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Experiment Design: genomic DNA of breast cancer cell lines were extracted and quantified by restriction enzyme digestion for quality and purity of DNA. The polymorphisms of p53 gene were detected by using 9 specific primers in PCR methods.
Result: the analysis of RE digested DNA samples provided a sufficient quantity for DNA analysis although the purity for it was lower than the standard range. This might be due to artificial contamination during the experimental practice. Out of 9 polymorphisms, only 7 variants were seen in the PCR analysis with specific primers. The missing lane could be due to technical errors or improper annealing of primers to specific gene sequences and confirmation analysis needed to be progressed.
Conclusion: the experimental results from PCR method could not alone confirm the variants of p53 gene. Further analyses are required such as immunohistochemistry analysis, mutational analysis, and statistical analysis. The ultimate aim of this study is to experimentally and theoretically understand the PCR analysis methods and p53 polymorphisms.
The p53 gene, which produces p53 tumor suppressor protein, is the most frequently mutated gene in different human cancers. The P53 tumor suppressor gene, which is located on chromosome 17p13.1 and encodes for a 53 kD protein, plays a major key response to a variety of damaging including hypoxia, metabolic stress and oncogene activation, cytotoxic drug and protect the genome by coordinately blocking cell proliferation, stimulation DNA repair and promoting apoptotic cell death. Over the decade of studies in mutation of p53 gene has been demonstrated that abnormality of p53 gene are highly associated with variety of human malignancies such as breast, colon and lung cancers. The p53 protein is almost always vulnerable to a large range of single nucleotide alterations. Although most of these polymorphisms are phenotypically silent, occasionally, it can compromise the function of wild type protein and can even confer new oncogenic activities. These single nucleotide polymorphisms count for 25-35% of total mutations. The majority of the p53 polymorphisms occur in the non-coding sequences and only 19 exonic polymorphisms have been reported in the series of study. Study also suggested that alteration in the ability of p53 providing a possible molecular basis for the associated increase in risk of developing cancer (Catherine Whibley et al, 2009).
Breast cancer, the fifth most common cause of cancer death in the world today, is becoming the major concern in the clinical research and practices. Study has shown that the prognosis of breast cancer in young women is much better than older women. These findings postulate that breast cancer affecting older and younger women may have a different origin, possibly caused by diverse pathways of genetic transformation. With thousands of researches have been done on breast cancer registered that there is strong correlation of this variation with p53 polymorphism. Although most of the single nucleotide polymorphisms in p53 genes have been study, the functional consequences of most of these polymorphisms are still unknown. Hypothetically, variation in the function of genes responsible for DNA repair and cell cycle control in the presence of carcinogen-mediated damage is a reasonable and convincing mechanism for explaining the variation in individual susceptibility to breast cancer. This has been implied in the study of individual who carries different alleles of codon 72 polymorphisms shows different frequency of p53 mutation in breast cancer (Langerod et al, 2002).
MCF-7 breast cancer cell line, which was isolated from a 69 year old Caucasian woman with metastatic breast cancer, is the first effective cultured cell lines (Soule HD, 1973) used in in vitro breast cancer study as the cell line has conserved several ideal characteristics of mammary epithelium. . In this experiment, polymorphisms of p53 protein from MCF-7 breast cancer cell line at codons 47, 72, 217, 267, 278, 290, 360 (figure 1) are detected by using the polymerase chain reaction method with specific 9 sets of forward and backward primers (table 2).
Figure 1: p53 single nucleotide polymorphisms: locations in the p53 protein and DNA sequences showing 11 common polymorphisms in association with oncogenesis. Source: Nature Reviews Cancer 9, 95-107 (February 2009), cited at 12:57PM 15/11/2010
Material and methods
Extraction of DNA from MCF-7 breast cancer cell line
Commercial MCF-7 breast cancer cell line grown after several passaging is provided. Trypsinizing and extracting of the genomic DNA from cancer cells by using Wizard Genomic DNA purification kit (Promega, catalogue number A 1120) was done following the manufacture's recommended protocol.
