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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

GIPC1 RNA expression analysis in normal and neoplastic cell lines. Panel A: Northern blot analysis of total RNA was performed with RNA samples from a human microvascular endothelial cell line (HMEC), normal breast epithelium cell line HBL100 and breast cancer cell lines MCF-7, T47D, SK-BR-3, MDA231, MDA157 and MDA453. A probe for the GAPDH gene was used to normalize expression. Panel B: Densitometry analysis of the Northern blot was performed to quantitate the mRNA expression. The data indicates that the GIPC1 gene is upregulated in breast cancer cell lines.
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Figure 4: GIPC1 RNA expression analysis in normal and neoplastic cell lines. Panel A: Northern blot analysis of total RNA was performed with RNA samples from a human microvascular endothelial cell line (HMEC), normal breast epithelium cell line HBL100 and breast cancer cell lines MCF-7, T47D, SK-BR-3, MDA231, MDA157 and MDA453. A probe for the GAPDH gene was used to normalize expression. Panel B: Densitometry analysis of the Northern blot was performed to quantitate the mRNA expression. The data indicates that the GIPC1 gene is upregulated in breast cancer cell lines.

Mentions: The strong staining by the human anti-GIPC1 monoclonal antibodies, 27.B1 and 27.F7, on breast cancer cell lines and absent staining of normal cells [25] suggests that the protein might be up-regulated. To semi-quantitatively examine gene expression we performed Northern blot analysis to determine if a higher level of GIPC1 specific mRNA was indeed present in the breast cancer cell lines relative to normal cell lines. RNA from a variety of breast cancer cell lines along with non-neoplastic cell lines was blotted and GAPDH expression was monitored as an internal control to normalize expression. The results are depicted in Figure 4. They indicate that breast cancer cells indeed have increased expression of GIPC1 specific RNA relative to that of normal cell lines.


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)

GIPC1 RNA expression analysis in normal and neoplastic cell lines. Panel A: Northern blot analysis of total RNA was performed with RNA samples from a human microvascular endothelial cell line (HMEC), normal breast epithelium cell line HBL100 and breast cancer cell lines MCF-7, T47D, SK-BR-3, MDA231, MDA157 and MDA453. A probe for the GAPDH gene was used to normalize expression. Panel B: Densitometry analysis of the Northern blot was performed to quantitate the mRNA expression. The data indicates that the GIPC1 gene is upregulated in breast cancer cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2529336&req=5

Figure 4: GIPC1 RNA expression analysis in normal and neoplastic cell lines. Panel A: Northern blot analysis of total RNA was performed with RNA samples from a human microvascular endothelial cell line (HMEC), normal breast epithelium cell line HBL100 and breast cancer cell lines MCF-7, T47D, SK-BR-3, MDA231, MDA157 and MDA453. A probe for the GAPDH gene was used to normalize expression. Panel B: Densitometry analysis of the Northern blot was performed to quantitate the mRNA expression. The data indicates that the GIPC1 gene is upregulated in breast cancer cell lines.
Mentions: The strong staining by the human anti-GIPC1 monoclonal antibodies, 27.B1 and 27.F7, on breast cancer cell lines and absent staining of normal cells [25] suggests that the protein might be up-regulated. To semi-quantitatively examine gene expression we performed Northern blot analysis to determine if a higher level of GIPC1 specific mRNA was indeed present in the breast cancer cell lines relative to normal cell lines. RNA from a variety of breast cancer cell lines along with non-neoplastic cell lines was blotted and GAPDH expression was monitored as an internal control to normalize expression. The results are depicted in Figure 4. They indicate that breast cancer cells indeed have increased expression of GIPC1 specific RNA relative to that of normal cell lines.

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