Limits...
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.


Scanning electron microscopy images and resulting EDAX in H460 cells.Notes: Cells were labeled with gold toward their respective antibody treatment. (A) Images were obtained using a BSE. This detector is specialized to image heavy metals within samples and highlights enhanced gold within the sample. Any distinguishable large particles of gold represent a bound antibody enhanced with gold. (B) Images were also obtained with a GSE, which showed cell morphology to ensure correct cell structure and integrity. (C) EDAX analysis of each sample showed the gold elemental peaks for all the elements present within the sample. Silicon is the highest represented element because cells were mounted on silicon cover slips for analysis. The gold elemental peak is indicated with a gold error. Images obtained from this analysis show the exact location of the HPRT bound to the surface of the cell and show no clear pattern indicating a random distribution of the antigen across the surface of the cell.Abbreviations: BSE, back scatter electron; EDAX, energy-dispersive analysis X-ray; GSE, gaseous side electron; HPRT, hypoxanthine guanine phosphoribosyltransferase.
© Copyright Policy
Related In: Results  -  Collection

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

f5-ott-10-1921: Scanning electron microscopy images and resulting EDAX in H460 cells.Notes: Cells were labeled with gold toward their respective antibody treatment. (A) Images were obtained using a BSE. This detector is specialized to image heavy metals within samples and highlights enhanced gold within the sample. Any distinguishable large particles of gold represent a bound antibody enhanced with gold. (B) Images were also obtained with a GSE, which showed cell morphology to ensure correct cell structure and integrity. (C) EDAX analysis of each sample showed the gold elemental peaks for all the elements present within the sample. Silicon is the highest represented element because cells were mounted on silicon cover slips for analysis. The gold elemental peak is indicated with a gold error. Images obtained from this analysis show the exact location of the HPRT bound to the surface of the cell and show no clear pattern indicating a random distribution of the antigen across the surface of the cell.Abbreviations: BSE, back scatter electron; EDAX, energy-dispersive analysis X-ray; GSE, gaseous side electron; 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
Scanning electron microscopy images and resulting EDAX in H460 cells.Notes: Cells were labeled with gold toward their respective antibody treatment. (A) Images were obtained using a BSE. This detector is specialized to image heavy metals within samples and highlights enhanced gold within the sample. Any distinguishable large particles of gold represent a bound antibody enhanced with gold. (B) Images were also obtained with a GSE, which showed cell morphology to ensure correct cell structure and integrity. (C) EDAX analysis of each sample showed the gold elemental peaks for all the elements present within the sample. Silicon is the highest represented element because cells were mounted on silicon cover slips for analysis. The gold elemental peak is indicated with a gold error. Images obtained from this analysis show the exact location of the HPRT bound to the surface of the cell and show no clear pattern indicating a random distribution of the antigen across the surface of the cell.Abbreviations: BSE, back scatter electron; EDAX, energy-dispersive analysis X-ray; GSE, gaseous side electron; HPRT, hypoxanthine guanine phosphoribosyltransferase.
© Copyright Policy
Related In: Results  -  Collection

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

f5-ott-10-1921: Scanning electron microscopy images and resulting EDAX in H460 cells.Notes: Cells were labeled with gold toward their respective antibody treatment. (A) Images were obtained using a BSE. This detector is specialized to image heavy metals within samples and highlights enhanced gold within the sample. Any distinguishable large particles of gold represent a bound antibody enhanced with gold. (B) Images were also obtained with a GSE, which showed cell morphology to ensure correct cell structure and integrity. (C) EDAX analysis of each sample showed the gold elemental peaks for all the elements present within the sample. Silicon is the highest represented element because cells were mounted on silicon cover slips for analysis. The gold elemental peak is indicated with a gold error. Images obtained from this analysis show the exact location of the HPRT bound to the surface of the cell and show no clear pattern indicating a random distribution of the antigen across the surface of the cell.Abbreviations: BSE, back scatter electron; EDAX, energy-dispersive analysis X-ray; GSE, gaseous side electron; 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.