Objective—To evaluate splenic mast cell tumors
(MCT) of cats for activating mutations in the protooncogene
Sample Population—10 formalin-fixed, paraffinembedded
splenic MCT from cats in the pathology
database of the Veterinary Medical Teaching Hospital
at the University of California, Davis.
Procedure—Genomic DNA was isolated from tumor
specimens, and the polymerase chain reaction (PCR)
procedure was performed for exons 11, 12, and 17.
The PCR products were analyzed by use of agarose
gel electrophoresis and then directly sequenced.
Results—We did not identify mutations in the juxtamembrane
domain (encoded by exons 11 and 12) or
catalytic domain (encoded by exon 17) of c-kit in any
of the splenic MCT specimens.
Conclusions and Clinical Relevance—Although
mutations in the proto-oncogene c-kitoccur frequently
in naturally developing MCT in dogs and aggressive
mastocytosis in humans, the data reported here documented
that dysregulation of Kit function through
activating mutations is unlikely in splenic MCT of cats.
Therapeutic strategies aimed at inhibiting Kit signaling
(ie, kinase inhibitors such as imatinib [STI571]) may
not be of benefit for the treatment of this disease in
cats. (Am J Vet Res 2002;63:1129–1133).
Objective—To determine the prevalence of activating
internal tandem duplications (ITDs) in exons 11 and 12
of c-kit in mast cell tumors (MCTs) of dogs and to correlate
these mutations with prognosis.
Sample Population—157 formalin-fixed, paraffinembedded
MCTs from dogs in the pathology database
of the Veterinary Medical Teaching Hospital at
the University of California, Davis.
Procedure—Genomic DNA was isolated from tumor
specimens and a polymerase chain reaction procedure
was performed to determine whether there
were ITDs in exons 11 and 12.
Results—We identified ITDs in 1 of 12 (8%) grade-I,
42 of 119 (35%) grade-II, and 9 of 26 (35%) grade-III
tumors (overall prevalence, 52 of 157 [33%]). Logistic
regression analysis revealed that the odds of grade-II
and -III tumors possessing an ITD were approximately
5 times greater than that for grade-I tumors,
although these odds did not differ significantly.
Although MCTs possessing an ITD were twice as likely
to recur after excision and twice as likely to result
in metastasis as those without an ITD, these values
also did not differ significantly.
Conclusions and Clinical Relevance—These results
provide evidence that ITDs in c-kit occur frequently in
MCTs of dogs. The high prevalence of c-kit activating
mutations in MCTs of dogs combined with the relative
abundance of mast cell disease in dogs provide
an ideal naturally developing tumor in which to test
the safety and efficacy of novel small-molecule kinase
inhibitors such as imatinib mesylate. (Am J Vet Res
Objective—To evaluate in vitro biological activity of gemcitabine, alone and in combination with Pamidronate or carboplatin, against canine osteosarcoma (OSA) cell lines.
Sample Population—In vitro cultures of OSA cell lines OSA8, OSA16, OSA32, and OSA36.
Procedures—Cell lines were treated with gemcitabine alone or in combination with pamidronate or carboplatin. Cell viability was assessed with the water soluble tetrazolium-1 (WST-1) assay, cell cycle distribution was evaluated by means of propidium iodide staining, and apoptosis was assessed by measuring caspase-3/7 activity. Synergy was quantified by use of combination index (CI) analysis.
Results—For all of the cell lines, treatment with gemcitabine induced growth inhibition, cell cycle arrest, and apoptosis. No synergistic or additive activity was identified when OSA cell lines were treated with gemcitabine in combination with pamidronate. However, when OSA cell lines were treated with gemcitabine in combination with carboplatin, a significant decrease in cell viability was observed, compared with treatment with carboplatin alone, and the drug combination was determined to be synergistic on the basis of results of CI analysis. For 3 of the 4 cell lines, this activity was greater when cells were treated with carboplatin prior to gemcitabine rather than with gemcitabine prior to carboplatin.
Conclusions and Clinical Relevance—Gemcitabine exhibited biological activity against canine OSA cell lines in vitro, and a combination of gemcitabine and carboplatin exhibited synergistic activity at biologically relevant concentrations. Findings support future clinical trials of gemcitabine alone or in combination with carboplatin for the treatment of dogs with OSA.
Objective—To determine whether exposure of canine cancer cells to histone deacetylase (HDAC) inhibitors S(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)benzamide (OSU-HDAC42) or suberoylanilide hydroxamic acid (SAHA) results in increased histone acetylation and decreased cell viability and whether any changes in viability involve induction of apoptosis or alterations in progression of the cell cycle.
Sample Population—9 canine cancer cell lines.
