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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 7  |  Issue : 2  |  Page : 42-49

Pattern of chromosomal aberrations and expression profile of p53ser15 and BAX protein in peripheral blood lymphocytes of healthy subjects and cancer patients


1 Department of Human Genetics, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India
2 Department of Medical Oncology, SRMC and RI, Sri Ramachandra University, Porur, Chennai, Tamil Nadu, India

Date of Web Publication7-Oct-2016

Correspondence Address:
Venkatachalam Perumal
Department of Human Genetics, Sri Ramachandra University, Porur, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-0168.191706

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  Abstract 

Introduction: Chemotherapy is an important treatment option which is used for all cancer types. The basic mechanism of action of chemotherapy is that the drugs cause damage to the cancer cells by breaking down DNA, interfere with replication, or enhance the cell killing. Emerging studies have shown that despite tremendous improvements on the therapeutic options, benefit derived from the therapy is not desirable. It is because, interindividual variations among the patient's response to therapy as well as complex signaling molecules and mechanism involved, determining the final outcome of the therapy. Therapeutic efficacy can be improved by predicting a patient response to that agent, adopting a suitable marker.
Materials and Methods: This study involves analysis of the frequencies of chromosomal aberrations and micronucleus, expression profile of p53 ser15 and BAX in healthy subjects and cancer patients, to identify a novel marker to predict their response to chemotherapy agents. For this, peripheral blood sample (4 ml) from cancer patients (solid tumors) was obtained before and after chemotherapy (n = 20). The change in those marker in cancer patients were compared with age- and sex-matched healthy subjects (n = 20).
Results: The present study results indicated substantial increase in all four biomarkers for postchemotherapy compared to that obtained before therapy; however, the increase was not significant (P > 0.05), whereas a significant increase (P < 0.05) was observed in all markers from cancer patients compared to that of healthy volunteers relate the genetic instability to the disease status. Furthermore, on comparison, the levels of all those changes are increased in samples obtained posttherapy, despite the magnitude of BAX expression is considerably higher when compared to other markers.
Conclusion: Therefore, the study results implied that BAX can be used as a better marker to predict the patient response to chemotherapy.

Keywords: BAX, chromosomal aberration, p53ser15 , solid tumors


How to cite this article:
Gulawani S, Raavi V, Suresh S, Perumal V. Pattern of chromosomal aberrations and expression profile of p53ser15 and BAX protein in peripheral blood lymphocytes of healthy subjects and cancer patients . J Radiat Cancer Res 2016;7:42-9

How to cite this URL:
Gulawani S, Raavi V, Suresh S, Perumal V. Pattern of chromosomal aberrations and expression profile of p53ser15 and BAX protein in peripheral blood lymphocytes of healthy subjects and cancer patients . J Radiat Cancer Res [serial online] 2016 [cited 2019 Jan 16];7:42-9. Available from: http://www.journalrcr.org/text.asp?2016/7/2/42/191706


  Introduction Top


Globally, cancer is considered as a major public health problem. It is a group of diseases, results in the expression status of multiple genes, which maintain normal cell structure and regulate essential cellular functions. Despite the enormous developments in the available treatment options, it has shown that the mortality ratio is higher in developing countries including India (68%) rather than developed countries such as North America and Europe (40%). The reason for poor survival was attributed to late diagnoses and inadequate treatment. [1] Nonetheless, the major objectives of the treatment of cancer are cure, improvement of survival quality, and prolongation of life. Treatment modalities available to treat/manage cancer are by different means to regulate the cell division. [2] On exposure to those agents, it can alter the biomolecules and can be related to the patient response to therapy; those changes are known as biomarkers or response. Undesirably, similar biomarkers are also used as screening or diagnostic markers for different types of cancer. Another challenge is to relate that cancer biomarker to its clinical pathology as well as to detect in the early stage. There is a variety of biomarkers, which can include chromosome abnormalities, nucleic acids, antibodies, and peptides, among other categories. [3] Biomarkers can be detected by different means such as from circulation, excretions, secretions, tissue-derived and also they are excellent candidates to evaluate the progression of disease, response to therapeutic drugs, and normal tissue responses for better therapeutic intervention. Thus, both prognostic and diagnostic biomarkers are helpful tools to identifying that are at risk, early stage diagnosis, narrow down a treatment modality, and monitor response to treatment. [4]

