Accepted 24 July, 2013
Mutation analysis of c-Myc gene family in patients with primary breast carcinoma in Turkey
population
Hasibe Cingilli Vural1, Nesrin Turaçlar2*, Şahande Elagöz3 and Sadettin Ünsal1
1Department
of Biology, Selcuk University, Molecular Biology, 42079 Selçuklu, Konya, Turkey,.
School of Health Services, Selcuk University, Konya, Turkey.
3Department of Pathology, Cumhuriyet Medical Faculty, Cumhuriyet University, Sivas, Turkey.
2Vocational
*Corresponding author. E-mail: drnesrinturaclar@yahoo.com.
ABSTRACT
The Myc cancer gene contains instructions for the production of the c-Myc protein. The c-Myc
protein is known as a transcription factor or a regulator of other genes. It is a protein that binds
Deoxyribonucleic acid (DNA) at specific sites and instructs genes whether or not they should be
transcribed into messages for cells to make additional or other new proteins. Therefore, we used
Polymerase chain reaction (PCR)-based assays that can target either DNA (the genome). For this
reason, the aim of this study was to assess the contribution of c-Myc gene to commonly cancer
types in the Turk population and investigate mutation of c-Myc gene in breast cancer. In our study,
DNA was obtained from 50 breast cancer samples, amplified, screened for 2 exons (exon 1 and 2)
of the c-Myc gene by PCR-SSCP and then confirmed by sequencing. There was only one sample
that presented an alteration and that was a transversion. Our results corroborate the hypothesis that
somatic alterations in the c-Myc gene are rare events in breast cancer. Furthermore, inherited
changes in several other genes, including c-Myc have been found to increase the risk of developing
breast cancer and endometrium cancer. This case affirmed that, for establishment of a correct
diagnosis, especially for significantly clinically overlapping syndromes, molecular testing is usually
the only reliable method. Myc was located to the nucleus of breast tumour epithelial cells and was
found to be significantly associated with living cells (P<0.0001). These data provide evidence that
growth factors can signal through the transcription factor c-Myc in human breast cancer.
Key words: c-Myc gene, breast cancer, mutation.
INTRODUCTION
Cancer evolves from the accumulation of mutations and the deregulation of two classes of genes,
oncogenes and tumor suppressor genes. The large majority of breast cancers arise in the epithelia
of the breast, and they are thought to evolve from hyperplasia with atypia to carcinoma in situ,
invasive carcinoma, and, finally, metastatic disease.
Breast cancer is the most common cancer among Turkish women, accounting for 24.1% of all
cancer in women (Turkish Republic Ministry of Health, 2002). The molecular mechanisms leading to
breast malignancies are unclear, but many genetic abnormalities and epigenetic factors have been
implicated, including changes affecting known tumor suppressor genes (p53, BRCA1, BRCA2,
PTEN/MMAC1/TEP1) and protooncogenes (neu/ErbB2/HER2, ErbB1/EGFR, PRAD-1/ cyclin D1,
Mdm2, and c-myc) (Ingvarsson, 1999).
The c-Myc gene is located on human chromosome 8q24, consisting of three exons. Its
transcription may be initiated at one of three promoters. Translation at the AUG start site in the
second exon produces a major 439 amino acid, 64 kDa c-Myc protein. The c-Myc gene is mapped
to this region of chromosome, and the gene family appears to play an important role in the
regulation of cellular proliferation and differentiation.
Over-expression of the c-Myc oncoprotein is observed in a large number of hematopoietic
malignancies and have revealed a potent role for c-Myc in the generation of leukemias and
lymphomas. However, the reason for high c-Myc protein levels in most cases is unknown. The
fundamental
importance
of
nucleic
acid
amplification
methods
in
basic
research,
pharmacogenomics and molecular diagnostics (Schweitzer and Kingsmore, 2001) continues to
direct efforts aimed at improving current methodologies as well as, the development of novel
technologies.
The Myc cancer gene contains instructions for the production of the c-Myc protein. The c-Myc
protein is known as a transcription factor or a regulator of other genes. It is a protein that binds DNA
at specific sites and instructs genes whether or not they should be transcribed into messages for
cells to make additional or other new proteins. Therefore, we used Polymerase chain reaction
(PCR)-based assays that can target either DNA (the genome).
For this reason, the aim of this study was to assess the contribution of c-Myc gene to commonly
cancer types in the Turk population and investigate mutation of c-Myc gene in breast cancer. In our
study, DNA was obtained from 50 breast cancer samples, amplified, screened for 2 exons (exon 1
and 2) of the c-Myc gene by PCR-SSCP and then, confirmed by sequencing. There was only one
sample that presented an alteration and that was a transversion. Our results corroborate the
hypothesis that somatic alterations in the c-Myc gene are rare events in breast cancer.
