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Analysis of the Repair of
Topoisomerase II DNA Damage

By: Eric Goldstein | Mentor: Dr. Mark Muller

Results

HR analysis following VP16 drug treatments and transfection of ISce-I in HR HeLa cells

The treatment of HR HeLa cells with VP16 served as an initial and essential founding piece of information for the following results (Figure 5). The positive control, which includes the transfection of ISce-I with no subsequent drug treatment, illustrates that the reporter system is working and shows the potential of GFP expression. The negative control consists of HR cells, lacking ISce-I transfection and drug treatment. These cells provide a background reading of fluorescence that is ultimately subtracted from the corresponding ISce-I data points. Although the anticipated GFP expression for the negative control should be 0%, the 0.1% GFP finding is likely due to the nature of the integrated GFP cassettes or autofluorescence. An autofluorescence control was not obtained.

The combination of VP16 with the transfection of ISce-I increased the GFP expression at 72 hours by 1.3% in the 5 μM VP16 range. GFP expression at 72 hours steadily declined with VP16 concentrations higher than 5 μM (Figure 5). In addition, GFP expression gradually decreased at 24 and 48 hours with VP16 concentrations above 5 μM. This is likely due to toxicity of the VP16 drug (IC50 = 200 μM). GFP fluorescence in general is low due to the nature of exposing a poison to cells; the surviving, fluorescing cells are most likely survivors of the poison exposure.

Due to the low percentages and small size of the HR reporter cassette, a topoIIα targeting sequence was used. The RY-HR (Figure 4) cassette contains an ultra high affinity 56 base pair purine rich sequence for topoIIα[10]. This high affinity topoII binding site, the RY site, should attract the endogenous topoII and direct cleavages in this region 5' of the ISce-I site in the presence of a drug such as VP16. As a result, the cellular DNA damage repair system should be activated with the HR cassette. Two exposure times of VP16 were allotted for the RY-HR HeLa cells. One batch of cells was exposed to VP16 for five days. The second batch was exposed to VP16 for one hour and then allowed to recover for five days. The positive control is solely a transfection of ISce-I plasmid while the negative control represents background GFP expression in the absence of HR induction through ISce-I transfection or drug treatment. The results show that HeLa cells are able to repair the VP16 initiated DSB up to 5 μM concentration (Figure 6). There is a clear trend towards increasing GFP expression with increasing concentration of VP16 treatment in both the prolonged and short drug exposure time frames. During the five day exposure, there is an increase of GFP beyond the positive control. The one hour drug treatments also evinced a trend of increasing GFP expression with increased dosage of VP16. The data suggest that the presence of the RY element, the ultra high affinity topoIIα binding site, is stimulating HR in the GFP reporter through VP16 drug treatment. In this analysis, cells were treated at low to high levels of VP16 for a total of five days or with the same concentration for one hour followed by five days of recovery. In both cases, it is clear that VP16 is inducing expression of GFP through HR at drug concentrations (0.05 – 50 μM). This indicated that the repair of topoIIα induced DNA damage can occur through HR. Higher concentrations of VP16 (> 50 μM) were toxic to the cells. As a result of this, we decided that a morphological investigation of the GFP expressed in HR, RY-HR, and NHEJ HeLa cells was required.

Figure 5 - HR in VP16 Treated HR HeLa Cells. HR HeLa cells were transfected with ISce-I and treated with VP16 for 1 hour. A time course for recovery for 72 hours (**p<0.01) was read for GFP expression using FACS analysis. GFP expression is indicative of a HR event. Standard error bars symbolize data range after three replicated trials.

Figure 6 - HR in VP16 Treated RY-HR HeLa Cells. RY-HR HeLa cells were treated with VP16 for the indicated time frame (5d = 5 days, 1h = 1 hour) at the stated VP16 concentrations. One hour drug treated cells were incubated for the remaining 5 days in the absence of VP16. All cells were harvested simultaneously for GFP analysis via FACS (**p<0.4). Standard error bars symbolize data range after three replicated trials.

Figure 7 - Confocal Microscopy of RY-HR HeLa Cells. RY-HR HeLa cells were seeded over a cover slip and incubated for 24 hours. Following incubation, 5 μM VP16 (A) and 100 μM VP16 (B) were exposed to the cells for one hour. The cells were then incubated for two days without VP16 exposure. The cover slips were then removed and exposed to a PI staining solution for 30 minutes. Confocal microscopy was viewed with fluorescence for GFP and PI. PI stains the nucleus red and the GFP fluoresces green. GFP expression is indicative of a HR event. (A) and (B) were captured at 20X magnification.

