Abstract
Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.
Highlights
The transportation of macromolecules between the nucleus and cytoplasm has long been established to play a key role in normal cellular function and is often deregulated in cancer cells[1,2]
It is important to choose appropriate proteins as fraction markers: Figure 2C shows immunoblots of nuclear/cytoplasmic fractions prepared from MEC1 cells in which RNA polymerase II (Rpb[1] CTD; 250 kDa) and Lamin A/C were blotted as markers of nuclear fractions, while β-tubulin and γ-tubulin (50 kDa) were used as cytoplasmic markers
It is clear from the data that an average of five patient samples in the cytoplasmic fractions reached near significance
Summary
The transportation of macromolecules between the nucleus and cytoplasm has long been established to play a key role in normal cellular function and is often deregulated in cancer cells[1,2] Such deregulation can result from overexpression/mutation of proteins that control nuclear export. Further protein mislocalization can occur when microenvironmental signals impinge upon the cancer cells, leading to the activation of intracellular signaling pathways such as the phosphatidyl-inositol-3-kinase (PI3K)/Akt pathway, resulting in inactivation of FOXO family members and subsequent export from the nucleus[4,5] Such mislocalization of tumor suppressor proteins has been implicated in the progression of a number of hematological and solid tumors[1,2,6]. The use of primary samples from CLL patients described here have been approved by the West of Scotland Research Ethics Service, NHS Greater Glasgow and Clyde (UK) and all work was carried out in accordance with the approved guidelines
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