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p53 is the well known tumour suppressor gene because of its protective function on the mutation of the genes. But there are several polymorphisms in p53 gene and it is associated with cancer formation. Therefore, researchers are highly interested to detect the p53 polymorphism in many cancers. In our experiment, we also tried to evaluate the p53 polymorphism from MCF-7 breast cancer cell line by using PCR with known 9 sets of primers. Our data revealed that codon 47 and 360 region is found to be polymorphism in this breast cancer cell line. And then, we can conclude that these polymorphisms itself increase the breast cancer risk or by combination with other mutation in breast cancer specific gene like BRCA1, BRCA2.
Breast cancer is spreading out all over the world and it is the most common malignancy in women population. There are different types of breast cancer such as ductal, lobular and invasive, non-invasive. The incidence is varied depend on the geographic and ethnic group. The incidence is four fold higher in northern American than the Asians (Dale, 2003). Breast cancer is related with different genetic alterations such as mutations in oncogenes and tumor suppressor genes. Mutations in the p53 gene are considered to represent the most common genetic alteration in human cancer including breast cancer (Runnebaum et al., 1991). Moreover, p53 polymorphisms and their associated malignancies could be differing from one population to another because of their genetic background (Goldman and Shields, 1998).
p53 is a tumor suppressor gene and also known as guardian of genome. Similarly, it governs the major defenses against the tumor growth. If it is damaged either deletion or mutation can lead to induce tumor formation because it regulates normal cell growth and division, transcription of the gene, DNA repair and genomic stability (Haffner and Oren, 1995). In the state of stressful conditions such as hypoxia, heat shock, ionizing radiation, ultraviolet irradiation and chemotherapeutic agents that are related to activation of p53 gene. These stressful stimuli give rise to adaptive responses to apoptosis (Agarwal et al., 1998).
The protein product of p53 is phosphor-protein and not normally found because of their short life (Bond et al., 2005). The p53 protein is a potent transcriptional factor and in normal wild type stage, it is responsible for cell cycle arrest, apoptosis, inhibition of angiogenesis and cellular senescence by acting on CDKN1A, BBC3, BAX, PERP and THBS1 genes. Interaction of p53 DNA binding domain with genomic DNA is the critical in the majority of p53 mutations that frequently occurs in the human cancer (eL-Deiry et al., 1992).
Therefore, p53 protein can promote or repress the expression of target genes in response to DNA damage (Vogelstein et al., 2000 and Vousden & Lane, 2007). There are several polymorphisms in p53 gene and polymorphic alleles at codon 72 of the p53 gene plays a role in increased susceptibility of breast cancer (Costa et al., 2008). Most of these variations are intronic and no cancer related biological consequences. Only few of the polymorphism can alter the biochemical function on cancer risk population. Therefore, it is an ongoing challenge to select the variants in laboratory based studies (Boldrini et al., 2008).
In this experiment, we tried to locate the polymorphisms of p53 protein from MCF-7 breast cancer cell line at codons 47, 72, 1414, 217, 267, 278, 290, 360 using the polymerase chain reaction method with specific 9 sets of forward and backward primers.
Materials and methods
1. DNA extraction
We use the DNA from commercial MCF-7 breast cancer cell line. And then, trypsinise to remove the cells form culture flask and extracted the DNA by using Wizard Genomic DNA purification kit (Promega, catalogue number A 1120).
2. DNA quantification
Extracted DNA was read in spectrometer at wavelength 260nm and 280nm by making dilution of genomic DNA with water 1 in 100 times.
3. Restriction enzyme digestion for qualification
After quantified the DNA, we check the quality of DNA again before PCR run. The extracted DNA was digested with EcoRI restriction enzyme from Fermentas. And then, electrophosed on 1% agrose gel, stained with ethidium bromide and check the DNA by using UV light gel doc system.
4. Prepare 9 sets of primers for PCR
We selected these 9 sets of primers to detect the sites of polymorphisms in p53 gene
47 F - 47R
72 F - 72 R
72 F - 72_1414_R
217 F - 217 R
267 F - 267 R
278 F -278 R
290 F - 290 R1
290 F - 290 R2
360 F - 360 R
5. Polymerase chain reaction (Promega PCR master mix cat: M7502)
Polymerase chain reaction (PCR) was done with genomic DNA and 9 sets of primers by using PCR master mix catalog M7502 (Promega, Madison USA). Each reaction was carried out with 9.5ul of water, 12.5ul of PCR Master Mix (which consists of 50units/ml of Taq DNA polymerase, 400uM of dNTPs and 3mM of MgCl2), 1ul of forward primer, 1ul of backward primer and finally adds 1ul of genomic DNA. The total mixture will be 25ul. We prepared two sets of PCR machines with different annealing temperature: 55' C for one machine and 60'C for another one.
