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Year : 2010  |  Volume : 47  |  Issue : 4  |  Page : 412--417

Detection of B cell lymphoma 2, tumor protein 53, and FAS gene transcripts in blood cells of patients with breast cancer

M Jaberipour1, M Habibagahi2, A Hosseini1, M Abbasi1, A Sobhani-lari1, A Talei3, A Ghaderi4,  
1 Cancer Gene Therapy Laboratory, Shiraz Institute for Cancer Research, Shiraz, Iran
2 Immunotherapy Laboratory, Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran
3 Cancer Gene Therapy Laboratory, Shiraz Institute for Cancer Research; Department of Surgery, Shiraz University of Medical Sciences, Shiraz, Iran
4 Cancer Gene Therapy Laboratory, Shiraz Institute for Cancer Research; Department of Immunology, Shiraz University of Medical Sciences, Shiraz, Iran

Correspondence Address:
M Habibagahi
Immunotherapy Laboratory, Department of Immunology, Shiraz University of Medical Sciences, Shiraz


Background: Proteins encoded by FAS, BCL-2 and TP53 genes are major regulators of cellular survival and apoptosis. Results of recent investigations show remarkable biological features of these factors, which propose their role in the course of cancer. Therefore, it is plausible to test whether transcripts of these genes could be detected in the peripheral blood cells of patients with breast cancer. Materials and Methods: Real-time polymerase chain reaction assay was performed to detect FAS, BCL-2, and TP53 gene transcripts in the peripheral blood samples of 50 women with histologically confirmed infiltrative ductal carcinoma of the breast. Gene expression of patients was compared with 40 healthy women without history of malignancies or autoimmune disorders. Results: The relative overexpression of BCL-2 in the blood cells from patients of early stages (I and II), nonmetastatic and low-grade tumors compared with healthy individuals, was shown by measuring the gene transcript. Similarly, 3-4-fold higher expression of FAS was found in those patients. The measurement of TP53 transcripts also showed higher levels of gene expression in patients compared with healthy controls. BCL-2 gene expression showed a significant correlation with FAS, while such a correlation was not observed between BCL-2 and TP53 . Conclusion: It seems tumor cells overexpress BCL-2 to inhibit apoptosis and guarantee their cell survival. As a physiologic response, FAS and TP53 could be upregulated to suppress tumors. However, these pathways at early stages of disease may be inadequate and cause progressive malignancy.

How to cite this article:
Jaberipour M, Habibagahi M, Hosseini A, Abbasi M, Sobhani-lari A, Talei A, Ghaderi A. Detection of B cell lymphoma 2, tumor protein 53, and FAS gene transcripts in blood cells of patients with breast cancer.Indian J Cancer 2010;47:412-417

How to cite this URL:
Jaberipour M, Habibagahi M, Hosseini A, Abbasi M, Sobhani-lari A, Talei A, Ghaderi A. Detection of B cell lymphoma 2, tumor protein 53, and FAS gene transcripts in blood cells of patients with breast cancer. Indian J Cancer [serial online] 2010 [cited 2020 Jul 11 ];47:412-417
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Several mechanisms and multiple genes share to control cellular proliferation, growth, and apoptosis. The literature shows the importance of some pathways where expression of BCL-2, FAS, and p53 have major roles in their action.

FAS gene encodes the FAS protein, which is a member of the tumor necrosis factor superfamily and mediates apoptosis when cross-linked with FAS ligand (FASL) or agonistic anti-FAS antibody. [1] FAS-mediated apoptosis has been shown in several inflammatory diseases, autoimmune diseases, and cancers. [2],[3] Concentration of serum soluble form of FAS was shown to be associated with tumor progression and metastasis in patients. In this regard, the concentration of soluble FAS in serum has been suggested as a valuable prognostic factor in patients with breast cancer (BC). [4],[5]

BCL2 gene encodes the Bcl-2 protein, which blocks apoptosis mediated by physiologic stimuli or by multiple stress conditions. [6] Several studies have evaluated the overexpression of BCL-2 in BC patients, and have considered that as an independent prognostic marker. [7],[8],[9] Recently, it was shown that BCL-2 expression is highly associated with an increased risk of local recurrence in patients with early stage of BC and it might be useful to select optimal treatment options for these patients. [10]

TP53 encodes a phosphoprotein, which is a multifunctional transcription factor involved in the control of cell cycle, DNA repair after damage, and in apoptosis. [11] TP53 overexpression has been identified in 50-75% of BC cases, a frequency which highlights this gene as a useful tumor marker. [12] A large number of studies have shown the correlation of TP53 overexpression with high histologic grade of tumor, negative hormonal status, and higher death rate. [13],[14],[15], [16] Also, it was suggested that the detection of TP53 mutations in plasma DNA can be a useful prognostic factor and an early marker of recurrence or distant metastasis in BC. [17]

Due to biological impact of BCL-2, TP53 , and FAS, this study aimed to compare the expression levels of these genes in blood samples of patients with BC and healthy women.

