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.     

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.     

Detection of centrosome amplification as a surrogate marker of dysfunction in the p53 pathway –p53 gene mutation or MDM2 overexpression

In human and canine cancers, the inactivation of p53 protein as well as p53 gene mutation and MDM2 overexpression result in centrosome amplification that in turn contributes to chromosomal instability. To explore the usefulness of the detection of centrosome amplification as a surrogate marker of dysfunction in the p53 pathway, we systematically analysed centrosome amplification, p53 overexpression, p53 gene mutation and MDM2 overexpression in canine tumours. Centrosome amplification was detected in 16 of 51 (31%) naturally developing tumours in dogs. All the tumour specimens with aberrations in the p53 pathway, including p53 overexpression, p53 gene mutation or MDM2 overexpression, showed centrosome amplification, suggesting that the detection of centrosome amplification could serve as a preliminary surrogate marker of dysfunction in the p53 pathway.     

Immunohistochemical evaluation of p53 expression with different antibodies in malignant canine tumours with or without p53 gene mutation

Six monoclonal antibodies and a polyclonal antibody (CM1) were used to investigate the overexpression of p53 protein by immunohistochemistry (IHC) in six sarcomas and 21 mammary carcinomas from 27 dogs. IHC was compared with p53 gene mutation analysis performed on the same samples. Only the monoclonal PAb240, PAb421 and the CM1 antibodies were able to detect expression of canine p53 protein. CM1 was found to give the highest concordance (8/11) between positive expression of the p53 protein by IHC and the presence of a gene mutation. In the samples that were negative for p53 expression by IHC, but contained a p53 gene mutation according to DNA analysis, the mutation often affected the epitopes that could have been recognized by these antibodies. Only one out of 16 tumours without a p53 gene mutation had a weakly positive IHC result. These findings indicate that in these two types of canine tumours, IHC – particularly with CM1 – can detect many alterations in p53 expression owing to a gene mutation. False‐positive results were very infrequent.     

Reduced expression of cyclin-dependent kinase inhibitor p27Kip1 in feline lymphoma.

Expression of p27Kip1 was identified in feline lymphoid tissues by immunohistochemistry. In normal lymphoid tissues, p27Kip1 was detected as a distinct nuclear stain in lymphocytes of the follicular mantle zone and interfollicular small lymphocytes, whereas activated lymphoblasts in the germinal center were negative. Lymphoid hyperplasia was similarly immunolabeled but with an expanded mantle zone and marginal zone of p27Kip1-reactive lymphocytes. Both T- and B-cell lymphomas lacked p27Kip1 immunolabel and were determined to be proliferative based on immunohistochemical detection of the Ki-67 antigen. Scattered p27Kip1-immunolabeled lymphocytes were detected throughout the lamina propria of most specimens characterized as lymphoplasmacytic enteritis. The results of this study suggest that the antiproliferative effect of the cell cycle regulator p27Kip1 is abrogated in feline lymphoma, presumably allowing cells to bypass the G1-S checkpoint of the cell cycle.     

Immunohistochemical detection of Mdm2 and p53 in feline mammary gland tumors

The objective of this study was to evaluate nuclear reactivity of Mdm2 and p53 proteins by immunohistochemical means in feline mammary gland tumors; 12 adenomas which included 6 adenomatous lesions obtained from the tissue adjacent to adenocarcinomas, and 22 adenocarcinomas. Seven adenomas and 18 adenocarcinomas showed moderate or marked Mdm2 reactivity. Sixteen adenocarcinomas showed moderate to marked p53 reactivity, but 9 adenomas showed none. Discordant Mdm2 overexpression was found in 5 adenomas and 3 adenocarcinomas, although co-overexpression of Mdm2 and p53 was found in 15 adenocarcinomas. These results suggest that nuclear overexpression of Mdm2 is present in the tumors of early stage without p53 overexpression and related to feline mammary gland tumorigenesis. Nuclear overexpression of p53 is more frequent in adenocarcinomas, but not in adenomas.     

