Indirect immunotargeting of CIS‐PT to human epidermoid carcinoma KB using the avidin‐biotin system

Schechter, Bilha; Arnon, Ruth; Wilchek, Meir; Schlessinger, Joseph; Hurwitz, Esther; Aboud-Pirak, Esther; Sela, Michael

doi:10.1002/ijc.2910480203

Cited by 33 publications

(13 citation statements)

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“…Peptide Partitioning into FHK 12 Membrane-Partitioning of rhodamine-labeled ErbB1 to the inner membrane of E. coli was determined by confocal laser scanning microscopy. FHK 12 bacteria from the control of the association assay were diluted 1:10,000 and incubated with rhodamine-labeled ErbB1, final concentration of 500 nM, for 2 h. Samples were fixed by the addition of agarose gel at a final concentration of 0.25% in phosphate-buffered saline.…”

Section: Toxr-gpa Protein Expression Levels By Westernmentioning

confidence: 99%

“…We thank Dr. Langosch from Heidelberg University, who provided the ToxR-GPA and A 16 plasmids as well as the FHK 12 and PD28 E. coli strains. We thank V. Kiss from the Weizmann Institute for help with the confocal imaging.…”

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confidence: 99%

“…This single span TM receptor family (type I), which is involved in a variety of cell processes, is also implicated in several types of cancer (11,12). Homodimerization of these receptors has been known to be a prerequisite for signal transduction for over a decade (13,14).…”

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confidence: 99%

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Two Motifs within a Transmembrane Domain, One for Homodimerization and the Other for Heterodimerization

Gerber

Sal‐Man

Shai

2004

Journal of Biological Chemistry

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Protein assembly is a critical process involved in a wide range of cellular events and occurs through extracellular and/or transmembrane domains (TMs). Previous studies demonstrated that a GXXXG motif is crucial for homodimer formation. Here we selected the TMs of ErbB1 and ErbB2 as a model since these receptors function both as homodimers and as heterodimers. Both TMs contain two GXXXG-like motifs located at the C and N termini. The C-terminal motifs were implicated previously in homodimer formation, but the role of the Nterminal motifs was not clear. We used the ToxR system and expressed the TMs of both ErbB1 and ErbB2 containing only the N-terminal GXXXG motifs. The data revealed that the ErbB2 but not the ErbB1 construct formed homodimers. Importantly, a synthetic ErbB1 TM peptide was able to form a heterodimer with ErbB2, by displacing the ErbB2 TM homodimer. The specificity of the interaction was demonstrated by using three controls: (i) Two single mutations within the GXXXG-like motif of the ErbB1 peptide reduced or preserved its activity, in agreement with similar mutations in glycophorin A. (ii) A TM peptide of the bacterial Tar receptor did not assemble with the ErbB2 construct. (iii) The ErbB1 peptide had no effect on the dimerization of a construct containing the TM-1 domain of the Tar receptor. Fluorescence microscopy demonstrated that all the peptides localized on the membrane. Furthermore, incubation with the peptides had no effect on bacterial growth and protein expression levels. Our results suggest that the N-terminal GXXXG-like motif of the ErbB1 TM plays a role in heterodimerization with the ErbB2 transmembrane domain. To our knowledge, this is the first demonstration of a transmembrane domain with two distinct recognition motifs, one for homodimerization and the other for heterodimerization.Protein recognition within the membrane milieu is crucial for a wide range of processes in all organisms. Membrane proteins associate via their extracellular and transmembrane (TM) 1 or solely via their TM domains. Understanding the interactions involved in peptide-peptide recognition within the membrane milieu is therefore an important task. Despite advances, the limited number of studies conducted report mainly on homodimerization and not on heterodimerization of TM segments in vitro and in vivo (1-7). The ability of TM domains to homodimerize in vivo was reported using either the ToxR (2) system or the TOXCAT (8) system. To our knowledge, only a few studies reported on hetero-association of TM domains in vivo. These studies include: (i) introducing exogenous TM peptide mutants of glycophorin A (GPA) to the ToxR system, which expresses the TM domain of glycophorin A, and observing a dominant negative effect (9); and (ii) the study of hetero-association of the TM domain ␣ and ␤ integrin subunits, using the GALLEX system (10). Furthermore, a direct identification of two assembly motifs within a single TM domain has not been reported.For this purpose, we investigated the recognition patterns of the ErbB ...

