Antitumor effects of celecoxib in COX-2 expressing and non-expressing canine melanoma cell lines

Cyclooxygenase-2 (COX-2) is a potential target for chemoprevention and cancer therapy. Celecoxib, a selective COX-2 inhibitor, inhibits cell growth of various types of human cancer including malignant melanoma. In dogs, oral malignant melanoma represents the most common oral tumor and is often a fatal disease. Therefore, there is a desperate need to develop additional therapeutic strategies. The purpose of this study was to investigate the anticancer effects of celecoxib on canine malignant melanoma cell lines that express varying levels of COX-2. Celecoxib induced a significant anti-proliferative effect in both LMeC and CMeC-1 cells. In the CMeC cells, treatment of 50 μM celecoxib caused an increase in cells in the G0/G1 and a decreased proportion of cells in G-2 phase. In the LMeC cells, 50 μM of celecoxib led to an increase in the percentage of cells in the sub-G1 phase and a significant activation of caspase-3 when compared to CMeC-1 cells. In conclusion, these results demonstrate that celecoxib exhibits antitumor effects on canine melanoma LMeC and CMeC-1 cells by induction of G₁-S cell cycle arrest and apoptosis. Our data suggest that celecoxib might be effective as a chemotherapeutic agent against canine malignant melanoma.     

The therapeutic effects of SET/I2PP2A inhibitors on canine melanoma

Canine melanoma is one of the most important diseases in small animal medicine. Protein phosphatase 2A (PP2A), a well conserved serine/threonine phosphatase, plays a critical role as a tumor suppressor. SET/I2PP2A is an endogenous inhibitor for PP2A, which directly binds to PP2A and suppresses its phosphatase activity. Elevated SET protein levels have been reported to exacerbate human tumor progression. The role of SET in canine melanoma, however, has not been understood. Here, we investigated the potential therapeutic role for SET inhibitors in canine melanoma. The expression of SET protein was observed in 6 canine melanoma cell lines. We used CMeC-1 cells (primary origin) and CMeC-2 cells (metastatic origin) to generate cell lines stably expressing SET-targeting shRNAs. Knockdown of SET expression in CMeC-2, but not in CMeC-1, leads to decreased cell proliferation, invasion and colony formation. Phosphorylation level of p70 S6 kinase was decreased by SET knockdown in CMeC-2, suggesting the involvement of mTOR (mammalian target of rapamycin)/p70 S6 kinase signaling. The SET inhibitors, OP449 and FTY720, more effectively killed CMeC-2 than CMeC-1. We observed PP2A activation in CMeC-2 treated with OP449 and FTY720. These results demonstrated the potential therapeutic application of SET inhibitors for canine melanoma.     

Comparative study of anti-oncogenic microRNA-145 in canine and human malignant melanoma

MicroRNA-145 (miRNA-145; miR-145) is aberrantly expressed in most of human cancers and plays a significant role in carcinogenesis and cancer progression. In the current study, we focused on how miR-145 plays a role in canine and human malignant melanomas. MiR-145 was significantly downregulated in canine malignant melanoma tissues and canine melanoma cell lines, as well as human melanoma cell lines tested. The ectopic expression of miR-145 showed a significant growth inhibition in both canine and human melanoma cells tested, and the effect was achieved partly by suppressing c-MYC in canine melanoma LMeC and in human melanoma A2058 and Mewo cells. At the same time, a suppressive tendency on cell migration in canine melanoma KMeC cells and significant suppression of cell migration in human melanoma A2058 cells by suppressing FASCIN1 were also found. These findings suggest that miR-145 acts as a tumor suppressor in both canine and human malignant melanomas.     

Effect of retinoids on growth inhibition of two canine melanoma cell lines

Two new canine melanoma cell lines (CMM1 and CMM2) were established from the patients with oral malignant melanomas. Histopathological type of both CMM1 and CMM2 was a mixed cell type consisted of spindle-shaped cells, polygonal cells, and oval cells. Doubling time of CMMI and CMM2 were 18.4 +/- 1.96 hr and 21.0 +/- 0.73 hr, respectively. The effect of two kinds of retinoids (all-trans retinoic acid and 9-cis retinoic acid) on the proliferation of these cells were examined by morphological changes, proliferation assay and apoptosis assay. However, the retinoids did not suppress growth rate of these cells. This result suggests that retinoids used in this study did not induce differentiation, apoptosis, and growth inhibition of the canine melanoma cell lines.     