Canine histiocytic sarcoma cell lines with SHP2 p.Glu76Gln or p.Glu76Ala mutations are sensitive to allosteric SHP2 inhibitor SHP099

Some canine cases of histiocytic sarcoma (HS) carry an activating mutation in the src homology two domain‐containing phosphatase 2 (SHP2) encoded by PTPN11. SHP099 is an allosteric inhibitor of SHP2 that stabilizes SHP2 in a folded, auto‐inhibited conformation. Here, we examined the expression and mutation status of SHP2 in five canine HS cell lines and evaluated the growth inhibitory properties of SHP099 against these cell lines. All five of the canine HS cell lines expressed SHP2, with three of the lines each harbouring a distinct mutation in PTPN11/SHP2 (p.Glu76Gln, p.Glu76Ala and p.Gly503Val). In silico analysis suggested that p.Glu76Gln and p.Glu76Ala, but not p.Gly503Val, promote shifting of the SHP2 conformation from folded to open‐active state. SHP099 potently suppressed the growth of two of the mutant cell lines (harbouring SHP2 p.Glu76Gln or p.Glu76Ala) but not that of the other three cell lines. In addition, SHP099 suppressed ERK activation in the cell line harbouring the SHP2 p.Glu76Ala mutation. The SHP2 p.Glu76Gln and p.Glu76Ala mutations are considered to be activating mutations, and the signal from SHP2 p.Glu76Ala is inferred to be transduced primarily via the ERK pathway. Moreover, SHP099‐sensitive HS cells, including those with SHP2 p.Glu76Gln or p.Glu76Ala mutations, may depend on these mutations for growth. Therefore, targeting cells harbouring SHP2 p.Glu76Gln and p.Glu76Ala with SHP099 may be an approach for the treatment of canine HS.     

Inactivation of p53 and retinoblastoma family pathways in canine osteosarcoma cell lines

Canine osteosarcoma (OS) has been used as a model system for the study of cancer biology and treatment despite the lack of information regarding its pathogenesis. Expression of tumor suppressor genes known to participate in malignant transformation were studied in five different OS cell lines. Each of the cell lines exhibited properties of transformed cells, and those that were tested grew in soft agarose and formed osteoid-containing tumors when injected subcutaneously into nude mice. p53 function was determined to be defective in each cell line as indicated by the lack of induction of p53-responsive genes, p21 and mdm2, following treatment with 5-fluorouracil. p53 mRNA and protein levels were elevated in three cell lines and were extremely low in two cell lines. p53 protein overexpression correlated with the presence of mutations within the DNA binding domain. Four cell lines appeared to contain normal retinoblastoma (Rb) mRNA and Rb protein and no detectable p16 mRNA or protein. In contrast, the remaining cell line contained high levels of p16 mRNA and protein and significantly reduced levels of Rb, p107, and p130 proteins. These results underscore the importance of inactivating p53 and Rb family pathways in canine OS and suggest that unlike human OS, cells derived from canine OS contain mutations that simultaneously inactivate all three Rb family members.     

Comparison of chemotherapeutic drug resistance in cells transfected with canine ABCG2 or feline ABCG2

ABCG2 (ATP binding cassette subfamily G, member 2) mediates resistance to a variety of cytotoxic agents. Although human ABCG2 is well characterized, the function of canine ABCG2 has not been studied previously. Feline ABCG2 has an amino acid substitution in the adenosine triphosphate‐binding domain that decreases its transport capacity relative to human ABCG2. Our goal was to compare canine ABCG2‐mediated chemotherapeutic drug resistance to feline ABCG2‐mediated chemotherapeutic drug resistance. HEK‐293 cells stably transfected with plasmid containing canine ABCG2, feline ABCG2 or no ABCG2 were exposed to carboplatin, doxorubicin, mitoxantrone, toceranib or vincristine, and cell survival was subsequently determined. Canine ABCG2 conferred a greater degree of chemotherapy resistance than feline ABCG2 for mitoxantrone. Neither canine nor feline ABCG2 conferred resistance to doxorubicin, vincristine or toceranib. Canine, but not feline, ABCG2 conferred resistance to carboplatin, a drug that is not reported to be a substrate for ABCG2 in other species.     

