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A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen.

Rudchenko S, Scanlan M, Kalantarov G, Yavelsky V, Levy C, Estabrook A, Old L, Chan GL, Lobel L, Trakht I - BMC Cancer (2008)

Bottom Line: Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer.Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Physicians and Surgeons, Columbia University, 630 W, 168 St,, New York, NY 10032, USA. RudchenkoS@HSS.EDU

ABSTRACT

Background: We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.

Methods: The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.

Results: By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.

Conclusion: We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.

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Related in: MedlinePlus

The target antigen for human monoclonal 27.B1 is a GIPC1 protein. A. The target antigen for monoclonal 27.B1 in SK-BR-3 cells, breast cancer tissue and normal breast tissue was identified by Western blot and is displayed. Human Ig H and L chains are present in the tissue and are recognized by the secondary anti-human antiserum. The target antigen is detected as a doublet in both the breast cancer tissue and SK-BR-3 cell line but is not detected in normal breast tissue. Both bands in the doublet are present in all breast cancer cell lines analyzed by Western blot but their intensity is variable. B. Immunoblotting with 27.B1 antibody of total cell lysates prepared from SK-BR-3 cells. 27.B1 antibody was preincubated with recombinant GIPC1 protein prior to blotting (lane 1), and compared to non-preincubated control (lane 2).
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Figure 2: The target antigen for human monoclonal 27.B1 is a GIPC1 protein. A. The target antigen for monoclonal 27.B1 in SK-BR-3 cells, breast cancer tissue and normal breast tissue was identified by Western blot and is displayed. Human Ig H and L chains are present in the tissue and are recognized by the secondary anti-human antiserum. The target antigen is detected as a doublet in both the breast cancer tissue and SK-BR-3 cell line but is not detected in normal breast tissue. Both bands in the doublet are present in all breast cancer cell lines analyzed by Western blot but their intensity is variable. B. Immunoblotting with 27.B1 antibody of total cell lysates prepared from SK-BR-3 cells. 27.B1 antibody was preincubated with recombinant GIPC1 protein prior to blotting (lane 1), and compared to non-preincubated control (lane 2).

Mentions: To identify the size of the 27.B1 target protein, Western blot analysis was performed using whole cell lysates from different cell lines and tissues. Cell lysates prepared from human breast cancer, normal breast tissue, human prostate cancer and two human fibroblast cell lines were run on PAGE under reducing conditions and blotted with fhMAb 27.B1. The antibody reacted with a protein band of approximately 42 kDa molecular weight that is detectable primarily in breast cancer cells (see Figure 2-A). There was no detectable immunoreactivity with the human fibroblasts' lysates and prostate cancer cell line LnCaP whereas only traces of immunoreactivity were detected to prostate cancer cell lines PC3 and DU-145 (data not shown). The protein band revealed by 27.B1 appeared as doublet with a dominant band that migrates slower on a gel. The doublet pattern was not the same in all 27.B1 positive cells; MCF-7 cells displayed the higher molecular weight band in much greater abundance, whereas SK-BR-3 showed both bands in more equivalent intensity (data not shown). Western blot analysis of the same cell lysates under non-reducing conditions displayed no difference in staining pattern, indicating that accessibility of the epitope bound by 27.B1 is disulfide bond independent and likely conformation independent.


A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen.

Rudchenko S, Scanlan M, Kalantarov G, Yavelsky V, Levy C, Estabrook A, Old L, Chan GL, Lobel L, Trakht I - BMC Cancer (2008)

The target antigen for human monoclonal 27.B1 is a GIPC1 protein. A. The target antigen for monoclonal 27.B1 in SK-BR-3 cells, breast cancer tissue and normal breast tissue was identified by Western blot and is displayed. Human Ig H and L chains are present in the tissue and are recognized by the secondary anti-human antiserum. The target antigen is detected as a doublet in both the breast cancer tissue and SK-BR-3 cell line but is not detected in normal breast tissue. Both bands in the doublet are present in all breast cancer cell lines analyzed by Western blot but their intensity is variable. B. Immunoblotting with 27.B1 antibody of total cell lysates prepared from SK-BR-3 cells. 27.B1 antibody was preincubated with recombinant GIPC1 protein prior to blotting (lane 1), and compared to non-preincubated control (lane 2).
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Figure 2: The target antigen for human monoclonal 27.B1 is a GIPC1 protein. A. The target antigen for monoclonal 27.B1 in SK-BR-3 cells, breast cancer tissue and normal breast tissue was identified by Western blot and is displayed. Human Ig H and L chains are present in the tissue and are recognized by the secondary anti-human antiserum. The target antigen is detected as a doublet in both the breast cancer tissue and SK-BR-3 cell line but is not detected in normal breast tissue. Both bands in the doublet are present in all breast cancer cell lines analyzed by Western blot but their intensity is variable. B. Immunoblotting with 27.B1 antibody of total cell lysates prepared from SK-BR-3 cells. 27.B1 antibody was preincubated with recombinant GIPC1 protein prior to blotting (lane 1), and compared to non-preincubated control (lane 2).
Mentions: To identify the size of the 27.B1 target protein, Western blot analysis was performed using whole cell lysates from different cell lines and tissues. Cell lysates prepared from human breast cancer, normal breast tissue, human prostate cancer and two human fibroblast cell lines were run on PAGE under reducing conditions and blotted with fhMAb 27.B1. The antibody reacted with a protein band of approximately 42 kDa molecular weight that is detectable primarily in breast cancer cells (see Figure 2-A). There was no detectable immunoreactivity with the human fibroblasts' lysates and prostate cancer cell line LnCaP whereas only traces of immunoreactivity were detected to prostate cancer cell lines PC3 and DU-145 (data not shown). The protein band revealed by 27.B1 appeared as doublet with a dominant band that migrates slower on a gel. The doublet pattern was not the same in all 27.B1 positive cells; MCF-7 cells displayed the higher molecular weight band in much greater abundance, whereas SK-BR-3 showed both bands in more equivalent intensity (data not shown). Western blot analysis of the same cell lysates under non-reducing conditions displayed no difference in staining pattern, indicating that accessibility of the epitope bound by 27.B1 is disulfide bond independent and likely conformation independent.

Bottom Line: Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer.Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.

View Article: PubMed Central - HTML - PubMed

Affiliation: College of Physicians and Surgeons, Columbia University, 630 W, 168 St,, New York, NY 10032, USA. RudchenkoS@HSS.EDU

ABSTRACT

Background: We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.

Methods: The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.

Results: By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.

Conclusion: We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.

Show MeSH
Related in: MedlinePlus