Expression and significance of p53, rb,p21/waf-1, p16/ink-4a,and PTEN tumor suppressors in canine melanoma

The role of tumor suppressor genes in the pathogenesis of canine melanoma is incompletely understood. The genes encoding the tumor suppressors p53, Rb, p21 (waf-1), p16 (ink-4a), and PTEN have been postulated to contribute to the pathogenesis of melanoma in humans and experimental animal models. To assess whether inactivation of these genes similarly contributes to the origin and progression of canine melanoma, we examined their expression in seven distinct canine melanoma cell lines and in 31 retrospective samples (representing 29 dogs) of spontaneous canine melanoma. Various patterns suggestive of loss of tumor suppressor function emerged in these cell lines. The most frequently observed abnormality was loss or significant reduction of p16 expression in six of seven cell lines and in 21 of 26 tumor samples. Loss or significant reduction of PTEN expression was seen in four of seven cell lines and in 13 of 27 tumor samples. Although p53 was detectable in all the cell lines and in 24 of 30 tumors, exclusion of p53 from the nuclear compartment was observed in each of the cell lines and in 18 of 25 tumor samples. These results indicate that loss of function of these tumor suppressor proteins is a common occurrence that may contribute to the origin of canine melanoma. In our sample population, abnormalities in the expression or localization of one or more tumor suppressor proteins occurred with similar frequency in malignant and benign tumors; thus, additional work is necessary to determine how these proteins may impact disease progression and response to therapy.     

Expression of S100a, vimentin, NSE, and melan A/MART-1 in seven canine melanoma cells lines and twenty-nine retrospective cases of canine melanoma

We evaluated the expression of vimentin, S100a, and Melan A/MART-1 (melanoma antigen recognized by T cells 1) in seven cell lines established independently from dogs with canine melanoma. We also compared routine immunostaining of 29 clinical specimens from melanoma cases using vimentin, S100a, and neuron-specific enolase (NSE) with staining for Melan A/MART-1 as part of a diagnostic panel. All the cell lines were positive for expression of vimentin and S-100a. MelanA/MART-1 expression was seen consistently in only two of the seven cell lines. Staining for Melan A/MART-1 was most intense near areas of heavy melanin pigmentation. All except one of the clinical specimens were positive for vimentin. S 100a was expressed in the majority of both pigmented (15/20, 75%) and amelanotic (8/9, 88.8%) tumors. Seventeen of 29 (58.6%) tumors were positive for NSE. Melan A/MART-1 was expressed in 18/29 (62%) tumors, including 90% of pigmented tumors, but in no amelanotic tumors. Intensity of Melan A/MART-1 staining correlated positively with biologic behavior, with seven malignant tumors showing negative to weak staining and 10 benign tumors showing moderate to strong staining. Three malignant tumors showed moderate to intense staining for Melan A/ MART-1. Our results suggest that expression of Melan A/MART-1 may be unstable in cultured cell lines. Assessment of both S100a and Melan A/MART-1 expression is useful to confirm a diagnosis of canine melanoma, and Melan A/MART-1 may be especially informative regarding the biologic behavior of these tumors.     

Effect of transforming growth factor-beta1, insulin-like growth factor-I,and hepatocyte growth factor on proteoglycan production and regulation in canine melanoma cell lines

