Summary of DNA damage repair is unaffected by mimicked heterozygous levels of BRCA2 in HT-2 cells
Abstract:
This article is about, the human BRCA2 gene, that naturally functions as a tumor suppressor. It is believed that if one or both functional allele(s) of this gene is lost, tumor will be generated consequently. In this article, the scientists tried to prove that the above mentioned statement is true. So, they mimicked this human BRCA2 gene and result was quite amazing. They saw that DNA damage and repair system was not affected by this mimicked BRCA2 gene. Also they found out that, the level of p53 (a tumor suppressor protein) and p27 (cell cycle inhibitor protein) was increased when the BRCA2 function is lost.
Introduction:
The human BRCA2 gene encodes a nuclear protein of 3,418 amino acids, and is believed to play a pivotal role in DNA damage repair. In this experiment, BRCA2 protein has been shown to bind to RAD51, the mammalian homologue of the RecA recombinase, and thus is believed to be involved in the repair of DNA double-strand breaks. Moreover, BRCA2 regulates both the DNA binding ability of RAD51 and its intracellular location. The scientists have utilized HT-29 colon cancer cells in this experiment and they mimicked the heterozygous state of BRCA2 in these cells through RNA interference.
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Materials and methods:
1. Retroviral vectors:
They utilized pRETRO-SUPER plasmid for short hairpin RNA targeting of BRCA2 and confirmed all plasmids by DNA sequencing and/or restriction digestion.
2. Cell culture and retro viral infection:
Firstly, they grew HT-29 cells in Dulbecco’s modification of Eagle’s medium. Produced supernatant and then incubated them overnight. After that, infected them with empty vector, control retrovirus (control RNAi cells).
After 48 hours they passed infected HT-29 cells into selection media and continued selection process for 14 days.
3. Western blotting:
They rinsed the cells with PBS and 500 μl of lysis buffer in 100mm plates for all western blots and incubated for 30 minutes at 4°C. Then by using a plastic scraper they collected the cells and re-suspended by pipetting. After that, they cleaned the lysates by micro centrifugation for 15 minutes at 4°C and collected the supernatants to conduct a Bradford assay (Bio-Rad) so that the protein concentration could be determined. SDS-PAGE gel was run with cell lines and was later on transferred to a PVDF membrane for western blotting. Band intensities were quantified after that by using gel analyzer.
4. Sub-cellular fractionation:
They trypsinized the HT-29 cell lines and then collected them with fresh media, washed and recollected them in appendrof tubes. Then added 500 μl of homogenization buffer and mixed each tube thoroughly. Incubated the tubes on ice for 20 minutes and mixed after every 5 minutes. Cells were then collected from the bottom of the tubes and homogenized with a plastic pestle. Then they spun the appendrof tubes at 4°C for 10 minutes at 600xg, collected supernatant in separate tube and added lysis buffer to pellet in original tube. Then they incubated the tubes on ice for 15 minutes and mixed them after every 5 minutes. Later, in a refrigerated micro centrifuged they spun the tubes at 4°C for 15 minutes with rpm if 13,500 and collected the supernatant.
5. RAD51 focus assay:
Two days before doing immunofluorescence microscopy they passed the cells onto coverslips in a six-well plate. The following day cells were treated with MMC overnight and the next day they were fixed and blocked. After that cells were incubated with primary and secondary antibody. Then the cells were washed 6 times with PBS tween, stained with DAPI and lastly mounted on the microscope slides and were visualized on a Zeiss Axioskop fluorescence microscope.
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6. Clonogenic survival assay:
Cells were passed into 4 culture plates and incubated till 50% confluence was reached. Then they incubated cells in MMC media for 1hour. After that they removed the MMC media and added the fresh one. Next day re-suspended and counted the cells using hemacytometer. Then they took 1000 cells from each plate and added fresh media every 2 to 3 days. After 2 weeks cells were fixed and stained for 10 minutes. Then they rinsed the cells twice with water, dried and the colonies on the plates were counted to perform ultraviolet radiation DNA damage assay. For this, they removed the media from plates and struck the cells with ultraviolet radiation in a tissue culture hood.
7. Standard growth assay:
They trypsinized cells at 50 % confluence, re-suspended in fresh media and counted by hemacytometer. Then they took 30,000 from each plate and added them to new media plates. After that, fresh media was added to each culture every 3 days. Cells were then trypsinized and counted by hemacytometer at 3 days interval for up to 19 days. Between 12 to 15 days of growth cells reached confluency.
Results:
In comparison to the parental and control RNAi cell lines, the BRCA2 RNAi cells expressed BRCA2 protein levels that were approximately half of the parental and control cells, verifying the mimic of BRCA2 heterozygosity. There was no significant difference between the control RNAi and BRCA2 RNAi cell lines in either the nuclear or cytosolic levels of RAD51. A p27kip1 western was performed and levels of p27kip1 were found to be slightly increased in BRCA2 RNAi cells.
Discussion:
The results suggested that, the loss of the p53 checkpoint may be vital for tumor progression generated by mutations in BRCA2. Loss of p53 has been suggested to be necessary for breast tumor formation following BRCA2 loss. However, it is also possible that the effects of lower BRCA2 levels may differ depending on cell type. Investigations into the differential effects of BRCA2 levels on cell growth and p53 activation may provide insight into why BRCA2 mutations lead to a small subset of observed tumor types.
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New terminologies
1. BRCA2
BRCA2 (breast cancer type 2 susceptibility protein) is a protein found inside cells. In humans, the instructions to make this protein are carried by a gene, also called BRCA2. BRCA2 belongs to the tumor suppressor gene family. The protein encoded by this gene is involved in the repair of chromosomal damage with an important role in the error-free repair of DNA double strand breaks. The BRCA2 gene is located on the long (q) arm of chromosome 13 at position 12.3 (13q12.3) and is 84,188 base pairs long.
2. HT29 cells:
HT29 cells (Human colon adenocarcinoma grade II cell line) are human intestinal epithelial cells which produce the secretory component of Immunoglobulin A (IgA), and carcinoembryonic antigen (CEA).
Cells are used for tumourigenicity studies.
3. RAD51:
RAD51 is a eukaryote gene. The protein encoded by this gene is a member of the RAD51 protein family which assists in repair of DNA double strand breaks. RAD51 family members are homologous to the bacterial RecA and yeast Rad51. The protein is highly conserved in most eukaryotes, from yeast to humans.
4. p53:
p53 (also known as protein 53 or tumor protein 53), is a tumor suppressor protein that in humans is encoded by the TP53 gene. P53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer. As such, p53 has been described as “the guardian of the genome”.
5. p27:
The encoded protein binds to and prevents the activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus controls the cell cycle progression at G1. It is often referred to as a cell cycle inhibitor protein because its major function is to stop or slow down the cell division cycle.
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6. Bradford assay:
The Bio-Rad Protein Assay, based on the method of Bradford, is a simple and accurate procedure for determining concentration of solubilized protein. It involves the addition of an acidic dye to protein solution, and subsequent measurement at 595nm with a spectrophotometer or micro plate reader. Comparison to a standard curve provides a relative measurement of protein concentration.
References for terminologies:
1. http://en.wikipedia.org/wiki/RAD51
2. http://www.abcam.com/HT29-Human-colon-adenocarcinoma-grade-II-cell-line-Whole-Cell-Lysate-ab3952.html
3. http://hmg.oxfordjournals.org/content/12/20/2645.full
4. http://labs.fhcrc.org/fero/Protocols/BioRad_Bradford.pdf