Maintenance of genome structure and functions constant is an essential for regulated cell division. Genome destabilizing events are considered as an initiating event for carcinogenesis. To monitor the stability of genome, the integrity of chromosomes is observed by employing various assays; they are collectively known as cytogenetic markers. [5] Structural chromosomal aberrations are formed due to misrepair of DNA breaks induced by the various types of mutations. The structural variations results in the exchange of genetic material between the chromosomes (balanced or nonreciprocal) or amplified fragments (double minutes) which leads to the activation of genes which regulates the vital cellular functions. Some of the regions get amplified and these sequences incorporated into nearly contiguous homogeneously staining regions of tumor chromosome. Knuutila et al. have summarized the abnormalities reported in various solid tumors. [6] The cytogenetic analyses on tumor genomes show considerable variation among the type of aberration; it depends on , regions that are altered, anatomical location and genetic makeup of the individual. [7]

Of the several structural changes observed in solid tumors, few are recurrent and shown to be involved in solid tumor development, whereas recurrent chromosomal structural aberrations are major transforming events in sarcomas; both leukemias and lymphomas constitute almost 75% of the currently reported >27,000 cytogenetically aberrant cases in literature. [8] Much less cytogenetic information has been gathered for solid neoplasms; this lack of data was attributed to technical difficulties in culturing neoplastic epithelial cells and karyotypic complexity. Even though high levels of abnormalities were observed in solid tumors, the major challenge is to find or disfiguring critical and irrelevant aberrations as the diagnostic, prognostic, and therapeutic applications of cytogenetic aberrations are highly underappreciated. In line with the structural chromosomal aberrations, identification of the gene involved in recurrent aberrations will be an attractive choice for better therapies. [9]

Micronuclei are the small bodies derived from broken acentric fragment of a chromosome or whole chromosome lagging behind cell division, which can be seen budding of newly divided cell. The increased formation of micronuclei is usually an indication of increased DNA damage or mutation, characteristically found in the cancerous cells. The patients undergoing chemotherapy shows a higher micronucleus (MN) frequency in peripheral blood erythrocytes; MN scoring in these cells shows its potential to monitor clastogenic exposures, premalignant lesions, and cancer risk. [10] The differential response of the individuals exposing to genotoxic agents and therapy is reported in epidemiological studies. The difference in response may be due to the individual's efficiency of DNA repair and susceptibility to agents such as radiomimetic, clastogenic, alkylating, and dimer inducing agents. The radiosensitivity of the normal tissue in cancer patients is evaluated using MN assay. [11]

Tumor protein 53 (p53) is guardian of the genome in multicellular organisms, functions as tumor suppressor gene, and helps in cancer prevention. [12] The mutations in p53 gene were reported as predictor of resistance to therapy in patients undergoing clinical studies. Simultaneously, it also acts as regulator of cell cycle, DNA repair, and apoptosis. Inactivation of p53 leads to transformation of normal cells into cancerous cells. [13] The regulator of apoptosis BAX is also called as B-cell lymphoma 2 (BCL-2) like protein 4, which is encoded by gene BAX known to be regulated by p53. Cancer cell resistance to apoptosis is a hallmark of cancer, and the proteins involved in apoptosis have a good prognostic value as marker in the monitoring of patients response to therapy. The importance of these mediators has been investigated in the development and progression of several cancers. Hence, this is also considered as important biomarker in many cancers. [14]

Thus, the scrutiny of literature clearly demonstrates that chromosomal aberrations and protein expression profiles can be used as biomarkers to predict the patient's response to therapy. While isolated studies on those markers have been reported in many studies, none had shown a comprehensive analysis of all multiple biomarkers in patients before and after chemotherapy that too in solid tumors. Moreover, as preliminary investigation, it was of our interest to identify a promising marker over a range of tumor types. Therefore, we made an attempt and reported the pattern and frequency of chromosomal aberrations (CAs), micronuclei formation, and expression of phosphorylated form of protein p53 (p53 ser15 ) and BAX for the prediction of patient response to therapy.


