A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system

BACKGROUND: Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. RESULTS: In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems. CONCLUSION: This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo

Comprehensive characterization of PTEN mutational profile in a series of 34,129 colorectal cancers

Loss of expression or activity of the tumor suppressor PTEN acts similarly to an activating mutation in the oncogene PIK3CA in elevating intracellular levels of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), inducing signaling by AKT and other pro-tumorigenic signaling proteins. Here, we analyze sequence data for 34,129 colorectal cancer (CRC) patients, capturing 3,434 PTEN mutations. We identify specific patterns of PTEN mutation associated with microsatellite stability/instability (MSS/MSI), tumor mutational burden (TMB), patient age, and tumor location. Within groups separated by MSS/MSI status, this identifies distinct profiles of nucleotide hotspots, and suggests differing profiles of protein-damaging effects of mutations. Moreover, discrete categories of PTEN mutations display non-identical patterns of co-occurrence with mutations in other genes important in CRC pathogenesis, including KRAS, APC, TP53, and PIK3CA. These data provide context for clinical targeting of proteins upstream and downstream of PTEN in distinct CRC cohorts.Loss of the tumour suppressor gene PTEN leads to the activation of pro-tumourigenic signalling pathways. Here, the authors analyse sequencing data from a large cohort of colorectal cancer patients harbouring PTEN mutations and identify distinct patterns of associations with genomic and clinical features

Candidate Variants in DNA Replication and Repair Genes in Early-Onset Renal Cell Carcinoma Patients Referred for Germline Testing

Background: Early-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined. Methods: Here, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes. Results: Analysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of [Formula: see text]H2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased [Formula: see text]H2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities. Conclusions: Together, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC

A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system-1

<p><b>Copyright information:</b></p><p>Taken from "A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system"</p><p>BMC Biotechnology 2004;4():9-9.</p><p>Published online 29 Apr 2004</p><p>PMCID:PMC420247.</p><p>Copyright © 2004 Sudomoina et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p>; the total amount of plasmid DNA was constant at 0.5 μg per test point. As graphically represented, at each apex of the triangle shown, only one of the test plasmids is present (100% of mix), while opposite the matrix this plasmid is absent (0% of mix), with the two other plasmids present in equal proportions. White circles indicate ratios of plasmids evaluated for degree of induction of SEAP activity. Degree of induction reflects difference between growth without dimerizer and with tetracycline (OFF), versus with dimerizer and without tetracycline (ON). Based on the ON:OFF ratios obtained, the software program "Statistica" modeled the optimal response surface, shown as a contour plot on the triangle. Faded edges of the triangle represent limitations of the model, because it is meaningless whenever one of the components is absent. The optimal ratio for plasmid transfection with the DDC system was 1.5:1.5:1 (pCN-RS/ZF3:pZBS-Tet-OFF:pBI-EGFP-SEAP). This contrasted with a ratio of 3:1 (activator proteins:reporter) used for the original ARIAD system

A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system-3

<p><b>Copyright information:</b></p><p>Taken from "A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system"</p><p>BMC Biotechnology 2004;4():9-9.</p><p>Published online 29 Apr 2004</p><p>PMCID:PMC420247.</p><p>Copyright © 2004 Sudomoina et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p>imerizer only; scale is on the right Y-axis) and the SEAP gene (tTA-dependent transcription expression responsive to dimerizer AND tetracycline; scale is on the left Y-axis). All cells contain pCN-RS/ZF3, pZI-hGH-2, pZBS-Tet-OFF, and pBI-EGFP-SEAP. See Results for details of manipulations A-D; briefly, A was grown in continuous dimerizer-/tetracycline+; B-D were initially in dimerizer+/tetracycline+. At 48 hours after transfection with plasmids, A and B were harvested, while C and D were grown for 48 more hours in dimerizer+/tetracycline+ (C) or dimerizer+/tetracycline- (D) medium before harvesting. Results of one typical experiment, with four parallel transfections for each data point, are shown. Each experiment was repeated at least three times

Acetyl-CoA Counteracts the Inhibitory Effect of Antiandrogens on Androgen Receptor Signaling in Prostate Cancer Cells

The commonly used therapeutic management of PC involves androgen deprivation therapy (ADT) followed by treatment with AR signaling inhibitors (ARSI). However, nearly all patients develop drug-resistant disease, with a median progression-free survival of less than 2 years in chemotherapy-na&iuml;ve men. Acetyl-coenzyme A (acetyl-CoA) is a central metabolic signaling molecule with key roles in biosynthetic processes and cancer signaling. In signaling, acetyl-CoA serves as the acetyl donor for acetylation, a critical post-translational modification. Acetylation affects the androgen receptor (AR) both directly and indirectly increasing expression of AR dependent genes. Our studies reveal that PC cells respond to the treatment with ARSI by increasing expression of ATP-citrate lyase (ACLY), a major enzyme responsible for cytosolic acetyl-CoA synthesis, and up-regulation of acetyl-CoA intracellular levels. Inhibition of ACLY results in a significant suppression of ligand-dependent and -independent routes of AR activation. Accordingly, the addition of exogenous acetyl-CoA, or its precursor acetate, augments AR transcriptional activity and diminishes the anti-AR activity of ARSI. Taken together, our findings suggest that PC cells respond to antiandrogens by increasing activity of the acetyl-coA pathway in order to reinstate AR signaling