Quantification of DNA
Make 1:100 dilution of genomic DNA by mixing with 1495ul of ddH2O with 5ul of DNA and read in spectrophotometer at wavelength of 260nm and 280nm respectively.
260nm = Optical density (OD260) x 50 x DF
= ______ ng/ml (average concentration = 3000ng/ml)
Purity = 260nm/280nm = ______ (normal range = 1.8-2)
Restriction Enzyme Digestion
The genomic DNA are digested with EcoR I provided by Fermentas. The procedure strictly followed the New England Biolabs protocol. The total amount of 25ul of sample is prepared which included 3ul of DNA (1000ng/ul), 2.5ul of required buffer-3, 18ul of water. Keep the reaction in a water bath at 37Ë™C for 3 hours.
Dilutes 1L of 1xTAE buffer from given 50xTAE buffer by mixing 20ml of 50xTAE buffer with 980ml of ddH20. Prepare 1% agarose gel by using 100ml of 1xTAE buffer, 1g of agarose powder and 6ul of ethidium bromide. Cast the gel in the gel rig and add 700ml of remaining 1xTAE buffer. Load 1kb DNA ladder genomic DNA, concentrated genomic DNA extract, 10x diluted DNA, EcoRI digested genomic DNA samples, and 1kb plus DNA ladder in the wells of the gel (table 1). 5ul of loading dyes is used for each sample. Run the gel in 120V till the lowest band passes through half of the gel. Visualize and take photo of the gel under UV light with gel doc system.
1kb DNA ladder
10x diluted genomic DNA
RE digested DNA
1kb plus DNA ladder
1ul + 5ul loading dye
15ul (500ng) + 5ul loading dye
25ul + 5ul of loading dye
8ulTable 1: loading sequences and concentration for quantitative gel electrophoresis.
Polymerase Chain Reaction
PCR were performed in a final volume of 30 ul containing 1ul of extracted DNA,1ul of forward and 1ul of backward primers, 12.5 ul of PCR Mater Mix from Promega (catalogue number: M7502), 9.5 ul of H2O and 5ul of loading dye. Similar procedure is run for genomic DNA using 9 sets of primers (table 2). Melting temperature for genomic DNA was calculated and annealing temperature for primers and DNA was adjusted by using mathematically methods [(Tm 1 +Tm2/2)-3]. Because of limited number of PCR machines, prepare two sets of PCR machines with different annealing temperature; 55Ë™C for one machine and 60Ë™C for another one. Add reaction 1, 6, 7 and 8 to 55Ë™C set up machine and reactions 2, 3, 4, 5 and 9 to another machine. Firstly, DNA is denatured at 94Ë™C for 4 min and additional 45 sec for denaturation. Secondly, the temperature is set at 55Ë™C or 60Ë™C for 50sec for annealing, followed by 72Ë™C for 1 min and additional 10 min after the cycles for extension. The repeated times of the cycle is 35 times and stores the samples at 4'C for overnight.
Running PCR products on agarose gel: Prepare 1% agarose gel to run gel electrophoresis. Load 1kb DNA ladder and 9 sets of primers. Loading dye is used for PCR products. Run the gel at 150V until the lowest band pass through half of the gel. Take off the gel and visualize and take photo under UV light with gel doc system.
Table 2: Forward and Reverse primers sequences of 9 specific polymorphisms in p53 gene
Source: Oligo@base-asia.com, assessed on 25th Nov 2010.
Quantitative analysis of target DNA samples
We identified the quantity of DNA samples extracted from breast cancer cell lines shown in figure 2. DNA quantification under Abs 260 and Abs 280 showed that the sample provided 3795ng/ml concentration of DNA and purity of 1.5 (standard= 1.8-2) respectively. RE digested DNA sample was analyzed on 1% agarose gel, and visualized with UV light under gel doc system which gave a smear pattern (fig 2: lane 4) ranging from 200 to 700 kb. Two DNA ladders (1kb and 1kb plus from Fermentas, fig 3 and 4) were also run as control sample. Interestingly, reading of genomic DNA extract in the lane 2 showed high density band due to the massive base pair of genomic DNA unable it to run through the gel. The electrophoresis showed that the quantitative base pairs for the tested sample are suitable for DNA analysis.
