Using CometChip to Characterize Extracellular Regulators of DNA Repair: Does CD73 Levels in Cancer Cells Affect DNA Repair by Regulating Levels of Intracellular NAD+?

Thu, 08/26/2021 - 11:50

3D rendering of CometChip as a banner image.

By Natalia Gurule, PhD


Historically, DNA repair pathways have been viewed from a tumor cell centric vantage point. Currently, the tumor microenvironment (TME) is recognized as having the potential to be a source of components that can participate in how a cancer cell responds to DNA damage. Within the TME, there are metabolites that can promote tumor cell growth and modulate immune cell function, as well as influence tumor cell metabolism through mechanisms including metabolic coupling or metabolic plasticity.

Download Bio-Techne’s DNA Repair Brochure

NAD+ and CD73 are Extracellular Metabolites That are Potentially Linked Through DNA Repair in Cancer Cells

One key metabolite found within the TME is nicotinamide adenine dinucleotide (NAD+), which is a critical coenzyme that is found in every cell in the body. There are two general sets of reactions that are regulated by NAD+: 1) it helps turn nutrients into energy and 2) it acts as a helper molecule for proteins that control other cellular functions. NAD+ is an essential substrate for signaling molecules that influence cell death, stress responses, genome stability, chromatin status, and mitochondrial function. Decreases in intracellular NAD+ levels are associated with aging as well as pathological conditions such as obesity, neurodegenerative disease, hearing loss, and cancer. Many of these conditions have been associated with genome instability and DNA repair defects. Molecularly, defects in DNA repair pathways cause increases in Poly (ADP-Ribose) Polymerase 1 (PARP1) activity which, in turn, leads to depletion of NAD+.

Another important extracellular molecule found within the TME is CD73, which is an ecto-5’-nucleotidase that is expressed by a majority of cells. Within the context of metabolic signaling pathways, CD73 cleaves NAD+ to generate nicotinamide mononucleotide (NMN) plus adenosine monophosphate (AMP). It has also been proposed that CD73 can hydrolyze both AMP and NMN, causing an accumulation of adenosine and nicotinamide riboside (NR). In cancer, CD73 has been linked with immunosuppression because of its role in generating adenosine, which suppresses T cell function. Although the role of CD73 in exerting enzymatic function in NAD+ biosynthesis has been demonstrated in bacteria, it is poorly defined in mammalian cells.

Immunohistochemical probing human cervical cancer tissue section with Mouse Anti-CD73 Monoclonal Antibody, followed by IgG Secondary Antibody [HRP Polymer], and stained with DAB and hematoxylin. Western Blot highlighting lysates from HEK293T parental cell line and HEK293T PARP knockout (KO) cell line probed with Mouse Anti-PARP Monoclonal Antibody resulting in a specific band in the parental cell line but not in the KO cell line.

(Left) CD73 detected in human cervical cancer tissue probed with Mouse Anti- 5'‑Nucleotidase/CD73 Monoclonal Antibody (MAB57951) followed by Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (VC001). Then, tissue was stained with DAB (brown) and nuclei were counterstained with hematoxylin (blue). Staining was localized to cytoplasm and cell membranes. (Right) Western blot depicting lysates from HEK293T human embryonic kidney parental cell line and HEK293T PARP knockout cell line (KO). The membrane was probed first with Mouse Anti-Human PARP Monoclonal Antibody (MAB8095) and then with HRP-conjugated Anti-Mouse IgG Secondary Antibody (HAF018). A band for PARP was detected at ~120 kDa in the parental cell line, but was not detected in KO cell line. GAPDH (MAB5718) was used as a loading control. 

Cancer Cell DNA Repair is Enhanced by Extracellular NAD+ Levels, But Not CD73 Status

Recently, a group of researchers from University of South Alabama’s Mitchell Cancer Institute sought to investigate the role of CD73 in NAD+ uptake and biosynthesis from exogenous precursors. Specifically, they asked whether CD73 status in cancer cells impacts DNA repair processes by regulating intracellular NAD+ levels. The researchers utilized a novel high throughput method for quantifying DNA damage and DNA repair based on a comet assay and single cell gel electrophoresis (SCGE) called the CometChip Platform, that was previously described by Dr. Sobol’s research group in Sykora et al.

Human Nalm6 cells untreated and visualized using CometChip Kit with no comet tail observed. Human Nalm6 cells treated with etoposide and visualized with CometChip Kit showing comet tail indicative of DNA damage.

