Molecular cloning of feline lung resistance-related protein (LRP) cDNA and its expression in a feline lymphoma cell line and adriamycin-resistant subline

Molecular cloning of feline lung resistance-related protein (LRP) was performed to evaluate the relationship between its expression level and drug resistance against chemotherapeutics. The nucleotide sequence of the coding region of feline LRP cDNA was found to be 2670-bp long and to show 84.2-92.6% homology to its human, mouse, and rat counterparts. The expression level of feline LRP mRNA was relatively high in lung, jejunum, and colon. An adriamycin (ADM)-resistant feline lymphoma subline, FT-1/ADM, showed a high level of MDR1 mRNA expression compared with parental FT-1 cells. However, no relationship was observed between the drug-resistant phenotype and the LRP mRNA expression level. Although no direct contribution of LRP to the development of the drug-resistant phenotype was observed, further investigation is advisable.     

Induction of Chemoresistance by Transfection of the Full‐Length Canine mdr1 Gene in Canine Cell Lines

Introduction:  In the chemotherapy for treatment of lymphoid tumors in dogs, myelosuppression is a frequently encountered dose‐limiting factor. One possible approach to overcome myelosuppression is induction of chemoresistance in hematopoietic stem cells through expression of the mdr1 gene. A full‐length canine mdr1 cDNA clone was isolated in our laboratory. The present study was carried out to assess whether it confers multidrug resistance in canine cell lines.
Materials and methods:  The full‐length canine mdr1 cDNA was inserted into an expression plasmid vector. A canine mammary tumor cell line (CIPP) and osteosarcoma cell line (OOS) were transfected with the canine mdr1 expression vector. Expression of P‐gp was examined by immunoblotting. Function of ATP‐dependent drug efflux was measured by flow cytometric analysis using Rhodamine 123. Sensitivity to chemotherapeutic drugs was shown by estimation of 50% inhibitory concentrations (IC₅₀) of vincristine or doxorubicin.
Results:  Immunoblotting of the transfected cells revealed a strong band of P‐gp detected by a monoclonal antibody directed to P‐gp. Rhodamine 123 efflux test showed an apparent drug efflux activity in the transfected cells. From the IC₅₀ of the chemotherapeutic agents, the transfected cells showed a remarkable increase (20 to 60‐fold) in the resistance to vincristine or doxorubicin.
Conclusion:  Transfection of canine mdr1 gene induced P‐gp expression and strong drug resistance in canine cell lines.     

Expression of Bcl-2 in feline lymphoma cell lines

BACKGROUND: The Bcl-2 gene is a member of the rapidly expanding Bcl-2 family of genes that regulate apoptosis. Bcl-2 has been shown to repress cell death triggered by a diverse array of stimuli, including chemotherapy and gamma irradiation. OBJECTIVE: The purpose of this study was to determine feline Bcl-2 expression level in feline lymphoma cells using an immunoblot assay with anti-human and anti-canine Bcl-2 monoclonal antibodies. METHODS: About 708 base pairs containing the coding sequence of the feline Bcl-2 gene were transformed into Escherichia coli. The recombinant Bcl-2 was used as a positive control for an immunoblot assay using mouse monoclonal antibodies against human and canine Bcl-2. An immunoblot assay using the monoclonal antibodies was carried out to determine the level of feline Bcl-2 expression in lymphoma and lymphocytic leukemia cell lines. RESULTS: The recombinant feline Bcl-2 protein produced in E. coli had a molecular weight of about 26 kDa and was detected by immunoblot assay by using anti-human Bcl-2 mouse monoclonal antibody. Feline Bcl-2 expression was high in lymphoma cell lines (FL-74-UDC-1 and FT-1) and low in the cell line from peripheral blood mononuclear cells from a healthy cat (FeTJ-1) but not low in freshly isolated peripheral blood mononuclear cells from a healthy cat. The anti-human Bcl-2 mouse monoclonal antibody was found to cross-react with feline Bcl-2. CONCLUSIONS: These results confirm the expression of Bcl-2 in T-cell lymphoma cell lines and indicate that it is suitable to detect feline Bcl-2 using an immunoblot assay. Pending further evaluation, Bcl-2 expression might be useful in the differential diagnosis of feline tumors.     

