Generation of monoclonal antibodies against tumor-associated antigen MX35/sodium-dependent phosphate transporter NaPi2b.

Tumor-associated antigen MX35, which is overexpressed in 70-90% of epithelial ovarian cancers, has been recently identified as phosphate transporter NaPi2b. This finding has raised significant interest in understanding NaPi2b function under physiological conditions and its deregulation in human pathologies, such as cancer. As a member of the sodium-dependent phosphate transporter family, NaPi2b is primarily involved in the maintenance of phosphate homeostasis in the human body. The role of NaPi2b in oncogenic transformation and malignant growth is not well understood. To date, several monoclonal antibodies specific to NaPi2b have been reported. However, available monoclonal antibodies are not very efficient in recognizing endogenous NaPi2b under reducing conditions. In addition, these antibodies could not recognize the mutant form of transporter (NaPi2b-T330V). In this study we describe the production of monoclonal antibodies raised against the N-terminal region of NaPi2b. One of them, designated N-NaPi2b(15/1), possesses very useful immunological characteristics. We found that N-NaPi2b(15/1) specifically recognizes NaPi2b protein in immunohistochemical analysis and immunoprecipitation assay. Importantly, N-NaPi2b(15/1) antibody detects very efficiently endogenous and expressed wild-type and mutant forms of NaPi2b under both reducing and non-reducing conditions in Western blot analysis. These features make N-NaPi2b(15/1) antibody a very useful tool for studying the pattern of NaPi2b expression in health and pathologies.

Monoclonal antibodies to Ki-67 protein suitable for immunohistochemical analysis.

Detection of cell proliferation index is widely used in experimental and clinical research. Earlier it was shown that nuclear Ki-67 protein expression is strictly related to cell proliferation. It was revealed during all active phases of the cell cycle in mammals but was absent in G0 phase, so Ki-67 presence in cell nuclei reflects a potential growth fraction of whole cell population. The main area of Ki-67 antibody application is in immunocytochemical and immunohistochemical analyses. The aim of our work was to generate mouse monoclonal antibodies for Ki-67 antigen detection in mammalian tissues and in cultured cells. His-tagged fragment of Ki-67 expressed in bacteria was used as an antigen. Antibody-producing hybridoma cells were generated by standard procedure by fusing SP2/0 myeloma cells with splenocytes of immunized mice. Monoclonal antibodies were analyzed using paraffin-embedded human melanoma tissue samples and breast cancer cell line MCF-7. It was shown that generated anti-Ki-67 antibodies revealed proliferating cells in MCF-7 culture and after heat-induced epitope retrieval on paraffin sections of human melanoma tissue. In summary, generated antibodies could be useful for detection of proliferating cells in immunohistochemical and immunofluorescence studies of mammalian cells and tissues.

Monoclonal antibodies with selective specificity towards different glycosylation isoforms of FGFR1.

Fibroblast growth factor receptor 1 (FGFR1) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes including cell proliferation, differentiation, survival, and tumorigenesis. This diversity is possibly mediated by the existence of multiple FGFR1 isoforms, generated by alternative splicing and post-translational modifications, mainly through glycosylation. In this study we report the generation and characterization of a panel of monoclonal antibodies directed towards FGFR1. To achieve this, we used as an antigen a fragment of FGFR1, corresponding to loop II-III of the extracellular domain, which shares low homology to other members of the FGFR family and possesses numerous antigentic determinants. Two rounds of ELISA screening and Western blot analysis allowed us to isolate a panel of monoclonal antibodies, which recognize specifically recombinant FGFR1 loop II-III. The ability of generated antibodies to recognize endogenous FGFR1 was examined in 3T3 L1 cells, which are known to express FGFR1, but not other members of FGFR family. Immunoblot analysis of 3T3 L1 cell lysates with hybridoma media of selected clones revealed a different, but overlapping pattern of immunoreactive bands, which might represent splicing and post-translationally modified forms of FGFR1. Furthermore, we also tested the cross-reactivity of generated antibodies towards recombinant full-length FGFR3 and their ability to recognize FGFR1 in 3T3 L1 cells by cyto- and immunocytochemistry. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR1 in normal and pathological tissues.

Development of monoclonal antibodies specific for the human sodium-dependent phosphate co-transporter NaPi2b.

Homeostasis of inorganic phosphate in the human body is maintained by regulated absorption, metabolism, and excretion. Sodium-dependent phosphate transporters (NaPi) mediate the transport of inorganic phosphate (P(i)) in cells in response to dietary phosphate consumption, hormones, and growth factors. NaPi2b is a member of the sodium-dependent phosphate transporter family, with a distinct pattern of expression and regulation. Signaling pathways activated by mitogens, glucocorticoids, and metabolic factors have been implicated in regulating P(i) transport via NaPi2b. Inactivation of NaPi2b function by mutations has been linked to human pathologies, such as pulmonary alveolar microlithiasis. In this study, we describe the generation and characterization of monoclonal antibodies against human NaPi2b. The monoclonal antibodies were shown to recognize specifically transiently overexpressed and endogenous NaPi2b in commonly used immunoassays, including Western blotting, immunoprecipitation, and immunohistochemistry. These properties make them particularly valuable reagents for elucidating NaPi2b function in health and disease.

