p53, cistron known as tumor suppresser cistron, is indispensable cistron in commanding cellular unity, proliferation and programmed cell death. It serves as possible barrier for assorted sorts of malignances. Mutant in p53 develops multiple malignant neoplastic diseases syndrome ( Li-Fraumeni Syndrome ) . It is loss of map mutant, so need to mutate in two allelomorphs. Recently, apart from mutant, incidences of the malignant neoplastic disease hazard are significantly high that are related with polymorphisms of this cistron. There are more than 200 of course occouring individual nucleotide polymorphisms ( SNP ) of p53 cistron so far in the general population that are expected to impact on p53 map. Relationship with our survey, there are 9 p53 polymorphisms related with chest malignant neoplastic disease. These polymorphisms addition the hazard of chest malignant neoplastic disease by itself or combination with other mutant in specific cistrons such as BRCA1, BRCA2 etc. So, in our research, we tried to observe which polymorphism is associated with MCF-7 chest malignant neoplastic disease cell line by utilizing known primers. Finally, we realized that polymorphisms found at codon 47 and 360 part in this malignant neoplastic disease cell line.
Keywords: chest malignant neoplastic disease ; p53 polymorphisms ; programmed cell death ; SNP ; codon 47 and 360
Breast malignant neoplastic disease is the most common dangerous malignant neoplastic disease among the adult females population worldwide. Over the past 25 old ages, incidence of chest malignant neoplastic disease is significantly risen particularly western states. Breast malignant neoplastic disease may be caused by both non-genetic and familial factors. Non-genetic factors include alterations in generative forms, dietetic alterations, intoxicant consumption, and physical activity whereas familial constituent predispose constellating in the household members ( Richards et al. , 2000 ) . Recently, familial constituent is more interesting apart from the epigenetic factors. Indeed, mutant in p53 cistron, tumour suppresser cistron located in chromosome 17p, is the commonest familial change in chest tumor cells ( 50 % ) ( Greenblatt et al. , 1994 ) . p53 mutant besides found in other types of tumors such as ovary malignant neoplastic disease, colorectal malignant neoplastic disease, lung malignant neoplastic disease etc. p53, guardian angel cistron, regulates the cell rhythm control, DNA fix and programmed cell death ( Lane, 1992 ) . Interestingly, apart from mutant, incidence of chest malignant neoplastic disease is besides increased in p53 cistron polymorphisms. Polymorphisms seem to change the messenger RNA processing which may alter the cellular maps ( Gemignani et al. , 2004 ) . Furthermore, p53 polymorphisms may do alterations the amino acid sequences changing acknowledgment motives for post- translational alteration, protein stableness or interaction with other proteins ( Li and Prives, 2007 ) .
p53 polymorphisms take topographic point in intronic ( 90 % ) every bit good as exonic sites ( Olivier et al. , 2002 ) . Although most polymorphisms are in noncoding DNAs, 16bp noncoding DNA 3 polymorphism merely increases the hazard of chest malignant neoplastic disease ( Wang et al. , 1999 ; Coasta et al. , 2008 ) . However, there are 9 exonic p53 polymorphisms detected so far which all are associated with chest malignant neoplastic disease. These polymorphous sites are codon 47, 72, 72 with fragment 1414, 217, 267, 278, 290 ( two types ) and 360. Among them, sufficient molecular groundss are found in codon 47, 72, 217 and 360. P47S is attendant from permutation of C to thymadine in codon 47. It substitutes serine blink of an eye of proline. It is rare discrepancies and merely reported in African population ( Bosco et al. , 1993 ) . Replacement of P47 reduces the activity of programmed cell death associated cistrons taking to protract cell endurance and potentially increase the chest malignant neoplastic disease hazard ( Feng et al. , 2006 ; Kurihara et al. , 2007 ) . Furthermore, R72P polymorphism besides increases malignant neoplastic disease hazard. Arginine in codon 72 is the susceptible venue for chest malignant neoplastic disease ( Easton et al. , 2007 ) . Fortunately, p53-R72 genotype addition endurance rates after chemo- and radiation therapy ( Xu et al. , 2005 ) . Another V217M polymorphism is the lone polymorphism located in the Deoxyribonucleic acid adhering sphere ( DBD ) of p53 ( exon 6 ) so that it straight manipulate the cistron written text and their merchandises. Similarly, G360A polymorphism affects in exon 10 and appears to damage the p53 response ( Kato et al. , 2003 ) . Therefore, despite p53 polymorphism frequences are low in general population ; they have important consequence on the development of the malignances including carcinoma of chest ( Garcia-Closas et al. , 2008 ) . Furthermore, p53 polymorphisms and their associated malignances could be differing from one population to another because of their background genetic sciences ( Goldman and Shields, 1998 ) .