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Section: Toxr-gpa Protein Expression Levels By Westernmentioning

confidence: 99%

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confidence: 99%

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Two Motifs within a Transmembrane Domain, One for Homodimerization and the Other for Heterodimerization

Gerber

Sal‐Man

Shai

2004

Journal of Biological Chemistry

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“…Use of the biotion-(strept)avidin system in vivo is not unprecedented, as it has been applied in a number of in vivo radioapplications, including radioimmunodetection and therapy of cancer in humans 46 and immunotargeting of cytotoxic molecules and cells to tumor cells. [47][48][49][50][51] An additional consideration for in vivo use of the contrast agent is method of delivery. We previously explored methods to administer the contrast agent topically, rather than through i.v.…”

Section: Discussionmentioning

confidence: 99%

Real‐time detection of epidermal growth factor receptor expression in fresh oral cavity biopsies using a molecular‐specific contrast agent

Hsu

Gillenwater²,

Hasan

et al. 2006

Intl Journal of Cancer

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Early diagnosis of individuals with high risk of developing head and neck squamous carcinoma should lead to decreased morbidity and increased survival. To aid in noninvasive early detection of oral neoplasia in vivo, we have developed a molecular-specific fluorescent contrast agent, consisting of a far-red fluorescent dye coupled to a monoclonal antibody targeted against the epidermal growth factor receptor. In our study, we used organ cultures of normal and neoplastic human oral tissue to evaluate the capabilities of using this contrast agent to enhance clinical diagnosis. Fresh tissue sections were prepared from 34 biopsies of clinically normal and abnormal oral mucosa from 17 consenting patients. Samples were exposed to contrast agent, rinsed and the presence of bound agent was detected using fluorescence confocal microscopy. Simple assays to assess cytotoxicity of the dye used in the agent and to determine labeling efficacy at physiologic temperatures were also performed. Results indicate that the mean fluorescence intensity (MFI) of samples with dysplasia and cancer are higher than that of the normal sample from the same patient, and that this increase in fluorescence could potentially be used in the early detection and delineation of premalignant lesions. Normal tissue could be distinguished from cancer or moderate dysplasia, using either the ratio of the MFI of abnormal to normal tissue or the MFI obtained from the epithelial surface. No detrimental effects from the dye were observed over a 4-day period. These results indicate that the use of this optical contrast agent could yield important clinical advantages for noninvasive early detection and molecular characterization of oral mucosa. ' 2006 Wiley-Liss, Inc.Key words: fluorescent contrast agent; epidermal growth factor receptor; molecular-specific contrast agentThe 5-year survival rate for oral cancer has not improved significantly in the last 3 decades, despite numerous advances in treatment modalities.

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“…The high-affinity biotin-(strept)avidin system has been used in vivo in radioapplications, including radioimmunodetection and therapy of cancer in humans (45) and immunotargeting of cytotoxic molecules and cells to tumor cells (37,(46)(47)(48)(49). The possible drawbacks in using streptavidin for in vivo applications are that it tends to be sequestered quickly by the kidneys (50), which could limit the amount of the streptavidin-conjugated compound delivered to the target, and it cannot be humanized, potentially making it more immunogenic.…”

Section: Discussionmentioning

confidence: 99%

A Far-red Fluorescent Contrast Agent to Image Epidermal Growth Factor Receptor Expression

Hsu

Anslyn

Dharmawardhane

et al. 2004

Photochem Photobiol

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Recent developments in optical technologies have the potential to improve the speed and accuracy of screening and diagnosis of curable precancerous lesions and early cancer, thereby decreasing the costs of detection and management of epithelial malignancies. The development of molecular-specific contrast agents for markers of early neoplastic transformation could improve the detection and molecular characterization of premalignant lesions. In the oral cavity, epidermal growth factor receptor (EGFR) overexpression has been identified in early stages of premalignant lesions of the oral squamous cell carcinoma; therefore, real-time assessment of EGFR expression could serve as a biomarker for oral neoplasia. The purpose of our study was to develop a molecular-specific optical contrast agent targeted against EGFR for in vivo assessment of epithelial neoplasia using a monoclonal antibody and the far-red fluorescent dye, Alexa FluorÒ 660 streptavidin. In addition to demonstrating the specificity of the contrast agent for EGFR in cell lines, we document the ability to achieve penetration through 500 lm thick epithelial layers using multilayer tissue constructs and permeability-enhancing agents. Finally, using the fluorescence intensity of the contrast agent on fresh oral cavity tissue sections, we were able to distinguish abnormal from normal oral tissue. This contrast agent should have important clinical applications for use in conjunction with fluorescence spectroscopy or imaging (or both) to facilitate tumor detection and demarcation.

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Indirect immunotargeting of CIS‐PT to human epidermoid carcinoma KB using the avidin‐biotin system

Cited by 33 publications

References 12 publications

Two Motifs within a Transmembrane Domain, One for Homodimerization and the Other for Heterodimerization

Two Motifs within a Transmembrane Domain, One for Homodimerization and the Other for Heterodimerization

Real‐time detection of epidermal growth factor receptor expression in fresh oral cavity biopsies using a molecular‐specific contrast agent

A Far-red Fluorescent Contrast Agent to Image Epidermal Growth Factor Receptor Expression

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