Immunohistochemical studies of epithelial cell proliferation and p53 mutation in bovine ocular squamous cell carcinoma

Bovine ocular squamous cell carcinoma (OSCC) is the second most common cause of rejection due to neoplasia in slaughterhouses on Sao Miguel Island, Azores, and accounts for significant economic losses. To obtain a better insight into the genesis and neoplastic transformation process of bovine OSCC, abnormal protein expression and proliferation index were assessed by the immunohistochemical evaluation of p53 and Ki67, respectively. OSCC samples were collected from 15 bovines and were classified histologically according to the degree of differentiation into three categories: poorly, moderately, and well differentiated. Immunohistochemistry using polyclonal anti-human p53 antibody and polyclonal anti-human Ki67 antibody was performed. Ten of 15 tumors tested were immunoreactive for p53. Twelve tumors demonstrated Ki67 expression. As in human squamous cell carcinoma, p53 overexpression is frequent in bovine OSCC, providing support for a possible role of the protein in the pathogenesis of this neoplasia. No correlation between the percentage of p53 stained nuclei and the degree of differentiation was observed, although different patterns of staining were seen according to the degree of keratinization of the tumor cells. With the exception of the moderately differentiated OSCC group, Ki67 index showed significant correlation with the histologic pattern, increased proliferation being found in poorly differentiated OSCC (P = 0.013).     

In vitro effects of the tyrosine kinase inhibitor, masitinib mesylate, on canine hemangiosarcoma cell lines

This study evaluated the in vitro activity of masitinib mesylate against canine hemangiosarcoma (HSA) cell lines after treatment with increasing concentrations of masitinib mesylate (0.01-100?μM) for 24, 48 and 72 h. Results indicated that masitinib mesylate caused a dose- and time-dependent decrease in HSA cell proliferation. The 50% inhibitory concentration (IC(50) ) at 72 h for three HSA cell lines (DEN, Fitz and SB) was found to be 8.56, 9.41 and 10.65?μM, respectively. Further investigation demonstrated that masitinib mesylate induced apoptosis in all HSA cell lines, including activation of caspase-3/7. Measurement of VEGF levels in cell supernatant found a statistically significant increased VEGF in close proximity to the IC(50) of each cell line followed by a decline back towards baseline. These findings indicate that masitinib mesylate causes dose-dependent HSA cell death in vitro and supports future clinical trials of masitinib for canine HSA.     

Somatic alterations of the p53 tumor suppressor gene in vaccine-associated feline sarcoma

OBJECTIVE: To determine somatic alterations in p53 in vaccine-associated feline sarcoma (VAFS). Animals-27 domestic shorthair cats undergoing first surgical treatment for primary VAFS with no history of chemotherapy or gamma radiation. PROCEDURES: Sequence analysis was performed on the genomic sequence of p53 (between exons 5 through 9) from tumor and blood samples obtained from the cats. Cats were monitored for 3 years and disease-free intervals and survival times calculated. RESULTS: Eight single nucleotide polymorphisms were detected within the genomic sequence of p53, with 20 of 27 cats (74%) having heterozygosity at > or = 1 polymorphic site. Somatic loss of heterozygosity at p53 was detected in the primary tumors of 12 of these 20 (60%) cats. Such allelic deletion was significantly associated with rapid tumor recurrence and reduced overall survival. Point mutations were rare, occurring in 3 of 27 primary tumors. The finding of malignant cells in the surgical margins was significantly associated with disease recurrence, but clear margins (with no detectable malignant cells) were not predictive of positive outcome. CONCLUSIONS AND CLINICAL RELEVANCE: p53 status is an indicator of postsurgical recurrence and overall survival in cats with VAFS. Careful follow-up is important in treating vaccine-site tumors containing allelic deletion of p53, whereas aggressive surgical treatment may be sufficient to control primary vaccination site tumors without the allelic loss.     

Comparison of T-cell subpopulations in cats naturally infected with feline leukaemia virus or feline immunodeficiency virus