In our experiment, we tried to happen out which p53 polymorphisms are associated with this MCF-7 chest malignant neoplastic disease cell line. Therefore, we used the appropriate known 9 sets of primers for observing these polymorphisms.
2. Materials and Method
2.1 DNA isolation and quantification
Deoxyribonucleic acid is extracted from the MCF-7 ( Michigan malignant neoplastic disease foundation ) chest malignant neoplastic disease cell line by wizard® genomic DNA purification kit ( merchandise of Promega catalog A1120 ) harmonizing to maker ‘s standard protocol. The concentration of genomic DNA extracted was measured utilizing Spectrophotometer at 260 and 280 nanometer.
2.2 DNA making
Extracted genomic Deoxyribonucleic acid was digested with limitation enzyme EcoRI from Fermentas ( Thermo Fisher Scientific, Waltham ; Massachusetts, USA ) . Deoxyribonucleic acid fragments were stained with ethidium bromide and separated on 1 % agrose gel by mean of cataphoresis. Then, they were visualized by UV visible radiation gel physician system and taken the exposure with Polaroid certification camera.
2.3 Primers design and polymerase concatenation reaction ( PCR )
For designation of the polymorphisms in the p53 cistron, known primers sets ( including frontward and change by reversal ) were used to magnify the several site of polymorphisms. The nucleotide places of all primers were designed harmonizing to the p53 cistron sequence in the NCBI database. Detail sequences of all primers ( 1st Base, Malaysia ) were provided in table 1.
Polymerase concatenation reaction ( PCR ) was performed with genomic DNA and 9 sets of primers by utilizing PCR maestro mix catalog M7502 ( Promega: Madison, Wisconsin, USA ) . Each reaction was carried out with 9.5ul of H2O, 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 eventually adds 1ul of genomic DNA. The entire mixture will be 25ul. Primer sets 1, 6, 7, 8 were applied at 55E™ C whereas sets 2, 3, 4, 5, 9 were applied at 60E™ C malting temperature. All reactions were ab initio denatured with 94 & A ; deg ; C at 4 proceedingss. Subsequent denaturations were done at 94 & A ; deg ; C for 45 seconds followed by 60 & A ; deg ; or 55 & A ; deg ; C for 45 seconds and so, 72 & A ; deg ; C for 1 minute. These rhythms were repeated 35 times. Last, concluding extension at 72 & A ; deg ; C for 10 proceedingss was recommended.
Table 1: Primers used in this survey
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 Confirmation with gel cataphoresis
All PCR merchandises were verified by gel cataphoresis once more after staining with 6ul of intercalating agent ( ethidium bromide ) . Besides, DNA ladder was besides used to corroborate the figure of basal braces of the PCR merchandises. Then, they were visualized by UV visible radiation under gel physician system and taken the exposure with Polaroid camera. Strict handling and disposal of ethidium bromide is indispensable because it is carcinogenic.
First of all, genomic Deoxyribonucleic acid was extracted from MCF-7 chest malignant neoplastic disease cell line and so, quantified by spectrophotometer. Under 260nm, the optical denseness ( OD ) was 0.253 and under 280nm, OD was 0.175. Therefore, the ration of optical density under 260 and 280nm was 1.44. The genomic DNA has sum of 1.26 ug/ul since 1 optical denseness at Absorbance 260nm is 50ug/ml of Deoxyribonucleic acid.
Figure 1: Polaroid exposure of agrose gel under gel physician system demoing EcoRI digested Deoxyribonucleic acid
After digestion with limitation enzyme EcoRI, genomic DNA, diluted genomic DNA and digested DNA were separated on the 1 % agrose gel by mean of cataphoresis. Two Deoxyribonucleic acid ladders were added in lane 1 and 5. There was no set in lane 2 containing undigested genomic DNA nevertheless ; diluted genomic Deoxyribonucleic acid in lane 3 was banded. EcoRI restricted DNA in lane 4 besides appears as a set on the Polaroid exposure.
Figure 2: Photograph of agrose gel with PCR fragments Figure3: standard DNA ladder
Merchandises of PCR were stained with ethidium bromide and separated on the 1 % agrose gel by cataphoresis. Then, when they were visualized with UV visible radiation under gel physician system, amplified fragments were seen as sets in their several lanes. Furthermore, two Deoxyribonucleic acid ladders were besides run in lane 1 and 11. There were no sets in lane 2 and 10. This meant that there was no amplified PCR fragment in those lanes. Hence, codon 47 part and codon 360 part could non be amplified. Lane 3 incorporating 72 codon showed the set about 300 base brace ( bp ) whereas lane 4 incorporating codon 72 with fragment 1414 displayed the set merely below 1500 base brace ( bp ) . Codon 217 in lane 5 besides appear the set about 496 bp. Lane 6 with codon 267 showed a set with its actural size ( 778 base brace ) . Another amplified fragment in lane 7 ( codon 278 ) contained a set about 300 bp but it did non retain its actural size 479 bp. Likewise, lane 8 ( codon 290 ) besides showed the set about 500 bp while 2nd last lane of all primers ( lane 9 ) appears a set merely above the old lane 8 set about 569 bp.
