Cancer Specificity of Promoters of the Genes Controlling Cell Proliferation

Abstract

Violation of proliferation control is a common feature of cancer cells. We put forward the hypothesis that promoters of genes involved in the control of cell proliferation should possess intrinsic cancer specific activity. We cloned promoter regions of CDC6, POLD1, CKS1B, MCM2, and PLK1 genes into pGL3 reporter vector and studied their ability to drive heterologous gene expression in transfected cancer cells of different origin and in normal human fibroblasts. Each promoter was cloned in short (335-800 bp) and long (up to 2.3 kb) variants to cover probable location of core and whole promoter regulatory elements. Cloned promoters were significantly more active in cancer cells than in normal fibroblasts that may indicate their cancer specificity. Both versions of CDC6 promoters were shown to be most active while the activities of others were close to that of BIRC5 gene (survivin) gene promoter. Long and short variants of each cloned promoter demonstrated very similar cancer specificity with the exception of PLK1-long promoter that was substantially more specific than its short variant and other promoters under study. The data indicate that most of the important cis-regulatory transcription elements responsible for intrinsic cancer specificity are located in short variants of the promoters under study. CDC6 short promoter may serve as a promising candidate for transcription targeted cancer gene therapy.

Construction of a Combinatorial Library of Chimeric Tumor-Specific Promoters

“…To prepare the library, cancer specific promoters CDC6 (803 bp), POLD1 , (568 bp), CKS1B , (334 bp), MCM2 (367 bp), PLK1 (439 bp) (12), BIRC5 (269 bp), TERT, (243 bp) (20), and the strong universal PCNA promoter (389 bp) (22) were used. Promoters were fragmented by restriction digestion, and fragments were ligated together to generate random chimeric promoter combinations.…”

“…Promoters from the CDC6 , POLD1 , CKS1B , MCM2 , PLK1, and PCNA genes were amplified from pAL-TA-based plasmid clones using Pfu DNA polymerase (Thermo Fisher Scientific, Inc.; Waltham, MA) as described earlier (12,22). All primers and PCR protocols are presented in Supplementary Table S1 and the Supplementary Materials and Methods.…”

“…In all experiments, co-transfection with plasmid pRL-TK (Promega) was used as an internal control. Parallel transfections of the cells with pGL3 Basic Vector (pGL3-BV); pGL Promoter Vector (pGL3-PV) (Promega); pGL3-CMV Pr/Enh Vector [pGL3-pCMV, containing an Ase I/ Bgl II fragment of the CMV promoter of immediate early genes from the pEGFP-N1 plasmid (Clontech Laboratories, Inc., Mountain View, CA)]; as well as with the original MCM2 cloned in pGL3 (12); were performed in each experiment. The intensity values for firefly luciferase luminescence were normalized to those for Renilla luciferase, and data from duplicate wells were averaged.…”

“…Even relatively strong tumor-specific promoters with a comparatively wide spectrum of activity, such as the phSurv promoter from the BIRC5 gene for the apoptosis inhibitor survivin (57) or the TERT gene promoter (phTERT) from the human telomerase catalytic subunit gene (810), are not active in all types of cancer, and their relative activity significantly varies between tumor cell lines. For instance, BIRC5 promoter activity varies from 0.06%16% (7,11,12) to 10%40% (13) of CMV promoter activity.…”

“…Promoters in this library contained various combinations of randomly ligated fragments of eight human promoters involved in control of cell proliferation. Promoters of the CDC6, POLD1, CKS1B, MCM2, PLK1, BIRC5, TERT , and PCNA genes, which were studied in our lab earlier (12,20,22), were used in the construction of this library. All promoters except the PCNA promoter were shown to be tumor-specific but rather weak (12,20), whereas the PCNA promoter is relatively strong (up to 30-fold compared with an SV40 promoter) and active in both normal and tumor cells of different origins (22).…”