 Materials and Methods

The participants in this study were 50 women with histopathologically confirmed infiltrative ductal carcinoma of the breast. The patients were referred to our laboratory from breast clinics during July to December 2008. All the patients provided their informed consent to take part in the study and the study was approved by the ethical committee of the university. Peripheral venous blood samples (2 mL) were collected before any therapeutic intervention on the day of surgery by venipuncture, using ethylenediaminetetraacetic acid as anticoagulant. None of the patients had received chemotherapy, radiotherapy, or immunotherapy at the time of sampling. Blood samples from 40 healthy women without history of malignancies or autoimmune disorders were also obtained as controls. Mean ages of the patients and healthy controls were 50 years (range, 22-81) and 55 years (range, 24- 70), respectively.

Total RNA was prepared from blood cells after lysis with ammonium chloride and TRizol reagent (Invitrogen, Paisley, UK) treatment according to the manufacturer's instructions. The quantity and quality of the extracted RNA samples were estimated by spectrophotometry at 260 and 280 nm. RNA was treated with DNase I (Invitrogen- Gibco, Paisley, UK) before cDNA synthesis to avoid DNA contamination. Complementary DNA was synthesized from 5μg of total RNA using the RevertAid First Strand cDNA Synthesis Kit (Fermentase, Vilnius, Lithuania).

The abundance of FAS, BCL-2, and TP53 gene transcripts were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) using Bio-Rad Chromo 4 Real-time PCR Detector system (Bio-Rad, CA, USA) with Syber Green PCR Master Mix (Applied Biosystems, CA, USA) in triplicates. Expression of a β-actin housekeeping gene was used as a reference for the level of target gene expression. [Table 1] shows sequences of primer pairs for each gene. {Table 1}

PCR reactions were performed in a final volume of 25 μL and contained 0.5 μg of the cDNA product, 4.0 pmol of each primer, and 1Χ reaction mix consisting of FastStart DNA polymerase reaction buffer, dNTPs, and SYBR green I (Applied Biosystems, CA, USA).

Thermal cycling for all genes was initiated with a denaturation step at 95C for 10 min, followed by 30 cycles (denaturation at 95C for15 s, annealing at 60C for 30 s, and elongation at 60C for 34 s, when fluorescence appeared). The qRT-PCR amplification products were assessed by melting curve analysis and by electrophoresis on 1% agarose gel (data not shown).

For each target gene, the efficiency of the real-time PCR reaction was calculated from the slope of the standard curve. Standard curves were plotted by Ct values of serial dilutions of plasmids containing the genes of interest against the logarithm concentration of input template DNA. Accordingly, the efficiency of the FAS, BCL-2, and TP53 PCR reactions were calculated as 94%, 95.7%, and 97%, respectively. The relative amounts of FAS, BCL-2, and TP53 transcripts were determined from the ΔCt and 2 (−∆ct) formulas. Target-to-reference gene ratios were calculated with the Pfaffl method. [18]

The numbers of FAS, BCL-2, and TP53 transcripts in the peripheral blood were compared with the corresponding values from control samples with the nonparametric Mann-Whitney U test using SPSS software v. 11.5 (IL, USA). Graphs were plotted and analyzed using Prism 4 software (CA, USA).


The following parameters were obtained from the hospital records of the 50 patients: age, tumor size, tumor histology, lymph node metastases, clinical stage, histological grade, and presence of metastases. Tumor stage was determined with the tumor-node-metastasis classification. [Figure 1] summarizes the frequency of women with different clinical classifications. {Figure 1}

Quantitative real-time PCR assay was used to detect and compare the expression of these genes in the peripheral blood cells of 50 patients and 40 healthy control women.

[Figure 1] shows significantly higher expression of FAS in patients compared with healthy control women (P = 0.0178). Among patients in the early stages of the disease (stage I-II), the relative expression level of FAS was about 3-fold as high as in the control group (P = 0.0043). Similarly, FAS expression in patients with the diagnosis of nonmetastatic and low-grade tumors showed up to 4- and 3-fold increases, respectively [P =0.019, [Figure 2]]. Due to limited numbers of patients with high-grade tumor, comparison of FAS gene expression between groups of patients was not possible although less FAS gene expression was detected in patients with higher grade of tumor. {Figure 2}

Real-time PCR results showed significantly higher expression of BCL-2 in patients compared with healthy women (P = 0.0045). The greatest difference appeared in patients of stages I and II, in whom relative expression (target-to-reference gene ratio) was 2.89 vs 0.19 in the control group (P = 0.0025). Women with nonmetastatic and low-grade BC also had higher BCL-2 expression than healthy women [P = 0.0157 and P = 0.0248, respectively; [Figure 3]]. Significant overexpression of BCL-2 was also found in metastatic patients compared with the nonmetastatic group (P = 0.0381). However, no significant difference was found between the expression of BCL-2 in patients with low-grade and high-grade tumors.{Figure 3}

Comparison of TP53 gene expression in the patients and healthy controls showed up to 10-fold increase of gene expression in patients (P = 0.0068). The patients in early stages of the disease showed a significant increase in the mean relative TP53 gene expression (P = 0.0018). Increased expression of TP53 was also found in nonmetastatic patients (P = 0.009) and those with a low-grade tumor burden (P = 0.0012) [Figure 4]. Interestingly, when TP53 expression was compared between low-grade and high-grade patients, significantly more TP53 gene transcripts were detected in blood cells of patients with low-grade BC (P = 0.0004). In addition, the patients of stages I and II of the disease showed significantly higher expression of TP53 transcripts than late-stage patients (P = 0.018). {Figure 4}

Comparing expression levels of the genes showed a significant correlation between BCL-2 and FAS (r = 0.518, P = 0.001) and between TP53 and FAS (r = 0.318, P = 0.023). However, there was not such association between BCL-2 level and TP53 expression (r = 0.153, P = 0.281) (data not shown).