  Materials and Methods Top


Study population

The study subjects consist of two groups, namely (i) apparently healthy volunteers as controls (n = 20) and (ii) patients with solid tumor at different sites as cases (n = 20). The present study was approved by the Institutional Ethics Committee of Sri Ramachandra University (CSP/16 JAN/45/50). The following criteria were adopted to include the subjects for the study.

Control

Inclusion criteria

  • Volunteers who are more than 18 years are included in the study
  • Without any known medication in the last 6 months.


Exclusion criteria

  • Age lesser than 18 years
  • Pregnancy.


Cases

Inclusion criteria

  • Biopsy-confirmed patients with solid tumor
  • Without starting any chemo treatment.


Exclusion criteria

  • Patient diagnosed with leukemia
  • Patient undergone radiotherapy.


Sample collection

Three study subjects were divided into three groups: (i) Group-I (healthy volunteers), (ii) Group-II (cancer patients before therapy), and (iii) Group-III (same cancer patients after the first dose of chemotherapy). Approximately 3-4 ml peripheral blood was collected in heparin vacutainers from each subject after obtaining informed consent. The samples were divided into two aliquots and one aliquot was used to prepare chromosomes and cytokinesis arrested binucleated cells. The second aliquot was used to isolate the lymphocytes followed by flow cytometric analysis of the expression of p53 ser15 and BAX proteins.

Chromosomal aberration assay

The spontaneous and chemotherapy drug-induced DNA damages were measured from the blood samples using chromosomal aberration assay as described earlier. [15] About 1 ml of peripheral blood was cultured with 80% RPMI 1640 medium (GIBCO), 20% of fetal bovine serum (GIBCO) supplemented, and 0.4% of PHA and incubated at 37°C in 5% CO 2 atmosphere. One set of the culture was used for the chromosomal aberrations assay and the second set of culture was used for MN assay. For chromosomal aberration analysis, the lymphocytes were arrested at metaphase by adding 0.02 μg/ml of colchicine at 24 th h, the cells were harvested at 48 th h using hypotonic solution (0.075 M KCl), fixed and washed twice using carnoys fixative (methanol:acetic acid, 3:1). The cell pellet was placed on a prechilled glass slide, air-dried, and stained with Giemsa. Chromosomal aberrations were scored using light microscopy with × 100 oil immersion (~250 metaphases per sample). [16],[17] The recorded aberrations include breaks, acentrics, minutes, gaps, and dicentrics.

Micronucleus assay

To the second set of culture, 6 μg/ml of cyto-b was added aseptically at 44 th h and the cultures were incubated for 28 h. At 72 h, the lymphocytes were harvested with prechilled hypotonic solution (0.075M) and fixed in Carnoy's fixative (5:1, methanol:acetic acid). For each dose, multiple slides were prepared, air-dried, and coded; then, the slides were stained with Giemsa (8%) and scored manually. The MN frequency was calculated which is present in the BN cell with an intact cytoplasm and as per detailed criteria described earlier [18] (2007).

Expression analysis of p53 ser15 and BAX

Lymphocytes were isolated from the whole blood using Ficoll Histopaque 1077 density gradient solution (GIBCO). The lymphocytes were washed with phosphate buffered saline (PBS), and the lymphocytes pellets were resuspended in 1 ml of RPMI (GIBCO) medium and processed for immunostaining of BAX and p53 ser-15 . The lymphocytes were fixed with 2% para formaldehyde (2%) for 20 min, permeabilized using 0.25% triton x-100 for 8 min, fixed with 2% bovine serum albumin-PBST for 30 min. All the incubations were performed at room temperature. The lymphocytes were incubated with 150 μl of primary antibody (BAX [abcam] [rabbit polyclonal IgG] and primary antibody of p53 [abcam] [rabbit polyclonal antibody to p53 (phospho s-15)]) for 2 h at room temperature. Then, the excess primary antibody was washed twice with PBS and the cells were incubated with 150 μl of secondary antibody (2 μg/ml) (abcam) (goat polyclonal IgG with fluorescein isothiocyanate conjugate) at room temperature for 1 h in dark. The excess secondary antibody was washed with PBS, and the lymphocyte pellet was resuspended in 300 μl of PBS and analyzed using BD Accuri (Becton Dickinson) flow cytometry. For each sample, 10,000 events were recorded using C6 software.

