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

Figures & data

Figure 1 Flow cytometry analysis of the salvage pathway enzymes in H460 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 control), mouse IgG (nonspecific binding control), rabbit IgG (isotype control), NF-κB (cytosolic protein control), and CD44 (positive surface antigen). (A) When anti-APRT antibody (green) was used to treat cells, a resulting insignificant shift in the population was observed upon comparing the histogram diagrams to controls. This insignificant shift is also shown in the lack of movement from Q3 in isotype controls to Q3 in APRT-treated cells (equaling only 4%). Cells treated with APRT have an insignificant level of binding compared to isotype controls (P=0.224). (B) Cells treated with anti-DCK antibody (purple) had an even smaller shift in the fluorescent population compared to APRT. No cells from Q3 in the mouse IgG control moved to Q3 in the DCK-treated cells, indicating a complete lack of the DCK antigen on the surface of H460 cells. Statistical analysis reveals no presence of DCK on the surface of H460 cells (P=0.106). (C) When treated with anti-HPRT antibody (pink), the histogram representation of the cell population showed a definitive shift in the population toward a higher fluorescence. This was confirmed when >20% of the population from Q3 in the mouse IgG control shifted to Q4 upon HPRT treatment. Of the three salvage pathway enzymes evaluated, only HPRT had a significant movement of the cell population toward a higher fluorescence, indicating the presence of HPRT on the surface of H460 cells. Statistical analysis shows significant HPRT binding on the surface of H460 cells (P=0.0036).
Abbreviations: APRT, adenine phosphoribosyltransferase; DCK, deoxycytidine kinase; HPRT, hypoxanthine guanine phosphoribosyltransferase.

Figure 2 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.

Figure 3 Levels of HPRT expression compared between A549 and H460 cells. Notes: While both A549 and H460 cells show a statistically significant increase in the surface expression of HPRT, H460 cells had a significantly higher expression (P<0.0001). H460 cells are a faster growing cell line, with a growth rate almost double that of A549 cells. As a result, HPRT expression on the surface of non-small-cell lung cancer cells may directly correspond to cell proliferation. ****P<0.0001.

Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.

Figure 4 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.

Figure 5 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.

Figure 6 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.

Figure 7 Evaluation of HPRT expression within patient tissue.

Notes: All tissues were stained with a monoclonal anti-HPRT antibody. The gray plots for each of the tissues are imaged below. (A) Tissue from a 69-year-old female patient with stage III basaloid carcinoma and (B) normal tissue from a 59-year-old female patient. The malignant tissue is significantly darker than the corresponding normal tissue. These tissues show an upregulation of HPRT within malignant cells.
Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.

Figure 8 Statistical analysis of HPRT expression within patient tissue.

Notes: Quantification of tissue was conducted utilizing a gray scale. The lower the gray value, the darker the tissue is stained. (A) There is a statistically significant presence of HPRT in approximately half of the tissues obtained from patients. This increased expression shows that in some patients there is an upregulation of the protein. As HPRT is a housekeeping gene, there is a basal level of expression present within the tissue. An isotype control was run to establish the gray value of unstained tissue and to account for nonspecific antibody binding. (B) Of the tissues evaluated there was a significant difference in HPRT presence in stage III tissue, indicating an increase in HPRT presence as cancer progressed and proliferated. *P≤0.05; ***P≤0.001; ****P<0.0001.
Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.

Figure S1 Salvage pathway expression on the surface of H460 cells.

Notes: CD44 is displayed in these diagrams in order to show a protein that is expressed on >99% of the cell population. (A) APRT surface expression is evaluated against a CD44 (blue) positive control and an NF-κB (aqua) negative control. (B) DCK (purple) surface expression in comparison with a CD44 (blue) positive control. (C) Anti-HPRT treated cells (pink) shift in the population in relation to CD44 (blue).

Abbreviations: APRT, adenine phosphoribosyltransferase; DCK, deoxycytidine kinase; HPRT, hypoxanthine guanine phosphoribosyltransferase.

Figure S2 HPRT surface expression on A549 non-small-cell lung cancer cells.

Notes: When cells were exposed to anti-CD44 antibody (blue), ∼98% of the population was positive for surface expression. HPRT binding in A549 cells is compared to CD44 surface expression along with all controls.

Abbreviation: HPRT, hypoxanthine guanine phosphoribosyltransferase.