Furthermore, inherited changes in several other genes, including c-Myc have been found to
increase the risk of developing breast cancer and endometrium cancer. This case affirmed that, for
establishment of a correct diagnosis, especially, for significantly clinically overlapping syndromes,
molecular testing is usually the only reliable method. Myc was located to the nucleus of breast
tumour epithelial cells and was found to be significantly associated with living cells (P<0.0001).
These data provide evidence that growth factors can signal through the transcription factor c-Myc in
human breast cancer.
MATERIALS AND METHODS
Cases
We enrolled 50 cases with primary breast carcinoma and 100 healthy controls in this study. Tissue
samples were also collected at the same time as the blood samples for control and were processed
within 5 h of collection. The study was approved by the medical ethics board of Cumhuriyet
University, Sıvas, Turkey.
Histopathology study
In the research, (from 2002 to 2008) 50 new cases which were sent to Cumhuriyet University
Medical Faculty, Department of Pathology, with the prediagnosis of malign tumors and whose fresh
tissue samples had been investigated were started to be studied. Fresh tissue samples about 3 mm.
were taken from the tumoral pieces aiming to have molecular genetic analysis and they were put
into deep freezer (at -20°C).
For histopathologic investigation, tissues were fixed in10% formal and were embedded in parafine,
and were then prepared in 5 µm cross sections painted with rutine haematoxylin-eosin dye. The
sample histopathological images of different cancer types (stained using hematoxylin-and-eosin
technique) are shown in Figure 3 and Table 1.
Patients and DNA isolation
The DNA used for polymorphic analysis was isolated from the biopsy samples of patients with
cancers by using DNA isolation kit purchased from nucleic acid isolation kit (Oiagen, Germany) the
manufacturer’s instructions (Figure 1). Isolated DNA was stored at -20°C till use. The control group
consisted of healthy unrelated volunteers without a medical history of cancer or other chronic
diseases. All patients and controls were of Turkish population.
Molecular materials
We have described a RT-qPCR reference assay, which we have named c-Myc primer pairs. It
identifies inhibitors of the reverse transcription or PCR steps by recording the Cts characteristic of a
defined number of copies of a sense-strand amplicon: an artificial amplicon (c-Myc) is amplified
using two primers (c-Myc F) and (c-Myc R).
In this study, PCR primers for the c-Myc gene target were as follows:
c-Myc forward primer : 5'-TCAAGAGGTGCCACGTCTCC-3' and
c-Myc reverse primer : 5'-TCTTGGCAGCAGGATAGTCCTT-3'.
In this study, cDNA were used for SYBR green quantitative PCR (Quantifast SYBR green PCR kit;
QIAGEN, Switzerland) and c-Myc primer pairs, for control λDNA (bacteriophage-ROCHE) to form
standard curve. We used Eppendorf Mastercycler Realple × 2S equipment for quantification of being
isolated DNA (By Biorobot EZ1, QIAGEN) to determination of Myc gene activation in the individuals
with leukemia patient. The cDNA was then diluted to 100 μl with water and stored at -20°C.
DNA quality
DNA quality and quantity encompasses both its purity (absence of protein and RNA contamination,
absence of inhibitors) and its integrity. Traditionally, DNA quality has been determined by analysis of
the A260/A280 ratio and/or analysis of the cDNA bands on agarose gels. In other words, five
microliter of each DNA was analyzed on a 1% agarose gel (TAE buffer), including a molecular
weight marker (Figure 1) and stained with ethidium bromide (0.5 µg/ml) for 30 min and then, with
agarose gel washed in double-distilled and UV-irradiated H2O.
Analysis of DNA fragmentation was performed by ethidium-bromide stained agarose gel
electrophoresis. The ethidium bromide luminescence from the CCD camera is integrated for 1 to 2 s
into the computer memory directly from the gel on the UV Transilluminator using Gel Doc 1000
system (Bio Rad). One of the most common methods for nucleic acid detection is the measurement
of solution absorbance at 260 nm (A260) due to the fact that nucleic acids have an absorption
maximum at this UV wavelength. Although, a relatively simple and time-honored method, A260
suffers from low sensitivity and interference from nucleotides and single-stranded nucleic acids.
