Dinámica temporal de las poblaciones de aves rapaces rupícolas en Sierra Nevada

The following chapter summarizes the results of the functional characterization of newly established cell lines harboring an Idh1R132H _{mutation with or without a knock-out of Tp53.}

The findings are related to cell lines with alterations induced and validated two weeks before the start of the experiments, which will be referred to as short-term.

3.3.1 Cell viability

An increase of cell viability of induced cells could not be observed. Experiments show that the induction of the Idh1R132H _{mutation in inducible cell lines and subsequent production of 2-}

HG decreases cell viability up to 44.3 ± 20.2%, compared to respective controls (Figure 13 A). In line with this, measurements of cell viability across 5 days show a decreased slope of

control induced Idh1R132H Cic-/- Anti-CIC Anti-IDH1R132H Anti-β Actin 164 kDa 47 kDa 42 kDa + - Idh1R132H Cic-/- Cic-/- + - A B C + - Cic-/- CreERT2+/- Cic-/- CreERT2+/+ + - 4-OHT 347 bp cDNA Anti-IDH1R132 Anti-CIC

day 5, Idh1R132H mutated cells grow on average 7.8% per day. In contrast, control cells have a growth increase of 18.7% daily (Figure 13 B). The cell number after induction of both alterations is slightly increased compared to Idh1R132H alone, 70.3 ± 9.8% vs. 44.3 ± 20.2%. This is however still a significant decrease compared to controls. The proliferation of p53-/- cells is 58.6 ± 23.8% compared to control cells (Figure 13 A). The slope of the growth curve of p53-/- cells is slightly decreased (11.6%) compared to respective controls (19.4%). Same observations can be made for double mutant cells that also have a decreased growth behavior compared to controls (12.8% vs. 19.2% daily).

Figure 13. Induction of Idh1R132H and subsequent production of 2-HG significantly

decreases cell viability. A After induction and validation of the genotypes, 5000 cells were

seeded per well in 96-well plate format and measurements using the CellTiter-Glo 3D Cell Viability Assay were performed five days after seeding. Idh1R132H leads to a significant decrease of cell viability. A significantly reduced cell number is also observed when a knock- out of Tp53 was induced alone or in the presence of Idh1R132H, which is however weakened compared to Idh1R132H alone. Bar plot of CellTiter-Glo3D assay data, normalized to average

A

control. B Time series of the CellTiter-Glo 3D Cell Viability Assay reveals that cells harboring the Idh1R132H mutation show a distinctive growth disadvantage compared to a single knock-out of Tp53 or double mutants. Measurements were performed after 0, 3 and 5 days of incubation. Diagram of the CellTiter-Glo 3D Cell Viability Assay, normalized to average control on day 5. (Indicated significances: two-sided unpaired students t-test, * = p<0.05, n = 3).

3.3.2 Colony formation in soft agar

Anchorage-independent growth is described as a hallmark of carcinogenesis, as transformed cells have the ability to grow independently of a solid surface. The soft agar assay is a well described method to check for this potential in vitro and is reported to be one of the most important tests to examine malignant transformation in cells (Borowicz et al., 2014). To check for this ability, cells were embedded in soft agar layers and were allowed to grow for 7 days in 96-well plates before quantification using the resazurin-based CellTiter-Blue Cell Viability assay. Experiments indicate that cells harboring an Idh1R132H mutation only, show a significantly reduced colony number compared to controls (88.9 ± 3.9%). In contrast, if cells harbor a Tp53 knock-out only or in combination with the Idh1R132H _{mutation, sphere}

formation is slightly increased (107.7 ± 6.3% and 107.1 ± 7.3% respectively) compared to controls, and significantly increased to Idh1R132H _{alone (Figure 14).}

Figure 14. Induction of Idh1R132H significantly decreases colony formation rate in soft

agar. Previously induced and validated cell lines were used to seed 2000 cells per well

embedded in a soft agar matrix in 96-well plates. After 7 days of incubation, measurements were performed using the CellTiter-Blue Assay. Idh1R132H alone leads to a significant decrease of colony formation. In contrast, if cells harbor a single knock-out of Tp53 or a combination of both alterations, there is a tendency towards an increase of colony formation rate. Bar plot of the soft agar assay data, normalized to average control after one week of

3.3.3 Analysis of apoptotic proteins

Differences in the phenotype of single and double mutants can be detected with cell viability and colony formation analysis. As a next step, it was aimed to understand these differences. It is of interest whether Idh1R132H cells with a decreased proliferative capacity have a higher rate of apoptosis. To analyze the level of apoptosis, proteins were analyzed that are described to play an important role during later steps of apoptosis. The apoptotic marker proteins cleaved Caspase 3 (clC3), cleaved Caspase 7 (clC7) and cleaved Poly (ADP) ribose Polymerase (clPARP) were analyzed in Western blot. Results show that apoptosis is slightly increased in cells harboring the Idh1R132H mutation compared to controls. Expression of the apoptotic proteins clC3, clC7 and clPARP is on average 127.9 ± 138.7%, 320.2 ± 250.4% and 95.1 ± 51.7%, respectively. In contrast, a knock-out of Tp53 shows a tendency towards a decrease of the level of apoptosis. The level of clC3 is 77.5 ± 24.9%, clC7 44.7 ± 6.3% and clPARP 59.5 ± 58.6%. A reduction of apoptosis could also be observed in double mutant cells, which is comparable to the Tp53 knock-out cells. Abundance of clC3 protein is reduced to 66.2 ± 82.6% compared to controls, as well as 65.2 ± 61.1% and 75.7 ± 60.0% for clC7 and clPARP respectively (Figure 15).

Figure 15. Induction of Idh1R132H slightly enhances levels of apoptosis. Whole cell lysates

were prepared from 300 µl cell suspension taken from previously induced and validated cell lines. After BCA, lysate was subjected to Western blot analysis with antibodies to the apoptotic proteins clC3, clC7 and clPARP. Expression of Idh1R132H shows a tendency towards an increase of apoptosis. The presence of a knock-out of Tp53, either alone or in combination with the Idh1R132H _{mutation results in a decreased level of apoptosis, observed in all proteins}

tested. Bar plot of the expression of apoptotic proteins, normalized to average control. (Indicated significances: two-sided unpaired students t-test, n = 3 and n = 2 for clC3).

3.3.4 Production of 2-HG

The intracellular level of 2-HG produced after induction of the Idh1R132H mutation was detected by an enzymatic 2-HG quantification assay. Cells harboring an Idh1R132H mutation produce high amounts of 2-HG, which are not altered when Idh1R132H is accompanied by a knock-out of Tp53(Figure 16). Mean intracellular 2-HG concentrations across three replicas are 2.7 ± 0.7 mM for single Idh1R132H _{mutated cells and equally 2.7 ± 0.7 mM for cells}

harboring both alterations. In contrast, p53-/- cells do not produce 2-HG (0.4 ± 0.1 mM), which is below the quantification limit of the assay.

Figure 16. Cells harboring an Idh1R132H mutation produce 2-HG, which is not altered by

a concomitant knock-out of Tp53. 300 µl of previously induced and validated cells was

taken for lysate production and protein concentration was determined with BCA. 2-HG concentration produced after induction of the alteration was determined with a 2-HG quantification assay. The presence of Idh1R132H alone leads to a significant production of 2- HG, whereas this is absent in single Tp53 knock-out cells. A combination of both alterations induces a similar production of 2-HG, compared to cells with Idh1R132H _{only. Bar plot of the}

2-HG assay carried out in triplicates, normalized to average control. (Indicated significances: two-sided unpaired students t-test, * = p<0.05, n = 3).