OBJECTIVE:To identify extracellular proteoglycans produced by canine melanoma cell lines and analyze the effect of transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor-I (IGF-I), and hepatocyte growth factor (HGF) on these proteoglycans. SAMPLE POPULATION: 3 canine melanoma cell lines (ie, CML-1, CML-6M, and CML-10c2). PROCEDURE: Extracellular proteoglycans were analyzed by use of metabolic labeling and western immunoblot analysis. The effect of TGF-beta1 on cell proliferation was determined by incorporation of 5-bromo-2'-deoxyuridine. RESULTS: The CML-1 and CML-6M melanoma cell lines produced 2 main extracellular proteoglycans. One of them was identified as versican, a proteoglycan found in undifferentiated human melanoma cell lines. The CML-10c2 cells produced a small amount of extracellular proteoglycans. Addition of TGF-beta1 (1.25 to 6.25 ng/ml) increased the release of sulfated proteoglycans into the medium. The TGF-beta1 had mainly a posttranslational effect, because it increased the molecular mass of the sulfated bands. Addition of IGF-I (50 ng/ml) slightly increased production of proteoglycans in the CML-6M cell line, whereas HGF (50 ng/ml) did not have any effect on proteoglycan production. CONCLUSIONS AND CLINICAL RELEVANCE: The proteoglycan content and response toTGF-beta1 treatment for CML-1 and CML-6M canine melanoma cell lines are similar to that for undifferentiated human melanoma cell lines. In contrast, CML-10c2 cells produced a low amount of proteoglycans with high molecular weight. Because these extracellular proteoglycans are involved in the control of cell adhesion, proliferation, and migration, they may play an important role in the progression of melanomas in dogs.     

Equine basal cell tumors: 6 cases (1985-1999)

Basal cell tumors are rare benign tumors in horses. Over a 15-year period, 6 horses were diagnosed with basal cell tumors. The tumors were well-circumscribed. freely moveable, firm, raised papules, nodules, or masses that ranged from 0.6 to 5 cm in diameter. Five of the 6 tumors were ulcerated. Based on gross appearance, the tumors were diagnosed as sarcoids, and 1 was diagnosed as a melanoma. The range of age of affected horses was 6-26 years. The tumors were identified clinically 1 week to 3 years before excision. In 4 horses for which information was available, complete surgical excision was curative with no recurrence 4 months to 2 years after removal.     

Immortalized goat milk epithelial cell lines replicate CAEV at high level

Primary milk epithelial cells were isolated from CAEV-uninfected goats and three cell lines designated TIGMEC-1, TIGMEC-2 and TIGMEC-3 were established. The three cell lines retained their morphological characteristics of epithelial cells and expressed specific epithelial cytokeratin marker as well as the immortalizing SV40 large T antigen. The kinetics of growth of TIGMEC1, TIGMEC2 and TIGMEC3 cell lines showed a doubling time of 24-48 hours while the parental cell lines became senescent after the passage 6 in cell culture. Like the parental primary cells, the three cell lines were found to be highly sensitive to CAEV-pBSCA, an infectious molecular clone of CAEV-CO strain, and to a French isolate CAEV-3112. TIGMEC cell lines infected with CAEV-pBSCA became chronically infected producing high virus titers in absence of cytopathic effects. These cell lines may be useful for study of the possible physiological alterations in mammary epithelial cells infected with small ruminant lentiviruses and their consequences on milk quality. On an other hand, these cell lines can be used to study their role in virus transmission and pathogenesis.     

Results of hyperamplification of centrosomes in naturally developing tumors of dogs.

OBJECTIVE: To evaluate results of centrosome hyperamplification in naturally developing tumors of dogs. SAMPLE POPULATION: Tumor specimens from 9 dogs with tumors (rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myxosarcoma, and mammary gland tumor) and 2 canine osteosarcoma cell lines. PROCEDURE: 3 antibodies for centrosome proteins (ie, anti-gamma-tubulin, anti-BRCA1, and anti-pericentrin) were used for immunohistochemical analysis. Double immunostaining for centrosomes was used to confirm the specificity of these antibodies for centrosomes. Mutational analysis of the canine p53 gene was carried out by polymerase chain reaction-single-strand conformation polymorphism analysis, and expression of canine MDM2 protein was evaluated by use of immunohistochemical analysis, using anti-MDM2 antibody. RESULTS: Immunohistochemical analysis of dog osteosarcoma cell lines with apparent aneuploidy revealed frequent hyperamplification of centrosomes in the osteosarcoma cell lines. Similar hyperamplified centrosomes were detected in the tumor tissues from all of the 9 tumors. The frequency of cells with hyperamplified centrosomes (3 to 20/cell) in each tumor tissue ranged from 9.50 to 48.1%, whereas centrosome hyperamplification was not observed in normal lymph nodes from these dogs. In 8 of the 9 tumors, mutation of p53 gene or overexpression of MDM2, or both, was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Various types of naturally developing tumors in dogs often have hyperamplification of centrosomes associated with chromosome instability. Hyperamplification of centrosomes is a novel tumor marker for use in cytologic and histologic examinations of clinical specimens obtained from dogs.     

