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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.


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HPRT surface expression on A549 non-small-cell lung cancer cells.Notes: The following samples were utilized in order to evaluate the expression of APRT, DCK, and HPRT on the surface of H460 cells: unstained (autofluorescence), mouse IgG (nonspecific binding), rabbit IgG (isotype control), NF-κB (cytosolic protein control), and CD44 (positive surface antigen). (A) Although not as prominent as the population shift in H460 cells (Figure 1C), A549 cells treated with anti-HPRT antibody (pink) have a clear shift in the population toward a higher fluorescent value, indicating the presence of HPRT antigen on the surface of A549 cells. (B) When treated with anti-HPRT antibody there is a shift in the cell population from Q4 to Q3 of an average of 8% when populations are compared to unstained and mouse IgG Q3 populations. (C) Statistical analysis reveals significant HPRT binding on the surface of A549 cells (P=0.0245) when compared to controls. ***P≤0.001.Abbreviations: APRT, adenine phosphoribosyltransferase; DCK, deoxycytidine kinase; HPRT, hypoxanthine guanine phosphoribosyltransferase.
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f2-ott-10-1921: HPRT surface expression on A549 non-small-cell lung cancer cells.Notes: The following samples were utilized in order to evaluate the expression of APRT, DCK, and HPRT on the surface of H460 cells: unstained (autofluorescence), mouse IgG (nonspecific binding), rabbit IgG (isotype control), NF-κB (cytosolic protein control), and CD44 (positive surface antigen). (A) Although not as prominent as the population shift in H460 cells (Figure 1C), A549 cells treated with anti-HPRT antibody (pink) have a clear shift in the population toward a higher fluorescent value, indicating the presence of HPRT antigen on the surface of A549 cells. (B) When treated with anti-HPRT antibody there is a shift in the cell population from Q4 to Q3 of an average of 8% when populations are compared to unstained and mouse IgG Q3 populations. (C) Statistical analysis reveals significant HPRT binding on the surface of A549 cells (P=0.0245) when compared to controls. ***P≤0.001.Abbreviations: APRT, adenine phosphoribosyltransferase; DCK, deoxycytidine kinase; HPRT, hypoxanthine guanine phosphoribosyltransferase.

Mentions: When treated with anti-HPRT fluorescent antibodies, both A549 and H460 cancer cells had an increase in the fluorescent population (Figures 1C and 2). A 28% shift in the population is observed in H460 cells (Figure 1C), while a 12% shift is observed in A549 cells (Figure 3). Statistical analysis comparing anti-HPRT-treated cells with isotype IgG controls showed a statistically significant difference in H460 and A549 cells (Figures 1C and 2C). Thus, these data show a significant association between HPRT and the surface of non-small-cell lung cancer cells. This analysis also revealed a significantly higher HPRT surface expression in H460 cells when compared to A549 (Figure 3).


Non-small-cell lung cancer cell lines A549 and NCI-H460 express hypoxanthine guanine phosphoribosyltransferase on the plasma membrane
HPRT surface expression on A549 non-small-cell lung cancer cells.Notes: The following samples were utilized in order to evaluate the expression of APRT, DCK, and HPRT on the surface of H460 cells: unstained (autofluorescence), mouse IgG (nonspecific binding), rabbit IgG (isotype control), NF-κB (cytosolic protein control), and CD44 (positive surface antigen). (A) Although not as prominent as the population shift in H460 cells (Figure 1C), A549 cells treated with anti-HPRT antibody (pink) have a clear shift in the population toward a higher fluorescent value, indicating the presence of HPRT antigen on the surface of A549 cells. (B) When treated with anti-HPRT antibody there is a shift in the cell population from Q4 to Q3 of an average of 8% when populations are compared to unstained and mouse IgG Q3 populations. (C) Statistical analysis reveals significant HPRT binding on the surface of A549 cells (P=0.0245) when compared to controls. ***P≤0.001.Abbreviations: APRT, adenine phosphoribosyltransferase; DCK, deoxycytidine kinase; HPRT, hypoxanthine guanine phosphoribosyltransferase.
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f2-ott-10-1921: HPRT surface expression on A549 non-small-cell lung cancer cells.Notes: The following samples were utilized in order to evaluate the expression of APRT, DCK, and HPRT on the surface of H460 cells: unstained (autofluorescence), mouse IgG (nonspecific binding), rabbit IgG (isotype control), NF-κB (cytosolic protein control), and CD44 (positive surface antigen). (A) Although not as prominent as the population shift in H460 cells (Figure 1C), A549 cells treated with anti-HPRT antibody (pink) have a clear shift in the population toward a higher fluorescent value, indicating the presence of HPRT antigen on the surface of A549 cells. (B) When treated with anti-HPRT antibody there is a shift in the cell population from Q4 to Q3 of an average of 8% when populations are compared to unstained and mouse IgG Q3 populations. (C) Statistical analysis reveals significant HPRT binding on the surface of A549 cells (P=0.0245) when compared to controls. ***P≤0.001.Abbreviations: APRT, adenine phosphoribosyltransferase; DCK, deoxycytidine kinase; HPRT, hypoxanthine guanine phosphoribosyltransferase.
Mentions: When treated with anti-HPRT fluorescent antibodies, both A549 and H460 cancer cells had an increase in the fluorescent population (Figures 1C and 2). A 28% shift in the population is observed in H460 cells (Figure 1C), while a 12% shift is observed in A549 cells (Figure 3). Statistical analysis comparing anti-HPRT-treated cells with isotype IgG controls showed a statistically significant difference in H460 and A549 cells (Figures 1C and 2C). Thus, these data show a significant association between HPRT and the surface of non-small-cell lung cancer cells. This analysis also revealed a significantly higher HPRT surface expression in H460 cells when compared to A549 (Figure 3).

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.


Related in: MedlinePlus