Epigenome alterations modulate the cell cycle

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Chapter 2 Materials and methods

Materials

The cell lines, chemical compounds, reagents, equipment and software were purchased as listed in tables 2.1 to 2.4.
ATCC (American Type Culture Collection), VA (Virginia), USA (The United States of America), NCI (National Cancer Institute), NIH (National Institute of Health), MD (Maryland), CA (California).
CAPE (caffeic acid phenethyl ester), PMA (phorbol myristate acetate), SAHA (suberoylanilide hydroxamic acid), TSA (trichostatin A), ACSRC (Auckland Cancer Society Research Centre), NZ (New Zealand), MO (Missouri).

Methods

Cell growth inhibition of DU145, PC3 and LNCaP cancer cell lines

To determine the 50% inhibitory concentrations (IC50) for the different drugs with the three prostate cancer cell lines, DU145, PC3 and LNCaP, a 96 well plate dosing assay was carried out at the different time points of 24 hours, four and seven days incubation. The three cell lines were harvested and the concentration of cells was determined by using a haemocytometer. For the three incubation times, 96 well plates were seeded at varying cell numbers according to the doubling times for each cell line (table 2.5).
To each well of a 96 well plate (except the last column) 150 µL of cell suspension was added to give the cell density required for cell line and incubation time. To the last column was added no cells (only medium), to act as a blank (figure 2.1). The plates were then incubated for 24 hours at 37oC in 5% CO2 to allow the cells to attach to the bottom of the wells. The appropriate concentration of drug was then diluted to a final volume of 150 µL in culture medium for 4 replicates and transferred to the first 4 wells of each of 4 rows, allowing for 2 compounds per 96 well plate. A multichannel pipette was used to mix the added drug solution and 150 µL transferred to the next well to give a 1:2 serial dilution. This was continued along the row, leaving the second to last column without drug to act as an untreated control. Where necessary, a 3:4 serial dilution was carried out instead. The 96 well plates were then incubated at 37oC in 5% CO2 for the appropriate incubation time.
The blank well contains medium only. The negative control well contain cells without drug treatment.
Following incubation, the plates were fixed by adding 50 µL of cold 40% trichloroacetic acid (TCA) to each well and left at 4oC for 1 hour. After the plates were washed with tap water and briefly air dried, 100 µL of 0.4% Sulforhodamine B (SRB) with 1% acetic acid was added to each well and the plates were placed in the dark for 30 minutes to allow staining of cell protein. After staining, the plates were washed four times in 2 litres of tap water containing 1% glacial acetic acid then dried in a Contherm Digital Series incubator at 37oC for 30 minutes. Furthermore, 100 µL of 10 mM un-buffered Tris was added to each well, the plates protected from light and placed on an Open-Air Platform Shaker Max-Q 2000 at 150 rpm for 1 hour. Once the stained protein was re-solubilised the plates were read on a Bio Tek EL808 Absorbance Microplate Reader at 490 and 450 nm wavelengths. The percentage survival values as compared to the untreated control values against drug concentration were plotted on Sigma Plot and IC50 concentrations established for each cell line at each incubation time.

Storage and maintenance of cell lines

Cryopreservation of cell lines

Cells were grown in a T75 flask. When this contained 70 – 80% of confluent cells, these were harvested by aspirating the cell medium, and washed once with 3 mL of 1x PBS. Thereafter, 2 mL of trypsin was added, and the flask was incubated for 3-6 minutes at 37oC until the cells were dislodged from the bottom of the flask. The resulting cell suspension was added to 8 mL of α-MEM medium, and the cells were counted on a haemocytometer. The cell suspension was then centrifuged (Heraeus Multifuge 1S) at 1000 rpm for 5 minutes to pellet the cells. The cell pellet was re-suspended in freeze medium to give a final concentration of 5-7×106 cells/mL, and 1mL aliquots were added to labelled 1.8 mL cryovials. The cryovials were then placed in a Mr. Frosty™ Freezing Container and stored at -80°C overnight before being transferred to liquid nitrogen for long term storage. The freeze medium was prepared by adding DMSO (10% v/v) and FBS (40% v/v) to un-supplemented α-MEM medium, and cooled before adding to the cell pellet.

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Counting of cells

A haemocytometer was used to count cells before cryopreservation and seeding for experimental use (figure 2.2)
The cells were counted by adding 20 µL of Trypan Blue to 20 µL of cell suspension to give a two-fold dilution and transferring 20 µL of this suspension to a haemocytometer for counting. Cell number/mL was determined by:
Total number of cells in haemocytometer grid/9x 2 (dilution factor) x 104 = cells/mL

