Inducible expression and phosphorylation of coactivator BOB.1/OBF.1 in T cells.

BOB.1/OBF.1 is a transcriptional coactivator that is constitutively expressed in B cells and interacts with the Oct1 and Oct2 transcription factors. Upon activation of Jurkat T cells and primary murine thymocytes with phorbol esters and ionomycin, BOB.1/OBF.1 expression and transactivation function were induced. BOB.1/OBF.1 was phosphorylated at Ser184 both in vivo and in vitro, and this modification was required for inducible activation. Mutation of Ser184 also diminished transactivation function in B cells, suggesting that the activating phosphorylation that is inducible in T cells is constitutively present in B cells. Thus, BOB.1/OBF.1 is a transcriptional coactivator that is critically regulated by posttranslational modifications to mediate cell type-specific gene expression.

CRISP-3, a protein with homology to plant defense proteins, is expressed in mouse B cells under the control of Oct2.

The Oct2 transcription factor is expressed throughout the B-lymphoid lineage and plays an essential role during the terminal phase of B-cell differentiation. Several genes specifically expressed in B lymphocytes have been identified that contain a functional octamer motif in their regulatory elements. However, expression of only a single gene, the murine CD36 gene, has been shown to date to be dependent on Oct2. Here, we present the identification and characterization of a further gene, coding for cysteine-rich secreted protein 3 (CRISP-3), whose expression in B cells is regulated by Oct2. We show that CRISP-3 is expressed in the B-lymphoid lineage specifically at the pre-B-cell stage. By using different experimental strategies, including nuclear run-on experiments, we demonstrate that this gene is transcriptionally activated by Oct2. Furthermore, analysis of CRISP-3 expression in primary B cells derived from either wild-type or Oct2-deficient mice demonstrates the dependence on Oct2. Two variant octamer motifs were identified in the upstream promoter region of the crisp-3 gene, and Oct2 interacts with both of them in vitro. Cotransfection experiments with expression vectors for Oct1 and Oct2 together with a reporter driven by the crisp-3 promoter showed that transcriptional activation of this promoter can only be achieved with Oct2. The C-terminal transactivation domain of Oct2 is required for this activation. Finally, introducing specific mutations in the two variant octamer motifs revealed that both of them are important for full transcriptional activation by Oct2.

Lymphoid- and myeloid-specific activity of the PU.1 promoter is determined by the combinatorial action of octamer and ets transcription factors.

The putative oncogene PU.1/Spi-1 is a member of the ets-family of transcription factors normally expressed in a subset of hematopoietic cell types. Here we have characterized the role of the PU.1 promoter region for the cell-type specific expression. The proximal 120 bp are sufficient to mediate a high level of activity specifically in B cells and macrophages. Three important motifs could be identified within this region. Two of them, an ets binding site (EBS) and a variant octamer motif were most important for cell-type-specific promoter activity in B cells and macrophages. An additional Sp1 motif stimulates basal activity of this promoter element. The relative contribution to overall activity of octamer motif and EBS differs in B cells and macrophages. In B cells, both octamer motif and EBS combine to mediate high level activity, whereas in macrophages the EBS predominantly confers promoter activity. Both the Oct1 and Oct2 transcription factors, presumably in combination with a B-cell-restricted coactivator, are responsible for the activity of the variant octamer motif in B cells. Interestingly, the PU.1 transcription factor can functionally interact with the EBS in its own promoter, suggesting a positive feedback regulation.

Identification of target genes of the lymphoid-specific transcription factor Oct2.

The Oct2 transcription factor is expressed predominantly in B lymphocytes and plays an essential role during the terminal phase of B cell differentiation. The regulatory regions of several genes specifically expressed in B cells contain functional binding sites for Oct2. Nevertheless, none of the genes originally thought to be regulated by Oct2 were affected in their expression in Oct2-deficient B cells. In an attempt to find such elusive Oct2 target genes and to understand the molecular function of Oct2 in B cell development, we isolated cDNAs for Oct2 target genes. So far, we have identified five potential targets for Oct2: the membrane glycoprotein CD36, the cysteine-rich secreted protein 3 (CRISP-3), a mouse homolog of the human monocyte/neutrophil elastase inhibitor (mEI) and two unknown cDNA sequences Nov1 and Nov2. These target genes show quite distinct expression patterns demonstrating that transcription factors in addition to Oct2 are involved in their regulation. Whereas CD36 and mEI were expressed in all hematopoetic cell lines containing Oct2,. CRISP-3 is pre-B cell-specific, Nov1 is plasma B cell-specific and Nov2 is B cell-specifically expressed.