Furthermore, compounds commonly used in the preparation of nucleic acids absorbed at 260 nm
led to abnormally high quantitation levels. However, these interference and preparation compounds
also absorbed at 280 nm led to the calculation of DNA purity by performing ratio absorbance
measurements at A260/A280.
c-Myc gene amplification by polymerase chain reaction
Amplification of c-Myc was assessed by the PCR assay. The reaction mixture contained 50 mM
Tris-HCl, pH 9.0, 20 mM ammonium sulfate, 100 to 500 ng of template, 0.50 μM each of 5′ and 3′
primers, 0.25 μM of each dNTP, 2.5 mM MgCl2 and 1 units of Taq DNA polymerase (Fermentase).
The PCR reaction was performed for 35 cycles of 94°C 2 min, 95°C 1 min, 62°C 1 min and 72°C 2
min.
For
c-myc
amplification,
5'-TCAAGAGGTGCCACGTCTCC-3'
and
5'-
TCTTGGCAGCAGGATAGTCCTT-3' primers were used. This defines a DNA fragment of 258 base
pairs. The c-Myc primers were based on the gene nucleic acid sequence (GeneBank, accession No.
J00120). The PCR products were separated by 2% agarose gel electrophoresis and assessed by
quantitative densitometry of ethidium bromide-stained bands with the use of Gel Doc 2000 system
(Bio-Rad) and the computer program Quantity One (Bio-Rad). All PCRs were performed in
triplicates (Figure 2).
SSCP analysis
Eight percent neutral polyacrylamide gel electrophoresis was performed as previously described
(Amati et al., 1998). In brief, 3 ml 40% acrylamide solution, 3 ml 5 × TBE solution, 3 ml 50%
glycerin, 6 ml ddH2O, 75 μl 10% ammonium presulfate and 7 μl TEMED were blended adequately
and poured into the gel, then concreted for 1 h at room temperature. 4 μl PCR products and 6 μl formamide samples were mixed. The mixture was centrifuged for 15 s, denatured at 95°C for 10 min,
bathed in ice for 10 min, put on an 8% neutral polyacrylamide gel, and electrophoresed with 1 × TBE
buffer for 8 h at 300 V. The fixation solution was infused into a flat utensil, into which gel was
immerged, vibrated for 10 min, and washed thrice (2 min each time) with ddH2O.
The gel was immerged into a staining solution, vibrated for 10 min, washed thrice (20 s each time)
with ddH2O. The gel was then immerged into a display solution, vibrated until the sample signal
became brown and the background became transparent yellow, and rinsed with tap water to stop
display. The staining results were observed and photographs were taken. According to the PCRSSCP results of genome DNA, the difference in the single strand strip number and electrophoresis
transference location, also known as the mobility shift and was considered PCR-SSCP positive
(Figure 4).
RESULTS AND DISCUSSION
In 1992, researchers started to realize that aberrant expression ofc- Myc could cause apoptosis
(Schweitzer and Kingsmore, 2001; Evan et al., 1992), although, the phenomenon had actually been
observed much earlier (Wurm et al., 1986).
Studies in recent years have further shown that the c-Myc gene regulates growth, both in the
sense of cell size and context of tissue differentiation (Gandarillas and Watt, 1997; Iritani and
Eisenman, 1999; Johnston et al., 1999; Schmidt, 1999; Schuhmacher et al., 1999a). Thus, it is now
known that the c-Myc gene participates in most aspects of cellular function, including replication,
growth, metabolism, differentiation and apoptosis (Schuhmacher et al., 1999b; Hoffman and
Liebermann, 1998; Dang, 1999; Dang et al., 1999; Elend and Eilers, 1999; Prendergast, 1999).
In physiological situations, the central role ofc-Myc may be its promotion of cell replication in
response to extracellular signals, by driving quiescent cells into the cell cycle. This function was
originally thought to be elicited mainly (Amati et al., 1998), in principle, promotion of cell cycle
progression by c-Myc can also be achieved by suppression of transcription of growth inhibitory
genes (Alexandrow and Moses, 1998).
Transgenic mice have been generated to target the c-Myc gene to the mammary glands by placing
the transgene under the control of the long terminal repeat of the mouse mammary tumor virus
(MMTV) or the whey acidic protein (WAP) promoters (Amundadottir et al., 1996a; Nass and
Dickson, 1997).
In MMTV-c-Myc transgenic mice, the transgene is expressed at high levels, specifically in the
mammary and salivary glands of females (Stewart et al., 1984). Spontaneous carcinomas develop
in mammary glands at a frequency of roughly 50% at about one year of age in the virgin females
and distant metastasis is rare (Amundadottir et al., 1996a; Amundadottir et al., 1995; Amundadottir
et al., 1996b; Rose-Hellekant and Sandgren, 2000).
