The research presented in this dissertation focuses on the effects of expression of two different genes in cancer. First is a study on the loss of expression of the tumor suppressor gene, E-cadherin, in canine mammary cancer. The loss of E-cadherin has been associated with increased tumor invasiveness and poor prognosis in a number of neoplasias including human pancreatic, colorectal and breast cancer. This loss is most frequently attributed to mutations, deletions and hypermethylation of the E-cadherin promoter. The mechanism for the loss of E-cadherin expression in canine mammary cancer is not well understood. To examine the role of methylation in E-cadherin gene silencing, bisulfite sequencing was used. First, expression of E-cadherin in normal, hyperplastic, benign and malignant canine mammary tissue was determined using immunohistochemistry. The mammary gland was then isolated using laser capture microdissection and the DNA was purified. The purified DNA underwent bisulfite treatment and subsequent bisulfite sequencing to determine the degree of methylation within each sample. The immunohistochemistry confirmed a correlation between tissue diagnosis and the level of membranous staining for E-cadherin. A correlation between methylation and this difference in gene expression could not be determined, however.
The second gene examined in this dissertation is the viral ongogene, Tax. Tax is encoded in the regulatory pX region of the Human T Lymphotrophic Virus Type-1 (HTLV-1) genome. HTLV-1 is the causative agent of adult T-cell leukemia/lymphoma (ATL) and HTLV-1 Tax has been shown to be necessary and sufficient for viral oncogenesis. To study the effect of Tax in oncogenesis and cancer progression, we examined transgenic mice that express HTLV-1 Tax under regulation of the human granzyme B promoter as well as double transgenic mice that express HTLV-1 Tax and are also deficient in IFN-γ (Tax +/IFN-γ-/-). Through gross examination, histology, flow cytometry, immunohistochemistry and imaging, we determined that these mice develop histiocytic sarcomas that arise from tendon sheaths and invade bone resulting in osteolysis.
To further understand the function of HTLV-1 Tax in tumor formation and progression, three cell lines were derived from tumors that spontaneously formed in Tax+/IFN-γ-/- mice. These cell lines, T94, 501 and Tom3 were all immortal in culture. T94 cells were determined to be a poorly differentiated sarcoma and 501 and Tom3 cells were determined to be macrophage/monocyte origin based on flow cytometry. All three cell lines formed tumors when injected subcutaneously, intratibially and bone adjacent in nude mice. Mice injected with T94 and 501 cells developed humoral hypercalcemia of malignancy (HHM) which correlated well with increased expression of PTHrP in these cell lines. Mice injected with Tom3 cells developed splenomegaly as the result of increased myelopoiesis and marked neutrophilic infiltration into the tumors. This finding corresponded to a marked increase in granulocyte-colony stimulating factor expression by this cell line compared to the other two. Finally, an increase in ex vivo osteolysis was found in 501 and Tom3 cells. This finding correlated with increased expression of IL-1α and IL-1β in these cell lines.
In summary, in this dissertation research, we found a correlations between loss of E-cadherin expression and invasive mammary cancer in dogs, and increased expression of PTHrP, IL-1 and G-CSF that correlated to the development of HHM, osteolysis and neutrophilic infiltration in Tax-induced malignant cells in vivo and in vitro. These findings support further use of these model systems for mechanistic and therapeutic studies of breast cancer and ATL.
|Commitee:||La Perle, Krista, Niewiesk, Stefan, Stromberg, Paul|
|School:||The Ohio State University|
|School Location:||United States -- Ohio|
|Source:||DAI-B 78/11(E), Dissertation Abstracts International|
|Keywords:||Canine mammary, E-Cadherin, HTLV-1 Tax, Mouse model, Retrovirus|
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