Induction of chemoresistance in a cultured canine cell line by retroviral transduction of the canine multidrug resistance 1 gene

OBJECTIVE: To induce chemoresistance in a normal canine cell line through the transduction of the canine multidrug resistance 1 gene (mdr1). SAMPLE POPULATION: Madin-Darby canine kidney (MDCK) epithelial cell line. PROCEDURES: The full-length canine mdr1 cDNA clone isolated in our laboratory was inserted into a Moloney murine leukemia virus-based vector to construct the retroviral vector, pLNC-cMDR1. After retroviral transduction of pLNC-cMDR1 into MDCK cells, the expression and function of the P-glycoprotein, a product of mdr1, were assessed by immunoblotting, measurement of rhodamine123 (Rh123) retention, and drug sensitivity assays. RESULTS: P-glycoprotein was strongly expressed in cells transduced with pLNC-cMDR1. This P-glycoprotein was fully functional, as demonstrated by the decreased Rh123 retention and the increased resistance to chemotherapeutic drugs. Measured as 50% inhibitory concentrations, resistance increased 59 times to vincristine and 25 times to doxorubicin in MDCK cells after transduction of pLNC-cMDR1. CONCLUSIONS AND CLINICAL RELEVANCE: Transduction of canine mdr1 is an effective method for inducing chemoresistance in normal canine cells. This system may be applicable to the induction of drug resistance in hematopoietic cells.     

Canine mdr1 gene mutation in Japan

Frequency of the 4-bp deletion mutant in canine mdr1 gene was examined in 193 dogs of eight breeds in Japan. The mutant allele was found in Collies, Australian Shepherds, and Shetland Sheepdogs, where its respective frequencies were 58.3%, 33.3%, and 1.2%. The MDR1 protein was detected on peripheral blood mononuclear cells (PBMC) from a MDR1/MDR1 dog, but not on PBMC from a mdr1-1Delta/mdr1-1Delta Collie. Rhodamine 123 was extruded from MDR1/MDR1 lymphocytes. That excretion was inhibited by a MDR1 inhibitor, verapamil. On the other hand, Rh123 excretion was not observed from lymphocytes derived from a mdr1-1Delta/mdr1-1Delta Collie. These results indicated that the mutant mdr1 allele also existed in Collie-breed dogs in Japan at high rates and that mdr1-1Delta /mdr1-1Delta dogs have no functional MDR1.     

Multi-Drug Resistance Genes in the Management of Neoplastic Disease

P-gp can function as an ATP-dependent cytotoxic drug-efflux pump. In normal tissues, protein expression is localized to cell surfaces that face excretory lumina; hence, P-gp may function as a toxic-waste disposal system. Tumors that are derived from these tissues can be high expressors of P-gp, and these tumors tend to display intrinsic chemoresistance. Other non-expressing tumors can become P-gp positive after treatment or at relapse, suggesting that mdr may be involved in acquired resistance. The use of MDR-modifying agents has had some clinical success, and further trials of chemosensitizers are proceeding. P-gp overexpression does not explain how clinical resistance to anthracyclines, alkylating agents, and cis-platinum can arise simultaneously. In these cases, multiple genetic mechanisms of resistance may coexist. Eventually, mdr status can be used to select the most effective chemotherapy protocol for the individual. Currently, conversion of a previously mdr negative tumor to mdr expression, in the face of clinical resistance, justifies changing to a non-MDR drug protocol, or if not feasible, the use of MDR sensitizers.     

