The ability to detect antigens immunologically is limited by the affinity of the antibodies and the amount of antigens. We have now succeeded in creating a modular, facile amplification system, termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). Such a system can detect protein targets specifically at subfemtomolar levels ( approximately 0.08 fM). We describe here the detection of Her2 (also known as Neu) from rodent and human sera. FACTT is adaptable to high-throughput screening and automation and provides a practical method to enhance current ELISAs in medical practice.

Ubiquitously expressed transcript (UXT) is a prefoldinlike protein that has been suggested to be involved in human tumorigenesis. Here, we have found that UXT is overexpressed in a number of human tumor tissues but not in the matching normal tissues. We demonstrate that UXT is located in human centrosomes and is associated with ␥-tubulin. In addition, overexpression of UXT disrupts centrosome structure. Furthermore, abrogation of UXT protein expression by small interfering RNA knockdown leads to cell death. Together, our findings suggest that UXT is a component of centrosome and is essential for cell viability. We propose that UXT may facilitate transformation by corrupting regulated centrosome functions.

Insight into the molecular mechanisms of malignant transformation is changing the way cancer is being treated. Conventional treatment strategies target the DNA of all dividing cells, resulting in a significantly increased risk of collateral toxicity. In addition, the accumulation of multiple mutations leads to drug resistance in many cancer cells. Targeted strategies have now been developed that specifically disrupt oncogenically active cell surface receptors and endogenous signaling molecules. These agents have a much greater selectivity for tumor tissue and decreased risk of side effects. Increased signaling through ErbB receptors via gene amplification, overexpression, and mutation has been implicated in many human cancers and associated with poor prognosis. Interruption of this process has been shown to cause antitumor effects. Downregulation of the ErbB receptors, HER-2/neu, and later EGFR, with monoclonal antibodies was the first demonstration of targeted therapy. Subsequently, the ErbB tyrosine kinase domain has been successfully targeted with small molecule inhibitors. The development of novel ErbB-directed entities is ongoing, with particular promise being shown by strategies targeting receptor interaction in oligomeric complexes.

The anti-p185 her2/neu peptidomimetic (AHNP) is a small exo-cyclic peptide derived from the anti-p185 her2/neu rhumAb 4D5 (h4D5). AHNP mimics many but not all of the antitumor characteristics exhibited by h4D5. However, the pharmacokinetic profiles of AHNP are less than optimal for therapeutic or diagnostic purposes. To improve the binding affinity to p185 her2/neu and the antitumor efficacy, we have engineered a fusion protein containing AHNP and a nonimmunoglobulin protein scaffold, streptavidin (SA). The recombinant protein, AHNP-SA (ASA) bound to p185 her2/neu with high affinity, inhibited the proliferation of p185 her2/neu -overexpressing cells, and reduced tumor growth induced by p185 her2/neutransformed cells. These data suggest that the bacterially produced tetrameric ASA can be used as an antibodysurrogate molecule. This class of molecule will play a role in the diagnosis and treatment of p185 her2/neu -related tumors. Our studies establish a general principle by which a small biologically active synthetic exo-cyclic peptide can be engineered to enhance functional aspects by structured oligomerization and can be produced recombinantly using bacterial expression.

The centrosome functions as the major microtubule-organizing center and plays a vital role in guiding chromosome segregation during mitosis. Centrosome abnormalities are frequently seen in a variety of cancers, suggesting that dysfunction of this organelle may contribute to malignant transformation. In our efforts to identify the protein components of the centrosome and to understand the structure features involved in the assembly and functions of this organelle, we cloned and characterized a centrosome-associated protein called Su48. We found that a coiled coil-containing subdomain of Su48 was both sufficient and required for its centrosome localization. In addition, this structure also modulates Su48 dimerization. Moreover, ectopic expression of Su48 causes abnormal mitosis, and a mutant form of Su48 disrupts the localization of ␥-tubulin to the centrosome. Finally, by microinjection of an anti-Su48 antibody, we found that disruption of normal Su48 functions leads to mitotic failure, possibly due to centrosome defects or incomplete cytokinesis. Thus, Su48 represents a previously unrecognized centrosome protein that is essential for cell division. We speculate that Su48 abnormalities may cause aberrant chromosome segregation and may contribute to aneuploidy and malignant transformation.cancer ͉ coiled coil ͉ mitosis

ADCC Antibody dependent cellular cytolysis ADCP Antibody dependent cellular phagocytosis CDR Complementarity Determining Region EGFr Epidermal growth factor receptor HAMA human anti-mouse antibodies response MAb Monoclonal Antibody ScFv single chain Fragment v. This work was supported by grants from Abramson Institute to MIG.