T-cell subsets were studied by flow cytometry in 58 feline leukaemia virus (FeLV)-positive cats with naturally acquired FeLV infection to determine whether the changes in CD4⁺ or CD8⁺ T cell populations differed from those observed in 55 feline immunodeficiency virus (FIV)-positive cats with naturally acquired FIV infection. The sole criterion for inclusion into the study was seropositivity. Mean (SD) CD4⁺ T cell values of FeLV positive cats were decreased to 31·1 (8·0) per cent and their CD8⁺ T cell values were increased to 22·8 (6·3) per cent in comparison with uninfected control cats (37·9 [9·5] per cent CD4⁺; 15·2 [6·3] per cent CD8⁺). The CD4⁺/CD8⁺ ratio was reduced to 1·5 (0·7), compared with 3·0 (1·5) in 39 FeLv- and FIV-negative control cats. Differences from control values were significant, but there was no significant difference between CD4⁺ and CD8⁺ lymphocytes of FeLV- versus FIV-infected cats. These findings indicate that FeLv and FIV have similar effects on T lymphocyte subsets. Both retrovirus infections can induce immunodeficiency, both viruses infect a broad range of lymphohaemopoietic cells, despite having different primary target cells, and can induce the killing of lymphocytic cells in vitro. It is concluded that a decreased CD4⁺/CD8⁺ ratio is not restricted to FIV infections but may also occur in FeLv infection.     

Loss of retinoblastoma protein, but not p53, is associated with the presence of papillomaviral DNA in feline viral plaques, Bowenoid in situ carcinomas, and squamous cell carcinomas

Although papillomaviral (PV) DNA is frequently present in feline cutaneous squamous cell carcinomas (SCCs), a causative association cannot be proven. Oncogenic human PVs cause neoplastic transformation by inhibiting retinoblastoma (pRb) and p53 activity. Therefore, absence of pRb and p53 immunostaining, along with increased p16 immunostaining, indicates a PV cause in some human SCCs. If PVs cause cutaneous feline SCCs, it was hypothesized that a similar immunohistochemistry profile, along with PV DNA, would be detectable. This was investigated using 5 feline viral plaques, 10 Bowenoid in situ carcinomas, 19 SCCs from ultraviolet-exposed (UV-exposed) skin, and 11 SCCs from UV-protected skin. Papillomaviral DNA was amplified by polymerase chain reaction from 30 of 45 lesions. Reduced pRb immunostaining was present in 26 of 45; increased p16 immunostaining was in 30; and p53 immunostaining was in 19. Both reduced pRb immunostaining and increased p16 immunostaining were more frequent in lesions containing PV DNA. In contrast, no association was observed between p53 immunostaining and the presence of PV DNA. SCCs from UV-protected skin more frequently contained PV DNA, reduced pRb, and increased p16 than UV-exposed SCCs. UV exposure was not associated with p53 immunostaining within the SCCs. These results suggest that feline PVs alter cell regulation by degrading pRb. Unlike oncogenic human PVs, there was no evidence that feline PVs degrade p53. These results provide further evidence that PVs may cause feline cutaneous SCCs, especially those in UV-protected skin, and they suggest a possible mechanism of this oncogenic action.     

Immunohistochemical expression of p53, fibroblast growth factor-b, and transforming growth factor-alpha in feline vaccine-associated sarcomas

Fifty feline sarcomas associated with vaccine-site injection were evaluated to determine the immunohistochemical expression of p53 protein, basic fibroblast growth factor (FGF-b), and transforming growth factor-alpha (TGF-alpha). Forty-one tumors (82%) were fibrosarcomas (FS), eight (16%) were malignant fibrous histiocytomas (MFH), and one (2%) was a chondrosarcoma (CS). Overexpression of p53 protein was observed in the nuclei of tumor cells in 28 (56%) sarcomas; FGF-b expression was found in the cytoplasm of tumor cells in 40 (80%) feline sarcomas, but the staining was more intense in the spindle-shaped cells of FS than in polygonal or round cells of MFH. The single CS faintly expressed FGF-b. The majority of feline vaccine-associated sarcomas (43 of 50, 86%) expressed moderate or intense staining for TGF-alpha in the cytoplasm of tumor cells. Heterogeneous immunolabeling for p53, FGF-b, and TGF-alpha was present in neoplastic, multinucleated giant cells. Intense expression of FGF-b was statistically associated with younger cats (P < 0.01) and with tumors with nodular growth patterns (P = 0.02). In addition, sarcomas negative for p53 protein expressed FGF-b more frequently than did p53-positive tumors (P = 0.04). The frequency of FGF-b immunostaining was significantly higher in sarcomas with intense expression of TGF-alpha (P = 0.05). Immunohistochemical detection of p53 protein, FGF-b, and TGF-alpha suggests that these growth-regulating proteins may play different roles in the development of sarcomas associated with vaccine sites.