Capítulo IV: Evaluación Interna
4.1 Análisis Interno AMOFHIT
4.1.2 Marketing y ventas (M)
In agreement with previous reports, of Pavon and colleagues showing that mild overexpression of tRNAiMet is sufficient to increase metabolic activity and proliferation rates, we observed that increased expression of tRNASer(WT) and expression of a misreading tRNASer(Ala) boosted protein synthesis rates in normal epithelial cells, and increased cellular proliferation rates when stably expressed 12. In the present study, we have gone beyond previous observations and studied the effect of increasing the expression of WT Ser tRNA overexpression and expressing a mutant tRNASer(Ala) in tumorigenic initiation. Both tRNAs increased colony formation capacity in an anchorage-dependent colony formation assay, which is an indicator of increased tumorigenic ability 153. We also studied the activation of the PERK branch of
the UPR and found that in both conditions the levels of phosphorylated eIF2α were Figure 4-7: Tumors developed by BEAS-2B-derived cell lines. A) Tumors derived from BEAS tRNASer(WT) and BEAS Mock cells inoculated in the same animal. Left panel shows an example of an
animal that developed tumors derived from both cell lines where the size of the Mock tumor is almost negligible relative to the tRNASer(WT) expressing tumor; the right panel shows the tumor of an animal
that only developed a tumor expressing the tRNASer(WT) construct. B) Tumor of the animal that only
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lower than the control, which has been associated to cellular transformation 125. In the cell line overexpressing the WT Ser tRNA, there was upregulation of GADD34, which explains the decrease in eIF2α-P levels, but GADD34 was not upregulated in the cell line expressing the misreading tRNA. In other words, those cell lines achieved the same eIF2α phosphorylation outcome through different molecular mechanisms. This cell line may instead increase CReP expression levels, an homolog of GADD34, which can also bind and direct PP1α towards eIF2α 154, but this hasn’t been confirmed. The reduced
levels of eIF2α-P are also positively correlated with the increased protein synthesis rate observed in both cell lines. Activation of the transcription factor ATF4 was also increased, suggesting that transcription of GADD34 and genes that are relevant for adaptation under hypoxic conditions, oxidative stress and nutrient deprivation, which are hallmarks of tumors, were upregulated as well 155–157. Moreover, BiP was
upregulated in the tRNASer(WT) cell line, which have been detected in head and neck
cancer initiating cells and in lung pre-malignant lesions 158,159.
The low number of mice used in our in vivo pilot assay and the erratic nature of the results prevented us to demonstrate that those tRNAs contribute to tumor initiation. In the mice harboring tRNASer(WT) tumors, the data indicate that overexpression of the tRNASer(WT) may indeed trigger formation of slow growing tumors. This experiment need to be repeated to clarify the role of those tRNAs in tumor initiation.
4.5. Materials and Methods
4.5.1. Cell Culture
BEAS-2B cell line was kindly provided by Professor Maria Carmen Alpoim, from IBILI, University of Coimbra. BEAS-2B cells were cultured in LHC-9 medium (Gibco, Life Technologies) supplemented with 1% of Penicillin-Streptomycin (Pen/Strep) (Gibco, Life Technologies). Cells were maintained in an incubator at 37ºC with 5% CO2 and 95% relative humidity. To execute the following procedures, cells
were detached using Tryple Xpress (Gibco, Life Technologies). Cells were regularly tested for mycoplasma contamination.
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4.5.2. Generation of stable cell lines
BEAS-2B stable cell lines were generated through electroporation (three independent times). BEAS-2B cells were seeded in 100mm dishes and cultured until 70- 90% of confluence was reached. Cells were detached and the pellet was resuspended in Hepes Buffered Saline (HBS) solution to improve the transfection efficiency. Then, 4mm electroporation cuvettes were prepared with 10µg of plasmid and 0.5ml of cell suspension was added, mixing carefully. For each sample, the voltage applied was 230V, with capacitance of 1500µF and resistance of 125Ω. This step was performed using ECM Electro Cell Manipulator (BTX, Harvard Apparatus). Immediately after the electroporation, 1ml of LHC-9 culture medium was added to the cuvette, cells were then carefully homogeneized and transferred to 60mm dishes. Stable cell lines were obtained by selection with 200µg/ml of G418 for three weeks.
4.5.3. Extraction and Quantification of gDNA
To ensure the plasmid did not acquire mutations when integrated in the genome, gDNA was extracted for Sanger sequencing. The NZY Tissue gDNA Isolation Kit was used following instructions recommended by the manufacturer. gDNA concentration was determined using a NanoDrop.
4.5.4. Cellular Viability Assay
1.5x105 cells/well (BEAS-2B and NCI-H460 derived cell lines) were seeded in a 24-well plate. After 48h, cells were detached and equal volumes of cell suspension and trypan blue were mixed. Finally, cell viability (%) was obtained by counting the live and death cells using a TC10Tm Automated Cell Counter (Bio-Rad). This assay was performed with triplicates and repeated three times.
