Abstract 835: Functional studies of the hematopoietic-specific tubulin isotype Hβ-1

The tissue-specific expression of tubulin isotypes has been well described, although–with few exceptions_unique roles for the tubulin isotypes within their respective tissues have not been found. β-1 tubulin (Hβ-1 or Class VI in humans) is a hematopoietic-restricted tubulin isotype restricted to platelets and megakaryocytes in the mouse; human expression has not been fully studied. Hβ-1 tubulin is unique among β-tubulin isotypes, with the most sequence diversity. This diversity lies mainly in the C-terminus where microtubule-associated proteins (MAPs) are hypothesized to interact. Furthermore, Hβ-1 has known small nucleotide polymorphisms (SNPs), some of which have been shown to have profound physiologic effects. These SNPs include the Q43P substitution with a phenotype including spherocytosis, impaired platelet aggregation, and possible cardioprotection against thrombosis, and R318W which is implicated in macrothrombocytopenia. Other SNPs have undetermined clinical significance. Given these properties of Hβ-1 tubulin, as well as the diverse clinical manifestations of leukemias and myeloproliferative neoplasms, we have begun to investigate the possible roles of Hβ-1 tubulin and its associated SNPs in these diseases. We have developed a highly specific polyclonal antibody against the Hβ-1 tubulin C-terminus. We found that in addition to megakaryocytes and platelets, Hβ-1 is expressed in CD34+ cells and in mature cells of myelogenous origin, but not in lymphocytes. Using the CML-derived K562 cell line, we performed indirect immunofluorescence for Hβ-1. Although Western blot confirmed the presence of Hβ-1 expression in this cell line, immunofluorescence did not show the expected microtubule network pattern, but rather a more diffuse, punctate cytoplasmic distribution similar to that seen in CD34+ and myeloid cells. To further explore this unique pattern, we transfected wild-type Hβ-1 tubulin into K562 cells as well as PC3 cells, a prostate cancer-derived cell line which has no detectable native Hβ-1 expression. Similar to previously published data, Hβ-1 tubulin labeling in PC3 cells showed a tubular pattern overlapping with the native MT network, suggesting free incorporation into the native MT network in PC3 cells. In K562, however, Hβ-1 immunofluorescence was punctate and did not completely co-localize with the native MT network, even with variations in the methods of cell fixation and permeabilization. This suggests that Hβ-1 tubulin may be sequestered from free incorporation into the native MT network in this malignant cell line, a novel process never previously described for Hβ-1 or other tubulin isotypes. Further studies are ongoing to elucidate the unique role of Hβ-1 tubulin in these malignant cells as well as clinical correlative studies to investigate the possible roles SNPs within Hβ-1 may play in disease manifestations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 835.