Abstract
The p53 gene, metaphorically named “the Guardian of the Genome“ by David Lane in 1992, is one of the most important decision makers inside the cell. By interacting with an array of downstream genes, p53 can regulate cell fate, either by precipitating events leading to cell death by apoptosis, or by acting as a regulatory factor during G1/S phase allowing the cell to repair minor damage to DNA and proceed to the next stage of cell division. Playing such a pivotal role within the cell, p53 itself is subjected to a tight and orchestrated control, creating a network of positive and negative regulations via a number of interacting proteins (MDM-2, Wip-1, Cyclin G, p14ARF). Yet another level of p53 regulation is represented by post-translational modifications, modulating its transcriptional/transactivational ability. These modifications include phosphorylations on serines 15, 18 and 20 or acetylations at C-terminal lysines of p53. Recently, it has been found that the regulation of p53 is also substantially controlled at the transcriptional level. They identified nine different splicing variants of p53 with distinct biological characteristics, resulting from combinations of an alternative splicing of intron 2, 9 and/or aberrant transcription, starting at so-far unrecognized cryptic promoter in intron 4. Herein we present evidence of a novel splicing variant of p53 gene, termed delta ex6 that is differentially expressed in patients with chronic lymphocytic leukemia (CLL) as compared to healthy donors. The delta ex6 variant was identified in 109 out of 127 (86%) CLL patients, while in healthy individuals it was not detected. Delta ex6 variant is devoid of transactivational activity as determined in vitro by FASAY (Functional Analysis of Separated Alleles in Yeast). To test the biological properties of the delta ex6 variant we have cloned its whole coding sequence and transfected the p53-double-knock-out model cell line H1299 to produce stable integrants. Stable H1299 cell lines expressing the delta ex6 variant were distinguished by a remarkable loss of intercellular contacts and semi-suspension growth properties, in contrast to the strictly adherent growth of the parental cells and mock-transfected cells. Four stable delta ex6 producing H1299 cell lines, as well as control cell lines in doublets (parental H1299 harboring the cloning vector, and H1299 stably transfected with wild type p53) were subjected to the Affymetrix GeneChip Human Exon 1.0 ST array expression analysis. The microarray data corroborated the accented and proliferative phenotype as observed in vitro in the tissue culture: overexpression of a number of cyclins (A1, G1, G2, F, I, B2, A2, T2), matrix metalloproteinases, hyaluronidases and caspase inhibitors; and downregulation of adhesion molecules and molecules of the intercellular matrix. Our data on the presence of the delta ex6 p53 variant in CLL patients supports the recent evidence on dysregulation of p53 splicing pattern in malignancies. Moreover, as assessed in vitro, overexpression of the delta ex6 variant leads to an accented and proliferative phenotype, a finding further supporting the biological role of the novel delta ex6 p53 variant in vivo.
Ligand-based pharmacophore model for the discovery of novel CXCR2 antagonists as anti-cancer metastatic agents