Canine testicular tumors in descended and cryptorchid testes

A comparative study of canine testicular tumors in descended and cryptorchid testes was performed in order to associate pathologic features with clinical signs. Observation on 80 canine testicular tumors revealed the following distribution: in undescended testes, 4 tumors were classified as Seminomas (mean age: 8.8 +/- 3 years) and 14 as Sertoli cell tumors (8.7 +/- 1.7 years); in descended testes, 21 tumors were classified as seminomas (8.8 +/- 3 years), 13 as Sertoli cell tumors (9.8 +/- 1.8 years), 22 as Leydig cell tumors (11.5 +/- 2 years), 5 multiple primary tumors and 1 as an immunoblastic lymphoma. Histological features were studied and correlated with other clinical parameters: feminization manifestations, prostatic disease, perineal hernia and perianal gland tumor.     

Analysis of transtracheal aspirates and pleural fluid from clinically healthy llamas (Llama glama)

Seventeen clinically healthy adult llamas were used to study the characteristics of transtracheal aspirates (TTA) and pleural fluid samples. Results of complete blood counts, fibrinogen determination and thoracic radiographs were within normal limits prior to sampling. Cytologic evaluation of TTA revealed the majority of cells were vacuolated macrophages (60-100%), with 0-40% neutrophils, and fewer lymphocytes (0-1%), eosinophils (0-3%), and ciliated respiratory epithelial cells (0-10%). In TTA from 10 of 17 llamas, neither aerobic nor anaerobic bacteria were isolated. Bacteria isolated in pure culture from TTA were similar to isolates found in clinically healthy animals of other species, and included Acinetobacter sp., Staphylococcus sp. and Bacillus sp. Results (mean +/- SD) of pleural fluid analyses were: total nucleated cell count 576 +/- 361/microliter; specific gravity 1.0133 +/- 0.002; glucose concentration 135.1 +/- 9.02 mg/dL; and lactate concentration 2.95 +/- 1.34 mg/dL. Pleural fluid total protein concentrations determined by refractometry ranged from < 2.5 to 3.5 g/dL. The refractive index ranged from 1.3396 to 0.0013. In pleural fluid, small lymphocytes were the predominant cell type.     

The oncolytic effects of reovirus in canine solid tumor cell lines

Oncolytic virotherapy is a new strategy for cancer treatment for humans and dogs. Reovirus has been proven to be a potent oncolytic virus in human medicine. Our laboratory has previously reported that canine mast cell tumor and canine lymphoma were susceptible to reovirus. In this study, canine solid tumor cell lines (mammary gland tumor, osteosarcoma and malignant melanoma) were tested to determine their susceptibility towards reovirus. We demonstrated that reovirus induces more than 50% cell death in three canine mammary gland tumors and one canine malignant melanoma cell line. The reovirus-induced cell death occurred via the activation of caspase 3. Ras activation has been shown to be one of the important mechanisms of reovirus-susceptibility in human cancers. However, Ras activation was not related to the reovirus-susceptibility in canine solid tumor cell lines, which was similar to reports in canine mast cell tumor and canine lymphoma. The results of this study highly suggest that canine mammary gland tumor and canine malignant melanoma are also potential candidates for reovirus therapy in veterinary oncology.     

Congenital and acquired melanocytomas (benign melanomas) in eighteen young horses.

