Romanian Biotechnological Letters Vol. 18, No. 5, 2013 [601805]

Romanian Biotechnological Letters Vol. 18, No. 5, 2013
Copyright © 2013 University of Bucharest Printed in Romania. All rights reserved
ORIGINAL PAPER
 
Romanian Biotechnological Letters, Vol. 18, No. 5, 2013 8603 Chromosome aberrations, telomere a nd telomerase dysfunction after beta
irradiation in human lymphocytes

Received for publication, June 20, 2013
Accepted, September 6, 2013

POJOGA (USURELU) MARIA DANIELA1*, MANAILA ELENA2,
CONSTANTIN NICOLETA1, DUTA CORNESCU GEORGIANA1,
CIMPONERIU DANUT 1, SIMON-GRUITA ALEXANDRA1
1 Department of Genetics, Faculty of Biology, University of Bucharest , 1-3 Aleea
Portocalilor, Sector 5, 060101, Bucharest, Romania
2 National Institute of Research-Development for Laser, Plasma and Radiation Physics
Bucharest, 409 Atomistilor St., Bucharest-Magurele, Romania * Corresponding author: Pojoga (Usurelu) Maria Daniela, Faculty of Biology, University of
Bucharest, 1-3 Aleea Portocalilor, Sector 5, 060101, Bucharest, Romania, e-mail:
[anonimizat]

Abstract
The ionizing radiations used in radiotherapy are producing DNA damages in both the directly
irradiated and the adjacent cells. The effect of various beta radiation doses on human lymphocytes was
investigated using classic and molecular cytogenetic methods. At chromosome level, complex chromosomal interchanges, acentric fragments and dicentric chromosomes were the most frequent
chromosomal rearrangements observed after exposure to 7 Gy and respectively 14 Gy. The number of
chromosomal abnormalities increases with the radiation dose. After hybridization with the telomeric probes, a partial or total loss of fluorescence signal was recognized. The analysis of hTERC and hTERT
genes, that encoded the RNA and reverse transcriptase components of telomerase showed in a limited
number of metaphases an amplification phenomenon. These results proved that classic and molecular cytogenetic methods are suitable for the screening of the bystander effect induced by the anticancer therapy with beta radiations.

Key words: beta radiation, telomere, te lomerase, Fluorescence in s itu hybridization (FISH)

Introduction

Ionizing radiations (alpha and beta particles, X and gamma rays, protons, neutrons etc.)
are known to cause DNA damage and chromosome aberrations, being linked to cancer and
non-cancer disease development, premature sene scence or cell death [1, 2]. Genuinely, a
major event caused at the DNA level induced by irradiation is chromatid breakage (single strand breakage – SSB or double strand breakage – DSB). The DSB is the lesion responsible
for most visible chromosomal aberrations obser ved at the metaphase after irradiation of
normal cells [3, 4]. Thus, these chromosome abe rrations are very useful biomarkers in the
evaluation of the harmful biological effects of ionizing radiation [5].
The radiation effects target the telomeres, which are known to play an essential role in
linear chromosome stability and replication. These functions are mediated by the highly
conserved repeats which consists of th e (TTAGGG)n sequence in humans and other
vertebrates [6]. The number of telomeric repeat s decreases with cell divisions and age, so
telomere shortening acts as a mitotic clock in normal somatic cells. Telomeric repeats are
synthesized by telomerase, an enzyme with two components: a RNA template ( hTR or
hTERC ) and a catalytic protein ( hTERT ) [7]. In humans, telomerase activity is widespread in
many tissues throughout fetal development, bu t becomes repressed in most somatic cells
before or shortly after birth. Without telomerase activity, somatic cells exhibit progressive

POJOGA (USURELU) MARIA DANIELA, MANAILA ELENA, CONSTANTIN NICOLETA,
DUTA CORNESCU GEORGIANA, CIMPONERIU DANUT, SIMON-GRUITA ALEXANDRA

