Abstract
Abstract DNA salvage pathway enzymes are known to be upregulated both within cancer cells and in the serum of cancer patients. These enzymes include TK1, APRT, HGPRT, and dCK. TK1 has been extensively studied and is known to be a cytosolic enzyme. However, relatively few studies have investigated the localization of the other three salvage pathway enzymes in cancer cells. The purpose of this study was to determine the localization of these salvage pathway enzymes in various types of cancer cells. Cancer cells and normal lymphocytes were embedded and prepared for immunolabeling transmission electron microscopy (TEM). Raji cells, a Burkitt's lymphoma cell line known to express high levels of TK1, were used to optimize the TEM protocol. Using rabbit polyclonal antibodies, we were able to determine where the salvage pathway enzymes are localized in cancer cells. Cancer cell lines studied included Raji, Jurkat (acute T cell leukemia), HL60 (acute promyelocytic leukemia), HT29 (colorectal adenocarcinoma), SW620 (colorectal adenocarcinoma from lymph node), and H460 (large cell lung carcinoma). Normal lymphocytes (both unstimulated and stimulated with PHA) were also embedded and prepared for labeling. An antibody control, and anti-Macrophage marker, was used as an isotype control to detect non-specific binding. We observed that DNA salvage pathway enzymes were localized in distinctive parts throughout the cytoplasm, the cell membrane, and the nucleus. The expression of APRT appeared very similar to the expression of TK1 and it was visualized in approximately the same patterns. The same appeared to be true for HGPRT and dCK, but with much lower level of expression than TK1 and ARPT. In several cases, these salvage pathway enzymes appeared to localize near the nuclear pores, as well as other parts of the nuclear membrane. These enzymes also appeared dispersed throughout the cytoplasm, and in some cases formed clusters, especially near the cellular membrane, possibly indicating exocytosis routes. There was no significant difference between the various types of cancer cell lines in the localization of these enzymes Our normal lymphocyte controls show markedly lower expression, while the secondary only and isotype controls showed no staining. Further research is needed to determine the mechanisms of this localization and the role of these enzymes in tumor cell growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 905. doi:10.1158/1538-7445.AM2011-905
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