p53 Sequencing as a Diagnostic Tool for Possible Carcinomas




Yan Nikhamin

Introduction

The tumor suppressor gene p53 is a critically important negative regulator of the cell cycle progression in humans and other mammals. p53 regulates several important genes in cell cycle control p21, apoptosis, and DNA repair [1]. The p53 protein is organized into several functional domains. The amino-terminal domain contains an acidic region required for transactivation, while the core domain contains sequence-specific DNA binding capability [2]. Point mutations within this gene, especially within the DNA binding domain, are associated with the loss of tumor suppressor activity and result in gain of oncogenic function [3]. Mutations in different exons lead to specific carcinomas. Mutations in exon 5 of the p53 tumor suppressor gene lead to lung cancer [4], burkitt lymphoma [5], thyroid carcinoma [6], T-cell leukemia [7], gastric carcinoma [8], as well as breast carcinoma [9] and myeloid leukemia [10]. This information is extremely useful and may be used for diagnostic testing for genetic predisposition to develop cancer. More importantly this would allow us to diagnose exactly the type of cancer a person may develop. DNA sequencing has become a very accessible technique. It is, in many institutions, automated and is very accurate. This will prove to be a valuable tool in early diagnosis. It may be possible to screen the sequence of the p53 by sequencing it from individuals and comparing that sequence to those with known mutations.

Figures


Figure 1-PCR gel electrophoresis. PCR was performed as described and run on a 0.8% Agarose gel. PCR products from YDNA (lanes 1 & 5), ADNA (lanes 2 & 6), U937 (3 & 7), and SK-Br3 (lanes 4 & 8) with either p53e51 & p53e52 primers (lanes 1 - 4) or actin specific primers (lanes 5 - 8) were run alongside a 100 bp ladder (lane L). The negative controls contained no template DNA (lanes 9 - 12).


Figure 2-The nucleotide sequence of exon 5 of p53. DNA was sequenced as described. Mismatched bases are indicated by bold-type and X (YDNAseq). Indistinguishable bases are indicated by N (YDNAseq). Bases present that are reported as mutations are represented in italics (U937seq). All sequences were compared to the normal exon 5 of p53 (p53exon5) using Blast at http://www.ncbi.nlm.nih.gov/BLAST and Human p53; accession No. HSU94788. The sequences of exon 5 of p53 of the DNA from the blood samples are labeled YDNAseq and ADNAseq respectively. Similarly the sequences from the DNA isolated from cell lines are labeled U937seq and SK-Br3seq respectively.


Figure 3-Sequence autoradiograph of YDNA derived PCR product. The sequencing was performed using either p53e51 primer (forward) or p53e52 primer (reverse). The samples were arranged in the following order: G (ddGTP), A (ddATP), T (ddTTP), and C (ddCTP).


Figure 4-Sequence autoradiograph of PCR products. The reaction sets were loaded in the following order: ADNA, U937, and SK-Br3 and forward or reverse primers as labeled. The sequencing was performed using either p53e51 primer (forward) or p53e52 primer (reverse). The samples were arranged in the following order: G (ddGTP), A (ddATP), T (ddTTP), and C (ddCTP).


Results 


PCR was performed as described in Materials and Methods and products were run on a 0.8% Agarose gel (Figure 1). The electrophoresis showed 3 bands in YDNA (lane 1) and a single band in ADNA (lane 2), U937 (lane3), and SK-Br3 (lane 4) derived DNA. The actin specific primers generated a product and served as a positive control (lanes 5 to 8). The PCR products were purified as described in Materials and Methods.
The purified DNA was sequenced as described in Materials and Methods. The nucleotide sequence was compared to the reported normal exon 5 of the p53 gene. The sequence of exon 5 is from base 13055 to 13238. The aim was to compare the sequence of the blood samples with the cell lines and to establish the presence or absence of the mutations reported. Sequencing and comparison to the sequence of the normal exon 5 of p53 was performed. The sample from the blood of subject 1 (YDNA) was sequenced and compared (Figure 2). Some bases in the beginning of exon 5 sequence were unreadable as indicated by N (Figure 2, A). However, in the comparison with the sequences of the cell line samples no point mutations were observed. The sample from the blood of subject 2 (ADNA) was sequenced and aligned perfectly with the sequence of the normal exon 5 (Figure 2, B). The cell line U937 sample was sequenced and compared. It was expected that there would be a point mutation (G-->A) in the first base of intron 5 [10], however no mutation was observed as represented in italics. However, an insertion of a C at position 32 of exon 5 was present (Figure 2, C). Sequencing of the sample from cell line SK-Br3 DNA confirmed the presence of a point mutation at base 149, replacing a G with an A as indicated in bold type (Figure 2, D). The autoradiograph of the sequencing gels was performed overnight (Figures 3 & 4).


Discussion

Alteration of the p53 tumor suppressor gene is present in more than 70% of all human cancers. Many such mutations have been reported [4-10]. The number of mutations depends on the type of cancer however a point mutation creates a carcinogenic p53 [12]. The objective of the experiment was to examine the possibility of using the reported sequence of the p53 tumor suppressor gene as a diagnostic tool. Having the ability to perform such genetic testing allows for improved early detection of possible carcinomas. Here, exon 5 of p53 isolated and sequenced to, ultimately, compare it to the reported sequence in an attempt to provide a diagnosis. PCR was utilized for amplification of exon 5 from pure DNA of blood samples of two human subjects (Figure 1, YDNA & ADNA) and cell cultures of two cell lines (Figure 1, U937 & SK-Br3). Once the amplified region was purified it was sequenced and compared to the reported normal exon 5.
Although sequencing of about 15 bases in the beginning of YDNA exon 5 is inconclusive, the comparison of the latter portion of the exon to the mutated version of the cell lines provided a tentative diagnosis that this person does not have the mutations which would suggest a possibility of myeloid leukemia [10] or breast carcinoma [9] (Figure 2, A, C &D). Sequence of the sample from the second subject was 100% identical to the normal exon 5, which allows the conclusion that all mutations involving exon 5 of p53 of this individual are not present (Figure 2, B). However, contrary to what has been reported of the mutation in the U937 cell line [10] the sequence of this sample does not show a point mutation at position one of intron 5 but revealed an insertion of a C (Figure 2, C). Since point mutations in only one allele of p53 are sufficient for its mutagenic properties it is possible that the sequence during this experiment is of the other, "normal", allele. On the other hand, with respect to the insertion, U937 is a cell line which is already transformed and not without capability to acquire new mutations. Either way, further inquiry into the sequence of U937 must be made in order to establish its value as a useful tool in comparative genetic diagnosis for myeloid leukemia. Finally, the sequence of SK-Br3 cell line is characteristic of breast cancer [9], and the findings from this project confirm the presence of the point mutation at base 149 (Figure 2, D). This mutation changes codon 145 from an Arginine (CGC) to a Histidine (CAC) rendering p53 carcinogenic.
Sequencing DNA has become a very powerful tool in medical diagnosing. It can also be used for early detection of possible cancers involving genes such as p53, which are primarily involved in cell cycle regulation and carcinoma formation.

Acknowledgments

I thank Dr. Rubin for providing the lab, resources and the opportunity to perform this research.  I also thank Rocco and Sabrina for their patience, help, and support throughout this project.  I also thank Dr. Sylvia Anderson for growing the cell cultures, and Ms. Avakyants for, so bravely, donating 200ul of her blood for the project.



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