Statistical analysis

The aberration frequency of CA, MN was calculated from the number of aberrations to that of total cells scored. The mean fluorescence intensity (MFI) of BAX and p53 ser15 from the samples obtained before and after chemotherapy were compared using Student's t-test. The mean frequencies of aberration and MFI of protein expression observed before and after the procedure as well as from healthy volunteers were compared using Student's t-test. All graphs were drawn using Microsoft Excel software.


  Results Top


Demographic details of study subjects

A total of twenty healthy volunteers were recruited for the study to compare those parameters obtained from cancer patients. The mean age of healthy individuals is 25.8 ± 2.94 years. The mean age of cancer patients is 49 ± 2.94 years, of which 35% are male and 65% are female. Cancer patients with the habit of chewing tobacco or smoking and alcoholics are 16%. The cancer patients included in the present study had cancer at different tissues; ovary, breast, salivary gland, rectum, hypopharynx, bladder, lung, stomach, maxilla, colon, and placental site trophoblastic tumor (postpregnancy). The stages of tumor in the cancer patients were either IIIA or IIIB. The cancer patients were treated mainly with either cisplatin alone or in combination with oxaliplatin and paclitaxel. The demographic details of cancer patients involved in the present study are given in [Table 1].
Table 1: Age, gender, stage, and type of tumor and treatment drug used among the cancer patients


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Frequency of chromosomal aberration in cancer patients before and after chemotherapy

CAs and MN assays are used as markers for genotoxicity. The CA includes dicentrics, acentric fragments, chromatid breaks, and gaps were scored [Figure 1]. The mean frequency of CAs cancer patients before therapy is 0.060 ± 0.017, whereas in patients treated with chemotherapy, the mean CA frequency is 0.073 ± 0.017. The frequencies of CA obtained from subjects differ before and after therapy. [Figure 2] shows an overall response of CA frequency obtained in all cancer patients before and after therapy. This shows that the chemotherapy drugs induced increase in the amount of CAs, but it is not significant (P > 0.05). The variation in pattern compared before and after chemotherapy shows increased number of both chromatid breaks and chromosome breaks after chemotherapy than that of before.
Figure 1: Representative images of chromosomal aberrations (dicentric chromosomes, chromatid breaks) observed in cancer patients undergoing chemotherapy

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Figure 2: Frequency of chromosomal aberrations observed in cancer patients undergoing chemotherapy (pre and post)

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Frequency of micronucleus in cancer patients before and after chemotherapy

Frequency of MN and nucleoplasmic bridges formation is also measured in cancer patients before and after therapy [Figure 3]. The frequency of MN obtained from cancer patients before the therapy is 0.035 ± 0.009, whereas in patients treated with chemotherapy, the mean MN frequency is 0.043 ± 0.011 [Figure 4]. Similar to CA, the frequency of chemotherapy drug-induced MN also shows an intraindividual variation. Thus, the overall results show that the chemotherapy drugs induced an increase in the amount of micronuclei formation.
Figure 3: Representative images of the binucleated cell and binucleated cell with micronucleus obtained from the cancer patients undergoing chemotherapy

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Figure 4: Frequency of micronucleus observed in cancer patients undergoing chemotherapy (pre and post)

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Expression levels of protein markers in cancer patients before and after chemotherapy

The frequencies of cytogenetic damages measured after therapy are higher when compared to that of obtained before therapy among the cancer patients. To confirm the results further, expression levels of p53 Ser-15 and BAX were quantified in cancer patients. A representative image of the dot blots obtained from the flow cytometry and bar diagrams for MFI observed for few patients before and after therapy for those proteins is shown in [Figure 5]. Thus, the expression levels of both proteins show interindividual variations while majority of the cases showed an increase at posttherapy few subjects did not. However, the pooled data for all the study subjects did not show a significant increase in their expression profiles [Figure 6].
Figure 5: Representative image of the immunocytochemistry BAX and p53 obtained from flowcytometry and the individual values of 12 study subjects

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Figure 6: Pattern of the expression of p53ser15 (a) and BAX (b) proteins obtained from the cancer patients undergoing chemotherapy (pre and post)

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Comparison of genotoxicity and protein expression among healthy volunteers and cancer patients

