Nimotuzumab is an EGFR-targeting antibody that has demonstrated encouraging clinical results in the absence of severe side-effects observed with other approved anti-EGFR antibodies. We investigated whether different clinical behavior of nimotuzumab is related to its bivalent/monovalent binding profile. Binding properties of nimotuzumab and cetuximab, the most development of anti-EGFR antibodies, were studied in vitro using chip surfaces and cells with varying EGFR expression levels. Experimental observations demonstrated that in contrast to cetuximab, the intrinsic properties of nimotuzumab required bivalent binding for stable attachment to the cellular surface, leading to nimotuzumab selectively binding to cells that express moderate to high EGFR expression levels. At these conditions, both antibodies bound bivalently, and accumulated to similar degrees. When EGFR density is low, nimotuzumab monovalent interaction was transient, whereas cetuximab continued to interact strongly with the receptors. We compared the in vitro anti-tumor efficacy of nimotuzumab and cetuximab. Cetuximab decreased the cell viability and induced apoptosis for all the tested cell lines, effects which did not depend on EGFR expression level. In contrast, nimotuzumab also provoked significant anti-cellular effects, but its anti-tumor capacity decreased together with EGFR expression level. Cetuximab Fab fragment was able to impact tumor cell survival, whereas nimotuzumab fragment totally lost this effect. Tumor-xenograft experiments using cells with a high EGFR expression revealed similar tumor growth inhibiting effects for both antibodies. This study suggests an explanation for nimotuzumab clinical profile, whereby anti-tumor activity is obtained in absence of severe toxicities due to its properties of bivalent binding to EGFR.

Proteomic analyses of the proliferative and secretory phases of the human endometrium were carried out to identify proteins and discover differentially expressed proteins using isotope-coded affinity tags, three stages of chromatographic separation and online tandem mass spectrometry (MS/MS). From an initial list of 346 proteins identified by ProICAT, manual inspection of MS/MS spectra and confirmatory searches pared the list down to 119 positively identified proteins. Only five of the proteins showed consistent differential expression. The utility of some of these proteins as indicators of true differential expression in the endometrium is open to discussion. The two proteins with unquestionable differential expressions in the secretory endometrium are: glutamate NMDA receptor subunit zeta 1 precursor and FRAT1. Some of the proteins that show no differential expression have previously been examined in gene-expression studies with similar conclusions.

The phosphorylation status of the myocyte enhancer factor 2 (MEF2) transcriptional regulator is a critical determinant of its tissue-specific functions. However, due to the complexity of its phosphorylation pattern in vivo, a systematic inventory of MEF2A phosphorylation sites in mammalian cells has been difficult to obtain. We employed modern affinity purification techniques, combined with mass spectrometry, to identify several novel MEF2 phosphoacceptor sites. These include an evolutionarily conserved KSP motif, which we show is important in regulating the stability and function of MEF2A. Also, an indirect pathway in which a protein kinase casein kinase 2 phosphoacceptor site is phosphorylated by activation of p38 MAPK signaling was documented. Together, these findings identify several novel aspects of MEF2 regulation that may prove important in the control of gene expression in neuronal and muscle cells. Myocyte enhancer factor 2 (MEF2)1 is a transcriptional regulatory complex mediating diverse cellular functions in neurons (1, 2), skeletal (reviewed in Ref.3) and cardiac muscle (4 -6), and T cells (7,8). It is now well established that MEF2 plays a role in the differentiation of these cell types as well as functioning in a protective role against neuronal apoptosis.To respond to diverse developmental and physiological cues, MEF2 is structurally organized to receive and respond to multiple signals from several intracellular signaling pathways (reviewed in Refs. 3 and 9). In this regard, perhaps the best characterized is the p38 MAPK-MEF2 axis, in mammals (10, 11) and in yeast (12), although other kinase-catalyzed cascades mediated by big MAP kinase (13,14), protein kinase C (10), and protein kinase CK2 (15) are known to target MEF2. Moreover, consistent with its role as a signal sensor, putative phosphoacceptor motifs in the carboxyl terminal MEF2 transactivation domain may prove to further modulate MEF2 function in response to extracellular cues.Given that MEF2, and the biological processes it regulates, are intrinsically governed by MEF2 phosphorylation status, we undertook to systematically document MEF2 phosphorylation patterns in mammalian cells; previous phosphopeptide mapping studies used in vitro phosphorylated MEF2 protein. The purpose thus being to detect physiologically relevant, and possibly novel, in vivo MEF2 phosphorylation sites. To accomplish this we used several state-of-the-art mass spectrometric techniques to detect phosphorylation sites from MEF2 expressed in mammalian cells. To this end, we have made use of a mammalian tandem affinity purification (TAP) method (16, 17) for low-abundance nuclear transcription factors that allows purification to homogeneity and provides amounts compatible with mass spectrometric analysis of phosphorylation sites.In these studies, we have identified two important and novel aspects of MEF2 regulation. One is a highly conserved phosphoacceptor motif that regulates MEF2 stability and function. The second is an indirect pathway of MEF2 regulation by p38 MAPK me...

Endometrial carcinoma is a common malignancy in women, being exceeded in incidence only by that of breast, lung, and colorectal cancers. At present, no serum tumor markers are available for the monitoring of endometrial carcinoma patients, and patients with recurrent disease are detected only following the development of symptoms or abnormalities in imaging assessments. Similarly, no screening tools are available for endometrial carcinoma. Protein profiling by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a sensitive and fast method of analysis for small proteins or peptides to yield specific biomarkers. In this study, a variety of normal and malignant endometrial tissue samples were fractionated and analyzed by SELDI-TOF MS (SELDI is a version of MALDI utilizing protein "chips"). A number of proteins displayed differential expression in malignant endometrial tissues. One of the prominent proteins fractionated by weak cation exchange chromatography and displaying enhanced expression in these malignant tissues was identified as chaperonin 10. The increased expression of chaperonin 10 in malignant endometrial tissues was further confirmed by parallel Western blot and immunohistochemistry analyses.