|Year : 2014 | Volume
| Issue : 4 | Page : 1008-1012
Absence of p53 gene mutations in mice colon pre-cancerous stage induced by o-nitrotoluene
Nahed A Hussien
Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt; Department of Biology, Faculty of Science, Taif University, Taif, Saudi Arabia
|Date of Web Publication||9-Jan-2015|
Nahed A Hussien
Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt
Source of Support: None, Conflict of Interest: None
p53 gene is one of the most frequently mutated genes found in the human colonic tumors. Mice have been used as an experimental model to study the pathogenesis of colon cancer in humans. The alterations in cancer genes and proteins found in the mouse large intestinal tumors included mutations which are hallmarks of human colon cancer, probably contributed to the pathogenesis of the large intestinal carcinomas in mice following o-nitrotoluene (o-nt) exposure.
Aim of Study: Detection of p53 gene mutations in colon precancerous stage.
Materials and Methods: In this study, mice colon precancerous stage induced by o-nt were examined for the presence of point mutations in highly conserved coding region (exons 5-8) and outside it (exons 10, 11) using a single-strand conformation polymorphism assay (SSCP).
Results: SSCP analysis showed no differences in banding patterns between the normal negative control group and o-nt-induced precancerous stage in mice colon.
Conclusion: The results from the present study indicate that point mutations in the p53 gene, in the coding region (exons 5-8) and outside it (exons 10, 11), are not involved in the development of the colon precancerous stage induced by o-nt in mice.
Keywords: Colon precancerous stage, mice, o-nitrotoluene, p53, single-strand conformation polymorphism
|How to cite this article:|
Hussien NA. Absence of p53 gene mutations in mice colon pre-cancerous stage induced by o-nitrotoluene. J Can Res Ther 2014;10:1008-12
| > Introduction|| |
Colon cancer is a serious health problem in most developed countries and is the third leading cause of cancer mortality throughout the world.  In the last few years, attention has focused on understanding the molecular pathogenesis of this disease. Colorectal carcinogenesis is a multistage process, involving multiple genetic changes that are identified at various stages of the tumor development. Several reports suggest that the accumulation of mutations in oncogenes and in different tumor suppressor genes may be critical to the full conversion of normal colonic mucosal cells to malignancy. ,,
In the previous study, immunohistochemical detection of cyclooxygenase-2 (COX-2) expression was used as a marker for early detection of colon cancer in mice treated by o-nitrotoluene (o-nt).  Early detection markers indicate the existence of cancer or that cancer will occur with nearly a 100% certainty within a specified time interval. 
In addition, induction of large intestine carcinomas in mice by the administration of o-nt has been used as an experimental model to study the pathogenesis of colon cancer in humans. The recently completed o-nt study provided the first cecal tumor response and an opportunity to evaluate the morphology and molecular profile of oncogenes and tumor suppressor genes that are relevant to humans. 
Over the last few years, attempts have been made to investigate whether the mice colon cancer model recapitulates the same genetic steps which occur during human colon carcinogenesis. Sills et al. identified alterations in cancer genes and proteins found in mice o-nt induced large intestinal tumors, included mutations that activate signal transduction pathways (Kirsten Ras [K-ras] and beta-catenin) and changes that disrupt the cell-cycle and bypass G1 arrest (p53, cyclin D1). These alterations, which are hallmarks of human colon cancer, probably contributed to the pathogenesis of the large intestinal carcinomas in mice following o-nt exposure.
Point mutations in the K-ras and p53 genes are two of the most commonly observed genetic alterations in human colorectal carcinogenesis. ,,,, The p53 gene codes for a nuclear protein whose functionality appears to be very important in the negative regulation of the cell cycle. , During the development of colonic neoplasms, point mutations in the K-ras oncogene are localized in codon 12, while mutations in the p53 gene seem to cluster predominantly within its highly conserved coding region (i.e. exons 5-8).
The purpose of this investigation was to determine the frequency for mutation of the p53 gene within its highly conserved coding region (exons 5-8) and outside it (exons 10, 11) in precancerous stage in mice colon induced by o-nt.
| > Materials and methods|| |
Albino male mice (Mus musculus; 8-10 weeks old; 26-30 g, body weight [b.w.]) were used as experimental animals. They were purchased from National Research Center animal house (Dokki, Giza, Egypt). They were housed in plastic cages for 7 days to be accommodated with our laboratory conditions. Food and water were presented ad libitum. Animals received care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals".