Males do not develop the tumors. Multiple pregnancies significantly increase the incidence and
shorten the tumor latency (Nass and Dickson, 1997; Stewart et al., 1984; Amundadottir et al., 1995;
Amundadottir et al., 1996b), indicating that certain physiological growth stimuli to the mammary
gland, such as estrogen or progesterone, may serve as promoters of carcinogenesis.
Female WAP-c-Myc mice, on the other hand, must undergo pregnancy to develop mammary
carcinomas (Sandgren et al., 1995 25). The incidence and tumor latency are thus, likely to depend
on the rounds of pregnancies. In the WAP-c-Myc model, pregnancy is required for activation of the
promoter, but it complicates the model as well, since pregnancy may also provide additional
promotion of carcinogenesis, as seen in MMTV-c-Myc mice.
A recent report reveals that only about 22% of the tumor cases showed increased c-Myc mRNA
expression, and the over-expression was rarely due to the gene amplification (Bieche et al., 1999).
Liao et al. (2000) noticed that in some of the relatively larger (>1 cm in diameter) mammary tumors,
there are focal areas of tumor cells that are both hematoxylin (H)- and eosin (E)-phobic on routine
H-E stained sections (Liao et al., 2000).
In these focal lesions, the number of apoptotic cells are much fewer, while the number of
proliferating cells are much greater compared with the surrounding tumor areas. Although, these foci
show a clear boundary of demarcation from surrounding tumor areas, they are not encompassed by
connective tissue capsules. Usually, some portion of each focus exhibits infiltration into the adjacent
tumor areas, a typical feature of invasive growth. All these morphological properties of the ‘tumorwithin-a tumor’ foci in c-Myc tumors suggest that they may belong to a tumor phenotype that is more
aggressive than their adjacent tumor area, and may thus, represent a second step of tumor
progression (Liao and Dickson, 2000).
Our present study was aimed at analyzing c-Myc gene mutations in breast carcinomas of women.
We also investigated correlations between the observed defects and clinicopathological features of
tumors. To our knowledge, this is the first report analyzing the relations between alterations in the
aforementioned c-Myc gene mutations in breast carcinomas and we hope it will lead to greater
understanding of the pathogenetic pathways of these neoplasms.
In summary, fifty cases of carcinoma breast were analyzed by PCR-SSCP for mutations in exon 1
and 2 of c-Myc gene family in the present study. No mutation was found in fifty of the cases. This is
in contrast to the findings of the previous studies where the mutation frequency has been reported to
be 13 to 30% by molecular analysis. This suggests that the occurrence of c-Myc gene mutations is
relatively low in Turkey women with breast cancer as there are only few other reported studies about
the mutation frequency in Myc gene in the Turkey population.
ACKNOWLEDGEMENTS
We are grateful to Dr. Şahande Elagöz for his assistance in obtaining the clinical material used in
this study and to Dr. Hasibe Cingilli VURAL for her support of this research program.
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Table 1. Patients with various cancer risks were analyzed with respect to the histologic diagnoses.
Tissue
Breast
Breast
Breast
Breast
Breast
Breast
Breast
Breast
Breast
Breast
Breast
Histopatolojik diagnosis
Apocrine CA
Apocrine CA
Mix invasive ductal+lobular CA
Invasive ductal CA
Invasive ductal CA
Invasive ductal CA
Invasive ductal CA
Invasive ductal CA
Invasive ductal CA
Inflammatory CA
Invasive lobular CA
Numbers of samples
5
3
1
4
4
5
2
5
4
13
4
Stage
IIA
I
IIIA
IIA
I
IIA
IIIA
IIA
IIIC
IIIB
IIA
TNM
T2N0MX
T1N0MX
T3N2MX
T2N0MX
T1N0MX
T1N1MX
T2N2aMx
T2N0Mx
T3N3Mx
T4dN2Mx
T1cN1aMx
Figure 1. Genomic DNA s were loaded in a 1% agarose gel and seperated by electrophoresis and visualized by ethidium bromide
staining with transillumination; respectively, lane 1 to 17 genomic DNAs isolated from breast tumor tissues with Rio Robot EZI.
Figure 2. PCR amplification of c-Myc gene family in patients with primary breast carcinoma.
Figure 3. Invasive ductal carcinoma of breast (HE × 100).
Figure 4. Showing SSCP results in c-Myc gene in patients with primary breast carcinoma.