hTERT and BIRC5 gene promoters for cancer gene therapy: A comparative study

Abstract

Abstract. Human telomerase reverse transcriptase (hTERT) and survivin (BIRC5) gene promoters are frequently used for transcriptional targeting of tumor cells, yet there is no comprehensive comparative analysis allowing rational choice of a promoter for a particular therapy. In the current study, the transcriptional activity of hTERT, human BIRC5 and mouse Birc5 promoters and their modifications were compared in 10 human cancer cell lines using the luciferase reporter gene activity assay. The results revealed that BIRC5-and hTERT-based promoters had strikingly different cell specificities with comparable activities in only 40% of cell lines. Importantly, relative hTERT and BIRC5 transcript abundance cannot be used to predict the most potent promoter. Among the hTERT-based promoters that were assessed, modification with the minimal cytomegalovirus promoter generally resulted in the most potent activity. Mouse Birc5 and modified human BIRC5 promoters were superior to the unmodified human survivin promoter; however, their tumor specificities must be investigated further. In summary, the present results emphasize the desirability for construction of more universal tumor-specific promoters to efficiently target a wide spectrum of tumor cells. IntroductionTranscriptional targeting in gene therapy is an approach based on the use of tissue-specific or tumor-specific promoters (TSPs) to direct the expression of therapeutic genes specifically to a tumor (1). It is assumed that it must meet the requirements of transgene expression in tumor tissues but not in normal tissues. This combination of 'tumor on' and 'normal tissue off' profile can result in an increase in the therapeutic index and limit the toxicity of vectors and transgenes in vivo. Numerous promoters have already been evaluated for transcriptional targeting in cancer gene therapy [for reviews, see (2-4)], such as the α-fetoprotein promoter for hepatic carcinoma, tyrosinase gene promoter for melanoma, prostate-specific antigen promoter for prostate cancer, cyclooxygenase-2 promoter for gastrointestinal cancer, midkine promoter for Wilms' tumor or neuroblastoma, chemokine (C-X-C motif) receptor 4 promoter for breast cancer and melanoma, hypoxia-inducible promoter for hypoxic tumor-targeting gene therapy, promoter of the carcinoembryonic antigen gene (5), and numerous others. However, many of the gene promoters used for this goal have been shown to be abundantly expressed in a variety of normal tissues (4), such that their usage also affects normal host cells, thus increasing the risk of unwanted side effects. TSPs that are expressed in a wide variety of tumors and have low expression in normal tissue are highly desirable to meet gene therapy demands. Two gene promoters frequently used for the gene therapeutic purposes appear to most completely satisfy the requirements: The human telomerase reverse transcriptase (hTERT) promoter, and the promoter driving the expression of BIRC5, encoding the apoptosis inhibitor survivin.Survivin is one of the central players of c...

Multidimensional integrative analysis uncovers driver candidates and biomarkers in penile carcinoma

“…This gene plays an important role in cell proliferation and apoptosis inhibition 29 . Due to the overexpression of BIRC5 during carcinogenesis, treatment targeting this gene has been increasingly recognized as a promising therapy to various cancers 30 – 32 . In our study, BIRC5 gene copy number gain and downexpression of hsa-miR-135a and hsa-miR-320 were significantly associated with increased expression levels of BIRC5 suggesting that multiple events could be involved with the aberrant activity of this gene in penile cancer.…”

Xenobiotic response elements (XRE) from human CYP1A1 gene enhance the hTERT promoter activity

“…Плазмиды, несущие кДНК репортёрного гена люциферазы под контролем двух (p2XRE), четырёх (p4XRE) и шести (p6XRE) копий элементов XRE, создавали на основе вектора pGL3-Basic ("Promega", США), последовательно клонируя по сайтам KpnI-SacI двухцепочечные олигонуклеотиды, полученные путём отжига одноцепочечных комплементарных олигонуклеотидов 5′-ccggagctcgcccaggcgttgcgtgagaa g g a c c t g c c c a g g c g t t g c g t g a g a a g g a c c c a g c t -3 ′ и 5′-gggtccttctcacgcaacgcctgggcaggtccttctcacgcaacgcctgggcgagctccgggtac-3′ (полужирным шрифтом выделена консенсусная последовательность XRE). Плазмиды, несущие кДНК люциферазы под контролем гибридного промотора (состоит из промотора hTERT и двух (p2XRE-hTERT), четырёх (p4XRE-hTERT) и шести (p6XRE-hTERT) копий элементов XRE), получали путём клонирования промотора hTERT из плазмиды phTERT-Luc [5] по сайтам XhoI-HindIII в 3′-область относительно элементов XRE в плазмиды p2XRE, p4XRE и p6XRE соответственно.…”

“…В работах [3,4] было показано, что слияние с синтетическим ТАТА-боксом или с минимальным промотором цитомегаловируса (CMV) увеличивает активность промотора hTERT. Однако активность этого промотора сильно варьируется в зависимости от происхождения опухолевых клеточных линий, что нивелирует преимущество его универсальности [5]. Всё это определяет необходимость поиска новых подходов к повышению активности промотора hTERT в опухолевых клетках с сохранением его опухолевой специфичности.…”

A Novel Hybrid Promoter ARE-hTERT for Cancer Gene Therapy

“…Since the hTERT promoter is "TATA-less," two modifications were proved to increase promoter activity: linking the promoter to a synthetic TATA-box or to a minimal early/immediate cytomegalovirus promoter to provide conventional basal promoter elements [8,9]. However, the activity of the hTERT promoter varies greatly among different tumor cell lines, which might compromise the advantage of its universality [10]. Therefore, taking into account the above considerations, there is a definite need to increase hTERT promoter activity in tumor cells while retaining its tumor cell specificity.…”