Limited expression of non-integrating CpG-free plasmid is associated with increased nucleosome enrichment

CpG-free pDNA was reported to facilitate sustained transgene expression with minimal inflammation in vivo as compared to CpG-containing pDNA. However, the expression potential and impact of CpG-free pDNA in in vitro model have never been described. Hence, in this study, we analyzed the transgene expression profiles of CpG-free pDNA in vitro to determine the influence of CpG depletion from the transgene. We found that in contrast to the published in vivo studies, CpG-free pDNA expressed a significantly lower level of luciferase than CpG-rich pDNA in several human cell lines. By comparing novel CpG-free pDNA carrying CpG-free GFP (pZGFP: 0 CpG) to CpG-rich GFP (pRGFP: 60 CpGs), we further showed that the discrepancy was not influenced by external factors such as gene transfer agent, cell species, cell type, and cytotoxicity. Moreover, pZGFP exhibited reduced expression despite having equal gene dosage as pRGFP. Analysis of mRNA distribution revealed that the mRNA export of pZGFP and pRGFP was similar; however, the steady state mRNA level of pZGFP was significantly lower. Upon further investigation, we found that the CpG-free transgene in non-integrating CpG-free pDNA backbone acquired increased nucleosome enrichment as compared with CpG-rich transgene, which may explain the observed reduced level of steady state mRNA. Our findings suggest that nucleosome enrichment could regulate non-integrating CpG-free pDNA expression and has implications on pDNA design.

Expression of dbat and dbtnbt Genes Involved in Paclitaxel Biosynthesis during the Growth Cycle of Taxus baccata L. Callus Cultures

The time-course of expression of dbat and dbtnbt genes involved in the later steps of paclitaxel biosynthesis and the intracellular taxane accumulation were investigated through a 64-day subculture interval of VI/M1 and VI/M2 Taxus baccata callus cultures. HPLC proved traces of baccatin III and an intracellular content of paclitaxel up to 90 μg/g DW. The steadystate of the respective gene transcripts was measured by quantitative real-time RT-PCR. The expression profile of dbat and dbtnbt genes was slightly different and varied within the subculture. The highest level of dbat expression was detected 24 h after inoculation followed by a decrease in both cultures. In contrast with dbat no substantially high expression of the dbtnbt gene after inoculation was observed. The impact of the conditions during inoculation on gene expression is discussed. Although the increase in transcriptional activity of both genes positively correlated with callus growth, the intracellular accumulation of paclitaxel varied during subculture with the maximum in the stationary (VI/M1) or at the end of the linear (VI/M2) phase. The increase of the steady-state mRNA level of the dbtnbt gene was followed by paclitaxel accumulation with a delay of approx. 28 (VI/M1) and 14 days (VI/M2).

Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs). Thus, we studied the effects of TGF-beta1 on IGFs and IGFBPs in human marrow stromal (hMS) osteoblast precursor cells. TGF-beta1 increased the steady-state mRNA level of IGF-I up to 8.5+/-0.6-fold (P<0.001) in a dose- (0.1-10 ng/ml), and time-dependent (12-72 h) manner. No significant effects on IGF-II gene expression were detectable. Employing RNase protection and nuclear run-on assays, these effects on IGF-I were found to take place at the transcriptional level and were not dependent on de novo protein synthesis. Using the transient transfection of various fragments of the IGF-I promoter 1, we found that TGF-beta responsive elements were present in a promoter fragment ranging from-65 bp to+55 bp relative to the major transcription start site in exon 1. In addition, TGF-beta1 treatment resulted in a dose- and time-dependent increase (2-fold) in the IGFBP-3 steady-state mRNA level as well as in protein production but did not affect IGFBP-2 or IGFBP-4 at mRNA or protein levels. Our results indicate that TGF-beta1 exerts significant effects on stimulatory components of the IGF-system and that may represent a mechanism mediating TGF-beta effects on the biological functions of osteoblasts.