Thawing and maintenance of cell lines

To initiate the growth of the prostate cancer cell lines, PC3, DU145 and LNCaP, the cryovials containing the cell lines were taken from liquid nitrogen storage and thawed for 3-5 minutes in a 37 °C water bath. The outside of each vial was then wiped with 70% ethanol to sterilise this before transferring to an Email Air Handling Cabinet. The thawed cells were transferred into a 15 mL Falcon tube with 10 mL of α10-MEM medium. This cell suspension was centrifuged at 1000 rpm for 5 minutes and medium aspirated. The cell pellet was re-suspended in 5 mL α10-MEM medium, transferred to a T25 flask and then incubated at 37oC in a Water Jacketed Incubator with 5% CO2 in air. After cells had been passaged twice, 1% Penicillin/Streptomycin/Glutamine was added to the culture medium for further passaging. Once the cells in the T25 flask showed 70-80% confluence, they were passaged by aspiration of the culture medium and washed once with 2 mL of 1x PBS. At that time, 1 mL of trypsin was added and the flask was then incubated at 37oC in an incubator for 3 minutes (DU145 and LNCaP), or 6 minutes (PC3), until the cells dislodged. This cell suspension was then transferred to 9 mL of α10-MEM medium, gently mixed and 200 µL of this cell suspension added to a new T25 flask containing 5 mL fresh α10-MEM medium.
Kit225 cells were grown in RPMI medium with 10% Fetal Calf Serum, 1% Penicillin/Streptomycin/L-Glutamine and cytokine IL2 (10 µg/mL). The cells were passaged 2 times a week and the medium was replaced once a week.
HEK-Blue™ hTLR2 and HEK-Blue™ hTLR4 cell lines growth were initiated as per the prostate cancer cell lines above. They were maintained in DMEM F+ medium (10% FBS + 1% Penicillin/ Streptomycin/ Glutamine) together with antibiotic HEK Blue Selection 2 mL/500 mL DMEM F+ medium. Cells were seeded into a T75 flask containing 20 mL medium and grown to 70-80% confluence. These cell lines were then passaged by aspirating the culture medium and washing cells with 3 mL of 1x PBS. Cells were then detached from the bottom of the flask by adding fresh medium and gently pipetting up and down while washing the flask surface. To 20 mL of fresh medium, 1 mL of this cell suspension was added to a T75 flask. When cells were used for experimental work, the medium was replaced with DMEM + 10% FBS only.

Global Methylation Analysis by High-performance Liquid Chromatography (HPLC)

Drug Treatment of DU145, PC3 and LNCaP

The three prostate cancer cell lines were seeded into duplicate p100 plates. Following 24 hours incubation of the cells at 37oC in 5% CO2, they were dosed at the 7 day IC50 concentrations of drug, and the plates were incubated for 7 days. LNCap was also dosed and incubated at the 4 day IC50 concentrations of drug for 4 days.

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 DNA Extraction

Following the required treatment, DNA was extracted from the cells of the dosing plates. For each plate the medium was aspirated, the plate washed with 1 x PBS, and the cells harvested with 2 mL of trypsin either for 3 minutes for DU145 and LNCaP, or 6 minutes for PC3 cells. The dislodged cells were added to 8 mL of fresh α10 MEM medium, centrifuged for 5 minutes at 1000 rpm and the cell pellet then washed twice by centrifugation with 1x PBS. Each cell pellet was re-suspended with 2.7 mL of TEN (50mM Tris, 1mM EDTA, 150mM NaCl) buffer followed by the addition of 300 µL of 10% SDS and 15 µL of a 10 mg/mL solution of Proteinase K, gently mixed and then incubated with shaking in a 55oC waterbath for 1 hour. From the lysed cell suspension, 970 µL was transferred to each of three 2 mL microcentrifuge tubes. To each tube an equal volume of phenol: chloroform: isoamylalcohol (25:24:1) was added and the contents mixed on a rotator for 3 minutes. The tubes were then centrifuged at 13000 rpm for 15 minutes and the top layer carefully transferred to a 14 mL polypropylene round-bottom tube containing 7 mL of a 100% Ethanol: 7.5 M Ammonium Acetate (2:0.5) mixture. After inversion of the tubes 25 times to precipitate the DNA, the tubes were centrifuged at 13000 rpm for 10 minutes at 4oC to pellet the DNA. The supernatant was carefully removed and the DNA pellet washed twice with 1 mL of 70% ethanol and centrifuged at 13000 rpm for 5 minutes at 4oC. After carefully removing the supernatant, the DNA pellet was air dried at room temperature for 15 minutes before dissolving in 500 µL of milliQ water and leaving overnight at room temperature.

Abstract 
Acknowledgments
Chapter 1 Review of the literature 
1.1 Introduction
1.2 Epigenetic modifications
1.3 Epigenome alterations modulate the cell cycle
1.4 Epigenetics in health and disease
1.5 Hypotheses and aims
Chapter 2 Materials and methods 
2.1 Materials
2.2 Methods
Chapter 3 DNA methylation effects
3.1 Introduction
3.2 Aim
3.3. Cytotoxicity (IC50 and IC10 values) of compounds
3.4 Global DNA methylation
3.5 Discussions
Chapter 4 The regulation of HDAC activity 
4.1 Introduction
4.2 Aims
4.3 Specific HDAC activity
4.4 HDAC activity of prostate cancer cell lines
4.5 Discussion
Chapter 5 Effect of HDAC inhibitors on the cell cycle
5.1 Introduction
5.2 Aims
5.3 Drug stability
5.4 PC3 cell cycle analysis
5.5 DU145 cell cycle analysis
5.6 LNCaP cell cycle analysis
5.7 Discussion
Chapter 6 Analysis of the anti-inflammatory effects 
6.1 Introduction
6.2 Aims
6.3 IL23 anti-inflammatory assay
6.4 HEK-Blue 2 anti-inflammatory assay
6.5 HEK-Blue 4 anti-inflammatory assay
6.6 Discussions
Chapter 7 Modulation of gene expression by an HDAC inhibitor 
7.1 Introduction
7.2 Aims
7.3 Microarray experiment
7.4 qRT-PCR
7.5 Discussion
Chapter 8 Concluding discussion 
8.1 The outline of the thesis
8.2 SN30028 is an anti-inflammatory agent
8.3 Anti-cancer effect of SN30028
Appendix 
Reference list
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