Molecular principles of Oct2-mediated gene activation in B cells.

The octamer motif is a crucial regulatory element for immunoglobulin promoter and enhancer function. We have investigated the molecular mechanisms that underlie octamer-mediated gene activation in B cells. This B cell-specific transcriptional regulation is subject to a novel type of regulatory mechanism. We could demonstrate that octamer-dependent transcription is not only regulated by specific DNA-binding transcription factors, but in addition requires the activity of B cell-restricted cofactors. Both octamer-dependent promoter and enhancer activation depend on such a combination of transcription factor and cofactors. However, the exact requirements differ for these two situations. Promoter activity can be achieved with either one of two distinct transcription factors, Oct1 and/or Oct2, together with the cofactor OCA-B1. In contrast, only Oct2 in conjunction with an additional cofactor, OCA-B2, can confer enhancer activity.

A comparison of shock wave and sinusoidal-focused ultrasound-induced localized transfection of HeLa cells.

Both shock waves and sinusoidal continuous wave ultrasound can mediate DNA transfer into cells. The relative transfection efficiencies of different ultrasound modalities are unclear. The purpose of this paper is to compare the transfection efficiency of lithotripter shock waves and focused sinusoidal ultrasound in vitro. HeLa cells were transfected with beta-galactosidase and luciferase plasmid DNA reporter. Shock waves were generated by an electromagnetic sound source. Sixty to 360 pulses at 1 Hz pulse frequency were administered at 13, 16 or 19 kV capacitor voltage. Sinusoidal focused ultrasound was generated by a single focus piezoceramic air-backed disk transducer at a carrier frequency of 1.18 MHz operated in a pulsed mode. Compared to cells mixed with DNA only, shock waves induced up to eightfold more transfected cells at a cell viability of 5%, while sinusoidal-focused ultrasound induced up to 80-fold more transfected cells at a cell viability of 45%. The corresponding transfection efficiencies of the HeLa cells were 0.08% for shock waves and 3% for focused ultrasound. These results may contribute to the selection of the ultrasound modality as a localized, noninvasive and safe tool to mediate gene transfer.

Natural products in structure-assisted design of molecular cancer therapeutics.

Since the late 1990's, novel insights into molecular biology and carcinogenesis enabled the rational design of mechanism-based anticancer therapeutics. The large number of natural product (NP)-derived drugs currently under clinical evaluation and the recent approval of temsirolimus (Torisel) as a first mTOR protein kinase inhibitor indicate that NPs have to be considered not only as a seminal source of cytotoxic, but also as a source of molecularly targeted agents. Whereas molecular modeling is well established as an important and successful method to discover and rationalize bioactivities in medicinal chemistry research, its application has also proven to be also a powerful tool in the field of NPs. This review highlights the impact of computer-assisted approaches on NPs as molecularly targeted anticancer drugs. Examples of applications are provided focusing on innovative targets such as protein kinases, tumour vasculature, epigenetic modulators, heat shock protein (Hsp) 90, and direct apoptosis enhancers.

In vitro and in vivo transfection of plasmid DNA in the Dunning prostate tumor R3327-AT1 is enhanced by focused ultrasound.

Gene therapy as a form of molecular medicine is expected to have a major impact on medical treatments in the future. However, the clinical use of gene therapy today is hampered by inadequate gene delivering systems to ensure sufficient, accurate and safe DNA uptake in the target cells in vivo. Nonviral transfection methods might have the advantage of safe application, but it would be helpful to increase their transfection rates, especially in vivo. In this study, we show that focused ultrasound provides an enhanced transfer of DNA plasmids in vitro and in vivo. In vitro, the beta-galactosidase and luciferase DNA reporter plasmid were transfected into four cell lines (NIH 3T3 fibroblasts, malignant melanoma Mewo, HeLa, Dunning prostate tumor R3327-AT1). Ultrasound induced a 55- (Mewo) to 220-fold (AT1) stimulation resulting in transfection efficiencies in vitro between 2% (Mewo) and 12% (AT1). The in vivo stimulation was assessed in the Dunning prostate tumor R3327-AT1 implanted subcutaneously in Copenhagen rats using the beta-galactosidase reporter. After intratumoral DNA injection, focused ultrasound induced a 10-fold increase of beta-galactosidase positive cells in histology and a 15-fold increase of beta-galactosidase protein expression in the ELISA assay. In contrast, ultrasound was not found to enhance reporter gene expression after intravenous plasmid application. Because ultrasound waves can be focused on different anatomical locations in the human body without significant adverse effects, the control of DNA transfer by focused ultrasound is a promising in vivo method for spatial regulation of gene-based medical treatments.