Differential regulation of γ-glutamylcysteine synthetase heavy and light subunit gene expression

γ-Glutamylcysteine synthetase (GCS) is the rate-limiting enzyme in the biosynthesis of glutathione and is composed of a heavy and a light subunit. Although the heavy subunit is enzymically active alone, the light subunit plays an important regulatory role by making the holoenzyme function more efficiently. In the current study we examined whether conditions which are known to influence gene expression of the heavy subunit also influence that of the light subunit, and the mechanisms involved. Treatment of cultured rat hepatocytes with hormones such as insulin and hydrocortisone, or plating hepatocytes under low cell density increased the steady-state mRNA level of the heavy subunit only. Treatment with diethyl maleate (DEM), buthionine sulphoximine (BSO) and t-butylhydroquinone (TBH) increased the steady state mRNA level and gene transcription rates of both subunits. These treatments share in common their ability to induce oxidative stress and activate nuclear factor κB (NF-κB). Treatment with protease inhibitors 7-amino-1-chloro-3-tosylamido-2-heptanone (TLCK) or L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) had no influence on the basal NF-κB and GCS subunit mRNA levels, but blocked the activation of NF-κB by DEM, BSO and TBH, and the increase in GCS heavy subunit mRNA level by BSO and TBH. On the other hand, the DEM-, BSO- and TBH-induced increase in GCS light-subunit mRNA level was unaffected by TLCK and TPCK. Thus only the heavy subunit is hormonally regulated and growth sensitive, whereas both subunits are regulated by oxidative stress. Signalling through NF-κB is involved only in the oxidative-stress-mediated changes in the heavy subunit gene expression.

Expression of REX-1, a gene containing zinc finger motifs, is rapidly reduced by retinoic acid in F9 teratocarcinoma cells

In the presence of retinoic acid (RA), cultured F9 murine teratocarcinoma stem cells differentiate into nontumorigenic cells resembling the extraembryonic endoderm of the early mouse embryo. By differential hybridization screening of an F9 cell cDNA library, we isolated a 1,745-nucleotide cDNA for a gene, REX-1 (for reduced expression), whose steady-state mRNA level began to decline in F9 cells in monolayer culture within 12 h after the addition of RA. By 48 to 96 h after RA treatment of F9 cells in monolayer culture, the REX-1 steady-state mRNA level was more than sevenfold lower than the level in undifferentiated F9 stem cells. The REX-1 mRNA decrease did not result from the reduction in cell growth rate associated with the differentiation process, since the REX-1 mRNA level did not decline in F9 cells that were partially growth arrested after 48 h of isoleucine deprivation. The RA-associated REX-1 mRNA decrease resulted primarily from a reduction in the transcription rate of the REX-1 gene in the presence of RA. In contrast to results in F9 cells, we have been unable thus far to detect REX-1 mRNA in day 7.5 to 12.5 mouse embryo RNA samples or in the P19 teratocarcinoma stem cell line. The putative REX-1 protein identified by DNA sequence analysis contains four repeats of the zinc finger nucleic acid-binding motif and a potential acidic activator domain, suggesting that REX-1 encodes a regulatory protein. The REX-1 gene is not identical to the previously reported murine genes that encode zinc finger-containing proteins.

Expression of REX-1, a gene containing zinc finger motifs, is rapidly reduced by retinoic acid in F9 teratocarcinoma cells.

In the presence of retinoic acid (RA), cultured F9 murine teratocarcinoma stem cells differentiate into nontumorigenic cells resembling the extraembryonic endoderm of the early mouse embryo. By differential hybridization screening of an F9 cell cDNA library, we isolated a 1,745-nucleotide cDNA for a gene, REX-1 (for reduced expression), whose steady-state mRNA level began to decline in F9 cells in monolayer culture within 12 h after the addition of RA. By 48 to 96 h after RA treatment of F9 cells in monolayer culture, the REX-1 steady-state mRNA level was more than sevenfold lower than the level in undifferentiated F9 stem cells. The REX-1 mRNA decrease did not result from the reduction in cell growth rate associated with the differentiation process, since the REX-1 mRNA level did not decline in F9 cells that were partially growth arrested after 48 h of isoleucine deprivation. The RA-associated REX-1 mRNA decrease resulted primarily from a reduction in the transcription rate of the REX-1 gene in the presence of RA. In contrast to results in F9 cells, we have been unable thus far to detect REX-1 mRNA in day 7.5 to 12.5 mouse embryo RNA samples or in the P19 teratocarcinoma stem cell line. The putative REX-1 protein identified by DNA sequence analysis contains four repeats of the zinc finger nucleic acid-binding motif and a potential acidic activator domain, suggesting that REX-1 encodes a regulatory protein. The REX-1 gene is not identical to the previously reported murine genes that encode zinc finger-containing proteins.