Generation and characterization of monoclonal antibodies against tuberous sclerosis complex 2.

TSC1 and TSC2 are two recently identified tumor suppressor genes encoding hamartin and tuberin, respectively. They have been implicated in the pathogenesis of tuberous sclerosis, a neurological disorder linked with the development of hamartomas in numerous organs, including the brain, kidneys, heart, and liver. Both protein products of TSC1 and TSC2 form an intracellular complex exerting GTPase-activating (GAP) activity towards a small G protein Rheb (Ras homologue enriched in brain). Inhibition of Rheb is important for the positive regulation of mTOR pathway, while mutations of hamartin or tuberin result in uncontrolled cell cycle progression. Although the precise role for the TSC1/2 complex in tumor suppression is not clear, many studies have established a link with the regulation of transcription and protein biosynthesis, increasing susceptibility to apoptosis, cell differentiation, and cell cycle control. We describe the development of a monoclonal antibody specific towards TSC2/tuberin and characterize the suitability for Western blotting, immunoprecipitation, and immunofluorescent applications. The C-terminal region of TSC2 was expressed as a His-tag fusion protein in bacteria, affinity purified and used as an immunogen. Hybrid myelomas were produced from the spleenocytes of immunized mice and SP2/0 myeloma cells. Testing the specificity of cell culture supernatants from generated hybridomas towards recombinant His-TSC2C in ELISA assay allowed us to isolate a panel of positive clones. Further analysis of selected clones by Western blotting and immunoprecipitation revealed one clone, termed D6, which specifically recognized recombinant and endogenous TSC2. The specificity of generated antibody was also confirmed in TSC2(/) and TSC2(+/+) mouse embryo fibroblasts. In summary, the produced antibody is a useful tool in our research program and will be available for researchers investigating signal transduction pathways involving TSC1/2 signaling under physiological conditions and in human pathologies.

Immunohistochemical analysis of Ki-67, PCNA and S6K1/2 expression in human breast cancer.

AIM: To assess the correlation between the expression profiles of ribosomal protein S6 kinase (S6K1/2), Ki-67 nuclear antigen (Ki-67) and proliferating cell nuclear antigen (PCNA) in human breast adenocarcinomas. METHODS: The expression pattern of S6K1/2, Ki-67 and PCNA has been investigated by immunohistochemical analysis of formalin fixed paraffin embedded sections of 40 human breast adenocarcinomas. Analysis was performed using specific monoclonal and polyclonal antibodies directed against S6K2, Ki-67 and PCNA followed by semi-quantitative measurements. RESULTS: Positive correlation between nuclear staining for S6K2 and PCNA (p < or = 0.01) or Ki-67 (p < or = 0.01) in breast cancer cells has been detected. No significant correlation in nuclear staining was observed between S6K1 and PCNA or Ki-67. In addition, we have detected an increase in S6K2 expression in the nuclei of cancer cells localized predominantly in peripheral areas of the tumor contacting with normal tissue. CONCLUSION: The accumulation of S6K2, but not S6K1, in the nuclei of cancer cells and the correlation with the expression of PCNA and Ki-67 suggest the involvement of S6K2 in the regulation of malignant growth.

Immunohistochemical analysis of S6K1 and S6K2 expression in human breast tumors.

AIM: To express recombinant S6K2 in baculovirus expression system; to purify large quantities of recombinant S6K2 for biochemical studies; to generate and characterise specific MABs against recombinant S6K2; to study the patterns S6K1 and S6K2 expression and subcellular localization in normal, benign and malignant breast tissues. METHODS: Recombinant baculovirus, expressing wild type S6K2 was generated using Bac-to-Bac system (Invitrogen); recombinant S6K was purified from infected Sf9 cells using affinity purification approach; monoclonal antibodies against recombinant S6K2 were generated; the specificity of generated MABs towards recombinant and endogenous S6K2 were examined by ELISA, Western blotting, immunoprecipitation and immuhohistochemical staining; immunohistochemical detection of S6K1 and S6K2 in human breast tissues was performed using specific monoclonal antibodies towards S6K1 and S6K2. RESULTS: Large amounts of enzymatically active S6K2 were purified using baculovirus expression system; highly purified preparations of S6K2 were used to generate and characterize anti-S6K2 MABs; elevated levels of S6K1 and S6K2 were found in breast tumors when compared to normal breast tissues; S6K2 is frequently localized in the nuclei of adenocarcinoma tissues, but rarely in fibroadenoma or "normal" breast tissues. CONCLUSION: Production of recombinant S6K2 in large amount and generation of specific monoclonal antibodies towards S6K2 has provided us with excellent tools to study the function and regulation of this important signalling molecule in normal and cancer cells. Immunnohistochemical analysis of S6K1 and S6K2 expression in normal and malignant breast clearly indicates that both kinases are overexpressed in breast tumors, when compared to "normal" tissues. The retention of S6K2 in the nuclei of malignant cells may be caused by disregulation of nucleocytoplasmic shuttling and could subsequently affect cell growth and proliferation.

Molecular cloning of CoA Synthase. The missing link in CoA biosynthesis.

Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of approximately 60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.