There has been great advancement in the early detection of circulating tumor cells with new sensitive techniques. These methods use different markers to identify rare tumor cells in the blood circulation. [19],[20],[21] However, we sought to analyze other endogenous markers in blood circulation rather than those from tumor cells in patients with early stages of BC. In this regard, we found the overexpression of BCL-2, TP53, and FAS in the peripheral blood cells from women in early stages of BC in comparison with healthy subjects. Interestingly, the relative expression of these genes in patients with nonmetastatic and low-grade tumor was significantly higher than healthy controls.

FAS is an apoptotic factor that activates a cascade reaction of caspases and thus perpetuates the process of apoptosis. [22] Several studies on BC patients have indicated that FAS can be regarded as an informative prognostic marker, [23] although their conclusions are still debatable. A significant association of FAS and FASL expression with tumor stage of patients has been shown previously. [5] Moreover, data show that FAS expression could contribute to the generation and progression of BC, and the levels of expression of FAS were in correlation with metastasis of tumor into the axillary lymph nodes. [24] The results of another study of 108 patients with BC of stages I or II showed a significant association between the expression of FAS and the number of tumor recurrences and also lymph node metastasis. [25] Our data also showed that BC patients in early stages of the disease (I and II) had significantly higher levels of FAS transcripts than healthy women. However, in a study by Sjostrom-Mattson et al, on 59 patients with BC, the similarity between primary breast tumors and matching metastasis lymph nodes was examined and showed dissimilar expression of p53, bax, bcl-2 , FAS, and FASL in those lymph nodes. [26] On the other hand, studies by Puiu et al, elegantly showed the similarity between the ratio of the corresponding mRNA species of transmembrane vs soluble FAS isoforms in patients with breast cancer. [27] Accordingly, the prevention of FAS-mediated apoptosis in cancer cells, such as breast tumors, cannot entirely be explained by the possible changes in FAS expression, and hence other mechanisms should be investigated. Future studies should resolve the variations among results that exist about the expression of this proapoptotic marker.

Cell survival and apoptosis is a tightly regulated mechanism, which is controlled by multiple genes and BCL-2 is a major component of that. The overexpression of BCL-2 has been found in a variety of human tumors and lymphomas, where it acts as an oncogene protein. [28],[29] Several cross-sectional and meta-analysis studies verify BCL-2 as a favorable independent prognostic biomarker in BC. [30] A recent study by Yang et al showed that BCL-2 expression is highly associated with increased risk of local recurrence in patients with early stage of BC. [31] However, some other data indicate that a high Bcl-2 expression by tumor cells had no predictive value in BC patients. [32] More variations have been shown in serum content of bcl-2. In this regard, Andalib et al suggested the use of mRNA level rather than soluble form. [33] In this study, we measured the BCL-2 mRNA content in the peripheral blood of patients and found higher levels of the transcripts from patients with early stages of BC than in healthy women. The ease of such measurement may provide the opportunity to test more patients of different stages for a conclusive result.

TP53 has often been found disabled in cancer cells because of point mutation or gene deletion. [34] Different studies on patients with BC have shown the overexpression of TP53 by cancer cells and have regarded that as a significant prognostic marker. [35],[36] In a study by Gerson et al on patients of early stages of BC, Her-2− , estrogen receptor− , and progesterone receptor - (triple-negative tumor cells) showed high frequency of TP53 expression. [37] Despite all these evidence, the contribution of this tumor suppressor in BC has been difficult to evaluate. [38] Our data indicating overexpression of TP53 in peripheral blood cells of patients of early-stage, low-grade, and nonmetastatic BC may provide more support regarding the possible role of this marker in BC detection.

Putting all these data together, it could be suggested that tumor cells at the early stages of BC overexpress BCL-2 to secure their outgrowth and survival. However, this coincides with the activation of a collection of physiologic regulatory mechanisms, such as increased expression of TP53 and FAS, which try to stop tumor cells by inducing apoptosis. Outcompeting or inadequate actions of these mechanisms result in tumor progression and malignancy. However, more data of other immunologic conditions are necessary to examine this suggestion and to test the specificity of these changes for BC.


This work was funded by a grant from, Iranian Cancer Network, Shiraz Institute for Cancer Research (ICR-87-505) and Shiraz University of Medical Sciences (Grant No.87-4331). We also are grateful to the patients and healthy women who participated in this project.


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