1 2 3 4 5
Figure 2: Agarose gel photo under gel document system photographing showing the results of EcoR I restriction enzyme digested samples. Lane 1 and 5 showed the 1kb and 1kb plus DNA ladder as control. Lane 2 showed concentrated DNA extract from breast cell line and 10x dilution of it was shown in lane 3. RE digested DNA sample in smear pattern was demonstrated in lane 4.
Figure 4: 1 kb plus DNA ladder 75-20,000bps
Source: Fermentas, DNA electrophoresis
Figure 3: 1 kb DNA ladder 25-10,000 bps
Source: Fermentas, DNA electrophoresis
Restriction digest-genotyping test or Polymerase chain reaction analysis
The p53 mRNA variants were amplified specifically by PCR methods with 9 specific forward and reverse primers. Genotyping demonstrated that there were six polymorphisms in targeted p53 DNA samples (fig: 5). the lane 2 for codon 47 and lane 10 for 360 regions have no band. Gel electrophoresis for codon 72 with 72F and 72_1414_R primers showed the band approximately at 328bps and 1414 bps in lane 3 and 4 respectively. For codon 217, prominent band of approximately 496 bps was seen in lane 5. The polymorphism for codon 267, 278, 290 (290R1) and 290 (290R2) showed similar bands with 778,479 and 524 respectively.
1Kb plus DNA ladder
1Kb DNA ladder
1 2 3 4 5 6 7 8 9 10 11
Figure 5: Agarose gel under gel doc system photographing describing the bands of PCR products
Over 3.1 million sequence variations have been identified throughout the study of p53 gene. Although majority of polymorphisms are in non-coding region, polymorphism in coding regions highly correlated with the functional activity of p53 proteins. In the present study, the polymorphism of the p53 protein regions can be estimated by comparing the experimental results with that of normal wild type p53 proteins. Study was designed to detect the 9 nonsynonumous polymorphisms in coding sequence in p53 gene in MCF-7 cell lines.
Two consecutive steps was carried out in the experiment; quantifying the DNA samples and PCR amplification and detection of sequence variations in p53 genes. The experiment result showed that the original genomic DNA from MCF-7 cell has size larger than 20000 bps which is quantified enough for PCR analysis. Smear pattern showed in gel electrophoresis on RE digested DNA samples determined that there are many EcoR I restriction sites in the whole experimental genomic DNA. The figure describes the smear of only one third of the whole range meaning that the sample was not very purified. This data is corresponding with Abs260: Abs280 ratio result in DNA quantification (1.44).
Polymerase chain reaction was done in the second part of the experiment, which showed that there was missing bands in the result. This probably could indicate that there might be technical error during procedure or the primers cannot anneal to the DNA because of the presence of mutation in primer binding regions.
Codon 47 variants are one of the rare non-synonymous polymorphisms in TP53 coding sequences with mutation from proline to serine (CCG to TCG) (Caamano et al. 1991). This variant is associated with decreased phosphorylation by p38 MAPK and 5 fold decreases in apoptosis. Decreased in apoptosis in this mutation is due to decreased ability in transactivation of p53 response apoptotic genes AIP1 and PUMA (Li et al, 2005). This variant has been widely study in recent decades (Emanuela Felley-Bosco et al, 1993). Although the PCR analysis showed negative result in this experiment, this could not accurately say that there is polymorphism in target cell lines. In fact, primer error or technical error should be considered and further confirmation is needed to proceed for detection of the polymorphisms. The polymorphisms were confirmed by several study designs such as immunohistochemistry with monoclonal antibodies to demonstrate the p53 protein accumulation, frequency analysis by statistic, functional study by southern blot, transfection experiment, PCR-RFLP detection or oligonucleotide hybridization method.