[Figure 7] and [Figure 8] show the comparison on the frequencies of CA, MN levels of p53 Ser-15 and BAX obtained among the healthy subjects and cancer patients before and after chemotherapy. Although all the markers are significantly (P < 0.05) high in cancer patients compared to healthy individuals, the changes in BAX levels show higher fold of increase among the cancer patients before and after therapy. Thus, chemotherapy drugs have induced increase in both CA and MN formation. Furthermore, both protein markers (p53 Ser-15 and BAX) showed increase in expression after chemotherapy. However, the increase in all four end-points is found as nonsignificant.
Figure 7: Comparative analysis of the frequency of chromosomal aberrations and micronucleus obtained from the healthy subjects and cancer patients undergoing chemotherapy (pre and post)

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Figure 8: Comparative analysis of the expression of BAX and p53ser15 proteins obtained from the healthy subjects and cancer patients undergoing chemotherapy (pre and post)

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  Discussion Top


At present, many therapeutic strategies are available to manage cancer patients. Despite the recent molecular approaches, traditional chemotherapy remains a major modality of treatment; this is because of the cost, affordability as well as options to regulate a particular pathways. Various drugs exert their effects by functioning as antibiotics, alkylation agent, inhibitors of topoisomerases, and modifier of epigenetic mechanism on chromosome remodeling. The curative intent of chemotherapy requires multiple cycles of treatment and is given with the intention of destroying cancer cells any place they may exist in the body. Nonetheless, the reduction of toxicity of drugs to normal tissue due to multidrug administration and tumor killing. That is why targeted therapy to the tumor site remains an attractive option. Alternatively, a marker which can better predict a patient response to therapy can be utilized to increase the patient benefit by means of better tumor control while reducing the toxicity to surrounding normal cells.

An identification of reliable biomarkers has multiple applications such as cancer diagnosis, prognosis, and treatment, localize the tumor and determine its stage. Henceforth, identification of such signature can be exploited for the better management of cancer. Tumor cells display wide variety of alterations such as point mutations, gene rearrangements; amplification of genes involved in pathways that are regulating various cellular processes includes cell growth, survival, and metastasis. These alterations in tumor cells can be exploited for the identification of suitable biomarkers which can be used for diagnosis, prognosis, and therapy. [4] Being a less invasive sampling method and from the administrative route, it circulates throughout the body and reaches the target site; peripheral blood was used as representative sample for the entire body and indirectly provides information on the levels of toxicity. Therefore, we made an attempt to identify a marker to predict the patient with solid tumor at different sites and their response to therapy. For this purpose, extend type of DNA damages was measured using CA and MN formation. In addition to molecular markers, p53 ser15 and BAX level was measured from peripheral blood lymphocyte (PBL) of cancer patients before and after chemotherapy. In fact, there are reports that show a substantial increase in both sister chromatid exchanges and spontaneous chromosome breakage among the patients underwent chemotherapy. [19] It was worth mentioning that administration of uniform dose of a drug to a population of patients responded differently; it was attributed to the variables associated with drug response and genetic differences. Furthermore, the types of lesions induced by the therapeutic agents, their repair, are varied within tissues which determine the response of patients to therapy. Induction of DNA lesions is the common effect observed in cells exposed to therapeutic agents. Out of all these lesions, double-strand break is the most important lesion produced in chromosomes because, the cell killing is associated with double-strand breaks, as double-strand breaks can lead to CA, which are lethal to cells. Results obtained in the present study showed that the chemotherapy drugs induced variety of CA in PBL; (before therapy, 0.06 ± 0.017 and after therapy, 0.073 ± 0.017); of which 60-70% of them are dicentric chromosomes, which are the end products of mis repaired DNA double-strand breaks, simple chromosome, and chromatid exchanges. The obtained results in the present study are in agreement with the previous studies of bleomycin treatment of patients in which 90% of aberrations are dicentric chromosomes. [16],[20] The MN frequencies induced by bleomycin are less (~50%) when compared to CA, for the same concentration of the drug. This is probably due to the mechanism of formation of MN and its elimination. It has been demonstrated that MN is produced during mitosis from broken chromosomes or whole chromosome which fails to incorporate into daughter nucleus, damaged kinetochores, or spindle fiber defects. [21] The inclusion of MN into the daughter nucleus [22] or masking of MN by the daughter nucleus, [23] fusion of more than one damage in the formation of MN, [24] and elimination from cells [25] are the various factors attributed to the less yield of MN when compared to CA due to radiation exposure. Moreover, it was also shown that the content and amount of MN will differ depending on the DNA damages induced by the agent. [26]

