Vaccine-associated carcinogenesis in cats

Abstract

Animal models have been extremely valuable to cancer research for the basic study of carcinogenesis, evaluation of therapeutic approaches, and understanding the function of cancer related genes. There are scientific and ethical problems with traditional rodent models. As an alternative, spontaneously occurring tumors in pets have great potential to be informative regarding human cancer. The cell and molecular biology of pet animal tumors is relatively poorly defined, however. My commitment to the development of this research niche led me to pursue a PhD and is the basis for my thesis work. As a paradigm for the study of the carcinogenesis of companion animal tumors and the development of new cancer models, I investigated feline vaccine-associated sarcomas. Epidemiologic evidence strongly associates vaccination of cats for rabies and feline leukemia virus with the development of soft tissue sarcomas at the administration site. I investigated two etiologic theories for vaccine-associated sarcoma. In part I of my dissertation, I address the first, which is the induction of cancer by the vaccine itself. I found that feline vaccines containing aluminum adjuvant and aluminum adjuvant itself are cytotoxic and mutagenic in cultured mammalian cells, whereas vaccines that do not contain adjuvant do not induce killing or mutation. I further demonstrated that cytotoxicity and mutagenicity of adjuvanted vaccines depends upon induction by aluminum hydroxide of reactive oxygen species. Part II concerns my work to improve the existing AL mutagenicity assay that I used in evaluating feline vaccines. I created high throughput system that that saves a great deal of time and expense. The assay that I developed utilizes flow cytometry to phenotypically characterize and enumerate mutant cells and to rapidly provide mutational spectra. Finally in this part, I describe a method for comparing mutational spectra using the Euclidean distance formula and cluster analysis. In Part III, I have addressed a second etiological theory for vaccine-associated carcinogenesis, namely genetic susceptibility. Evidence supports the idea that certain cats are predisposed to develop this cancer. To investigate genetic susceptibility and determine genetic loci important in vaccine-associated carcinogenesis, I developed cytogenetic techniques for use in cats. The first step in cytogenetic studies is the preparation of metaphase nuclei for evaluation. Peripheral blood lymphocytes are frequently used for karyotyping because they are easy to collect and culture. I found, however, that feline lymphocytes were hypersensitive to apoptosis in vivo, making collection of mitotic cells more difficult than with other species. I was able to modulate apoptosis and obtain satisfactory metaphase yields by adding IL-2 to pokeweed-stimulated lymphocyte cultures. In part III, I also present the use of a novel form of fluorescent R-banding for karyotyping cats, by which I was able to identify a variable anomaly in the feline karyotype: the deletion of a band from one of the F2 homologues. I found this abnormality in 2 unrelated, clinically normal cats and in a normal feline cell line. Whether this lesion has biologic significance remains to be determined. I also karyotyped 3 cats with vaccine-associated sarcoma and failed to identify chromosomal aberrations that might be linked to cancer susceptibility. Additional work is required to reach any conclusions in this regard. Finally, I developed comparative genomic hybridization (CGH) for use in cats. CGH is a fluorescent in situ hybridization technique which can be used to rapidly screen the entire genome for numerical chromosome aberrations, for use in cats. I demonstrated the use of CGH to evaluate a tumor using a feline bronchoalveolar cell line. In summary, I have studied feline vaccine-associated sarcoma as a paradigm for studies of spontaneous cancer in companion animals and as a model for human disease. This work allowed me to develop a variety of techniques that I can apply to the study of this particular cancer as well as other animal tumors. In addition, I found that feline vaccines induce mutation through an ROS-mediated mechanism. Based upon these findings and the tentative suggestion that genetic susceptibility plays a role in vaccine-associated carcinogenesis, I propose, and am investigating, a new hypothesis: cats, some more than others, are sensitive to ROS-induced cancer. This system could provide a model to shed light on the relationship between ROS and carcinogenesis in humans.