Highly Specific Antibodies for Co-Detection of Human Choline Kinase α1 and α2 Isoforms

Abstract

BackgroundCholine kinase is the first enzyme in the CDP-choline pathway that synthesizes phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase exists as three isoforms (CKα1, α2, and β). Specific inhibition of CKα has been reported to selectively kill tumoral cells. Monoclonal and polyclonal antibodies against CKα used in previous studies to detect the level of this isozyme in different cellular or biochemical contexts were able to detect either the α1 or the α2 isoform.Methodology/Principal FindingsIn this study, an antiserum against CKα was produced by immunizing rabbits with denatured, purified recombinant CKα2 full-length protein. This antiserum was highly specific for CKα when tested with extracts from different cell lines, and there was no cross reactivity with purified CKβ and other related proteins like human ethanolamine kinases (EK) and yeast choline or ethanolamine kinases. The antiserum simultaneously detected both CKα1 and α2 isoforms in MCF-7 and HepG2 cell extracts, but not in HeLa, HCT-116, and mouse embryonic stem cell extracts. Subsequent protein dot blot assay of total CKα in a human normal/tumor protein array of 30 tissue samples by using the antiserum showed that CKα was not overexpressed in all tumor tissues when compared to their normal counterparts. Most striking differences between tumor and normal CKα expression levels were observed in kidney (11-fold higher in tumor) and liver (15-fold lower in tumor) samples.Conclusion/SignificanceApart from its high sensitivity and specificity, the antiserum produced in this work, which does not require further purification, has the advantage of co-detecting both α1 and α2 isoforms in cell extracts for direct comparison of their expression levels.

Balance of human choline kinase isoforms is critical for cell cycle regulation

“…These findings strongly support the role of CK in human cancer pathogenesis and suggest that the levels of CK and PCho could be used as tumor markers [36]. More recently, higher levels of CKα, but not of CKβ, were detected in multiple human breast cancer cell lines [37,38]. CKα was also considered as a potential prognostic factor for identifying nonsmall‐cell lung cancer patients with a high risk of recurrence [39].…”

“…The highly specific CKα polyclonal antibody [38] was used to detect CKα by indirect immunofluorescence (Fig. 1C).…”

“…Extracts of cytoplasm and nucleoplasm from HeLa cells were kindly provided by R. Luehrmann (MPIbpc, Goettingen, Germany). Polyclonal rabbit anti‐CKα serum was produced as described previously [38]. Polyclonal anti‐CKβ IgG was produced by immunizing rabbit with highly purified recombinant human CKβ protein [38] and the immunoglobulin was obtained by affinity purification.…”

Novel 4‐Amino Bis‐pyridinium and Bis‐quinolinium Derivatives as Choline Kinase Inhibitors with Antiproliferative Activity against the Human Breast Cancer SKBR‐3 Cell Line

“…[4,5] The present work focuses on the inhibitory interaction of novel compounds with human ChoKa2. We aimed to characterize their specific properties with respect to enzymatic mechanism, substrate selectivity, catalytically important residues, protein stability, and distinctive tertiary structure elements.…”

“…Families of bis-pyridinium compounds(1)(2)(3)(4)(5)7), and bis-quinolinium compounds (6) prepared by our research group.…”