The p53 gene is known as the guardian of genome, because of its vital role in regulating the cellular functions such as check point activation, DNA repair as well as the crucial role of tumor suppression. The above-mentioned cellular responses depending on the type and extend of DNA damages induced by the therapeutic agent. Owing to its multiple functions, it has been demonstrated that clinical studies in patients with various types of cancer have mutations in the p53 gene, and those mutations are used as predictor of patient's response to therapy. [27],[28],[29] Therefore, we have measured the level of a phosphorylated form of p53, a marker of DNA damage rather than cell cycle regulator. There was an interindividual variation on the changes in the biomarker [Figure 5]. The variation could be attributed to factors such as difference in tumor type, stage of the tumor, variation in the administered drug, and patient's sensitivity to the drug. Therefore, the comparisons were made before and after therapy by pooling the data. The obtained results show that an increased expression of p53 ser15 in the patients after therapy when compared to that of before exposure suggest that p53 is an important molecule can be used as a marker of patient response to treatment in far with the published literature.

Apoptosis is the regulated destruction of a cell, which is a sequential and regulated process mediated by multiple enzymes, by two distinct pathways: The cell death is either induced by receptor-mediated signaling at the plasma membrane (extrinsic pathway) or triggered by leakage of cytochrome-c from mitochondria (intrinsic pathway). The initiator caspases, namely, procaspase-8 and procaspase-9 activated the extrinsic and intrinsic pathways, respectively. Once activated, either of those caspases activates the effectors (caspase-3, -6, and -7); these downstream molecules cleave a variety of substrates, including the nuclease inhibitor, components of the cytoskeleton to give rise to internucleosomal DNA degradation, and characteristic morphological changes such as blebbing. Whereas, the upstream events that activate caspase-8 versus caspase-9 are very different. Thus, the mitochondria act as a central role in regulating the intrinsic pathway mediated cell death. An interruption of the mitochondrial pathway, which can occur by several different mechanisms; such a mechanism is the ratio on the levels of pro-apoptotic (BCL-2) and anti-apoptotic factors (BAX). Altered expression of those factors has been reported in acute myelogenous leukemia, intermediate grade lymphomas, and cancers of the prostate, ovary, and upper aerodigestive tract suggest that measurement of those changes can be exploited as marker to predict the patient response to therapy. [17] The results obtained in the study showed that the level of BAX, the mediator of apoptotic pathway is increased in posttherapy of all cancer patients. Our results support that expression levels of BAX can also be used to predict a patient response to chemotherapy. It is well established that p53 is the major regulator of intrinsic pathway mediated apoptosis. Using knockout studies, it has been suggested that mitochondrial p53 would bind anti-apoptotic BCL-2 proteins, thereby liberating pro-apoptotic proteins such as BAX to induce apoptosis. [17] Our study results showed that an increased expression of p53 ser15 could have increased the stability of p53 and then the BAX-mediated apoptosis.


  Conclusion Top


Chromosomal aberration, MN frequency, and expressions of p53 ser15 and BAX were quantified in healthy subjects and cancer patient before and after chemotherapy. On comparison, the levels of all those changes are increased in samples obtained posttherapy, despite the magnitude of BAX expression is considerably higher when compared to that of other markers. Therefore, our study results implied that BAX can be used as a better marker to predict the patient response to chemotherapy.

Acknowledgments

We sincerely acknowledge the financial assistance from the Department of Science and Technology, Government of India (SR-SO/HS-127/2012) and Atomic Energy Regulatory Board (AERB/CSRP/Proj. no. 58/04/2014).

Financial support and sponsorship

We are grateful to the financial support provided by the Department of Science and Technology, Government of India (SR-SO/HS-127/2012) and Atomic Energy Regulatory Board (AERB/CSRP/Proj. No. 58/04/2014).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1]


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