8604 Romanian Biotechnological Letters, Vol. 18, No. 5, 2013 loss of telomeric sequences with each cell divi sion because of “the end replication problem”
and enter senescence when a critical length of telomeres is reached [8]. Therefore, telomerase
activity was found in over 90% of tumor and in vitro -immortalized cells [6, 9].
There is less information on the effects of beta ionizing radiation on the telomeres and
telomerase as opposed to other radiation types [2]. Classical cytogenetic methods are not
powerful enough for the complex investigation of the damages caused by irradiation at this
chromosomal level requiring the use of more sensitive/adequate methodologies. Fluorescence
in situ hybridization (FISH) is widely applied for detecting genetic aberrations with clinical
significance [10], but only few studies have used this method for the analysis of the telomeres
or telomerase genes.
Due to the importance of the end chromosomal structures and telomerase in
carcinogenesis, cancer therapy and aging, the main objective of this study was to evaluate the
effects of beta radiations on human lymphocy tes by classical cytoge netic methods. Moreover,
using FISH methodology, the telomere integrity and telomerase genes before and after
irradiation were investigated.

Materials and Methods

Subjects. The peripheral blood was collected from 7 subjects of middle age (between 40
and 45 years old), healthy, non-smokers, non-al cohol consumers and without any history of
irradiation. For each subject, a personal identification code was assigned. All the subjects
were previously informed about the content of the research and gave their written consent for
participation in this study.
Three lymphocyte cultures per subject (one control and two for irradiation) were
initiated using the conventional Hungerford method [11] on 72 hrs short-term cultures, adapted for human chromosome investigation.
Irradiation procedure. Irradiations were performed 24 hr s after the culture initiation at
Electron Accelerators Laboratory, National Institute for Laser, Plasma and Radiation Physics
(INFLPR), Magurele-Bucharest, Romania, using an ALIN 10 linear electron accelerator (built
in Romania, at INFLPR). ALIN 10 is a linac type travelling wave, operating at 2.998 GHz,
6.5 MeV average energy, with a 0.1 mm Al foil exit window. Improved Fricke (ferrous
sulphate, cupric sulphate and sulphuric acid in triple distilled water) dosimetry system [12]
has been used to perform preliminary dose measur ements. The doses used in this study were 7
Gy and, respectively 14 Gy.
Classical cytogenetics : Microscopic slides were obtained from the lymphocyte cultures
according to standard procedures. The slides were stained with a 10% Giemsa solution and
100 metaphases for each sample were sc ored for chromosomal aberrations.
FISH analysis : For chromosome preparation, a special kit from Kreatech Diagnostics
(Netherlands) was utilized. In this stud y the following probes were used: (1) a MP
Biomedicals (USA) probe and a Dako PNA
(Denmark) one, for the telomeric repeats; (2) a
dual-color hTERC (3q26)/3q11 probe Kreatech Diagnostics (Netherlands) for RNA
telomerase component. The hTERC 3q26 region exhibits red signal, whereas the 3q11 region
probe which is included to facilitate chromoso me identification exhibits green signal; (3) a
dual-color hTERT (5p15)/5q31 probe Kreatech Diagnostics (Netherlands) for revers
transcriptase component. The hTERT 5p15 region exhibits red signal, and the control 5q31
(CDC25C/EGR1 genes) region probe shows green signal. At least 100 well–spread
metaphases were examined under Carl Zeiss Axiostar Plus microscope, both for experimental
and control samples.

Chromosome aberrations, telomere and telomerase dysfunction after beta irradiation in human lymphocytes

Romanian Biotechnological Letters, Vol. 18, No. 5, 2013 8605Results and Discussions

The assessment of ionizing radiation effect on living systems, including humans, both at
chromosomal and telomerase gene levels, is of much interest [13, 14], especially after the
recent atomic accident from Fukus hima I Nuclear Power Plant, Japan, and, also, in connection
with the development of radiotherapy used for the treatment of cancer and other diseases.
In this study, human genome reactivity to beta irradiation was investigated using an
experimental in vitro system (lymphocyte cultures) by scoring the chromosomal abnormalities
and by FISH identification of the telomere and telomerase gene response.

Classic cytogenetic
A number of 2100 metaphases, 700 for each i rradiation doses and 700 as controls (non
irradiated) were analysed microscopically (objective 60x and 100x).
The lymphocytes exposed to 7 Gy and, resp ectively, 14 Gy beta radiations showed a
large spectrum of chromosomal rearrangements: dicentric chromosomes, acentric fragments,
ring chromosomes, double minutes, complex chromosomal interchanges and endomitosis. In
Figure 1, A and B some representative chromosomal aberrations after exposure at 14 Gy are
presented.

