Surface expression of gpA33 is dependent on culture density and cell-cycle phase and is regulated by intracellular traffic rather than gene transcription.
The cell-surface marker, gpA33, a new member of the immunoglobulin superfamily, is expressed by gastrointestinal cells and by 95% of colon cancers. It has become a promising target of immunologic therapy strategies, but its biologic function and potential role in tumorigenesis are unknown. In this study, we have investigated the expression of gpA33 on the mRNA and cell-surface protein levels by quantitative reverse transcriptase polymerase chain reaction and flow cytometry, respectively, in response to cell density in the culture and to cell-cycle arrest in the G1, S, or G2/M phases. As internalization of the surface protein had previously been reported, we also investigated the binding and intracellular migration of an anti-gpA33 fluobody with green fluorescent protein (A33scFv::GFP) by laser confocal microscopy. Contrary to intuition, we found that gpA33 surface expression and mRNA levels do only partly correlate under the conditions tested. Dependent on cell density in culture, gpA33 surface expression peaked at the point of confluence. Dependent on cell-cycle phase, it peaked in the G2/M phase but was lowest in the S phase, whereas mRNA levels were highest in S, but almost absent in G1. Laser confocal microscopy clearly demonstrated the intracellular uptake of A33scFv::GFP and showed the migration of microvesicles over time. These findings are, in part, concordant with the putative role of gpA33 as an adhesion molecule. However, intracellular traffic and recycling to the cell surface appear to play a major role in its function and to have an influence on its surface density superseding translational regulation.
Frey
Dietmar
D
Centrum für Tumormedizin, Charité Universitätsmedizin Berlin, Berlin, Germany.
Coelho
Vania
V
Petrausch
Ulf
U
Schaefer
Michael
M
Keilholz
Ulrich
U
Thiel
Eckhard
E
Deckert
P Markus
PM
D002452:Cell Count; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000502:physiology*; D002465:Cell Movement; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D018613:Microscopy, Confocal; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C104258:GPA33 protein, human; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins