Nicotine-mediated signals modulate cell death and survival of T lymphocytes.

The capacity of nicotine to affect the behavior of non-neuronal cells through neuronal nicotinic acetylcholine receptors (nAChRs) has been the subject of considerable recent attention. Previously, we showed that exposure to nicotine activates the nuclear factor of activated T cells (NFAT) transcription factor in lymphocytes and endothelial cells, leading to alterations in cellular growth and vascular endothelial growth factor production. Here, we extend these studies to document effects of nicotine on lymphocyte survival. The data show that nicotine induces paradoxical effects that might alternatively enforce survival or trigger apoptosis, suggesting that depending on timing and context, nicotine might act both as a survival factor or as an inducer of apoptosis in normal or transformed lymphocytes, and possibly other non-neuronal cells. In addition, our results show that, while having overlapping functions, low and high affinity nAChRs also transmit signals that promote distinct outcomes in lymphocytes. The sum of our data suggests that selective modulation of nAChRs might be useful to regulate lymphocyte activation and survival in health and disease.

Influence of genetic background on tumor karyotypes: evidence for breed-associated cytogenetic aberrations in canine appendicular osteosarcoma.

Recurrent chromosomal aberrations in solid tumors can reveal the genetic pathways involved in the evolution of a malignancy and in some cases predict biological behavior. However, the role of individual genetic backgrounds in shaping karyotypes of sporadic tumors is unknown. The genetic structure of purebred dog breeds, coupled with their susceptibility to spontaneous cancers, provides a robust model with which to address this question. We tested the hypothesis that there is an association between breed and the distribution of genomic copy number imbalances in naturally occurring canine tumors through assessment of a cohort of Golden Retrievers and Rottweilers diagnosed with spontaneous appendicular osteosarcoma. Our findings reveal significant correlations between breed and tumor karyotypes that are independent of gender, age at diagnosis, and histological classification. These data indicate for the first time that individual genetic backgrounds, as defined by breed in dogs, influence tumor karyotypes in a cancer with extensive genomic instability.

MHC-dependent desensitization of intrinsic anti-self reactivity.

The survival of naive T cells is compromised in the absence of molecules encoded by the major histocompatibility complex (MHC) while antigen-experienced T cells survive. We hypothesized that survival pressures in an in vivo, MHC-deficient environment would permit enrichment of less frequent antigen-experienced autoreactive cells at the expense of the majority of antigen naive T cells. To test this hypothesis, we generated MHC class I- and class II-deficient mice in NOD and C57Bl/6 (B6) backgrounds, and examined the capacity of adoptively transferred autoimmune-prone NOD T cells, or non-autoimmune prone naive B6 T cells, respectively, to reject transplanted wild-type pancreatic islets or transplantable tumors in the MHC-deficient mice. In the MHC-deficient environment, CD4 T cells acquired self-hostile properties (islet rejection and tumor invasion) that were independent from their genetic propensity for autoreactivity, while CD8 T cells required appropriate prior exposure to antigen in order to survive and function (reject tumor) in this environment; however, disengagement of Tob1, a negative regulator of proliferation, led to a reverse phenotype with regard to persistence of CD4 and CD8 T cells in the MHC-deficient environment. Our data suggest that self-peptide/MHC interactions have dual roles to facilitate survival and restrain autoreactivity, thus acting as integral components of an intrinsic network of negative regulation that maintains tolerance.

Canine hemangiosarcoma originates from hematopoietic precursors with potential for endothelial differentiation.

OBJECTIVE: Two competing hypotheses can be formulated regarding the origin of canine hemangiosarcoma (HSA). One states HSA originates from differentiated vascular endothelial cells that undergo mutations which endow them with malignant potential. The other states HSA originates from transformed hemangioblastic stem cells. This study was designed to begin to distinguish between these possibilities, as well as to test if flow cytometry was sufficiently sensitive to detect malignant cells in blood samples from dogs with HSA. METHODS: We used multiparameter flow cytometry to examine expression of cell-surface determinants associated with hematopoietic precursors (c-kit, CD34, CD133, CD45) or with lineage-committed cells (CD3, CD11b, CD14, CD21, CD105, CD146, alphavbeta3-integrin) in HSA cell lines and in blood samples from healthy dogs or dogs with HSA. RESULTS: The data show that HSA cells coexpress surface markers associated with hematopoietic precursors and with commitment to endothelial lineage, providing a means to identify their presence in circulation and distinguish them from normal or malignant white blood cells. The percentage of cells that coexpressed these markers ranged from 0.5 to 1.25% for HSA dogs, and was less than 0.3% for unaffected dogs or dogs with HSA that had the tumors removed within 48 hours prior to obtaining samples. CONCLUSIONS: The results place the ontogeny of HSA with multipotential bone marrow-derived stem cells whose progeny arrest differentiation at the hemangioblast or angioblast stage. In addition, these expression patterns may assist to confirm an HSA diagnosis, monitor minimal residual disease, and detect the disease in early stages.

Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors.

Recognition of the domestic dog as a model for the comparative study of human genetic traits has led to major advances in canine genomics. The pathophysiological similarities shared between many human and dog diseases extend to a range of cancers. Human tumors frequently display recurrent chromosome aberrations, many of which are hallmarks of particular tumor subtypes. Using a range of molecular cytogenetic techniques we have generated evidence indicating that this is also true of canine tumors. Detailed knowledge of these genomic abnormalities has the potential to aid diagnosis, prognosis, and the selection of appropriate therapy in both species. We recently improved the efficiency and resolution of canine cancer cytogenetics studies by developing a small-scale genomic microarray comprising a panel of canine BAC clones representing subgenomic regions of particular interest. We have now extended these studies to generate a comprehensive canine comparative genomic hybridization (CGH) array that comprises 1158 canine BAC clones ordered throughout the genome with an average interval of 2 Mb. Most of the clones (84.3%) have been assigned to a precise cytogenetic location by fluorescence in situ hybridization (FISH), and 98.5% are also directly anchored within the current canine genome assembly, permitting direct translation from cytogenetic aberration to DNA sequence. We are now using this resource routinely for high-throughput array CGH and single-locus probe analysis of a range of canine cancers. Here we provide examples of the varied applications of this resource to tumor cytogenetics, in combination with other molecular cytogenetic techniques.