4.5.5. Cellular Proliferation Assay
To evaluate cellular proliferation, we used a colorimetric immunoassay ELISA, based on the measurement of BrdU incorporation during DNA synthesis (Roche, Cat.11647229001), following the manufacturer’s instructions. 5x103cells/well (BEAS-
2B and NCI-H460 derived cell lines) were plated in a 96-well and analysis was performed after 48h.
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4.5.6. Anchorage-Dependent Colony Formation Assay
300 cells per condition were seeded in 60mm dishes and maintained in culture for two weeks (BEAS-2B derived cell lines) or 9 days (NCI-H460 derived cell lines). The colonies were then fixed using ice cold methanol and incubated at -30ºC for 30min. Methanol was removed and a solution of 0.1% crystal violet in 20% methanol was added and the plates were incubated at room temperature for 30min. Plates were washed with H2O milliQ to remove excess dye and the colonies were counted. This assay was
performed in triplicates and repeated four times.
4.5.7. Protein synthesis rate determination
To determine protein synthesis rate, we used a non-reactive fluorescence- activated cell sorting-based assay called SUnSET, with few modifications. 1x106cells were plated in 60mm petri dishes and after 48h, 10% v/v puromycin (Sigma Aldrich) was added to each plate. Cells were then incubated for 15 min. Total protein lysates were obtained from cells with Lysis Buffer (0.5% Triton X-100, 50mM HEPES, 250mM NaCl, 1mM DTT, 1mM NaF, 2mM EDTA, 1mM EGTA, 1mM PMSF, 1mM Na3VO4 supplemented with a cocktail of protease inhibitors (Roche). Cells were sonicated with a probe sonicator in 5 pulses of 5 seconds. After centrifugation at 16000g for 30min, protein in the supernatants was quantified using the BCA assay (Thermo Fisher Scientific). 100 µg of protein was denaturated with loading buffer (6x) at 95ºC for 5 min, resolved in 10% SDS-PAGE and blotted onto nitrocellulose membranes (0.2µm) (GE Healthcare Life Sciences). Anti-puromycin, clone 12D10 (kindly given by Philippe Pierre) was used (1:2500) to detect the incorporation of puromycin in proteins. IRDye800 goat anti-mouse secondary antibody (Li-cor Biosciences, Cat.400-33) was used (1:10000 dilution) and detected in an Odyssey Infrared Imaging System (Licor Biosciences). Membranes were also probed with Anti-GAPDH (Santa Cruz Biotechnology) (1:1000) as a loading control.
4.5.8. Immunoblots
Total protein lysates were obtained with the same extraction method used with the SUnSET assay. 40µg of protein were immunoblotted onto nitrocellulose membranes with antibodies against eIF2α (1:1000; Cell signalling); phospho-eIF2α (1:1000; Abcam); ATF6 (1:400; Stressgen); GADD34 (1:1000 ThermoFisher Scientific);
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phospho-ATF4 (1:1000; tebu-bio); ATF4 (1:1000; tebu-bio) and β-tubulin (1:1000; Invitrogen). IRDye680 goat anti-rabbit or IRDye800 goat anti-mouse secondary antibodies (1:10000, Li-cor Biosciences) were used and the signal was detected using an Odyssey Infrared Imaging system (Li-cor Biosciences).
4.5.9. Tumor induction assay
Six-week-old male N:NIH(s)II:nu/nu nude mice were obtained previously from the Medical School, University of Cape Town in 1991 and then reproduced, maintained and housed at IPATIMUP Animal House at the Medical Faculty of the University of Porto, in a pathogen-free environment under controlled conditions of light and humidity. Male nude mice, aged 6-8 weeks, were used for in vivo experiments. Animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals, directive 2010/63/EU. To measure tumorigenic potential in vivo, BEAS-2B cell lines harboring the empty vector (Mock), the tRNASer(WT) and the misreading tRNASer(Ala) were subcutaneously injected in the dorsal flanks using a 25- gauge needle with 4.5x106 (BEAS-2B) of each cell line. A total of 4 mice per group
were used. Each mouse was injected in the left flank with the Mock variant and in the right flank with the cells misexpressing the WT or the misreading variants of each previously described clone. Mice were weighed, and tumor width and length were measured with calipers three times per week. Tumor volumes were calculated assuming ellipsoid growth patterns. Mice were humanely euthanized when tumors reached a median volume of 2000mm3 or whenever any signs of disease were detected. Tumors and lungs, were collected, fixed in 10% buffered formalin, paraffin embedded and then sectioned for histological examination. A part of each tumor was frozen in liquid nitrogen and stored at –80ºC until used.
4.5.10. Statistical analysis
For all the assays, except for the in vivo experiments, our data represents 3 independent biological replicates and 3-4 independent experiments. Average values are usually shown and error bars represent the standard error of the mean (SEM). Statistical significance was determined using One-way ANOVA with Dunnet’s post-test; Kruskal- Walis with Dunnet’s post-test or Two-Way ANOVA for the in vivo experiments.
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