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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.


Gold percentage of H460 cells.Notes: The gold elemental composition of each sample is denoted on the Y-axis. The increase in the gold percentage when cells were exposed to HPRT and CD44 shows a quantifiable increase in the gold present on the outside of the cell. Cells exposed to HPRT antibody had a gold weight of ∼10.4%, which is statistically significant to the IgG controls used for background binding (P=0.0159). These data indicate a statistically significant presence of HPRT on the surface of H460 cells. *P≤0.05; **P≤0.01.Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.
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f6-ott-10-1921: Gold percentage of H460 cells.Notes: The gold elemental composition of each sample is denoted on the Y-axis. The increase in the gold percentage when cells were exposed to HPRT and CD44 shows a quantifiable increase in the gold present on the outside of the cell. Cells exposed to HPRT antibody had a gold weight of ∼10.4%, which is statistically significant to the IgG controls used for background binding (P=0.0159). These data indicate a statistically significant presence of HPRT on the surface of H460 cells. *P≤0.05; **P≤0.01.Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.

Mentions: The location of the HPRT protein on the surface of H460 cells was also analyzed with scanning electron microscopy (Figure 5). The gold elemental peak along with the elemental composition of each sample reveals the changes in the surface gold percentages when cells are exposed to primary antibodies. Images obtained from this analysis show HPRT on the cell surface, but there is no apparent clustering of the antigen as gold particles are scattered across the cell randomly. EDAX analysis showed that cells treated with anti-HPRT antibody had an increase in the average gold weight percentage of 10.39% in comparison with only 8.75% for IgG controls. With a P-value of 0.012 (Figure 6), these data indicate a statistically significant presence of HPRT on the surface of H460 cells while also demonstrating that the antigen shows no patterns of expression.


Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane
Gold percentage of H460 cells.Notes: The gold elemental composition of each sample is denoted on the Y-axis. The increase in the gold percentage when cells were exposed to HPRT and CD44 shows a quantifiable increase in the gold present on the outside of the cell. Cells exposed to HPRT antibody had a gold weight of ∼10.4%, which is statistically significant to the IgG controls used for background binding (P=0.0159). These data indicate a statistically significant presence of HPRT on the surface of H460 cells. *P≤0.05; **P≤0.01.Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC5384690&req=5

f6-ott-10-1921: Gold percentage of H460 cells.Notes: The gold elemental composition of each sample is denoted on the Y-axis. The increase in the gold percentage when cells were exposed to HPRT and CD44 shows a quantifiable increase in the gold present on the outside of the cell. Cells exposed to HPRT antibody had a gold weight of ∼10.4%, which is statistically significant to the IgG controls used for background binding (P=0.0159). These data indicate a statistically significant presence of HPRT on the surface of H460 cells. *P≤0.05; **P≤0.01.Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.
Mentions: The location of the HPRT protein on the surface of H460 cells was also analyzed with scanning electron microscopy (Figure 5). The gold elemental peak along with the elemental composition of each sample reveals the changes in the surface gold percentages when cells are exposed to primary antibodies. Images obtained from this analysis show HPRT on the cell surface, but there is no apparent clustering of the antigen as gold particles are scattered across the cell randomly. EDAX analysis showed that cells treated with anti-HPRT antibody had an increase in the average gold weight percentage of 10.39% in comparison with only 8.75% for IgG controls. With a P-value of 0.012 (Figure 6), these data indicate a statistically significant presence of HPRT on the surface of H460 cells while also demonstrating that the antigen shows no patterns of expression.

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.