Figure 1 Chromosomal aberrations in human lymphocytes after 14 Gy beta irradiation:
A. a) DM; b) dicentrics; c) ring chromosomes; d) acentric fragments;
B. Complex chromosomal interchange in subject no. 5 after 14 Gy irradiation (x 100)

The results showed that the number of rearrangements is increasing with the irradiation
dose (from 438 abnormalities at 7 Gy to 677 at 14 Gy) (Table no. 1), excepting the double
minutes, all of this rearrangements being present, with different frequency, at all the subjects
independently of the irradiation doses.

POJOGA (USURELU) MARIA DANIELA, MANAILA ELENA, CONSTANTIN NICOLETA,
DUTA CORNESCU GEORGIANA, CIMPONERIU DANUT, SIMON-GRUITA ALEXANDRA

8606 Romanian Biotechnological Letters, Vol. 18, No. 5, 2013 Table 1 Type and frequence of chromosomal rearrangements at 7 and 14 Gy beta radiation doses
7 Gy
Subjects Chromosomal
interchanges Dicentric
chromosomes Acentric
fragments Ring
chromosomes Double
minutes Endomithosis
N* F** N* F** N* F** N* F** N* F** N* F**
1 15 0.43 9 0.26 5 0.14 4 0.11 1 0.03 1 0.03
2 22 0.27 20 0.25 23 0.28 14 0.17 2 0.02 0 0.00
3 30 0.34 32 0.36 22 0.25 4 0.05 0 0.00 0 0.00
4 19 0.43 13 0.30 5 0.11 6 0.14 0 0.00 1 0.02
5 25 0.33 32 0.42 13 0.17 5 0.07 1 0.01 0 0.00
6 27 0.35 22 0.28 11 0.14 10 0.13 4 0.05 4 0.05
7 10 0.28 16 0.44 3 0.08 5 0.14 0 0.00 2 0.06
14 Gy
1 22 0.36 11 0.18 12 0.20 12 0.20 2 0.03 2 0.03
2 31 0.28 25 0.23 30 0.27 21 0.19 3 0.03 1 0.01
3 56 0.42 33 0.25 34 0.26 8 0.06 1 0.01 1 0.01
4 22 0.34 17 0.26 12 0.18 9 0.14 2 0.03 3 0.05
5 39 0.34 44 0.38 21 0.18 7 0.06 3 0.03 2 0.02
6 45 0.36 37 0.29 25 0.20 10 0.08 5 0.04 4 0.03
7 17 0.26 22 0.34 7 0.11 15 0.23 2 0.03 2 0.03

* N= number of chromosomal aberrations ** F= frequency of chromosomal aberrations

Overall, the most represented abnormalities produced by beta irradiation are
chromosomal interchanges (0,34 at 7 and 14 Gy), dicentric chromosomes (0,33 at 7 Gy and
0,28 at 14 Gy) followed by acentric fragments (0,19 at 7 Gy and 0,21 at 14 Gy) and ring chromosomes (0,11 at 7 Gy and 0,12 at 14 Gy). Double minutes structures and endomithosis
phenomenon were observed too, but their frequen cy was very low (0,03 and respectively 0,02
at both doses).
The different types of rearrangements varied from one individual to another. For
example, chromosomal interchange frequency ranged from 0,27 to 0,43 at 7 Gy, similar with the values observed at 14 Gy (from 0,26 to 0,42). Notably, none of the chromosomal
aberrations can be specifically correlated with the increasing of the irradiation dose, although
at 14 Gy is observed a slightly higher frequency of the ring chromosomes, acentrics and DM
compared to 7 Gy.

FISH analysis
The fluorescence signal for telomere repeat s was detected in 98% of samples before
irradiation (Figure 2. A). After irradiation, part ial or total lack of the signal was noticed for
chromosomes involved in complex interchanges or telomere-to-telomere fusions, and, also at
one end of the acentric fragments and at the end of the ring chromosomes (Figure 2. B, C, D).
A higher number o telomere signals was scored for polyploid cells and endomithosis (Figure
2. E, F).

