Cancer specificity of promoters of the genes involved in cell proliferation control

ABSTRACT Core promoters with adjacent regions of the human genes CDC6, POLD1, CKS1B, MCM2, and PLK1 were cloned into a pGL3 vector in front of the Photinus pyrails gene Luc in order to study the tumor specificity of the promoters. The cloned promoters were compared in their ability to direct luciferase expression in differ-ent human cancer cells and in normal fibroblasts. The cancer-specific promoter BIRC5 and non-specific CMV immediately early gene promoter were used for comparison. All cloned promoters were shown to be substan-tially more active in cancer cells than in fibroblasts, while the PLK1 promoter was the most cancer-specific and promising one. The specificity of the promoters to cancer cells descended in the series PLK1, CKS1B, POLD1, MCM2, and CDC6. The bidirectional activity of the cloned CKS1B promoter was demonstrated. It apparently directs the expression of the SHC1 gene, which is located in a “head-to-head” position to the CKS1B gene in the human genome. This feature should be taken into account in future use of the CKS1B promoter. The cloned promoters may be used in artificial genetic constructions for cancer gene therapy.KEYWORDS promoter; cloning; cancer-specific; cancer gene therapy.ABBREVIATIONS TSS – transcription start site.INTRODUCTIONthe design of genetically engineered vectors that ex-press products that are toxic for tumor cells holds an important position among the topical directions in the development of antitumor agents. these vectors need to contain cancer-specific regulatory elements that can en-sure both the expression of the therapeutical gene in the maximum possible number of tumors and the absence of expression in normal tissues. today, the number of promoters known to have these properties is limited.While searching for new cancer-specific promoters, we have put forward a hypothesis that many promot-ers participating in DnA replication may exhibit tu-mor specificity, since the disturbed regulation of cell division is considered to be the common property of all tumors. In order to verify this hypothesis, we cloned the promoters of several genes participating in DnA synthesis and cell division and assessed the ability of these promoters to direct the expression of the reporter gene in normal and tumor cells of different origins. Pro-moters of the CDC6, POLD1, CKS1B, MCM2, and PLK1 genes were used for cloning.the CDC6 gene product is the homologue of Saccha-romyces cerevisiae cDc6, a protein essential for the initi-ation of DnA replication. cDc6 regulates the early stag-es of DnA replication and helps control the check-point determining the termination of DnA replication before mitosis begins. A disturbed regulation of CDC6 expres-sion is associated with a high risk of cancer development [1, 2]. the POLD1 gene encodes the catalytic subunit of DnA polymerase δ, which participates in the replication and reparation of human genomic DnA. this subunit exhibits polymerase (synthesis of DnA) and exonucle-ase (in the 3’–>5’ direction) activities. Moreover, POLD1 participates in the completion of the Okazaki fragments initiated by the DnA polymerase α/primase complex. the frequency of the development of spontaneous tu-mors is higher in mice with a deficient DnA polymer-ase δ function [3]. the cKS1B protein is a component of cDc28 protein kinase required for embryogenesis and correct alternation of the phases of the somatic cell cycle [4]. cKS1B forms a complex with the cDc2 protein and regulates the transcription of the CDC20 gene. the in-teraction between cKS1В and the SKP2–cyclin e-p27