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Identification of ATP synthase beta subunit (ATPB) on the cell surface as a non-small cell lung cancer (NSCLC) associated antigen.

Lu ZJ, Song QF, Jiang SS, Song Q, Wang W, Zhang GH, Kan B, Chen LJ, Yang JL, Luo F, Qian ZY, Wei YQ, Gou LT - BMC Cancer (2009)

Bottom Line: But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far.The monoclonal antibody 4E7 (McAb4E7) specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells.ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, 37 Guoxue Xiang Street, Chengdu 610041, Sichuan, PR China. luzejun.01@163.com

ABSTRACT

Background: Antibody-based immunotherapy has achieved some success for cancer. But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far. It is essential to identify more immunogenic antigens (especially cellular membrane markers) for tumor diagnosis and therapy.

Methods: The membrane proteins of lung adenocarcinoma cell line A549 were used to immunize the BALB/c mice. A monoclonal antibody 4E7 (McAb4E7) was produced with hybridoma technique. MTT cell proliferation assay was carried out to evaluate the inhibitory effect of McAb4E7 on A549 cells. Flow cytometric assay, immunohistochemistry, western blot and proteomic technologies based on 2-DE and mass spectrometry were employed to detect and identify the corresponding antigen of McAb4E7.

Results: The monoclonal antibody 4E7 (McAb4E7) specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells. By the proteomic technologies, we identified that ATP synthase beta subunit (ATPB) was the corresponding antigen of McAb4E7. Then, flow cytometric analysis demonstrated the localization of the targeting antigen of McAb4E7 was on the A549 cells surface. Furthermore, immunohistochemistry showed that the antigen of McAb4E7 mainly aberrantly expressed in tumor cellular membrane in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC). The rate of ectopic expressed ATPB in the cellular membrane in lung adenocarcinoma, squamous carcinoma and their adjacent nontumourous lung tissues was 71.88%, 66.67% and 25.81% respectively.

Conclusion: In the present study, we identified that the ectopic ATPB in tumor cellular membrane was the non-small cell lung cancer (NSCLC) associated antigen. ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

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MALDI-TOF-MS and MS/MS analysis of the antigen of McAb4E7. (A) Peptide mass fingerprinting (PMF) of the targeting protein recognized by McAb4E7. Black arrow in the image indicated the peak of ATPB (B) MS/MS spectrum of peak [M +2H]2+ at m/z 1439.77 from positive spot matched sequence RVALTGLTVAEYFRD of ATPB (C) Probability based mowse score.
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Figure 5: MALDI-TOF-MS and MS/MS analysis of the antigen of McAb4E7. (A) Peptide mass fingerprinting (PMF) of the targeting protein recognized by McAb4E7. Black arrow in the image indicated the peak of ATPB (B) MS/MS spectrum of peak [M +2H]2+ at m/z 1439.77 from positive spot matched sequence RVALTGLTVAEYFRD of ATPB (C) Probability based mowse score.

Mentions: The excised gel piece was destained and trypsinized into peptides for MS and MS/MS analysis. Mass spectra were acquired with a Q-TOF Premier Mass Spectrometer (Figure 5). The MS/MS data, including the mass values, the intensity and the charge of the precursor ions, were analyzed with a licensed copy of the Mascot 2.0 program against SWISS-PROT protein database. The charged ion of 1439.76 m/z and its fragment ions revealed the peptide sequence, R.VALTGLTVAEYFR.D, unique to ATPB (Figure 5A and 5B). The protein spot was identified as ATP synthase beta subunit (ATPB). The score was 62 (P < 0.05) (Figure 5C).


Identification of ATP synthase beta subunit (ATPB) on the cell surface as a non-small cell lung cancer (NSCLC) associated antigen.

Lu ZJ, Song QF, Jiang SS, Song Q, Wang W, Zhang GH, Kan B, Chen LJ, Yang JL, Luo F, Qian ZY, Wei YQ, Gou LT - BMC Cancer (2009)

MALDI-TOF-MS and MS/MS analysis of the antigen of McAb4E7. (A) Peptide mass fingerprinting (PMF) of the targeting protein recognized by McAb4E7. Black arrow in the image indicated the peak of ATPB (B) MS/MS spectrum of peak [M +2H]2+ at m/z 1439.77 from positive spot matched sequence RVALTGLTVAEYFRD of ATPB (C) Probability based mowse score.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654462&req=5

Figure 5: MALDI-TOF-MS and MS/MS analysis of the antigen of McAb4E7. (A) Peptide mass fingerprinting (PMF) of the targeting protein recognized by McAb4E7. Black arrow in the image indicated the peak of ATPB (B) MS/MS spectrum of peak [M +2H]2+ at m/z 1439.77 from positive spot matched sequence RVALTGLTVAEYFRD of ATPB (C) Probability based mowse score.
Mentions: The excised gel piece was destained and trypsinized into peptides for MS and MS/MS analysis. Mass spectra were acquired with a Q-TOF Premier Mass Spectrometer (Figure 5). The MS/MS data, including the mass values, the intensity and the charge of the precursor ions, were analyzed with a licensed copy of the Mascot 2.0 program against SWISS-PROT protein database. The charged ion of 1439.76 m/z and its fragment ions revealed the peptide sequence, R.VALTGLTVAEYFR.D, unique to ATPB (Figure 5A and 5B). The protein spot was identified as ATP synthase beta subunit (ATPB). The score was 62 (P < 0.05) (Figure 5C).

Bottom Line: But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far.The monoclonal antibody 4E7 (McAb4E7) specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells.ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, 37 Guoxue Xiang Street, Chengdu 610041, Sichuan, PR China. luzejun.01@163.com

ABSTRACT

Background: Antibody-based immunotherapy has achieved some success for cancer. But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far. It is essential to identify more immunogenic antigens (especially cellular membrane markers) for tumor diagnosis and therapy.

Methods: The membrane proteins of lung adenocarcinoma cell line A549 were used to immunize the BALB/c mice. A monoclonal antibody 4E7 (McAb4E7) was produced with hybridoma technique. MTT cell proliferation assay was carried out to evaluate the inhibitory effect of McAb4E7 on A549 cells. Flow cytometric assay, immunohistochemistry, western blot and proteomic technologies based on 2-DE and mass spectrometry were employed to detect and identify the corresponding antigen of McAb4E7.

Results: The monoclonal antibody 4E7 (McAb4E7) specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells. By the proteomic technologies, we identified that ATP synthase beta subunit (ATPB) was the corresponding antigen of McAb4E7. Then, flow cytometric analysis demonstrated the localization of the targeting antigen of McAb4E7 was on the A549 cells surface. Furthermore, immunohistochemistry showed that the antigen of McAb4E7 mainly aberrantly expressed in tumor cellular membrane in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC). The rate of ectopic expressed ATPB in the cellular membrane in lung adenocarcinoma, squamous carcinoma and their adjacent nontumourous lung tissues was 71.88%, 66.67% and 25.81% respectively.

Conclusion: In the present study, we identified that the ectopic ATPB in tumor cellular membrane was the non-small cell lung cancer (NSCLC) associated antigen. ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

Show MeSH
Related in: MedlinePlus