Codon 72, another example of non-synonymous polymorphism with change in amino acid, has a band of approximately 328 bps. The result was the same as in wild type p53 DNA PCR results which showed that this region has no obvious deletion or SNPs within primer amplified sites. Further confirmation needed to be considered as above. Several studies have found that the p53 codon 72 with mutation from Arginine to Proline (CGC to CCC) plays an important role in cancer such as the original publication on HPV-associated cervical cancer (Storey et al, 1998), cervical cancer (Dokianakis and Spandidos, 2000), lung (Papadakis et al, 2002), skin (Dokianakis et al, 2000), laryngeal (Sourvinos et al, 2001) and bladder cancer (Soulitzis et al, 2002). Report also found that these polymorphisms are functionally significant. Codon 72 of p53 can encode either arginine (the R72 form) or proline (the P72 form). In some studies (Storey et al, 1998) (Papadakis et al, 2002), an allele-specific PCR assay was used to detect either the Arg or Pro p53 allele revealed that the frequency of Arg/Arg genotype was considerably higher in patients although Pro/Pro homozygous patients have poorer overall survival than Arg/Arg or Arg/Pro genotype especially in patients receiving chemotherapy. Thus, study claimed that codon 72 polymorphism can be used as prognosis marker for chemotherapy response. (Toyama T et al, 2007). In 1999, M. Thomas also mentioned that Arg/Arg genotype has more apoptotic efficiency than Pro/Pro genotype.
In codon 217, there is a band of approximately size of 496bp which is the same with the product result of wild type p53. This has been illustrated in study of yeast which describes V217M polymorphism in codon 217 region of p53 (Kato, S. et al, 2003). However, this mutation is found to have more potent effect in cell cycle arrest and apoptosis genes such as CDKN1A, BAX and PMAIP1. Therefore, it probably has protective effect rather than transformation to cancer cells. Nevertheless, more wide and precise study is needed to do more.
Codon 278 contains length of approximately 497 bps. This result is the same with wild type p53 results mean that there is no polymorphism or only SNPs in this codon. One study shows a SNP in this codon mentioning that substitution of proline to threonine (CCT to ACT) on this codon is found in metastasis region but not in the primary gastric tumour. This raises the concept that this mutation may be associated with gastric tumour progression rather than the original tumour formation (Kim JH et al, 1993). The codon 267 product shows mutation in some of the studies. This codon mutation in exon 8 is found in breast cancer cells and white blood cells DNA of Li Fraumeni Syndrome patients (Prosser J et al, 1992).
Interestingly, we did not get visual band in codon 360 whereas the normal wild type p53 amplified with the same PCR primers shows a single band with 829 bps. This cannot be attributed to methodological problems. The technique used to type this polymorphism (i.e. allele specific PCR), may have tendency to over-estimate or under-estimate the heterozyogosity. It has been mentioned that G360A polymorphism of p53 protein at the linker region lie adjacent to tetramerization domain of p53. This type of polymorphism also shows decreased expression of pro apoptosis genes such as BAX, 14-3-3 σ and GADD 45. However, further mammalian cells studies are needed for confirm this result.
To conclude, p53 protein is crucial for maintaining of gene integrity and any polymorphism in its genome can leads to abnormal conditions especially cancer stage in most cases. As PCR cannot detect exactly the polymorphism types and specific changes in sequences, thus the applicability and the generalizability of this finding need to be confirmed for the polymorphisms in p53 genes. Study is primarily aimed to achieve in understanding of laboratory handling of DNA extraction, purification and analytical methods.
This study was in part sponsored by Management Development Institute of Singapore, School of Life Sciences by a grand from University of Bradford, UK. We are grateful to Dr. Godge Mandar Radhakisan, supervisor and lecturer of this practical work, for providing good supervision during the procedures, and also Dr Prickly for supporting during this work. Moreover, we thank for all the lab technicians who technically supported and provided basic requirements throughout the project.