**2.2 Detection of TOP1 activity in the Caco2 colon cancer cells: direct comparison of the fluorescent, chemiluminescent, and colorimetric readouts of the REEAD assay**

The well-defined colorectal cancer derived cell line Caco2 was used as a model cell line to demonstrate the functionality of the modified colorimetric/ECL REEAD assay and to investigate whether this readout method can be used instead of the fluorescence-microscope-based readout. Nuclear extract from increasing number of Caco2 cells (as indicated in **Figure 2**) was incubated with the TOP1-specific substrate and, upon hybridization to the surface-anchored primer on a glass slide, the generated closed DNA circles were amplified by RCA in the presence of fluorescently labeled nucleotides. The fluorescent RCPs were visualized using 60× magnification in a fluorescence microscope. Fifteen pictures per sample were taken and the number of RCPs was estimated by using Image J software [54]. **Figure 2A** and **B** show the results of such analysis. **Figure 2A** depicts representative microscopic images of the observed fluorescent signals in extracts from 0 to 10,000 Caco2 cells. Note that due to the high sensitivity of the assay, it was not possible to quantify the signals obtained

#### **Figure 2.**

*Analyses of TOP1 activity using a fluorescence microscopic readout. (A) Representative microscopic images obtained when analyzing TOP1 activity in cell extracts from 156, 312, 625, 1250, 2500, 5000, or 10,000 Caco2 cells. Each green dot corresponds to a single TOP1 cleavage-ligation reaction. (B) Graphical depiction of the results obtained when analyzing the TOP1 activity from 156 to 10,000 Caco2 cells as indicated on the figure. A negative control without cell extract was included. Plotted data represent average from two independent experiments.*

when using extract from >10,000 Caco2 cells, due to the abundance of signals that hinders the discrimination between the single RCPs in the image frame. **Figure 2B** shows a graphical depiction resulting from the quantification of the REEAD signals obtained from two independent experiments. As evident from the graphical depiction, the TOP1 activity increased as the amount of Caco2 cells increased. Hence, with this REEAD setup, it was possible to get a quantitative measure of the TOP1 activity in even a small number of cells, as low as 150–350 Caco2 cells. This high sensitivity of the assay, when performed in bulk, already proved the relevance of using the REEAD assay in the cancer research field as well as in cancer diagnostics and treatment-outcome prediction, where often the amount of cells in a biological sample is very limited [42, 52]. However, as described previously using the fluorescent readout requires time, training, and the use of an advanced fluorescence microscope.

To overcome the disadvantages of using a fluorescent readout in the REEAD assay, two new readout methods, chemiluminescent or colorimetric, were introduced (as schematically depicted in **Figure 1**). The closed DNA circles were obtained by incubating nuclear extracts from Caco2 cells with the TOP1-specific substrate, as described under **Figure 2**. The circles were hybridized into separated wells created onto the surface of a glass slide in a multi-well system, called Wellmaker in the following. In addition, two more sets of nuclear extraction from 0 to 40,000 Caco2 cells were included, and the TOP1 activity was then measured in four independent experiments. **Figure 3A**, left panel shows a representative image of the intensities of the biotin-containing RCPs when visualized using ECL. The quantitative depiction in **Figure 3A**, right panel, indicates a linear relationship between TOP1 activity and the increasing number of Caco2 cells. Similar results were obtained using the colorimetric readout with a TMB substrate that can be converted into an insoluble form that precipitates onto the slide upon HRP-mediated oxidation, as shown in **Figure 3B**. *Simple and Fast DNA-Based Tool to Investigate Topoisomerase 1 Activity, a Biomarker for Drug… DOI: http://dx.doi.org/10.5772/intechopen.105758*

