Maize transformation technology development for commercial event generation.

Maize is an important food and feed crop in many countries. It is also one of the most important target crops for the application of biotechnology. Currently, there are more biotech traits available on the market in maize than in any other crop. Generation of transgenic events is a crucial step in the development of biotech traits. For commercial applications, a high throughput transformation system producing a large number of high quality events in an elite genetic background is highly desirable. There has been tremendous progress in Agrobacterium-mediated maize transformation since the publication of the Ishida et al. (1996) paper and the technology has been widely adopted for transgenic event production by many labs around the world. We will review general efforts in establishing efficient maize transformation technologies useful for transgenic event production in trait research and development. The review will also discuss transformation systems used for generating commercial maize trait events currently on the market. As the number of traits is increasing steadily and two or more modes of action are used to control key pests, new tools are needed to efficiently transform vectors containing multiple trait genes. We will review general guidelines for assembling binary vectors for commercial transformation. Approaches to increase transformation efficiency and gene expression of large gene stack vectors will be discussed. Finally, recent studies of targeted genome modification and transgene insertion using different site-directed nuclease technologies will be reviewed.

Heregulin-induced activation of HER2 and HER3 increases androgen receptor transactivation and CWR-R1 human recurrent prostate cancer cell growth.

PURPOSE: The androgen receptor (AR) is a ligand-dependent transcription factor that mediates gene expression and growth of normal and malignant prostate cells. In prostate tumors that recur after androgen withdrawal, the AR is highly expressed and transcriptionally active in the absence of testicular androgens. In these "androgen-independent" tumors, alternative means of AR activation have been invoked, including regulation by growth factors and their receptors in prostate cancer recurrence. EXPERIMENTAL DESIGN AND RESULTS: In this report, we show that HER receptor tyrosine kinases 1 through 4 are expressed in the CWR-R1 recurrent prostate cancer cell line; their stimulation by epidermal growth factor (EGF) and heregulin activates downstream signaling, including mitogen-activated protein kinase and phosphatidylinositol-3 kinase and Akt pathways. We show that heregulin activates HER2 and HER3 and increases androgen-dependent AR transactivation of reporter genes in CWR-R1 cells. Tyrosine phosphorylation of HER2 and HER3, AR transactivation, and cell proliferation induced by heregulin were more potently inhibited by the EGFR/HER2 dual tyrosine kinase inhibitor GW572016 (lapatinib) than the EGFR-specific inhibitor ZD1839 (gefitinib). Basal proliferation in the absence of growth factors was also inhibited by GW572016 to a greater extent than ZD1839, suggesting that low level HER2/HER3 activation perhaps by an autocrine pathway contributes to the proliferation signal. CONCLUSIONS: These data indicate that heregulin signaling through HER2 and HER3 increases AR transactivation and alters growth in a recurrent prostate cancer cell line. Therefore, inhibition of low-level HER2 signaling may be a potential novel therapeutic strategy in prostate cancer.

Epidermal growth factor increases coactivation of the androgen receptor in recurrent prostate cancer.

Growth of normal and neoplastic prostate is mediated by the androgen receptor (AR), a ligand-dependent transcription factor activated by high affinity androgen binding. The AR is highly expressed in recurrent prostate cancer cells that proliferate despite reduced circulating androgen. In this report, we show that epidermal growth factor (EGF) increases androgen-dependent AR transactivation in the recurrent prostate cancer cell line CWR-R1 through a mechanism that involves a post-transcriptional increase in the p160 coactivator transcriptional intermediary factor 2/glucocorticoid receptor interacting protein 1 (TIF2/GRIP1). Site-specific mutagenesis and selective MAPK inhibitors linked the EGF-induced increase in AR transactivation to phosphorylation of TIF2/GRIP1. EGF signaling increased the coimmunoprecipitation of TIF2 and AR. AR transactivation and its stimulation by EGF were reduced by small interfering RNA inhibition of TIF2/GRIP1 expression. The data indicate that EGF signaling through MAPK increases TIF2/GRIP1 coactivation of AR transactivation in recurrent prostate cancer.

The growth of Drosophila bristles and laterals is not restricted to the tip or base.

The highly elongated bristles of Drosophila have proven to be a valuable model system for studying cellular morphogenesis. Extending bristles contain a series of large bundles of actin filaments juxtaposed to the plasma membrane and centrally located microtubules. Models to explain the extension of the bristle have principally focused on the assembly of actin filaments at the distal tip of the bristle. We have used time-lapse observations of wild-type and mutant bristles and the related arista laterals and come to the conclusion that growth takes place throughout the growing cellular extension. This distributed growth can explain the behavior of split laterals and the shape changes seen at the tip during bristle and lateral outgrowth. Inhibitor studies suggest that the microtubule cytoskeleton is essential for maintaining the highly biased axial growth of these structures. We have used fluorescence recovery after photo-bleaching to study the dynamics of the cytoskeleton during bristle growth. Our experiments show that actin bundles in growing bristles are quite stable and move in a retrograde fashion. The bristle microtubules are less stable. The retrograde movement of the peripheral actin appears to be counterbalanced by the distally directed movement of cytoplasm in the center of the bristle.