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

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

Expression of the MDR1 gene and P-glycoprotein in canine mast cell tumor cell lines

Cellular drug resistance to antineoplastic drugs is often due to the presence of a drug efflux pump that reduces intracellular drug accumulation and chemosensitivity. P-glycoprotein (P-gp), which is encoded by the MDR1 gene, is considered to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance. In the present report, we assessed the expression of MDR1 by RT-PCR in three canine mast cell tumor cell lines, TiMC, CoMS and LuMC, originating from a cutaneous tumor, an oral-mucosal tumor and a gastrointestinal tumor, respectively. P-gp expression was also examined by Western blot analysis, while the functional activity of P-gp was assessed by flowcytometric analysis of intracellular rhodamine-123 (Rhd-123) uptake. The results revealed that MDR1 gene and P-gp were both expressed in CoMS and LuMC cells, whereas neither was present in TiMC cells. In CoMS and LuMC cells, intracellular uptake of Rhd-123 increased in the presence of verapamil, a functional modulator of P-gp. In contrast, TiMC cells did not show any changes in the intracellular accumulation of Rhd-123 after the verapamil addition. These findings suggest that the expressions of MDR1 gene and P-gp probably contribute to cellular drug resistance in canine mast cell tumors.     

Haplotype analysis of the MDR1 flanking region in the dog breed Elo

A deletion mutation in the canine multidrug resistance (MDR1) gene provokes drug sensitivity in several dog breeds from the Collie lineage. A haplotype of four microsatellites containing this mdr1-1Delta mutation was conserved among affected breeds. In this study, we analysed the haplotypes of the MDR1 flanking region of 177 dogs of the breed Elo which is composed of several dog breeds including the Old English sheepdog from the Collie lineage. We detected a haplotype in the Elo breed which had previously been associated with the mutant mdr1-1Delta allele in Old English sheepdogs. Using a regression analysis for the probability of the haplotype on the proportion of genes of the founder breeds, we could exclude the Old English sheepdog as origin of this haplotype for the Elo breed. The MDR1 flanking region could be traced back to the Japanese Spitz as one of the founder dog breeds of the Elo and thus, the introgression of the mdr1-1Delta mutation into the dog breed Elo through the Collie lineage is very unlikely.     

Monoclonal antibodies to feline lymphocyte membranes recognize the leukocyte-common antigen (CD45R).

By immunization of BALB/c mice with a feline T lymphoblastoid cell line, MYA-1 cells, two types of lymphocyte-specific monoclonal antibodies (mAbs) were obtained. The 220/205/190 kd protein defined by 2F11 mAb is highly expressed on the surface of MYA-1 cells and another feline T lymphoma cell line, FL74 cells. The protein is also expressed on normal feline thymocytes, splenocytes and feline peripheral blood mononuclear cells (PBMCs). Another mAb, 17B10, caused similar results as those of 2F11 except for its low reactivity with FL74 cells. The second type of mAb, 15B3, defined the 220 kd protein. The reactivities of this mAb with MYA-1 cells, FL74 cells, PBMCs and feline splenocytes were lower than the former two mAbs, and did not react to feline thymocytes. On the other hand, 17B10 and 15B3 defined partial populations of MYA-1 and FL74 cells recognized by 2F11. The cells defined by the 2F11 and 17B10 are all leukocytes in spleen and lymph node. In contrast, 15B3 defined most of the cells in B cell area and partially in T cell area. These results suggested that 2F11 and 17B10 recognized the specific antigen of 220/205/190 kd of the leukocyte-common antigen (L-CA) family, CD45R, with different epitopes, and that 15B3 defined the distinct antigen of 220 kd on CD45R.     

Analysis of the canine mdr1-1Delta mutation in the dog breed Elo

A deletion mutation in the canine multidrug resistance gene, MDR1, is associated with drug sensitivity. This was shown for several purebred dog breeds from the Collie lineage such as the Collie (rough-coated and smooth-coated), the Australian Shepherd and the Old English sheepdog. To determine whether the mdr1-1Delta mutation could be found in the newly bred German dog breed Elo which is based amongst other breeds on Old English sheepdogs, 177 blood samples representative for the Elo breed were collected. After DNA extraction, a polymerase chain reaction-based method with subsequent polyacrylamide gel electrophoresis was used for detection of the mdr1-1Delta mutation. The mdr1-1Delta allele was not observed in the Elos investigated. The probability that the mdr1-1Delta allele originated in the Old English sheepdog breed is segregating in the Elo population was estimated at 3.68 x 10(-17).     