#### **Figure 3.**

*Analyses of TOP1 activity using the chemiluminescent and colorimetric readout. (A) Left panel: Image obtained after measuring TOP1 activity in Caco2 cells using the ECL readout REEAD. The number of cells in each sample is indicated to the left of the image. Right panel: Graphical depiction of the results obtained when analyzing the TOP1 activity from 156 to 40,000 Caco2 cells as indicated on the figure. A negative control without cell extract was included. Plotted data represent average from three independent experiments. Welch's t-test, p = 0.02. a.u: arbitrary units. (B) Same as A, except that TMB was used instead of ECL. Plotted data represent average from three independent experiments. Welch's t-test, p = 0.01. a.u: arbitrary units.*

This makes the RCPs permanently colored and visible to the naked eye. In both the ECL and the TMB readouts, the quantification can easily be performed by acquiring a picture with a CCD or a smartphone camera. Then, the Image J software can be used to determine the intensity of the rectangular-shaped areas of the slide, which correlate with the number of RCPs and in turn with the cleavage-ligation activity of

TOP1 in the cell extracts. As evident form **Figure 3**, both readouts allowed detection of TOP1 activity with a detection limit around 1000 cells for the ECL and around 300 cells for the TMB. When using the ECL-based readout (**Figure 3A**), it is evident that a higher number of cells is required to be able to detect the TOP1 activity as compared with the fluorescent readout (**Figure 2**). However, the ECL-based readout can easily be developed using chemiluminescent developer solutions and detected either by a CCD camera or by using X-rays films (such as Kodak) in a darkroom. Strikingly, the TMB-based readout resembled that of the fluorescence microscope-based readout, with a comparable detection limit of 312 Caco2 cells, as indicated in **Figure 3B**. Another advantage of the TMB-based readout is that it does not require any specific equipment, and it can hence be implicated in any relevant setting. In conclusion, both the colorimetric and chemiluminescent readout methods are excellent alternatives to fluorescence in the detection of TOP1 activity using the REEAD assay. Furthermore, these readout methods make the REEAD assay usable to any relevant setting.

### **3. Conclusion**

Chemotherapy is currently one of the most common treatment methods for colorectal cancers [55]. Frequently, treatment fails because of chemoresistance onset or due to poor prediction of the chemotherapy response. Especially for the most advanced stage of colorectal cancer, TOP1 has proved to be one of the best biomarkers and targets of chemotherapy [7, 56] thanks to the well-known and clinically used TOP1 poisons, CPTs. For instance, a study reported a borderline association between increased TOP1 gene-copy number and objective response to irinotecan [57]. This is in agreement with a previous clinical trial (FOCUS) where a significant association between immune-histochemistry-based assessment of TOP1 protein level and response to Irinotecan was reported [58]. However, a subsequent study from the same group (FOCUS3) and a large prospective trial (CAIRO) failed to confirm this findings [59, 60]. Indeed, in the clinical settings often it is the level of DNA-RNA or amount of the TOP1 that is measured, even though it is the TOP1 activity that determines the effect of an inhibitor.

In the case of TOP1, multiple factors can influence the activity and the drug sensitivity in the patients, and for this reason there is an increasing need of tools that allow the measure of TOP1 activity in samples with few hundreds of cells.

In this chapter, we described two alternative readout formats of the highly sensitive and fast, fluorescence-microscope-based REEAD assay, which has single-event sensitivity and recently proved to allow measurement of TOP1 activity in few cells, even a single cell [50, 51]. Even with such a great detection limit, the REEAD has the limitation of the need for skilled personnel and time-consuming image acquisition and analysis. For these reasons, the presented ECL and colorimetric readouts provide excellent alternatives. Both methods are fast, simple, and do not require expensive equipment or trained technicians. Especially the TMB-based method provides an excellent alternative as the limit of detection even resembles the very sensitive fluorescent readout. This modified REEAD assay provides a great platform for a fast and simple detection of TOP1 activity and for using TOP1 as a biomarker for drug susceptibility in cancer cells isolated from colorectal cancer patients. We believe that the presented results may pave the road for the use of the REEAD assay in the clinical setting for the identification of the best outcome for colon cancer patient treatment with CPTs.

*Simple and Fast DNA-Based Tool to Investigate Topoisomerase 1 Activity, a Biomarker for Drug… DOI: http://dx.doi.org/10.5772/intechopen.105758*
