4.4 Clonogenic Capacity
4.4.2 Clonogenic Capacity in Cells Treated with Cytotoxic Anti-Cancer Drugs
DRUGS
As no difference in clonogenic capacity was observed between non-target and RAI2-depleted cells under standard condition, next, the colony formation assay was performed with cells treated with different cytotoxic anti-cancer drugs. The used drugs have effects on different cell cycle steps.
4.4.2.1 CLONOGENIC CAPACITY IN ARRESTED CELLS
In order to arrest cells in S phase a wide range of drugs are available that inhibit the enzyme topoisomerases (TOPs). Topoisomerases are regulating the torsional stress of DNA by inducing transient DNA breaks by either inducing single-strand breaks (TOP I) or DSBs (TOP II) (Banerji and Los, 2006). Targeting TOP I with camptothecin leads to a stabilisation of the DNA TOP I complex and this inhibits the religation of the DNA after uncoiling, leading to an arrest of the cells in S phase. As a consequence, cell death is triggered by generating replication-mediated DSB (Pommier, 2006; Pommier et al., 2003). The response to camptothecin was tested in KPL-1 and MCF-7 cells after RAI2-depletion using colony formation assay. As shown in Figure 23, clonogenic capacity after treatment was not different between non-target and RAI2-depleted samples in both cell lines.
Figure 23: Clonogenic capacity in RAI2-depleted KPL-1 and MCF-7 cells after treatment with camptothecin. After induction of RAI2 depletion, cells were treated with 10 nM camptothecin for 4h and cultured for 12 days. a, Representative images showing colony growth of DMSO control and camptothecin-treated KPL-1 cells. b, Area percentage, which was covered by cells, was quantified using an Image J tool and normalised to DMSO control (Guzman et al., 2014). For p-values (Student’s t-test) a cut-off of 0.05 was applied.
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61 Cell were also treated with the TOP II inhibitor etoposide, which inhibits the religation of the TOP II cleavage complex from torsional-stressed DNA. Similar to camptothecin, etoposide leads to high levels of DSBs resulting in cell death (Fortune and Osheroff, 2000; Pommier et al., 2010).
After treatment with etoposide, no significant difference in clonogenic capacity was observed between control and after RAI2 depletion in both cell lines KPL-1 and MCF-7 (Figure 24).
Figure 24: Clonogenic capacity in RAI2-depleted KPL-1 and MCF-7 cells after treatment with etoposide. After induction of RAI2 depletion, cells were treated with 10 µM etoposide for 4h and cultured for 12 days. a, Representative images showing colony growth of DMSO control and etoposide-treated KPL-1 cells. b, Area percentage, which was covered by cells, was quantified using an Image J tool and normalised to DMSO control (Guzman et al., 2014). For p-values (Student’s t-test) a cut-off of 0.05 was applied.
Inhibition of both TOPs using doxorubicin, which additionally intercalate into DNA, negatively affects the functioning of helicases and generates reactive oxygen species (Banerji and Los, 2006;
Tacar et al., 2013), had no effect on clonogenic capacity in RAI2-depleted cell lines (Figure 25).
Thus, targeting TOPs had no significant effect on clonogenic potential in RAI2-depleted cells compared to non-target control cells.
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Figure 25: Clonogenic capacity in RAI2-depleted KPL-1 and MCF-7 cells after treatment with doxorubicin. After induction of RAI2 depletion cells were treated with 100 nM doxorubicin for 4h and cultured for 12 days. a, Representative images showing colony growth of DMSO control and doxorubicin-treated KPL-1 cells. b, Area percentage, which was covered by cells, was quantified using an Image J tool and normalised to DMSO control (Guzman et al., 2014). For p-values (Student’s t-test) a cut-off of 0.05 was applied.
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63 4.4.2.2 CLONOGENIC CAPACITY UPON OXIDATIVE STRESS
Oxidative stress is an imbalance in cell metabolism, where a high concentration of reactive oxygen species (ROS) is present in the cells that, among others, can lead to an oxidation of DNA bases.
The most common product, a fallout of oxidative stress, is 8-oxoguanine, which is mended through base excision repair (David et al., 2007). H2O2 belongs to ROS and can be used for cell culture experiments to mimic oxidative stress. In order to analyse clonogenic capacity upon oxidative stress, cells were treated with H2O2 and colony formation was measured. RAI2-depleted cells showed no significant difference compared to non-target control when analysing clonogenic capacity (Figure 26).
Figure 26: Clonogenic capacity in RAI2-depleted KPL-1 and MCF-7 cells after treatment with H2O2. After induction of RAI2 depletion cells were treated with 30 µM H2O2 for 4h and cultured for 12 days. a, Representative images showing colony growth of DMSO control H2O2-treated KPL-1 cells. b, Area percentage, which was covered by cells, was quantified using an Image J tool and normalised to DMSO control (Guzman et al., 2014). For p-values (Student’s t-test) a cut-off of 0.05 was applied.
4.4.2.3 CLONOGENIC CAPACITY UPON PARP INHIBITION
Inhibition of PARP is successful in treating patients with triple negative breast cancer that having germline mutation in BRCA1 or 2 gene. When PARP is inhibited by olaparib, spontaneous single-strand breaks occur during S phase which leads to an accumulation of DSBs because of a replication fork collapse. Normally, these DSBs are repaired by HR including functional activity of BRCA 1 and 2. As BRCA-deficient cells show an HR defect irreparable toxic DSBs accumulate upon olaparib treatment and lead to cell death. This effect is called synthetic lethality and is utilised in patients with BRCA deficiency as treatment opportunity (Bryant et al., 2005; Sunada et
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64 al., 2018). As RAI2 effects repair capacity, KPL-1 cells were treated with the PARP inhibitor olaparib and changes in clonogenic capacity upon RAI2 depletion was analysed. Again, even after this treatment, RAI2 depletion had no effect on colony formation (Figure 27).
Figure 27: Clonogenic capacity in RAI2-depleted KPL-1 and MCF-7 cells after treatment with olaparib. After induction of RAI2 depletion cells were continuously treated with 100 nM olaparib and cultured for 12 days. a, Representative images showing colony growth of DMSO control and olaparib-treated KPL-1 cells. b, Area percentage, which was covered by cells, was quantified using an Image J tool and normalised to DMSO control (Guzman et al., 2014).
For p-values (Student’s t-test) a cut-off of 0.05 was applied.
65