma/leukemia cases were examined for genetic rearrangements of immunoglobulin and T-cell receptor (TCR) genes in parallel with cell surface antigens. Ten of these 15 cases showed rearrangements of the immunoglobulin heavy chain (IgH) gene, while 4 cases displayed TCR beta-chain gene rearrangements on Southern blot analysis. All the cases with IgH gene rearrangements had multicentric form lymphoma, and 6 of the 10 cases were cell surface immunoglobulin-positive. On the other hand, the cases with TCR gene rearrangements included atypical lymphoma/leukemia cases, and 3 of the 4 cases were Thy-1 antigen-positive. Although the tumor cell lineage of a considerable number of lymphoma/leukemia cases could not be determined by phenotypic analysis, examination of the IgH and TCR gene rearrangements disclosed the lineages of 14 of 15 cases. Genetic analysis demonstrated that the tumor cells in most canine multicentric lymphomas were formed by clonal expansion of B-lymphocyte. These findings show that studies on the rearrangements of immunoglobulin and TCR genes are very useful for understanding the cellular origin, clonality and hierarchy of canine lymphoma/leukemia cells.