Journal of Cancer Research and Therapeutics

: 2014  |  Volume : 10  |  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

Correspondence Address:
Nahed A Hussien
Department of Zoology, Faculty of Science, Cairo University, Giza, Egypt


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.

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;10:1008-1012

How to cite this URL:
Hussien NA. Absence of p53 gene mutations in mice colon pre-cancerous stage induced by o-nitrotoluene. J Can Res Ther [serial online] 2014 [cited 2020 Jun 1 ];10:1008-1012
Available from:

Full Text


Colon cancer is a serious health problem in most developed countries and is the third leading cause of cancer mortality throughout the world. [1] 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. [2],[3],[4]

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). [5] Early detection markers indicate the existence of cancer or that cancer will occur with nearly a 100% certainty within a specified time interval. [6]

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. [7]

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.[7] 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. [2],[3],[4],[8],[9] The p53 gene codes for a nuclear protein whose functionality appears to be very important in the negative regulation of the cell cycle. [10],[11] 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.


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 [12],[13] ) 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]. [14],[15] 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.{Table 1}

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. [16]

Polymerase chain reaction products were denatured with TE buffer (diluted 1:10, pH 8.0 [17] ). 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. [18] 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).


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. [5] 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. [19]

[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}{Figure 2}

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}{Figure 4}{Figure 5}{Figure 6}{Figure 7}{Figure 8}


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, [20],[21] also mutations in the other exons account for negative SSCP pattern. Indeed, recent studies by Greenblatt et al. [22] and Hartmann et al. [23] 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. [24] 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. [25] 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. [26] 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, [27],[28],[30] 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. [31] Mutations may also occur in secondary genes whose products interact and transregulate p53. [32] 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.


Dr. Mai A. El-Watidy for technical help.


