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p53, gene known as tumour suppressor gene, is essential gene in controlling cellular integrity, proliferation and apoptosis. It serves as potential barrier for various kinds of malignancies. Mutation in p53 develops multiple cancers syndrome (Li-Fraumeni Syndrome). It is loss of function mutation, so need to mutate in two alleles. Recently, apart from mutation, incidences of the cancer risk are significantly high that are related with polymorphisms of this gene. There are more than 200 naturally occouring single nucleotide polymorphisms (SNP) of p53 gene so far in the general population that are expected to affect on p53 function. Relationship with our study, there are 9 p53 polymorphisms related with breast cancer. These polymorphisms increase the risk of breast cancer by itself or combination with other mutation in specific genes such as BRCA1, BRCA2 etc. So, in our research, we tried to detect which polymorphism is associated with MCF-7 breast cancer cell line by using known primers. Finally, we realized that polymorphisms found at codon 47 and 360 region in this cancer cell line.
Keywards: breast cancer; p53 polymorphisms; apoptosis; SNP; codon 47 and 360
Breast cancer is the most common life-threatening cancer among the women population worldwide. Over the past 25 years, incidence of breast cancer is significantly risen especially western countries. Breast cancer may be caused by both non-genetic and genetic factors. Non-genetic factors include changes in reproductive patterns, dietary changes, alcohol intake, and physical activity whereas genetic component predispose clustering in the family members (Richards et al., 2000). Recently, genetic component is more interesting apart from the epigenetic factors. Indeed, mutation in p53 gene, tumour suppressor gene located in chromosome 17p, is the commonest genetic alteration in breast tumour cells (50%) (Greenblatt et al., 1994). p53 mutation also found in other types of tumours such as ovary cancer, colorectal cancer, lung cancer etc. p53, guardian angel gene, regulates the cell cycle control, DNA repair and apoptosis (Lane, 1992). Interestingly, apart from mutation, incidence of breast cancer is also increased in p53 gene polymorphisms. Polymorphisms seem to alter the mRNA processing which may change the cellular functions (Gemignani et al., 2004). Moreover, p53 polymorphisms may cause changes the amino acid sequences altering recognition motifs for post- translational modification, protein stability or interaction with other proteins (Li and Prives, 2007).
p53 polymorphisms take place in intronic (90%) as well as exonic sites (Olivier et al., 2002). Although most polymorphisms are in introns, 16bp intron 3 polymorphism only increases the risk of breast cancer (Wang et al., 1999; Coasta et al., 2008). Nevertheless, there are 9 exonic p53 polymorphisms detected so far which all are associated with breast cancer. These polymorphic sites are codon 47, 72, 72 with fragment 1414, 217, 267, 278, 290 (two types) and 360. Among them, sufficient molecular evidences are found in codon 47, 72, 217 and 360. P47S is resultant from substitution of cytosine to thymadine in codon 47. It substitutes serine instant of proline. It is rare variants and only reported in African population (Bosco et al., 1993). Replacement of P47 reduces the activity of apoptosis associated genes leading to prolong cell survival and potentially increase the breast cancer risk (Feng et al., 2006; Kurihara et al., 2007). Moreover, R72P polymorphism also increases cancer risk. Arginine in codon 72 is the susceptible loci for breast cancer (Easton et al., 2007). Fortunately, p53-R72 genotype increase survival rates after chemo- and radiotherapy (Xu et al., 2005). Another V217M polymorphism is the only polymorphism located in the DNA binding domain (DBD) of p53 (exon 6) so that it directly manipulate the gene transcription and their products. Similarly, G360A polymorphism affects in exon 10 and appears to damage the p53 response (Kato et al., 2003). Therefore, despite p53 polymorphism frequencies are low in general population; they have significant effect on the development of the malignancies including carcinoma of breast (Garcia-Closas et al., 2008). Moreover, p53 polymorphisms and their associated malignancies could be differing from one population to another because of their background genetics (Goldman and Shields, 1998).
In our experiment, we tried to find out which p53 polymorphisms are associated with this MCF-7 breast cancer cell line. Thus, we used the appropriate known 9 sets of primers for detecting these polymorphisms.
