Michelle G. Ritt

Inactivation of the p16 cyclin-dependent kinase inhibitor in high-grade canine non-Hodgkin's T-cell lymphoma

The significance of p16/Rb tumor suppressor pathway inactivation in T-cell non-Hodgkins lymphoma (NHL) remains incompletely understood. We used naturally occurring canine NHL to test the hypothesis that p16 inactivation has specific pathologic correlates. Forty-eight samples (22 T-cell NHL and 26 B-cell NHL) were included. As applicable, metaphase- or array-based comparative genomic hybridization, Southern blotting, promoter methylation, and Rb phosphorylation were used to determine the presence, expression, and activity of p16. Fishers exact test was used to test for significance. Deletion of p16 (or loss of dog chromosome 11) was restricted to high-grade T-cell NHL (lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified). These were characterized by a concomitant increase of tumor cells with Rb phosphorylation at canonical CDK4 sites. Rb phosphorylation also was seen in high-grade B-cell NHL (diffuse large B-cell lymphoma and Burkitt-type lymphoma), but in those cases, it appeared to be associated with c-Myc overexpression. The data show that p16 deletion or inactivation occurs almost exclusively in high-grade T-cell NHL; however, alternative pathways can generate functional phenotypes of Rb deficiency in low-grade T-cell NHL and in high-grade B-cell NHL. Both morphologic classification according to World Health Organization criteria and assessment of Rb phosphorylation are prognostically valuable parameters for canine NHL.

Sustained nuclear localization of p21/WAF-1 upon growth arrest induced by contact inhibition

We assessed the expression and distribution of p21/Waf-1 in TLM1 melanoma cells that exhibit contact inhibition and require serum for growth. The growth stage of cells stimulated to enter the mitotic cell cycle synchronously and grow to confluence was characterized by distinct, yet consistent levels and patterns of distribution of p21/Waf-1. Significantly, sustained accumulation of p21/Waf-1 in the nuclear compartment was seen only after 4 days in culture when cell-to-cell contacts were established, leading to a diminished rate of cell growth. Overexpression of wild-type waf-1 in melanoma cells reduced growth of subconfluent cells, decreased Cdk4 activity with a concomitant increase in hypophosphorylated Rb, and promoted cell death by apoptosis. The data support the premise that cell-to-cell contacts provide signals that mediate sustained nuclear localization of p21/Waf-1 leading to cell growth arrest; furthermore, an elevation in the activity of this protein can lead to apoptosis.

Enhancing antimelanoma immune responses through apoptosis

We examined the feasibility of using tumor apoptosis at accessible sites to enhance antimelanoma immune responses in a model of spontaneous canine melanoma. We show that priming peripheral blood mononuclear cells with apoptotic melanoma cells significantly enhanced autologous and allogeneic lymphokine-activated killing of tumor cells. Since various pathways required for intrinsic apoptosis are often inactivated in melanoma, we used Fas ligand (FasL) overexpression to promote extrinsic apoptosis. FasL induced apoptosis in five of six cell lines. Each of the susceptible lines, but not the resistant one, expressed Fas mRNA. In addition, direct intratumoral administration of FasL DNA to tumor-bearing dogs was safe, with no adverse events reported over 7 days of observation. A reduction of tumor burden was seen in three of five dogs treated. The reduction of tumor volume was correlated with Fas expression by the tumors, although one dog with a Fas-negative tumor survived for 82 weeks after treatment. Our data show that overexpression of FasL is suitable to promote apoptosis of Fas+ melanomas, and support the notion that priming immune responder cells with apoptotic tumor cells may enhance antitumor responses. The results also suggest that intratumoral administration of FasL offers a safe route for therapeutic gene delivery.

Growth arrest of melanoma cells is differentially regulated by contact inhibition and serum deprivation.

Both growth-factor deprivation and contact inhibition suppress cell growth; however, the mechanisms by which they inhibit cell proliferation may not be identical. The function of antiproliferative genes and the induction of programmed cell death are among the potential differences between these growth-arrest mechanisms. Specifically, an inverse relation between the expression of cyclin-dependent kinase inhibitors (CDKIs) and the susceptibility to apoptosis has been reported. To test this relation, we examined the features of growth arrest in a canine melanoma cell line, TLM1. Both contact inhibition and serum deprivation halted cell-cycle progression of TLM1 cells in the G1 phase. Prolonged growth arrest of the cells without restimulation resulted in apoptosis; conversely, the cells reentered the cell cycle after release from contact inhibition or on restimulation with serum. Cell-to-cell contact, but not serum deprivation, led to the expression of p53 and p21/Waf-1. The expression of p21/Waf-1 did not prevent apoptosis. Moreover, the ectopic overexpression of CDKIs increased apoptosis. These results support the premise that growth arrest induced by contact inhibition and serum deprivation are mediated through distinct mechanisms. Furthermore, CDKIs are not universal inhibitors of apoptosis, and in some cases, they may initiate or enhance the apoptotic program.