In a retrospective study, cutaneous melanocytic tumors from 18 horses, less than 2 years old, were examined histopathologically and clinical follow-up requested. Melanocytomas (benign melanomas) occurred in a variety of breeds and in horses of varied coat color. The age of the horses at the time of biopsy ranged from 3 weeks old to 2 years old. Four melanocytomas were congenital, 11 melanocytomas were acquired by 1 year of age, and three were acquired prior to 2 years of age. Of the 18 horses, five were male, and 13 were female. All tumors were solitary and located on the legs or trunk; none were in the perineal region. Ulceration of the overlying epidermis was common. Tumors were generally localized and were not encapsulated. The tumors had a variety of cell patterns ranging from sheets, to streams, or nests of melanocytes. Cellular morphologic findings also ranged from epithelioid, to a mixture of epithelioid and spindle cells or to a spindle pattern. The nuclei were large and euchromatic, especially in the epithelioid cells. Several tumors had moderate cellular pleomorphism and binucleate cells. Mitotic activity was generally low (less than 1/high-powered field), but was readily detected (1-2/high-powered field) in bleached sections of four cases. Melanin pigmentation varied from mild to heavy. Melanophages were admixed with the tumor cells or in the adjacent tissue. Follow-up information was obtained on 15/18 horses and revealed that 14/15 horses were free of recurrence following excision. One neoplasm, that was poorly demarcated and had a spindle cell pattern, was not completely resected and continued to grow. These melanocytic tumors in young horses are distinct from melanomas in aged horses in their location, epithelial involvement, and age of horses affected. The majority of these tumors appear to be benign and share features of melanocytic nevi of human beings.     

Comparative susceptibility of Marek's disease cell lines to chicken infectious anemia virus

Chicken infectious anemia virus (CIAV) is known to infect and replicate in various Marek's disease chicken cell lines (MDCCs) derived from Marek's disease (MD) tumors. One line, MDCC-MSB1, has been the substrate used in most studies. We compared a total of 26 MDCCs, including two sublines of MDCC-MSB1, MSB1 (L) and MSB1 (S), four other MD tumor-derived lines, and 20 lines derived from MD virus-induced local lesions, for susceptibility to the Cux-1 and CIA-1 strains of CIAV. The cell lines represented six phenotypic groups of T cells based on the expression of CD4, CD8, and TCR-2 and -3 surface markers. Susceptibility was measured by the number of cells positive for viral antigen in immunofluorescence (IF) tests at 3-10 days postinfection. No clear-cut differences were found in susceptibility related to phenotype, although CD4-/8+ lines and CD4-/8- lines might be more susceptible than CD4+/8- lines. However, several individual lines were more susceptible to Cux-1 than the two MSB1 sublines tested. Contrary to an earlier report, cells of MDCC-CU147, a CD8+, TCR3+, local-lesion derived line, were found to be susceptible to CIA-1. In fact, CU147 was distinguished by very high susceptibility to both CIAV strains. In direct comparisons with MSB1, CU147 detected approximately 10-fold lower doses of virus. Also, virus spread was faster (P < 0.05) in CU147 than in MSB1 and other lines. Results from polymerase chain reaction (PCR) tests to detect infection in titrations were in general agreement with IF test results although PCR detected infection in a few terminal dilution cultures that were negative by IF.     

Epididymal interstitial (Leydig) cell tumors in B6C3F1 mice

Six primary interstitial cell tumors of the epididymis were identified from 46,752 male B6C3F1 mice used in chronic toxicity and carcinogenicity studies. Five of the tumors occurred at the end of 2-year studies; none were attributed to treatment. None of the mice with epididymal tumors had a primary testicular tumor. Histologically, tumors were characterized by a nodular or diffuse proliferation of tumor cells in the epididymal interstitium. Most cells were polygonal with highly vacuolated cytoplasm (vacuolated cells) or eosinophilic cytoplasm (eosinophilic cells). Smaller hyperchromatic cells with scant basophilic cytoplasm (basophilic cells) and cells with yellow-brown pigment characteristic of lipofuscin (pigmented cells) were less common. In each tumor two or more cell types were present. Extension of these tumors through the capsule, invasion of the testis, or metastasis did not occur. By electron microscopy both eosinophilic and vacuolated cell types had a large round or oval nucleus with sparse heterochromatin, abundant mitochondria with tubulovesicular cristae, and frequent desmosome structures between cell membranes. Vacuolated cells contained numerous lipid droplets. Morphological features of the epididymal tumors are similar to those of the testicular interstitial (Leydig) cell tumor in mice and rats.