All reactions were initially denatured with 94Â°C at 4 minutes. Subsequent denaturations were done at 94Â°C for 45 seconds followed by 60Â° or 55 Â°C for 45 seconds and then, 72Â°C for 1 minute. Lastly, final extension at 72Â°C for 10 minutes was recommended. These cycles were repeated 35 times and store the samples at 4'C for overnight.
6. Running the PCR products on gel electrophoresis
All these PCR products were run on 1% agarose gel after staining with ethidium bromide that intercalates with DNA. We used the DNA ladder to confirm the number of base pairs of PCR products. Run the gel at 120V until the lowest band pass through half of the gel and then visualized under UV light with gel doc system.
Findings and results
The extracted genomic DNA was quantified by spectrophotometer. Under 260nm, the optical density (OD) was 0.253 and under 280nm, OD was 0.175. Therefore, the ratio of absorbance under 260 and 280nm was 1.44. In 1 optical density, the amount of DNA is 500Î¼g/ml at Absorbance 260nm. So, the extracted genomic DNA has amount of 1.26 Î¼g/ml.
Figure 1: photo of agarose gel under gel doc system showing the results of EcoRI restriction enzyme digested symbols
As you seen in the picture, there was no band in the lane 2 where it contained undigested genomic DNA. In contrast, diluted genomic DNA in lane 3 was banded. And also EcoRI restricted DNA in lane 4 appeared as a band. Two DNA ladders were added in lane 1 and lane 5.
Figure 2: photo of agarose gel under doc system showing the bands of PCR products
Products of PCR were stained with ethidium bromide and separated on the 1% agarose gel by electrophoresis. Then, when they were visualized with UV light under gel doc system, amplified fragments were seen as bands in their respective lanes. Moreover, two DNA ladders were clearly seen in lane 1 and 11. But, there were no bands in lane 2 and 10. This meant that there was no amplified PCR fragment in those lanes. Hence, codon 47 region and codon 360 regions could not be amplified. Lane 3 containing 72 show the band approximately 328 base pairs (bp) whereas lane 4 containing codon 72 with fragment 1414 show the band but it does not reach their appropriate size. Codon 217 in lane 5 also appear the band of about 300 bp. Lane 6 with codon 267 show the actual size of 778 bp band. Another amplified fragment in lane 7 (codon 278) contain about 479 bp while lane 8 (codon 290) also show the band. Second last lane (lane 9) just appears the band, about 569 bp.
The regions of polymorphism in p53 gene from MCF-7 breast cancer cell line can be identified by comparing the results with normal wild type p53 proteins results. According to our experiment, amount of extracted DNA that we quantified in first step before PCR run is 3795ng. After that, we checked the purification of DNA by ration of A 260: A 280 and the result is about 1.44. It also reveals that the sample is purified enough according to the standard protocol that is 1.5.
In the figure 1, there are multiple bands in the lane 4, it means EcoRI digestion was achieved and EcoRI restricted the genomic DNA at the multiple sites. We added pure genomic DNA in lane 2 and diluted genomic DNA in the lane 3 as a control. There is a band in lane 3 and lane 2 shows no band. It is due to non-diluted DNA (pure genomic DNA) is slightly tough to run during electrophoresis. Positive bands in lane 3 mean that there is no error in gel preparation and electrophoresis.
In figure 2, there are no visible bands in lane 2 and 10 on the Polaroid photograph after PCR running. It means that the region of codon 47 and codon 360 may not be amplified. The result may be due to technical error or mutation and changing the base pair in this region. Some of the studies show that the mutation in the codon 47 from proline to serine (CCG to TCG) is associated with decreased phosphorylation in p38 MAPK and also decrease in apoptosis that leads to cancer formation (Kruse and Gu, 2008).
Similarly, primers of codon 360 did not anneal in the lane 10 because this region may be mutated. Mutation in codon 360 is associated with substitution of glycine to alanine, this is the linker region to domain of p53. In turn, results in decrease transactivation of pro-apoptotic genes (eg. BAX, MDM2).
The other lanes such as lane 3, 4, 5, 6, 7, 8, 9 show a band respectively. These results are the same with wild type p53 result meaning there is no polymorphism in these codon regions.
To conclude that p53 gene play a role in normal cell division and DNA repair and are critical for detecting inappropriate growth signals in cells. If these genes, as a result of inherited or acquired mutations, become unable to function, genetic mutations in other genes can precede unchecked, leading to neoplastic transformation. Therefore, p53 polymorphisms can be associated with not only breast cancer but also other types of tumor such as lung, colorectal and ovarian cancer (Sjalnder et al.,1995)
First, we are grateful to MDIS (Singapore) and University of Bradford (UK) for providing this experiment and special thanks to our supervisor Dr. Mandar Godge for supporting throughout the whole procedures. We also appreciate Dr. S. Picksley for visiting the lab and discussing the important informations with us.