2. Materials and Method
2.1 DNA isolation and quantification
DNA is extracted from the MCF-7 (Michigan cancer foundation) breast cancer cell line by wizard® genomic DNA purification kit (product of Promega catalog A1120) according to manufacturer's standard protocol. The concentration of genomic DNA extracted was measured using Spectrophotometer at 260 and 280 nm.
2.2 DNA qualification
Extracted genomic DNA was digested with restriction enzyme EcoRI from Fermentas (Thermo Fisher Scientific, Waltham; Massachusetts, USA). DNA fragments were stained with ethidium bromide and separated on 1% agrose gel by mean of electrophoresis. Then, they were visualized by UV light gel doc system and taken the photograph with Polaroid documentation camera.
2.3 Primers design and polymerase chain reaction (PCR)
For identification of the polymorphisms in the p53 gene, known primers sets (including forward and reverse) were used to amplify the respective site of polymorphisms. The nucleotide positions of all primers were designed according to the p53 gene sequence in the NCBI database. Detail sequences of all primers (1st Base, Malaysia) were provided in table 1.
Polymerase chain reaction (PCR) was performed with genomic DNA and 9 sets of primers by using PCR master mix catalog M7502 (Promega: Madison, Wisconsin, 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. Primer sets 1, 6, 7, 8 were applied at 55Ë™ C whereas sets 2, 3, 4, 5, 9 were applied at 60Ë™ C malting temperature. All reactions were initially denatured with 94°C at 4 minutes. Subsequent denaturations were done at 94°C for 45 seconds followed by 60° or 55 °C for 45 seconds and then, 72°C for 1 minute. These cycles were repeated 35 times. Lastly, final extension at 72°C for 10 minutes was recommended.
Table 1: Primers used in this study
5'-TGA GGA CCT GGT CCT CTG AC-3'
5'-GAG GAA TCC CAA AGT TCC AAA-3'
5'-TCC CCC TTG CCG TCC CAA GC-3'
5'-CGG CCA GGC ATT GAA GTC TCA TGG-3'
5'-CTC AGG CGG CTC ATA GGG C-3'
5'-CGC GTC CGC GCC ATG GCC -3'
5'-CGC CCA GCC AAG CAG GGG-3'
5'-CAC CCT GCA CAC TGG CCT-3'
5'-GCT ACA ACC AGG AGC CAT-3'
5'-CTT CTC CTC CAC CTA CCT-3'
5'-CAC TTG ATA AGA GGT CCC-3'
5'-CTT CTC CTC CAC CTA CCT-3'
5'-CAC TTG ATA AGA GGT CCC-3'
5'-AGT CAA GAA GAA AAC GGC-3'
5'-ACT TGA ACC CCA GAG GCG-3'
5'-CAC TCG CCT TGG CCT CCC-3'
2.4 Verification with gel electrophoresis
All PCR products were verified by gel electrophoresis again after staining with 6ul of intercalating agent (ethidium bromide). Besides, DNA ladder was also used to confirm the number of base pairs of the PCR products. Then, they were visualized by UV light under gel doc system and taken the photograph with Polaroid camera. Strict handling and disposal of ethidium bromide is essential because it is carcinogenic.
First of all, genomic DNA was extracted from MCF-7 breast cancer cell line and then, quantified by spectrophotometer. Under 260nm, the optical density (OD) was 0.253 and under 280nm, OD was 0.175. Thus, the ration of absorbance under 260 and 280nm was 1.44. The genomic DNA has amount of 1.26 ug/ul since 1 optical density at Absorbance 260nm is 50ug/ml of DNA.
Figure 1: Polaroid photograph of agrose gel under gel doc system showing EcoRI digested DNA
After digestion with restriction enzyme EcoRI, genomic DNA, diluted genomic DNA and digested DNA were separated on the 1% agrose gel by mean of electrophoresis. Two DNA ladders were added in lane 1 and 5. There was no band in lane 2 containing undigested genomic DNA however; diluted genomic DNA in lane 3 was banded. EcoRI restricted DNA in lane 4 also appears as a band on the Polaroid photograph.
