ABSTRACT
B23/NPM is a multifunctional nucleolar protein frequently overexpressed, mutated, or rearranged in neoplastic tissues. B23/NPM is involved in diverse biological processes and is mainly regulated by heteroligomer association and posttranslational modification, phosphorylation being a major posttranslational event. While the role of B23/NPM in supporting and/or driving malignant transformation is widely recognized, the particular relevance of its CK2-mediated phosphorylation remains unsolved. Interestingly, the pharmacologic inhibition of such phosphorylation event by CIGB-300, a clinical-grade peptide drug, was previously associated to apoptosis induction in tumor cell lines. In this work, we sought to identify the biological processes modulated by CIGB-300 in a lung cancer cell line using subtractive suppression hybridization and subsequent functional annotation clustering. Our results indicate that CIGB-300 modulates a subset of genes involved in protein synthesis (ES = 8.4, p < 0.001), mitochondrial ATP metabolism (ES = 2.5, p < 0.001), and ribosomal biogenesis (ES = 1.5, p < 0.05). The impairment of these cellular processes by CIGB-300 was corroborated at the molecular and cellular levels by Western blot (P-S6/P-4EBP1, translation), confocal microscopy (JC-1, mitochondrial potential), qPCR (45SrRNA/p21, ribosome biogenesis), and electron microscopy (nucleolar structure, ribosome biogenesis). Altogether, our findings provide new insights on the potential relevance of the CK2-mediated phosphorylation of B23/NPM in cancer cells, revealing at the same time the potentialities of its pharmacological manipulation for cancer therapy. Finally, this work also suggests several candidate gene biomarkers to be evaluated during the clinical development of the anti-CK2 peptide CIGB-300.
Subject(s)
Casein Kinase II/genetics , Neoplasms/genetics , Nuclear Proteins/metabolism , Ribosomes/genetics , Apoptosis/drug effects , Casein Kinase II/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Energy Metabolism/genetics , Humans , Neoplasms/drug therapy , Neoplasms/pathology , Nuclear Proteins/genetics , Nucleophosmin , Peptides, Cyclic/administration & dosage , Phosphorylation/drug effects , Ribosomes/metabolismABSTRACT
The SV40t polyadenylation and splicing signals of the pAEC plasmid vectors were replaced by synthetic intron and synthetic rabbit beta globin-based termination/polyadenylation sequences, and 5, 10, and 20 copies of the 5'-AACGTT-3' CpG motif were inserted. Balb/c mice were immunized by intramuscular injection of 200 microg of each plasmid, coding for the HIV-1 multiepitope TAB9, under the control of the human cytomegalovirus promoter. After three doses of DNA, a fourth boost with plasmid DNA or a TAB9-expressing recombinant fowlpox virus rFPTAB9LZ was administered. ELISA and ELISPOT assays were conducted for antibody and IFN-gamma-secreting cell-mediated responses' evaluation against the whole TAB9 and the TAB9's IIIB V3 peptide, respectively. Serum IgG antibodies were not detected. Effector IFN-gamma-secreting responses were only detected on the animals receiving the new set of DNA constructs, alone or in combination with a recombinant virus boost, with or without in vitro re-stimulation. The response was dependent on the new transcriptional unit and influenced by the number of CpG motifs. We showed that plasmid backbone optimization based on these two factors could enhance the response against a multiepitope-based DNA vaccine. A new family of plasmid vectors is also available for evaluation with desired antigens.
