Reevaluation of the 22-1-1 antibody and its putative antigen, EBAG9/RCAS1, as a tumor marker.
BACKGROUND
Tumor-associated antigens are appreciated as diagnostic markers, but they have also prompted tremendous efforts to develop tumor-specific immunotherapy. A previously cloned tumor-associated antigen, EBAG9, was initially defined by reactivity with the monoclonal antibody 22-1-1. Functionally, the EBAG9-encoded gene-product was believed to induce apoptosis in activated immune cells. However, using a cell-biological approach we identified EBAG9 as a Golgi-resident modulator of O-linked glycan expression, the latter product was then recognized by the 22-1-1 antibody. Secondly, EBAG9 expression was found physiologically in all murine tissues examined. This raised the question if EBAG9 is tumor-specific and mediates apoptosis itself or through O-linked glycans generated, among them the cognate 22-1-1 antigen Tn.


METHODS
We have used immunohistochemistry to detect the expression of 22-1-1 and EBAG9 in various tissues. Correlation between expression of both antigens in cell lines was analysed by immunoblot and flow cytometry. Apoptosis was studied by using flow cytometry and Caspase-Glo 3/7 assay kit. Cellular distribution of EBAG9 was analysed by electron and confocal microscopy.


RESULTS
Here, we compared expression of the 22-1-1 and EBAG9-defined antigens in normal and neoplastic tissues in situ. In contrast to 22-1-1 staining, EBAG9 is a ubiquitously expressed antigen in all normal and cancerous tissues. Functional studies on the role of 22-1-1 reactive material did not support any evidence for apoptosis induction. Employing electron and confocal microscopy, a refined subcellular localization of EBAG9 at the Golgi was obtained.


CONCLUSIONS
We suggest that the estrogen-inducible EBAG9 gene-product and the 22-1-1 defined antigen are structurally and functionally separate antigens.
Reimer
Tatiana A
TA
Department of Hematology, Oncology and Tumorimmunology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany. reimer@mdc-berlin.de
Anagnostopoulos
Ioannis
I
Erdmann
Bettina
B
Lehmann
Insa
I
Stein
Harald
H
Daniel
Peter
P
Dörken
Bernd
B
Rehm
Armin
A
D000230:Adenocarcinoma / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000737:chemistry; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000096:biosynthesis*; D020126:Brefeldin A / Q000494:pharmacology; D002277:Carcinoma / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000378:metabolism; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000096:biosynthesis; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D015179:Colorectal Neoplasms / Q000378:metabolism; D005434:Flow Cytometry; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007167:Immunotherapy / Q000379:methods; D008175:Lung Neoplasms / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009062:Mouth Neoplasms / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D011134:Polysaccharides / Q000737:chemistry; D011471:Prostatic Neoplasms / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000378:metabolism; D013347:Subcellular Fractions; D014018:Tissue Distribution
C099099:22-1-1 antigen; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; C120762:EBAG9 protein, human; D011134:Polysaccharides; D011500:Protein Synthesis Inhibitors; C040286:Tn antigen; D020126:Brefeldin A; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D015739:Nocodazole