Animals were administrated with o-nt (150 mg/kg b.w., dissolved in corn oil just before use , ) orally three times a week for 6 consecutive weeks then mice were sacrificed after 24 h or 1 month from the last treatment.
Frozen colon tissue (~0.5 cm 2 ) were minced and then suspended in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 10 mM ethylenediaminetetraacetic acid, and 1% sodium dodecyl sulfate, incubated with 50 μg/ml proteinase K at 56°C overnight, and then centrifuged at 10,000 rpm for 30 min. Soluble DNA in the resulting supernatant was precipitated with ethanol at −20°C, dissolved in sterile ddH 2 O. Electrophoresis was carried out in a 1.5% agarose gel containing ethidium bromide. The gel was examined and photographed under ultraviolet (UV) light.
The sequences of the primers are indicated in [Table 1]. , Polymerase chain reaction (PCR) mixture (20 μl) was setup: 7 μl sterile water, 1 μl (100 ng/1 μl) extracted DNA, 1 μl forward primer (50 pmole), 1 μl reverse primer (50 pmole), and then 10 μl 2x master mix ( Promega, USA) were added in 0.2 ml PCR eppendorf.
Cycling was started in Thermal Cycler (Programmable Thermal Cycler, PTC-100™ thermal cycler, Model 96 [MJ Research, INC., Watertown, MA, USA]), with the initial denaturation at 94°C for 5 min, DNA double stranded denaturation at 94°C for 30 s, primer annealing at 55°C (exons 10, 11) or 58°C (exons 5-8) for 1 min, and primer extension at 72°C for 1 min, for 30 cycles. Final extension at 72°C for 10 min was necessary for complete amplification.
Polymerase chain reaction products were separated and visualized by electrophoresis through 1.5% ethidium bromide-treated agarose gel (Sigma, St. Louis, MO, USA) using the standard protocol described by Sambrook et al. 
Polymerase chain reaction products were denatured with TE buffer (diluted 1:10, pH 8.0  ). 5 μl of each diluted solution was mixed with 5 μl of denaturing-loading dye (95% formamide, 4M urea, 0.1% bromophenol blue, 0.1% xylene cyanol FF, and 0.5 μl 15% ficoll) and the mixture was heated to 94°C for 5 min and then was chilled on ice for 10 min.  The denaturated PCR samples were subjected to 9% polyacrylamide gel electrophoresis (acrylamide: Bisacrylamide = 49:1 v/v). The gel was stained for 10 min in 100 ml of 1x Tris Borate EDTA TBE with 10 μl 10 mg/ml ethidium bromide to visualize the DNA bands with the aid of shaking. The gel was placed on a UV transilluminator (Stratagene, La Jolla, CA, USA) and a picture was taken with a polaroid camera (Polaroid MP4 Land Camera).
| > Results|| |
In the previous study, it was estimated that o-nt (150 mg/kg) administration for 6 consecutive weeks at 24 h and 1 month sampling time induce precancerous stage in mice colon. That was declared in the distortion of different histoarchitectural structures, the high expression of COX-2 protein, DNA damage represented by Comet assay.  It is widely accepted that alterations to COX-2 expression and the abundance of its enzymatic product prostaglandin E2 have key roles in influencing the development of colorectal cancer. 
[Figure 1] represents extracted DNA for control and o-nt treated groups. It shows unamplified nondegraded DNA with high molecular weight that was suitable for subsequent PCR amplification. [Figure 2] is a representative 1.5% agarose gel for PCR products of p53 exons (5-8, 10, 11). The figure shows a successful amplification process which yields the expected product size. The size of PCR product was 214 bp, 181 bp, 170 bp, 280 bp, 290 bp, and 249 bp for exons 5, 6, 7, 8, 10, and 11, respectively.
|Figure 1: Representative 1.5% agarose gel separating purified DNA (Lanes 1-5)|
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|Figure 2: Representative 1.5% agarose gel separating polymerase chain reaction (PCR) product for p53 exons. In which, Lane M, low molecular weight DNA marker (100-1500 bp); Lanes 1 and 2, PCR product for exon 5 (214 bp); Lanes 3 and 4, PCR product for exon 6 (181 bp); Lanes 5 and 6, PCR product for exon 7 (170 bp); Lanes 7 and 8, PCR product for exon 8 (280 bp); Lanes 9 and 10, PCR product for exon 10 (290 bp); Lanes 11 and 12, PCR product for exon 11 (249 bp)|
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Polymerase chain reaction - single strand conformation polymorphism (SSCP) patterns of p53 (exons 5, 6, 7, 8, 10, and 11) for o-nt groups at 24 h and 1 month sampling time show no difference from that of the negative control group as shown in [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7] and [Figure 8] respectively. This shows the absence of p53 gene mutations in mice colon precancerous stage induced by o-nt treatment.
