SMI of Bcl-2 TW-37 is active across a spectrum of B-cell tumors irrespective of their proliferative and differentiation status

The Bcl-2 family of proteins is critical to the life and death of malignant B-lymphocytes. Interfering with their activity using small-molecule inhibitors (SMI) is being explored as a new therapeutic strategy for treating B-cell tumors. We evaluated the efficacy of TW-37, a non-peptidic SMI of Bcl-2 against a range spectrum of human B-cell lines, fresh patient samples and animal xenograft models. Multiple cytochemical and molecular approaches such as acridine orange/ethidium bromide assay for apoptosis, co-immunoprecipitation of complexes and western blot analysis, caspase luminescent activity assay and apoptotic DNA fragmentation assay were used to demonstrate the effect of TW-37 on different B-cell lines, patient derived samples, as well as in animal xenograft models. Nanomolar concentrations of TW-37 were able to induce apoptosis in both fresh samples and established cell lines with IC50 in most cases of 165–320 nM. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 heterodimerization and induced apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg × 3 days) via tail vein. TW-37 failed to induce changes in the Bcl-2 proteins levels suggesting that assessment of baseline Bcl-2 family proteins can be used to predict response to the drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy regardless of the stage of B-cell differentiation

PDGF in gliomas: more than just a growth factor?

Platelet-derived growth factor B (PDGF-B) is a growth factor promoting and regulating cell migration, proliferation, and differentiation, involved in both developmental processes and in maintaining tissue homeostasis under strict regulation. What are the implications of prolonged or uncontrolled growth factor signaling in vivo, and when does a growth factor such as PDGF-B become an oncogene? Under experimental conditions, PDGF-B induces proliferation and causes tumor induction. It is not known whether these tumors are strictly a PDGF-B-driven proliferation of cells or associated with secondary genetic events such as acquired mutations or methylation-mediated gene silencing promoting neoplasia. If PDGF-B-driven tumorigenesis was only cellular proliferation, associated changes in gene expression would thus be correlated with proliferation and not associated with secondary events involved in tumorigenesis and neoplastic transformation such as cycle delay, DNA damage response, and cell death. Changes in gene expression might be expected to be reversible, as is PDGF-B-driven proliferation under normal circumstances. Since PDGF signaling is involved in oligodendrocyte progenitor cell differentiation and maintenance, it is likely that PDGF-B stimulates proliferation of a pool of cells with that phenotype, and inhibition of PDGF-B signaling would result in reduced expression of oligodendrocyte-associated genes. More importantly, inhibition of PDGF signaling would be expected to result in reversion of genes induced by PDGF-B accompanied by a decrease in proliferation. However, if PDGF-B-driven tumorigenesis is more than simply a proliferation of cells, inhibition of PDGF signaling may not reverse gene expression or halt proliferation. These fundamental questions concerning PDGF-B as a potential oncogene have not been resolved

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

BACKGROUND: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. METHODOLOGY/PRINCIPAL FINDINGS: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), β-galactosidase (β-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational up-regulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. CONCLUSIONS/SIGNIFICANCE: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium

De novo assembly of a transcriptome from the eggs and early embryos of Astropecten aranciacus

Starfish have been instrumental in many fields of biological and ecological research. Oocytes of Astropecten aranciacus, a common species native to the Mediterranean Sea and the East Atlantic, have long been used as an experimental model to study meiotic maturation, fertilization, intracellular Ca2+ signaling, and cell cycle controls. However, investigation of the underlying molecular mechanisms has often been hampered by the overall lack of DNA or protein sequences for the species. In this study, we have assembled a transcriptome for this species from the oocytes, eggs, zygotes, and early embryos, which are known to have the highest RNA sequence complexity. Annotation of the transcriptome identified over 32,000 transcripts including the ones that encode 13 distinct cyclins and as many cyclin-dependent kinases (CDK), as well as the expected components of intracellular Ca2+ signaling toolkit. Although the mRNAs of cyclin and CDK families did not undergo significant abundance changes through the stages from oocyte to early embryo, as judged by real-time PCR, the transcript encoding Mos, a negative regulator of mitotic cell cycle, was drastically reduced during the period of rapid cleavages. Molecular phylogenetic analysis using the homologous amino acid sequences of cytochrome oxidase subunit I from A. aranciacus and 30 other starfish species indicated that Paxillosida, to which A. aranciacus belongs, is not likely to be the most basal order in Asteroidea. Taken together, the first transcriptome we assembled in this species is expected to enable us to perform comparative studies and to design gene-specific molecular tools with which to tackle long-standing biological questions

Intracellular Trafficking Considerations in the Development of Natural Ligand-Drug Molecular Conjugates for Cancer

Overexpressed receptors, characteristic of many cancers, have been targeted by various researchers to achieve a more specific treatment for cancer. A common approach is to use the natural ligand for the overexpressed receptor as a cancer-targeting agent which can deliver a chemically or genetically conjugated toxic molecule. However, it has been found that the therapeutic efficacy of such ligand-drug molecular conjugates can be limited, since they naturally follow the intracellular trafficking pathways of the endogenous ligands. Therefore, a thorough understanding of the intracellular trafficking properties of these ligands can lead to novel design criteria for engineering ligands to be more effective drug carriers. This review presents a few commonly used ligand/receptor systems where intracellular trafficking considerations can potentially improve the therapeutic efficacy of the ligand-drug molecular conjugates