Cloning of the feline GADD45 cDNA and analysis of its mutation in feline lymphoma cell lines

Growth arrest and DNA damage-inducible protein 45 (GADD45) plays an important role in suppressing multistep carcinogenesis. In this report, we describe the isolation of the complete wild-type feline GADD45 cDNA from feline tissues. Expression of feline GADD45 mRNA was detected in the liver, spleen, kidney, lung, and testis. The predicted amino acid sequences encoded by the full-length feline GADD45 cDNA display sequence homology with those from other vertebrates, and as in the case of human GADD45, cell growth suppression was observed by ectopic expression of feline GADD45. However, no mutations were detected by sequence analysis of feline GADD45 in several feline lymphoma cell lines, indicating that the GADD45 mutation might be uncommon in feline oncogenesis.     

In vitro susceptibility of canine and feline Escherichia coli to fosfomycin

Therapeutic options for multi-drug resistant (MDR) Escherichia coli in dogs or cats are limited. The objective of this study was to establish in vitro susceptibility of canine and feline E. coli to fosfomycin. Two sources of isolates were categorized based on susceptibility as to no resistance (NDR), single drug resistance (SDR), multidrug resistance (MDR) or extreme drug resistance (XDR). Clinical isolates were collected from throughout the US from dogs (n=157) or cats (n=43) with naturally occurring infection between March 2008 and January 2010. Experimental isolates were collected from fecal samples of dogs treated with no drug (NDR), amoxicillin (expressing SDR) or enrofloxacin (expressing MDR or XDR). Fosfomycin minimum inhibitory concentrations (MIC) were determined using E-Test(?). For clinical isolates, most (165/200) originated from the urinary tract, with the number of isolates per resistant category being: NDR (N=44, 22%), SDR (N=65, 32.5%), MDR (N=74, 37%), and XDR (N=17, 8.5%). Of these isolates, 99% (197/200) were susceptible to fosfomycin with the MIC(90) and MIC(50) being 2 and 1 μg/ml, respectively (range: 0.25-196 μg/ml). The number of experimental isolates in each category was NDR (3), SDR (23), MDR (38), and XDR (11) (29.3, 44, and 14.7%, respectively). Of these, 100% were susceptible to fosfomycin with MIC(90) and MIC(50) being 1.5 and 1 μg/ml (range: 0.38-4 μg/ml), respectively. The susceptibility of canine and feline MDR and XDR E. coli to fosfomycin at concentrations well below the susceptible breakpoint supports further investigation for its use when treating E. coli resistant to alternative antimicrobials.     

Development of multidrug resistance in a canine lymphoma cell line

New multidrug resistant cell lines developed from the canine B cell lymphoma cell line (GL-1) were characterized in terms of chemosensitivity to some antineoplastics and P-glycoprotein (Pgp) expression. GL-1 was continuously exposed to a culture medium containing gradually increasing levels of doxorubicin and the cells that could grow in the presence of doxorubicin were obtained. Chemosensitivity of these cells to various antineoplastics were investigated with or without verapamil, which reversed Pgp-mediated drug resistance. The expression of Pgp on the cells was also examined by Western blot analysis. As a result, three kinds of resistant cell lines, designated as GL-DOX60, 300, and 4000 were obtained. These cell lines showed stable proliferation in the medium containing 60, 300, and 4000 ng/ml, respectively. These cells were much more resistant to vincristine than doxorubicin. This resistance was strongly reversed by the presence of verapamil. On the other hand, cisplatin was effective enough in killing these derived cells. In the Western Blot analysis, some bands that reacted to the anti-human Pgp monoclonal antibodies were observed in GL-DOX4000. The cells derived from GL-1 have multidrug resistance potential mediated by canine Pgp. The cells produced in this experimental trial are considered to be useful models for various investigations on canine multidrug resistance.