Mammalian cells constantly monitor and respond to a myriad of extracellular signals, often by using cell surface receptors. Two important classes of cell surface receptors include the receptor tyrosine kinases, which recognize peptide growth factors such as insulin, and the integrins, which most often mediate binding to components of the extracellular matrix. We report that the collagens serve as ligands for the previously orphan family of discoidin domain-containing receptor-like tyrosine kinases. The unexpected realization that an extracellular matrix molecule can directly serve as a ligand for receptor tyrosine kinases provides an example of ligands shared by integrins and receptor tyrosine kinases, and this finding seems likely to change prevailing views about the mechanisms by which cells perceive and respond to the extracellular matrix.

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Yin-Yang-1 (YY1) regulates the transcription of many genes, including the oncogenes c-fos and c-myc. Depending on the context, YY1 acts as a transcriptional repressor, a transcriptional activator, or a transcriptional initiator. The yeast two-hybrid system was used to screen a human complementary DNA (cDNA) library for proteins that associate with YY1, and a c-myc cDNA was isolated. Affinity chromatography confirmed that YY1 associates with c-Myc but not with Max. In cotransfections, c-Myc inhibits both the repressor and the activator functions of YY1, which suggests that one way c-Myc acts is by modulating the activity of YY1.

The c-Myc oncoprotein has previously been shown to associate with transcription regulator YY1 and to inhibit its activity. We show herein that endogenous c-Myc and YY1 associate in vivo and that changes in c-Myc levels, which accompany mitogenic stimulation or differentiation of cultured cells, affect the ratio of free to c-Myc-associated YY1. We have also investigated the mechanism by which association with c-Myc inhibits YYI's ability to regulate transcription. c-Myc does not block binding ofYY1 to DNA. However, protein association studies suggest that c-Myc interferes with the ability of YY1 to contact basal transcription proteins TATAbinding protein and TFIIB.

We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K(D)s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.

Purpose This study aimed to create new optical surgical navigation NIRF probes for prostate and breast cancers. Procedures IR800-linker-QWAVGHLM-NH2 with linker = GSG, GGG, and G-Abz4 were synthesized and characterized. IC50 for bombesin receptors (BBN-R) in PC-3 prostate and T47D breast cancer cells, fluorescence microscopy in PC-3 cells, and NIRF imaging in mice PC-3 tumor xenografts were studied. Results GGG, GSG, and G-Abz4 derivatives had IC50 (nM) for BBN-R+ PC-3 cells=187±31, 56 ±5, and 2.6±0.2 and T47D cells=383±1, 57.4±1.2, and 3.1±1.1, respectively. By microscopy the Abz4 derivative showed the highest uptake, was competed with by BBN, and had little to no binding to BBN-R− cells. In NIRF imaging the G-Abz4 probe was brighter than GGG probe in BBN-R+ tissues in vivo and tissues, tumors, and tumor slices ex vivo. Uptake could be partially blocked in BBN-R+ pancreas but not visibly in tumor. Conclusions Linker choice can dominate peptidic BBN-R binding. The G-Abz4 linker yields a higher affinity and specific BBN-R binder in this series of molecules.

Binding sites for three families of sequence-specific DNA-binding proteins, ,uE3, C/EBP, and OCT, are found in both the promoters and the intronic enhancer of the immunoglobulin heavy-chain gene. We have used a cotransfection system to investigate how proteins binding these promoter region. Studies supporting the scanning mechanism have typically introduced a barrier between a promoter and an enhancer region and found decreased enhancer activity (12,22). The looping model predicts that activators bound at a distance interact with proteins at the initiation site, with the concomitant looping out of the intervening DNA. There is extensive evidence supporting a looping mechanism in the regulation of prokaryotic genes (6, 61). In eukaryotic systems, loop formation in vitro can be mediated by higher-order multimers of such proteins as progesterone receptor (71), Spl (68), and bovine papillomavirus E2 protein (39) and by heteromeric complexes of Spl and E2 (43). The protein-protein interactions involved in loop formation have been shown to be functionally important for recruitment of proteins from a distal position in Spl-Spl and Spl-E2 associations. The looping model has also been supported by the demonstration of enhancer activation of a promoter through a protein bridge (48) and by the close association of the enhancer and promoter in chromatin-packaged prolactin gene substrates (23).We wished to explore mechanisms of eukaryotic enhancer action within the context of the IgH gene. Like all enhancers, the IgH enhancer can activate transcription from heterologous promoters. However, the IgH enhancer appears to be unusual in that it preferentially and synergistically activates VH promoters (33). The basis for this synergism is not understood. The IgH enhancer is a complex element composed of at least seven functionally important binding sites for activator proteins representing different structural classes (13, 64). The VH promoters that have been characterized are simpler than the enhancer. For example, the VH1 promoter contains three functionally important binding sites, all of which also occur in the enhancer: VE3, C/EBP (formerly called E), and OCT.The jxE3 site is recognized by a family of basic helix-loophelix-zipper (BHLHZIP)-containing DNA-binding proteins including

Receptor targeting ligands for imaging and/or therapy of cancer are limited by heterogeneity of receptor expression by tumor cells, both inter-patient and intra-patient. It is often more important for imaging agents to identify local and distant spread of disease than it is to identify a specific receptor presence. Two natural hormone peptide receptors, GRPR and Y1, are specifically interesting because expression of GRPR, Y1 or both is up-regulated in most breast cancers. We describe here the design and development of a new heterobivalent peptide ligand, truncated bombesin (t-BBN)/BVD15-DO3A, for dual-targeting of GRPR and Y1, and validation of its dual binding capability. Such a probe should be useful in imaging cells, tissues and tumors that are GRPR and/or Y1 positive and should target radioisotopes, for example, 68Ga and/or 177Lu, to more tumors cells than single GRPR or Y1 targeted probes. A GRP targeting ligand, J-G-Abz4-QWAVGHLM-NH2 (J-G-Abz4-t-BBN), and an Y1 targeting ligand, INP-K[ε-J-(α-DO3A-ε-DGa)-K]-YRLRY-NH2([ε-J-(α-DO3A-ε-DGa)-K]-BVD-15), were synthesized and coupled to produce the heterobivalent ligand, t-BBN/BVD15-DO3A. Competitive displacement binding assays using t-BBN/BVD15-DO3A against 125I-Tyr4-BBN yielded an IC50 value of 18 ± 0.7 nM for GRPR in T-47D cells, a human breast cancer cell line. A similar assay using t-BBN/BVD15-DO3A against porcine 125I-NPY showed IC50 values of 80 ± 11 nM for Y1 receptor in MCF7 cells, another human breast cancer cell line. In conclusion, it is possible to construct a single DO3A chelate containing probe that can target both GRPR and Y1 on human tumor cells.