1Wingo PA, Tong T, Bolden S. Cancer statistics, 1995. CA Cancer J Clin 1995;45:8-30.
2Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-67.
3Hamilton SR. Molecular genetics of colorectal carcinoma. Cancer 1992;70:1216-21.
4Fearon ER, Jones PA. Progressing toward a molecular description of colorectal cancer development. FASEB J 1992;6:2783-90.
5Hussien NA, Omara EA, El-Watidy MA, El-Ghor AA. Chemotherapeutic potential of grape seed extract (GSE) against experimentally induced precancerous stage in mice colon. J Asian Sci Res 2013;9:2335-46.
6Srivastava S, Verma M, Henson DE. Biomarkers for early detection of colon cancer. Clin Cancer Res 2001;7:1118-26.
7Sills RC, Hong HL, Flake G, Moomaw C, Clayton N, Boorman GA, et al. o-Nitrotoluene-induced large intestinal tumors in B6C3F1 mice model human colon cancer in their molecular pathogenesis. Carcinogenesis 2004;25:605-12.
8Bos JL, Fearon ER, Hamilton SR, Verlaan-de Vries M, van Boom JH, van der Eb AJ, et al. Prevalence of ras gene mutations in human colorectal cancers. Nature 1987;327:293-7.
9Forrester K, Almoguera C, Han K, Grizzle WE, Perucho M. Detection of high incidence of K-ras oncogenes during human colon tumorigenesis. Nature 1987;327:298-303.
10Diller L, Kassel J, Nelson CE, Gryka MA, Litwak G, Gebhardt M, et al. p53 functions as a cell cycle control protein in osteosarcomas. Mol Cell Biol 1990;10:5772-81.
11Levine AJ. The p53 protein and its interactions with the oncogene products of the small DNA tumor viruses. Virology 1990;177:419-26.
12Slaga TJ, Triplett LL, Smith LH, Witschi HP. Carcinogenesis of nitrated toluenes and benzenes. Skin and lung tumor assays in mice. Final report. ORNL/TM-9645. Oak Ridge National Laboratory, Oak Ridge, TN.
13Dunnick JK, Burka LT, Mahler J, Sills R. Carcinogenic potential of o-nitrotoluene and p-nitrotoluene. Toxicology 2003;183:221-34.
14Morimura K, Yamamoto S, Murai T, Mori S, Chen TX, Wanibuchi H, et al. LOH and mutational analysis of p53 alleles in mouse urinary bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. Carcinogenesis 1999;20:715-8.
15Kramata P, Lu YP, Lou YR, Singh RN, Kwon SM, Conney AH. Patches of mutant p53-immunoreactive epidermal cells induced by chronic UVB Irradiation harbor the same p53 mutations as squamous cell carcinomas in the skin of hairless SKH-1 mice. Cancer Res 2005;65:3577-85.
16Sambrook J, Fritsch EF, Maniatis T, editors. Molecular Cloning: A Laboratory Manual. 2 nd ed. NY: CSH Cold Spring Harbor Press; 1989.
17Dai J, Wei H, Xiao Y. PCR-RFLP analysis on mitochondrial DNA D-Loop area of six inbred mice. J Med Coll PLA 1999;21:709-11.
18Dai JG, Min JX, Xiao YB, Lei X, Shen WH, Wei H. The absence of mitochondrial DNA diversity among common laboratory inbred mouse strains. J Exp Biol 2005;208:4445-50.
19Greenhough A, Smartt HJ, Moore AE, Roberts HR, Williams AC, Paraskeva C, et al. The COX-2/PGE2 pathway: Key roles in the hallmarks of cancer and adaptation to the tumour microenvironment. Carcinogenesis 2009;30:377-86.
20Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science 1991;253:49-53.
21Caron de Fromentel C, Soussi T. TP53 tumor suppressor gene: A model for investigating human mutagenesis. Genes Chromosomes Cancer 1992;4:1-15.
22Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in the p53 tumor suppressor gene: Clues to cancer etiology and molecular pathogenesis. Cancer Res 1994;54:4855-78.
23Hartmann A, Blaszyk H, McGovern RM, Schroeder JJ, Cunningham J, De Vries EM, et al. p53 gene mutations inside and outside of exons 5-8: The patterns differ in breast and other cancers. Oncogene 1995;10:681-8.
24Fazeli A, Steen RG, Dickinson SL, Bautista D, Dietrich WF, Bronson RT, et al. Effects of p53 mutations on apoptosis in mouse intestinal and human colonic adenomas. Proc Natl Acad Sci U S A 1997;94:10199-204.
25Boland CR, Sato J, Appelman HD, Bresalier RS, Feinberg AP. Microallelotyping defines the sequence and tempo of allelic losses at tumour suppressor gene loci during colorectal cancer progression. Nat Med 1995;1:902-9.
26Okamoto M, Ohtsu H, Miyaki M, Yonekawa H. No allelic loss at the p53 locus in 1,2-dimethylhydrazine-induced mouse colon tumours: PCR-SSCP analysis with sequence-tagged microsatellite site primers. Carcinogenesis 1993;14:1483-6.
27Almoguera C, Shibata D, Forrester K, Martin J, Arnheim N, Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell 1988;53:549-54.
28Grünewald K, Lyons J, Fröhlich A, Feichtinger H, Weger RA, Schwab G, et al. High frequency of Ki-ras codon 12 mutations in pancreatic adenocarcinomas. Int J Cancer 1989;43:1037-41.
29Schaeffer BK, Zurlo J, Longnecker DS. Activation of c-Ki-ras not detectable in adenomas or adenocarcinomas arising in rat pancreas. Mol Carcinog 1990;3:165-70.
30Weghorst CM, Dragnev KH, Buzard GS, Thorne KL, Vandeborne GF, Vincent KA, et al. Low incidence of point mutations detected in the p53 tumor suppressor gene from chemically induced rat renal mesenchymal tumors. Cancer Res 1994;54:215-9.
31Scheidtmann KH, Haber A. Simian virus 40 large T antigen induces or activates a protein kinase which phosphorylates the transformation-associated protein p53. J Virol 1990;64:672-9.
32Quartin RS, Finlay CA, Levine AJ. The p53 gene and gene products. In: Sharp PA, editor. Nuclear Processes and Oncogenes, Bristol-Myers Squibb Cancer Symposia. Vol. 14. San Diego, California: Academic Press, Inc.; 1992. p. 87-104.