Confocal Microscopy

Confocal microscopy was performed to examine GFP expressing cells morphologically (Figure 7 – 8). The GFP protein appears to be well distributed throughout the cell in the RY-HR cultures treated with VP16 for one hour followed by a 48 hour recovery interval (Figure 7). At 100 μM VP16, there is an obvious cytotoxic effect on the cells using microscopic observations. Confocal analyses were performed on the doxycycline inducible NHEJ and HR reporter cells, to examine any cytological differences between these two reporter systems. The NHEJ reporter yielded cells with GFP distributed throughout the cell (Figure 8). In contrast, the GFP produced as a result of HR in the HR HeLa cells without the RY element appears largely in the nucleus, possibly due to a nuclear localization sequence on the GFP. GFP is indicative of a HR or NHEJ event; cells that do not express GFP have not expressed either of these pathways.

Figure 8 - Confocal Microscopy of HR and NHEJ HeLa Cells. Doxycycline inducible NHEJ and HR HeLa cell lines were seeded with an inherent slide base. Following one day of incubation, doxycycline was added to the stated wells. The cells were incubated with the doxycycline for three days. (A) Shows the uninduced HR HeLa cells at 20X zoom. (B) Shows doxycycline induced HR HeLa cells at 20X zoom. (C) Shows doxycycline induced NHEJ reporter system integrated HeLa cells at 20X zoom. (D) shows cells from (C) at 100X zoom. The confocal microscope was set to excite solely GFP. GFP expression coincides with the NHEJ or the HR repair pathway utilization.

Figure 9 - Cytotoxicity for NHEJ and RY-HR HeLa Cells. Cytotoxicity percentages were calculated for NHEJ reporter in HeLa cells and RY-HR HeLa cells. Drug treatments lasted for one hour with both VP16 and CPT (TopoI Poison). The cells were then washed and allotted a recovery interval of 48 hours. Cytotoxicity percentages were calculated using trypan blue exclusion assays.

Figure 10 - HR and NHEJ after VP16 Treatments (A). Illustrates GFP expression from NHEJ reporter cassettes in HeLa cells. (B) Shows GFP expression from RY-HR HeLa cells. Drug treatments lasted for one hour for both VP16 and CPT (TopoI poison). For the positive controls: ISce-I transfection lasted for four hours and three day exposure for the DOX inducible NHEJ system. The drug treated cells recovered for three days and were analyzed for GFP expression using FACS. Transfection efficiency was measured by pEGFP (18%).

Analysis of Toxicity

The results of the trypan blue exclusion assay shows that with increasing concentration of VP16, there is an increase in cytotoxicity as expected (Figure 9). VP16 is a known chemotherapeutic and thus should have a high toxicity rating as shown in these results. CPT, a highly specific TopoI poison, shows an increased cytotoxicity amongst the NHEJ reporter system with concentrations from 0.5 – 50 μM being toxic. The RY-HR HeLa cells show increased resistance to CPT. The RY-HR toxicity data show that VP16 is generally less cytotoxic than CPT with regard to HR, which indicates that HeLa cells may be able to repair more efficiently the damage caused by a topoIIα poison.

Analysis of Repair of Topoisomerase I and II DNA Damage by HR and NHEJ Pathways

There are two major DSB repair pathways in animal cells: HR and NHEJ. Since we have dedicated reporters for each pathway and given that the drugs are highly specific for topoI (CPT) or topoII (VP16) mediated DNA damage, the repair process was evaluated in each case (Figure 10). In figure 10-A, the NHEJ reporter system shows there is an increase in susceptibility in CPT damage and mild reparability in exposure to VP16 with dose dependent decrease in GFP. The positive control is noticeably elevated in comparison to the remaining samples. There is a decrease in NHEJ repair with an increase in VP16 concentration. Figure 10-B shows the effects of CPT and VP16 on RY-HR HeLa cells. There is an increase in HR with an increase in VP16 concentration and the opposite trend with CPT concentration. GFP expression percentage remains low in the RY-HR HeLa cells due to the cytotoxicity of the introduced poisons, and also the expression abilities of the clones as noted by the positive control. In both instances, 50 μM of CPT or VP16 caused toxicity in the culture (see also Figure 9). The data points show that VP16 and CPT HR repair gives a GFP reading close to that of the positive control, ISce-I transfection. These trends may mean that HR is the preferred pathway utilized by HeLa cells to repair from topoIIα and topoI poison damage.

Discussion >>