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Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane

View Article: PubMed Central - PubMed

ABSTRACT

In both males and females, lung cancer is one of the most lethal cancers worldwide and accounts for >30% of cancer-related deaths. Despite advances in biomarker analysis and tumor characterization, there remains a need to find suitable biomarker antigen targets for treatment in late-stage lung cancer. Previous research on the salvage pathway enzyme TK1 shows a unique relationship with cancer patients as serum levels are raised according to cancer grade. To expand this analysis, the other salvage pathway enzymes were evaluated for possible upregulation within lung cancer. Adenine phosphoribosyltransferase, deoxycytidine kinase, and hypoxanthine guanine phosphoribosyltransferase (HPRT) were assessed for their presentation on two non-small-cell lung cancer cell lines NCI-H460 and A549. In the present study, we show that deoxycytidine kinase and adenine phosphoribosyltransferase have no significant relationship with the membrane of NCI-H460 cells. However, we found significant localization of HPRT to the membrane of NCI-H460 and A549 cells. When treated with anti-HPRT antibodies, the average fluorescence of the cell population increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison with controls. To ensure that expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold-conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and energy-dispersive analysis X-ray. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (P=0.012) in comparison with isotype controls. Our results show that HPRT is localized on the surface of these non-small-cell lung cancer cell lines.

No MeSH data available.


Plasma membrane colocalization with HPRT in H460 cells.Notes:H460 cells were dyed with both a FITC dye and a Rhodamine Red membrane dye to label antibody treatments and the plasma membrane, respectively. Utilizing unstained cells, IgG-treated cells, and NF-κB-treated cells as controls, plasma membrane associations were evaluated to determine whether any of the treatments significantly bound to the membrane of H460 cells. (A) Each sample was analyzed and imaged by a 488 nm laser to illuminate FITC-positive cells. These images show the binding of the respective antigen treatment. (B) Samples were also imaged in a 594 nm laser to show rhodamine-positive cells. This dye binds to the plasma membrane of all cells. (C) The two images obtained from columns A and B were merged to show associations between treated antibodies and the plasma membrane of cells. These results show a clear overlap between cells treated with anti-HPRT antibody and those treated with the membrane dye. This demonstrates a clear association between HPRT and the plasma membrane of H460 cells.Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.
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f4-ott-10-1921: Plasma membrane colocalization with HPRT in H460 cells.Notes:H460 cells were dyed with both a FITC dye and a Rhodamine Red membrane dye to label antibody treatments and the plasma membrane, respectively. Utilizing unstained cells, IgG-treated cells, and NF-κB-treated cells as controls, plasma membrane associations were evaluated to determine whether any of the treatments significantly bound to the membrane of H460 cells. (A) Each sample was analyzed and imaged by a 488 nm laser to illuminate FITC-positive cells. These images show the binding of the respective antigen treatment. (B) Samples were also imaged in a 594 nm laser to show rhodamine-positive cells. This dye binds to the plasma membrane of all cells. (C) The two images obtained from columns A and B were merged to show associations between treated antibodies and the plasma membrane of cells. These results show a clear overlap between cells treated with anti-HPRT antibody and those treated with the membrane dye. This demonstrates a clear association between HPRT and the plasma membrane of H460 cells.Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.

Mentions: In order to confirm that HPRT was not bound to cytoplasmic protein, the surface expression of HPRT was further evaluated with confocal microscopy (Figure 4). Images obtained from cells treated with membrane dye and FITC antibody stain were overlapped to show colocalization of treated antigen on the plasma membrane of the cancer cell. When cells are treated with anti-HPRT antibody, a yellow pigment appears in the merged image, which indicates a direct relationship between the plasma membrane dye and the FITC dye. No other treatment experienced this same overlapped pigmentation, which confirms the relationship between HPRT and the plasma membrane of H460 cells.


Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane
Plasma membrane colocalization with HPRT in H460 cells.Notes:H460 cells were dyed with both a FITC dye and a Rhodamine Red membrane dye to label antibody treatments and the plasma membrane, respectively. Utilizing unstained cells, IgG-treated cells, and NF-κB-treated cells as controls, plasma membrane associations were evaluated to determine whether any of the treatments significantly bound to the membrane of H460 cells. (A) Each sample was analyzed and imaged by a 488 nm laser to illuminate FITC-positive cells. These images show the binding of the respective antigen treatment. (B) Samples were also imaged in a 594 nm laser to show rhodamine-positive cells. This dye binds to the plasma membrane of all cells. (C) The two images obtained from columns A and B were merged to show associations between treated antibodies and the plasma membrane of cells. These results show a clear overlap between cells treated with anti-HPRT antibody and those treated with the membrane dye. This demonstrates a clear association between HPRT and the plasma membrane of H460 cells.Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5384690&req=5

f4-ott-10-1921: Plasma membrane colocalization with HPRT in H460 cells.Notes:H460 cells were dyed with both a FITC dye and a Rhodamine Red membrane dye to label antibody treatments and the plasma membrane, respectively. Utilizing unstained cells, IgG-treated cells, and NF-κB-treated cells as controls, plasma membrane associations were evaluated to determine whether any of the treatments significantly bound to the membrane of H460 cells. (A) Each sample was analyzed and imaged by a 488 nm laser to illuminate FITC-positive cells. These images show the binding of the respective antigen treatment. (B) Samples were also imaged in a 594 nm laser to show rhodamine-positive cells. This dye binds to the plasma membrane of all cells. (C) The two images obtained from columns A and B were merged to show associations between treated antibodies and the plasma membrane of cells. These results show a clear overlap between cells treated with anti-HPRT antibody and those treated with the membrane dye. This demonstrates a clear association between HPRT and the plasma membrane of H460 cells.Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.
Mentions: In order to confirm that HPRT was not bound to cytoplasmic protein, the surface expression of HPRT was further evaluated with confocal microscopy (Figure 4). Images obtained from cells treated with membrane dye and FITC antibody stain were overlapped to show colocalization of treated antigen on the plasma membrane of the cancer cell. When cells are treated with anti-HPRT antibody, a yellow pigment appears in the merged image, which indicates a direct relationship between the plasma membrane dye and the FITC dye. No other treatment experienced this same overlapped pigmentation, which confirms the relationship between HPRT and the plasma membrane of H460 cells.

View Article: PubMed Central - PubMed

ABSTRACT

In both males and females, lung cancer is one of the most lethal cancers worldwide and accounts for >30% of cancer-related deaths. Despite advances in biomarker analysis and tumor characterization, there remains a need to find suitable biomarker antigen targets for treatment in late-stage lung cancer. Previous research on the salvage pathway enzyme TK1 shows a unique relationship with cancer patients as serum levels are raised according to cancer grade. To expand this analysis, the other salvage pathway enzymes were evaluated for possible upregulation within lung cancer. Adenine phosphoribosyltransferase, deoxycytidine kinase, and hypoxanthine guanine phosphoribosyltransferase (HPRT) were assessed for their presentation on two non-small-cell lung cancer cell lines NCI-H460 and A549. In the present study, we show that deoxycytidine kinase and adenine phosphoribosyltransferase have no significant relationship with the membrane of NCI-H460 cells. However, we found significant localization of HPRT to the membrane of NCI-H460 and A549 cells. When treated with anti-HPRT antibodies, the average fluorescence of the cell population increased by 24.3% and 12.9% in NCI-H460 and A549 cells, respectively, in comparison with controls. To ensure that expression was not attributed to cytoplasmic HPRT, confocal microscopy was performed to visualize HPRT binding on the plasma membrane. After staining NCI-H460 cells treated with both fluorescent antibodies and a membrane-specific dye, we observed direct overlap between HPRT and the membrane of the cancer cells. Additionally, gold-conjugated antibodies were used to label and quantify the amount of HPRT on the cell surface using scanning electron microscopy and energy-dispersive analysis X-ray. Further confirming HPRT presence, the gold weight percentage of the sample increased significantly when NCI-H460 cells were exposed to HPRT antibody (P=0.012) in comparison with isotype controls. Our results show that HPRT is localized on the surface of these non-small-cell lung cancer cell lines.

No MeSH data available.