Nephrotic Syndrome Resulting in Thromboembolic Disease and Disseminated Intravascular Coagulation in a Dog

Thromboembolic disease and progression to disseminated intravascular coagulation (DIC) are potential life-threatening complications for dogs with nephrotic syndrome. Platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), plasma concentration of fibrinogen degradation products (FDPs), antithrombin III (ATIII), protein C, and plasminogen were used to identify hemostatic abnormalities in a dog with nephrotic syndrome. Pulmonary thromboembolic disease was diagnosed by thoracic radiography, arterial blood gas analysis, and pulmonary scintigraphy. Prompt recognition and treatment of hemostatic complications is necessary in dogs with nephrotic syndrome.

Functional Loss of p21/Waf-1 in a Case of Benign Canine Multicentric Melanoma

Mutations of tumor suppressor genes remove mechanisms that normally arrest proliferation of transformed cells, resulting in tumor formation. The p53 gene product functions as a tumor suppressor that induces p21/Waf-1, the 21-kDa product of the waf-1/cip-1/mda-6 gene. p21/Waf-1 is a pan-cyclin-dependent kinase inhibitor that arrests cell cycle progression under a variety of circumstances. We examined tissues from a dog with multiple primary pigmented proliferative lesions (benign, multicentric melanoma consisting of three distinct dermal lesions and a matrical cyst) for p21/Waf-1 and p53 expression by immunohistochemistry and immunoblotting. p21/Waf-1 and p-53 proteins were undetectable in the tumor cells and in the cyst but were present in adjacent normal tissues. Abundant cyclin-dependent kinase 4 (Cdk4), a protein related functionally to p21/Waf-1, also was present in the cyst. A somatic mutation of the waf-1 gene or of the p53 gene may have resulted in the loss of p21/Waf-1 expression in a common precursor of pigment-producing cells from the affected dog. Furthermore, this functional loss of p21/Waf-1 may play an important role in the genesis of canine benign melanoma.

Stimulation with Concanavalin-A induces IL-17 production by Canine peripheral T cells

The characteristics of canine IL-17-producing cells are incompletely understood. Expression of mRNA encoding orthologs of IL-17 and the IL-17 receptor has been documented in tissues from dogs with arthritis, inflammatory bowel disease, and lymphoma; however, no associations have been found between IL-17 gene expression and disease phenotype in these conditions. Robust assessment of the role of IL-17-producing cells in dogs will require measuring the frequency of these cells in health and disease in balance with other lymphocyte subsets. The aim of this study was to confirm that the T-cell IL-17 response in dogs is evolutionarily conserved. Canine peripheral blood mononuclear cells were stimulated with Concanavalin A with or without polarizing cytokines. We used a canine specific IL-17 ELISA and flow cytometry to identify IL-17-producing T cells. Accumulation of intracellular IL-17 was observed in stimulated CD4 and CD8 T cells. The addition of pro-inflammatory cytokines appeared to enhance polarization of canine CD4 T cells to the Th17 phenotype. Conversely, the addition of IL-2 in the presence of TGF-β resulted in expansion of Treg cells. We conclude that canine IL-17-producing cells behave similarly to those from humans and mice when stimulated with mitogens and polarized with pro-inflammatory or immune regulatory cytokines.

Anti-Insulin Immune Responses Are Detectable in Dogs with Spontaneous Diabetes.

Diabetes mellitus occurs spontaneously in dogs. Although canine diabetes shares many features with human type-1 diabetes, there are differences that have cast doubt on the immunologic origin of the canine disease. In this study, we examined whether peripheral immune responses directed against islet antigens were present in dogs with diabetes. Routine diagnostics were used to confirm diabetic status, and serum samples from dogs with (N = 15) and without (N = 15) diabetes were analyzed for the presence of antibodies against islet antigens (insulin, glutamic acid decarboxylase, insulinoma-associated protein tyrosine phosphatase, and islet beta-cell zinc cation efflux transporter) using standard radioassays. Interferon-γ production from peripheral blood T cells stimulated by porcine insulin and by human insulin was tested using Elispot assays. Anti-insulin antibodies were detectable in a subset of diabetic dogs receiving insulin therapy. Pre-activated T cells and incipient insulin-reactive T cells in response to porcine or human insulin were identified in non-diabetic dogs and in dogs with diabetes. The data show that humoral and cellular anti-insulin immune responses are detectable in dogs with diabetes. This in turn provides support for the potential to ethically use dogs with diabetes to study the therapeutic potential of antigen-specific tolerance.