Targeting mTOR: prospects for mTOR complex 2 inhibitors in cancer therapy.

Small molecule inhibitors that selectively target cancer cells and not normal cells would be valuable anti-cancer therapeutics. The mammalian target of rapamycin complex 2 (mTORC2) is emerging as a promising candidate target for such an inhibitor. Recent studies in cancer biology indicate that mTORC2 activity is essential for the transformation and vitality of a number of cancer cell types, but in many normal cells, mTORC2 activity is less essential. These studies are intensifying interest in developing inhibitors that specifically target mTORC2. However, there are many open questions regarding the function and regulation of mTORC2 and its function in both normal and cancer cells. Here, we summarize exciting new research into the biology of mTORC2 signaling and highlight the current state and future prospects for mTOR-targeted therapy.

Age-dependent transformation frequency in elite wheat varieties.

Wheat is a major world crop and as such is a primary target for improvement of agronomic characteristics via genetic engineering. Optimization of transformation is essential in order to overcome the relatively low transformation frequencies encountered with wheat. Transformation of elite wheat varieties is not always successful due to variability in regeneration and transformation frequencies between varieties. In this work, two elite wheat varieties with a relatively high embryogenic capacity were transformed by particle bombardment. A strong correlation between transformation frequency and the age of wheat donor plants was observed in both varieties. The mean transformation frequency rose from 0.7% to 5% when using immature embryos from old and young donor plants, respectively. This was observed in both varieties, the best bombardments achieving up to 7.3% frequency. Using explants at an optimal developmental stage from donor plants grown under environmentally-controlled conditions has improved the reproducibility of transformation efficiency of elite wheat varieties and leads to the production of apparently phenotypically normal, fertile, transgenic plants.

Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis.

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.

Amorphous no longer: the centrosome comes into focus.

Recent genetic and biochemical studies have provided new insights into the molecular basis of centrosome-mediated microtubule nucleation. In addition, molecules and mechanisms involved in microtubule severing and stabilization at the centrosome, assembly of proteins onto centrosomes and regulation of centrosome duplication and separation are being defined. Characterization of centrosome function, together with studies implicating centrosomes in tumorigenesis and demonstrating that centrosomes are highly organized, are beginning to bring into focus an organelle once viewed as an 'amorphous cloud'.

Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome.

Pericentrin and gamma-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin-gamma-tubulin complex was distinct from the previously described gamma-tubulin ring complex (gamma-TuRC) as purified gamma-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and gamma-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.

Product of the oncogene-activating gene Tpr is a phosphorylated protein of the nuclear pore complex.

We have identified a component of the human nuclear pore complex and have shown that it is the product of a gene involved in oncogenic activation. A monoclonal antibody raised against purified nuclear matrix proteins recognizes a single protein with an electrophoretic mobility of approximately 300 kDa and stains the nuclear envelope in a punctate pattern typical of nuclear pores. The antibody was used to screen lambda gt11 human cDNA libraries, and the resulting clones were sequenced and compared to sequences in the Genbank database. An exact match was found with the human tpr (for translocated promoter region) gene, a gene shown previously to be involved in the oncogenic activation of several protein kinases. Double-label immunofluorescent microscopy with the anti-Tpr antibody and an antibody to the previously characterized nuclear pore complex protein nup153 confirms that Tpr is localized to the nuclear pore complex. Tpr is located on the cytoplasmic face of the nucleus, as demonstrated by immunofluorescent staining of cells permeabilized with digitonin. Tpr is a 2,349-amino acid protein with extensive coiled-coil domains and an acidic globular C-terminus. The protein contains 10 leucine zipper motifs and numerous sites for phosphorylation by a variety of protein kinases. Immunoprecipitation of Tpr from 32P-orthophosphate-labeled cells shows that it is a phosphoprotein. Potential functions for Tpr and possible mechanisms for the transforming activity of Tpr fusion proteins are discussed.

Phosphorylation of NUMA occurs during nuclear breakdown and not mitotic spindle assembly.

NuMA, the nuclear mitotic apparatus protein, is a component of the nuclear matrix at interphase that redistributes to the spindle poles at mitosis. While the function of NuMA is not known, it has been implicated in spindle organization during mitosis and nuclear reformation. Phosphorylation is thought to play a regulatory role in NuMA function. In this study, NuMA phosphorylation was examined through the cell cycle using highly synchronized cells. In intact cells labeled with 32P-orthophosphate, NuMA appeared as a 250 kDa phosphoprotein in interphase that shifted to a higher apparent molecular mass in mitosis. The shift was due to phosphorylation as shown by reduction of the shifted band to interphase mobility by phosphatase treatment. This phosphorylation event occurred roughly at the G2/M transition at the time of NuMA's release from the nucleus and its redistribution to the mitotic spindle. However, mitotic phosphorylation did not require spindle formation since the phosphorylated species was detected in nocodazole-treated cells lacking microtubule spindles. Dephosphorylation of NuMA occurred in two distinct steps, after lamin B assembled into the nuclear lamina, in early G1 and at the end of G1. Based on the timing of the phosphorylation and dephosphorylation observed in this study, we propose that they may play a role in nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G1/S.

Assignment of the nuclear mitotic apparatus protein NuMA gene to human chromosome 11q13.

A monoclonal antibody that was specific for a nuclear matrix protein was obtained and used to screen a human lambda gt11 expression library. Several partial cDNA clones were isolated and sequenced. The sequence for this protein was shown to be identical to that of NuMA, a 236-kDa nuclear mitotic spindle apparatus protein. NuMA has been recently characterized by two independent studies, and is thought to be part of a family of proteins that is required for the completion of mitosis. In this report, the chromosomal localization and copy number of the NuMA gene are analyzed using cDNA clones. High-resolution in situ hybridization reveals a single pair of signals on sister chromatids of human chromosome 11 at band q13. Stringent Southern analysis of human genomic DNA resulted in simple restriction patterns. These results together indicate that the NuMA gene is present as a single copy on human chromosome 11q13.

The measurement of steroid hormones in endometrial tissue: a comparison between two methods of extraction.

Concentrations of oestrone, oestradiol, 5-androstene-3 beta,17 beta-diol (androstenediol) and dehydroepiandrosterone (DHA) were measured in human endometrium by radioimmunoassay following extraction with either diethyl ether or ethanol-acetone. Our results demonstrate that there is no difference between values obtained using these methods of extraction.