|Figure 3: Representative 9% polyacrylamide gel showing polymerase chain reaction-single strand conformation polymorphism for p53 exon 5. Lanes 1 and 2 represent negative control group; Lanes 3-10 represent o-nitrotoluene treated groups at different sampling time|
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|Figure 4: Representative 9% polyacrylamide gel showing polymerase chain reaction-single strand conformation polymorphism for p53 exon 6. Lanes 1 and 2 represent negative control group; Lanes 3-10 represent o-nitrotoluene treated groups at different sampling time|
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|Figure 5: Representative 9% polyacrylamide gel showing polymerase chain reaction-single strand conformation polymorphism for p53 exon 7. Lanes 1 and 2 represent negative control group; Lanes 3-10 represent o-nitrotoluene treated groups at different sampling time|
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|Figure 6: Representative 9% polyacrylamide gel showing polymerase chain reaction-single strand conformation polymorphism for p53 exon 8. Lanes 1 and 2 represent negative control group; Lanes 3-10 represent o-nitrotoluene treated groups at different sampling time|
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|Figure 7: Representative 9% polyacrylamide gel showing polymerase chain reaction-single strand conformation polymorphism for p53 exon 10. Lanes 1 and 2 represent negative control group; Lanes 3-10 represent o-nitrotoluene treated groups at different sampling time|
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|Figure 8: Representative 9% polyacrylamide gel showing polymerase chain reaction-single strand conformation polymorphism for p53 exon 11. Lanes 1 and 2 represent negative control group; Lanes 3-10 represent o-nitrotoluene treated groups at different sampling time|
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| > Discussion|| |
The present study has examined the evolutionarily conserved portion of the coding region (exons 5-8) and outside it (exons 10, 11) of the p53 gene for mutations in mice colon precancerous stage induced by o-nt treatment. SSCP analysis failed to detect the presence of any mutation in the selected regions.
The present study focused on exons 5-8, where the majority of the mutations are thought to be localized, , also mutations in the other exons account for negative SSCP pattern. Indeed, recent studies by Greenblatt et al.  and Hartmann et al.  indicated that 13% and 22%, respectively, of the p53 gene mutations reported fell outside exons 5-8. A false negative SSCP result may occur when the tumor is not sufficiently represented in the tissue from which the DNA is extracted because colon tissue is still in the early cancer stage, and the tumor does not appear yet.
Fazeli et al.  concluded that, to the extent that p53 mutation plays a role in colon cancer progression, such a role becomes important only in later stages. In which, p53 mutations rarely occur in cells of human colonic adenomas having a low to the intermediate degree of dysplasia but frequently occur in adenomas as they progress to high grade of dysplasia.  This may conclude the absence of p53 mutation in the present study in different exons because the tissue is still in its early or precancerous stage.
In another explanation, the results of the present study clearly imply that mutations in the p53 gene might not involve in colonic tumor development in mice. A study has also reported the absence of p53 mutations in mouse colonic tumors induced by 1,2-dimethylhydrazine.  Similarly, absence of ras and/or p53 mutations have been reported in some target organs in rodents, even though these genetic alterations appear to play a major role in the same target organs in humans, ,, thereby suggesting the presence of alternative molecular pathways in mice that do not involve p53 mutations in the pathogenesis of colon cancer might be considerable.
Previous studies have shown that other possibilities exist that could result in indirect inactivation of the wild-type p53 protein without deletions, insertions, or point mutations within coding regions. For example, alternative posttranslational mechanisms like p53 phosphorylation have been shown to indirectly modulate p53 function.  Mutations may also occur in secondary genes whose products interact and transregulate p53.  All this evidence suggests that mutations upstream and downstream of the p53 signaling pathway can operationally substitute for mutations within the p53 gene itself. Thus, it is possible that one or more of such pathways is the target in the experimental system used in this study. It is also possible that mutations in the p53 gene could have been present in the region outside the translated area of the gene under investigation.
| > Acknowledgment|| |
Dr. Mai A. El-Watidy for technical help.
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