Procedures—Cells from 9 canine cancer cell lines were treated with dimethyl sulfoxide vehicle, OSU-HDAC42, or SAHA, then assays of cell viability were performed. Histone acetylation was assessed by use of western blot analysis. Apoptosis was assessed via ELISA to detect fragmentation of cytoplasmic nucleosomal DNA and western blot analysis to detect cleavage of caspase 3. Cell cycle analysis was performed by use of propidium iodide staining and flow cytometry.
Results—Concentrations of OSU-HDAC42 and SAHA required to achieve 50% inhibition of cell viability (IC50) were reached in cells of 6 and 4 canine cancer cell lines, respectively, and ranged from approximately 0.4 to 1.3μM for OSU-HDAC42 and 0.6 to 4.8μM for SAHA. Cells from T-cell lymphoma, mast cell tumor, osteosarcoma, and histiocytic sarcoma lines were most sensitive to HDAC inhibition, with IC50s of < 1μM for OSU-HDAC42 and < 5μM for SAHA. Induction of apoptosis was indicated via cleavage of caspase 3 and increases in cytoplasmic nucleosomes and the subG1 cell population.
Conclusions and Clinical Relevance—Micromolar concentrations of HDAC inhibitors OSU-HDAC42 and SAHA induced histone acetylation, cytotoxicity, and apoptosis in canine cancer cells. In general, OSU-HDAC42 was more potent than SAHA.
Procedures—Cell viability assays were performed on canine osteosarcoma cell lines OSCA2, OSCA16, OSCA50, and D17 after 24, 48, and 72 hours of treatment with dihydroartemisinin at concentrations of 0.1 to 100μM. Apoptosis was assessed by use of an ELISA for free nuclosomal DNA fragmentation and by western blot analysis for cleavage of caspase 3. Cell cycle analysis was performed by use of staining with propidium iodide and flow cytometry. Detection of reactive oxygen species (ROS) was conducted in the D17 cell line by use of 6-carboxy-2′,7′-dihydrofluorescein diacetate and flow cytometry.
Results—The concentration of dihydroartemisinin required for 50% inhibition of cell viability (IC50) was achieved in all 4 canine osteosarcoma cell lines and ranged from 8.7 to 43.6μM. Induction of apoptosis was evident as an increase in nucleosomal DNA fragmentation, cleavage of caspase 3, and an increase in the population in the sub G0/G1 phase of the cell cycle detected by flow cytometry. Exposure to dihydroartemisinin also resulted in a decrease in the G0/G1 population. Iron-dependent generation of ROS was detected in dihydroartemisinin-treated D17 cells; ROS generation increased in a dose-dependent manner.
Conclusions and Clinical Relevance—Incubation with dihydroartemisinin resulted in biological activity against canine osteosarcoma cell lines, which included induction of apoptosis and arrest of the cell cycle. Clinical trials of dihydroartemisinin in dogs with osteosarcoma should be conducted.
Objective—To evaluate canine histiocytic sarcoma cell lines and tumor samples for dysregulation of the Kit/stem-cell factor (SCF), Flt3/Flt3 ligand (Flt3L), and Met/hepatocyte growth factor (HGF) receptor tyrosine kinase signaling pathways, as these are known to contribute to the differentiation and survival of normal dendritic cells as well as malignant transformation of dendritic cells in mouse models.
Sample Population—4 histiocytic sarcoma tumor cell lines and 35 formalin-fixed histiocytic sarcoma specimens obtained from dogs.
Procedure—Histiocytic sarcoma cell lines were evaluated for expression of Kit/SCF, Flt3/Flt3L, and Met/HGF by use of reverse transcriptase-PCR procedures. Histiocytic sarcoma cell lines and tumor samples were evaluated for mutations in Kit, Flt3, and Met by use of PCR analysis of genomic DNA, followed by both sequencing and fluorescent PAGE for deletions or internal tandem duplications. The ability of the multitargeted split-kinase inhibitor SU11654 to block proliferation and induce apoptosis of histiocytic sarcoma cell lines was also evaluated.
Results—No mutations in Kit, Flt3, and Met were identified in any of the cell lines or tumor samples evaluated. Furthermore, SU11654 did not induce cellcycle arrest or apoptosis of histiocytic sarcoma lines, even at supratherapeutic doses.
Conclusions and Clinical Relevance—These data suggest that dysregulation of Kit/SCF, Flt3/Flt3L, and Met/HGF signaling pathways is unlikely to occur in histiocytic sarcomas of dogs and that inhibitors of the Kit, Flt3, and Met pathways are unlikely to provide clinical benefit to dogs with histiocytic sarcomas.