Figure 2: Photo of agrose gel with PCR fragments Figure3: standard DNA ladder
Products of PCR were stained with ethidium bromide and separated on the 1% agrose 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 also run in lane 1 and 11. 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 region could not be amplified. Lane 3 containing 72 codon showed the band approximately 300 base pairs (bp) whereas lane 4 containing codon 72 with fragment 1414 displayed the band just below 1500 base pairs (bp). Codon 217 in lane 5 also appear the band about 496 bp. Lane 6 with codon 267 showed a band with its actural size (778 base pairs). Another amplified fragment in lane 7 (codon 278) contained a band about 300 bp but it did not retain its actural size 479 bp. Likewise, lane 8 (codon 290) also showed the band approximately 500 bp while second last lane of all primers (lane 9) appears a band just above the previous lane 8 band about 569 bp.
This experiment evaluated which types of p53 polymorphisms are associated with given MCF-7 breast cancer cell line. Firstly, we extracted genomic DNA and quantified the amount of extracted DNA which is about 1.26 ug/ul. Then, we checked the purification of DNA by ratio of A260: A280. More than 1.5 is adequate for purification according to standard protocol however, our result is about 1.44. It may be due to little sample of DNA extracted or slight contamination.
According to electrophoresis result, we found that EcoRI digestion was achieved because lane 4 appears multiple bands. Regarding for positive control diluted genomic DNA run in 3rd lane on 1% agrose gel although pure genomic DNA in lane 2 did not appear the band. It is due to non-diluted DNA is slightly tough to run during electrophoresis. Positive band in lane 3 means that there is no error in gel preparation and electrophoresis. Multiple bands in lane 4 describe that EcoRI restricted the genomic DNA at multiple sites. EcoRI recognize the 5' G A A T T C 3' sequences and cut between guanine and adenosine (Griffin et al., 1974; Robberson et al., 1974). So, it can produce multiple fragments of genomic DNA. EcoRI restrictive enzyme digestion form multiple or smear pattern bands in electrophoresis. Therefore, we conclude that this genomic DNA is quite purified.
In figure 2, there is no visible band in lane 2 and 10 on the Polaroid photograph after finishing the polymerase chain reaction and gel electrophoresis. Therefore, codon 47 and codon 360 may not be amplified properly because primers (including forward and reverse) for these codons cannot anneal or amplify with their appropriate region in genomic DNA. The result may be due to technical fault or mutation and / or changing the base pair in this region. Technical error should not be entertained because other lanes show their appropriate amplified DNA fragments. So, we conclude that there are the polymorphisms in those codons (codon 47 and 360).
Mutation in codon 47 (changing CCG to TCG) substitute Proline amino acid to Serine at position 1. p38 and HIPK2 (homeodomain -interacting protein kinase 2) phosphorylate proline residue adjacent to S46 leading to increase apoptosis (Kruse and Gu, 2008). Serine instant of proline occurred in P47S polymorphism decrease the phosphorylation of p38 and HIPK2. Thus, reduction in phosphorylation of p38 and HIPK2 reduce pro-apoptotic genes expression and increase the potential risk of malignancies (Li et al., 2005).
Similarly, we found no band in the lane 10 on the UV light under gel doc system because codon 360 did not amplified appropriately. Primers of codon 360 did not anneal its respondent region as this region may be mutated. Substitution of glycine to alanine in the linker region of codon 360 adjacent to tetramerization domain of p53 decrease transactivation of pro-apoptotic genes (e.g. BAX, MDM2) resulting in prolong survivals and DNA damages (Kato et al., 2003; Kim et al., 1993).
Conclusively, p53 gene plays the centre role in cellular integrity, maintainance and cell death (apoptosis as well as autophagy) (Tasdemir et al., 2008). p53 gene polymorphisms associate with not only breast cancer but also other types of tumour such as lung (Papadakis et al., 2002), colorectal (Sjalander et al., 1995), ovarian cancer (Lancester et al., 1995). Moreover, polymorphisms promote the pre-existing risk of cancer which may be environmental factors or existing mutation in BRCA2 carrier (Osorio et al., 2006). In our experiment, we have a little bit controversial errors and technical faults such as contaminations, preparation in solution and staining dye. Anyway, we found out two p53 polymorphisms (P47S and G360A) in given breast cancer cell line. Therefore, we need further assessments and metaanalysis of the data to confirm the relationship between p53 polymorphisms and breast cancer.
We are grateful to our supervisor Dr. Mandar Godge for thankful guidance and assistance. Also, we appreciate Dr. S. Picksley pointing out important information. This work was supported partly by MDIS laboratory (Singapore) and University of Bradford (United Kingdom).