Identification of CD36 as the first gene dependent on the B-cell differentiation factor Oct-2.

The Oct-2 transcription factor is expressed predominantly in B lymphocytes and has been shown previously to be important for the terminal phase of B-cell differentiation in mice. A number of genes specifically expressed in B cells contain Oct-2-binding sites in their regulatory regions. However, the analysis of expression levels of these genes in Oct-2-deficient B cells revealed that they were unaffected. Hence, there were no genes known that critically depend on Oct-2 for their expression. To understand the molecular basis for the Oct-2 effect on B-cell development, we searched for Oct-2 target genes by subtractive cDNA cloning. We show here that expression of the murine CD36 gene in B cells and macrophages requires a functional Oct-2 protein. Nuclear run-on experiments demonstrate that this gene is regulated transcriptionally by Oct-2. Moreover, CD36 levels correlated with the levels of Oct-2 expression in several mouse B-cell and macrophage cell lines. Finally, compared to wild-type and heterozygous mice, CD36 mRNA levels were markedly reduced in spleens and B-cell-enriched splenocyte fractions from oct-2-/- mice. The data identify CD36 as the first target gene critically dependent on Oct-2 for its expression. Because CD36 expression is also dependent on Oct-2 in vivo, it is a candidate gene through which Oct-2 could affect B-cell differentiation.

Functional characterization of the murine homolog of the B cell-specific coactivator BOB.1/OBF.1.

B cell-specific transcriptional promoter activity mediated by the octamer motif requires the Oct1 or Oct2 protein and additional B cell-restricted cofactors. One such cofactor, BOB.1/OBF.1, was recently isolated from human B cells. Here, we describe the isolation and detailed characterization of the murine homolog. Full-length cDNAs and genomic clones were isolated, and the gene structure was determined. Comparison of the deduced amino acids shows 88% sequence identity between mouse and human BOB.1/OBF.1. The NH2-terminal 126 amino acids of BOB.1/OBF.1 are both essential and sufficient for interaction with the POU domains of either Oct1 or Oct2. This protein-protein interaction does not require the simultaneous binding of Oct proteins to DNA, and high resolution footprinting of the Oct-DNA interaction reveals that binding of BOB.1/OBF.1 to Oct1 or Oct2 does not alter the interaction with DNA. BOB.1/OBF.1 can efficiently activate octamer-dependent promoters in fibroblasts; however, it fails to stimulate octamer-dependent enhancer activity. Fusion of subdomains of BOB.1/OBF.1 with the GAL4 DNA binding domain reveals that both NH2- and COOH-terminal domains of BOB.1/OBF.1 contribute to full transactivation function, the COOH-terminal domain is more efficient in this transactivation assay. Consistent with the failure of full-length BOB.1/OBF.1 to stimulate octamer-dependent enhancer elements in non B cells, the GAL4 fusions likewise only stimulate from a promoter-proximal position.

Differential transactivation potential of Oct1 and Oct2 is determined by additional B cell-specific activities.

Cell type-specific transcriptional regulation is generally believed to be mediated by sequence-specific transcription factors that are specifically present in the corresponding cells. The interaction of the lymphoid-specific Oct2 transcription factor has been thought to be responsible for the B cell-specific activity of octamer-containing promoter and enhancer elements. Here we show that physiological concentrations of Oct2 do not suffice to generate octamer-dependent promoter activity in non-B cell lines. Furthermore, we have tested the activity of octamer-dependent promoter and enhancer elements in B cell lines that lack the endogenous Oct2 protein. Our results demonstrate that in these Oct2-deficient B cells the ubiquitous endogenous Oct1 protein is able to stimulate octamer-containing promoters to a level comparable with that of normal Oct2-positive B cells. However, reporter constructs bearing the octamer motif in a distal enhancer position are not stimulated by the Oct1 protein, but do require the presence of Oct2. The B cell-specific octamer-dependent promoter activity mediated by Oct1 correlates with the presence of a novel B cell-specific octamer-binding complex containing the Oct1 protein. From these results we conclude that B cells contain two different activities: one that interacts with both Oct1 and Oct2 and mediates promoter proximal activity of the octamer motif and a second that specifically interacts with Oct2 to confer function from a remote enhancer position.