(Left) Untreated human Nalm6 cells were loaded into a CometChip and visualized using the CometChip Kit (Catalog # 4260-096-K) under alkali conditions. Because the samples are untreated, no comet tail is observed. (Right) Human Nalm6 cells were treated with 5 µM etoposide for 30 minutes and then loaded into a CometChip. Cells were visualized using the CometChip Kit (Catalog # 4260-096-K) under alkali conditions. The comet tail is indicative of DNA damage while the comet head contains undamaged DNA.

Watch CometChip® Webinar

The research group utilized this CometChip technology to quantify DNA damage and repair in MCF-7 cells upon exposure to different agents that are known to induce DNA damage including methylnitrosoguanidine (MNNG), etoposide, methyl methanesulfonate (MMS), bleomycin, and cisplatin. They found that agents which induce DNA damage that require the base excision repair pathway (BER) to perform DNA repair are extremely dependent on levels of NAD+. Specifically, MCF-7 cells were treated with NAMPT inhibitor FK866, which prevents conversion of NAD+ to NMN. Then, MCF-7 cells were treated with etoposide or MMS and tail DNA was measured to determine the effect of these agents on DNA repair capacity. Etoposide induced 30% tail DNA while MMS treatment induced a dose dependent accumulation of tail DNA. In the etoposide treated cells, NAD+ levels had no effect on the amount of DNA damage or the DNA repair capacity. In contrast to this finding, NAD+ levels were correlated with a greater degree of DNA damage at low concentrations of MMS treatment. At high concentrations of MMS treatment, the level of DNA damage was not linked with NAD+ concentrations. Taken together, these results indicate that NAD+ availability impacts critical DNA repair processes that may play an important role in cellular responses to damaging gents such as chemotherapy and genotoxic exposure.


Data presented as % Tail DNA of DNA damage and repair in MCF-7 cells treated with varying concentrations of  methyl methanesulfonate (MMS) for 60 min, in the presence or absence of FK866, followed by a repair time-course to 3 hours.

Effect of NAD+ depletion on DNA damage and repair in MCF-7 cells as determined using the CometChip assay. Level of DNA damage and DNA repair capacity in MCF-7 cells exposed for 60 min to different MMS concentrations in the presence or absence of FK866 (FK), followed by a DNA repair course to 3 hrs. Data is expressed as % Tail DNA, with standard deviation. Plots are the average of 8–12 wells from two-three independent CometChips; >1000 comets per bar. Statistical analysis was performed using GraphPad Prism 7 and two-way ANOVA followed by post-hoc with Tukey’s multiple comparison test (**p<0.0029, ***<0.0008, ****<0.0001). Data image was cropped from Figure 1 from Wilk, A., Hayat, F., Cunningham, R. et al. Extracellular NAD+ enhances PARP-dependent DNA repair capacity independently of CD73 activity. Sci Rep 10, 651 (2020)., licensed by CC License.


Next, the researchers examined the levels of NAD+ upon expression of CD73. Using a biochemical assay, they found that CD73 is highly active against AMP, but did not result in any increases in processing of NAD+. Furthermore, their results demonstrate that CD73 processes NMN at a very low rate that is not physiologically relevant.

Overall, these findings are important because they expand upon our understanding of the role of CD73 in cancer and NAD+ biosynthesis in eukaryotic cells which was not previously characterized. The study also provides insight on the potential use of NAD+ supplementation to enhance cellular DNA repair capacity.


Note: Wilk et al. used the CometChip Electrophoresis Starter Kit (Catalog # 4260-096-ESK) available from R&D Systems, a Bio-Techne Brand.


Natalia Gurule HeadshotNatalia Gurule, PhD
Dr. Gurule is a postdoctoral fellow at National Jewish Health. She studies pathways that regulate inflammation upon infection in leukemia and myeloid dysplastic syndromes.  

Research in focus

Wilk, A., Hayat, F., Cunningham, R., Li, J., Garavaglia, S., Zamani, L., Ferraris, D. M., Sykora, P., Andrews, J., Clark, J., Davis, A., Chaloin, L., Rizzi, M., Migaud, M., & Sobol, R. W. (2020). Extracellular NAD+ enhances PARP-dependent DNA repair capacity independently of CD73 activityScientific reports.

Sykora, P., Witt, K. L., Revanna, P., Smith-Roe, S. L., Dismukes, J., Lloyd, D. G., Engelward, B. P., & Sobol, R. W. (2018). Next generation high throughput DNA damage detection platform for genotoxic compound screeningScientific reports.


Blog Topics