This experiment evaluated which types of p53 polymorphisms are associated with given MCF-7 chest malignant neoplastic disease cell line. First, we extracted genomic DNA and quantified the sum of extracted DNA which is about 1.26 ug/ul. Then, we checked the purification of Deoxyribonucleic acid by ratio of A260: A280. More than 1.5 is equal for purification harmonizing to criterion protocol nevertheless, our consequence is about 1.44. It may be due to small sample of DNA extracted or little taint.
Harmonizing to cataphoresis consequence, we found that EcoRI digestion was achieved because lane 4 appears multiple sets. Sing for positive control diluted genomic DNA tally in 3rd lane on 1 % agrose gel although pure genomic Deoxyribonucleic acid in lane 2 did non look the set. It is due to non-diluted DNA is somewhat tough to run during cataphoresis. Positive set in lane 3 means that there is no mistake in gel readying and cataphoresis. Multiple sets in lane 4 describe that EcoRI restricted the genomic Deoxyribonucleic acid at multiple sites. EcoRI acknowledge the 5 ‘ G A A T T C 3 ‘ sequences and cut between G and adenosine ( Griffin et al. , 1974 ; Robberson et al. , 1974 ) . So, it can bring forth multiple fragments of genomic DNA. EcoRI restrictive enzyme digestion signifier multiple or smear pattern sets in cataphoresis. Therefore, we conclude that this genomic Deoxyribonucleic acid is rather purified.
In figure 2, there is no seeable set in lane 2 and 10 on the Polaroid exposure after completing the polymerase concatenation reaction and gel cataphoresis. Therefore, codon 47 and codon 360 may non be amplified decently because primers ( including frontward and change by reversal ) for these codons can non temper or magnify with their appropriate part in genomic Deoxyribonucleic acid. The consequence may be due to proficient mistake or mutant and / or altering the base brace in this part. Technical mistake should non be entertained because other lanes show their appropriate amplified Deoxyribonucleic acid fragments. So, we conclude that there are the polymorphisms in those codons ( codon 47 and 360 ) .
Mutant in codon 47 ( altering CCG to TCG ) replacement Proline amino acid to Serine at place 1. p38 and HIPK2 ( homeodomain -interacting protein kinase 2 ) phosphorylate proline residue adjacent to S46 taking to increase programmed cell death ( Kruse and Gu, 2008 ) . Serine blink of an eye of proline occurred in P47S polymorphism decrease the phosphorylation of p38 and HIPK2. Thus, decrease in phosphorylation of p38 and HIPK2 cut down pro-apoptotic cistrons look and increase the possible hazard of malignances ( Li et al. , 2005 ) .
Similarly, we found no set in the lane 10 on the UV visible radiation under gel physician system because codon 360 did non amplified suitably. Primers of codon 360 did non temper its answering part as this part may be mutated. Substitution of glycine to alanine in the linker part of codon 360 adjacent to tetramerization sphere of p53 lessening transactivation of pro-apoptotic cistrons ( e.g. BAX, MDM2 ) ensuing in prolong endurances and DNA amendss ( Kato et al. , 2003 ; Kim et al. , 1993 ) .
Conclusively, p53 cistron plays the centre function in cellular unity, maintainance and cell decease ( programmed cell death every bit good as autophagy ) ( Tasdemir et al. , 2008 ) . p53 cistron polymorphisms associate with non merely breast malignant neoplastic disease but besides other types of tumor such as lung ( Papadakis et al. , 2002 ) , colorectal ( Sjalander et al. , 1995 ) , ovarian malignant neoplastic disease ( Lancester et al. , 1995 ) . Furthermore, polymorphisms promote the preexistent hazard of malignant neoplastic disease which may be environmental factors or bing mutant in BRCA2 bearer ( Osorio et al. , 2006 ) . In our experiment, we have a small spot controversial mistakes and proficient mistakes such as taints, readying in solution and staining dye. Anyhow, we found out two p53 polymorphisms ( P47S and G360A ) in given chest malignant neoplastic disease cell line. Therefore, we need farther appraisals and metaanalysis of the informations to corroborate the relationship between p53 polymorphisms and chest malignant neoplastic disease.