Chromosome aberrations, telomere and telomerase dysfunction after beta irradiation in human lymphocytes

Romanian Biotechnological Letters, Vol. 18, No. 5, 2013 8607

Figure 2. Human telomeres on normal and 14 Gy irradiated probes
A. Normal chromosomes in subject no. 5 (with Dako PNA probes); B. Complex interchange in subject no. 5
(with MP Biomedical probes); C. Acentric fragments in subject no. 3 (with Dako PNA probes); D. Ring
chromosomes in subject no. 3 (with Dako PNA probes); E. Endomithosis in subject no. 3 (with Dako PNA
probes); F. Polyploidy in subject no. 5 (with Dako PNA probes) (x 60)

Further, using FISH method the gene coding the RNA component of telomerase
(hTERC ) and the gene coding the reverse transcriptase component ( hTERT ) were investigated.
In both cases, four red signals in the corresponding chromosomes (chromosomes 3,
respectively 5) in the control samples (non i rradiated) were observed (Figure 3. A, B).
Similar results as in the case of controls were obtained at the 7 Gy exposures, nor
amplification phenomenon for hTERC or hTERT genes being detected. In case of hTERC
gene, in subjects no. 3 and no. 5, after exposure to 14 Gy, eight fluorescent red signals were
scored in those cells showing polyploidy or endomithosis as a response to irradiation (Figure
3. C, D).
Unlike those multiple signals above-mentioned, in the subject no. 5 for hTERC gene,
after 14 Gy irradiation, six red fluorescence signals were determined in 1% of the analyzed
metaphases, suggesting a gene amplification phenomenon (Figure 3. E).
In case of the gene encoding the reverse transcriptase compound ( hTERT ), the specific
red signal was detected on 5p. At 14 Gy ir radiation, different chromosomal aberrations
(polyploidy or endomithosis), as in the case of hTERC , generates changes in the number of
fluorescent signals. Signals repr esenting gene amplification for hTERT were also detected in
subject no. 5 in 2% of the analyzed metaphases. The amplified signal was scored once on a
dicentric chromosome (Figure 3. F) and the second time on a chromosome belonging to group
D (Figure 3. G).

POJOGA (USURELU) MARIA DANIELA, MANAILA ELENA, CONSTANTIN NICOLETA,
DUTA CORNESCU GEORGIANA, CIMPONERIU DANUT, SIMON-GRUITA ALEXANDRA

8608 Romanian Biotechnological Letters, Vol. 18, No. 5, 2013

Figure 3 hTERC and hTERT genes in human lymphocytes (normal and 14 Gy irradiated probes)
A. hTERC gene in normal metaphases (subject no. 5); B. hTERT gene in normal metaphases (subject no. 5); C.
Endomithosis in subject no. 5 ( hTERC gene); D. Polyploidy in subject no.5 ( hTERC gene);
E. hTERC amplification in subject no. 5; F., G. hTERT amplification in subject no. 5. (x 60)

It is well known that ionizing radiations are clastogenic, causing chromosomal
aberrations, whose incidence depends on radiation type and dose [15, 16, 17]. The complex
chromosomal interchanges, acentric fragments and dicentric chromosomes were the most obvious and frequent chromosomal rearrangemen ts observed in this study after beta
irradiation. These types of abnormalities are tr aditional used in biodosimetric studies and for
monitoring the people accidentally exposed to radiations. The dicent ric presence is a good
indicator of irradiation, especially when analys is is made within a short-period of time after
exposure [18].
Many of these chromosomal aberrations are a consequence of the damaged telomeres
after the exposure to beta radiations. In the irradiated cells, the eroded telomeres are often
improper recognized as double-break strands by the DNA repair machinery, generating
broken ends and determining chromosomal rearrangements such as telomere-telomere,

Chromosome aberrations, telomere and telomerase dysfunction after beta irradiation in human lymphocytes

Romanian Biotechnological Letters, Vol. 18, No. 5, 2013 8609telomere-DSB, DSB-DSB [19]. Thus, dicentric and ring chromosomes, well documented in
this study, are the result of the genomic instability due to the telomere radio-sensitivity [20].
This experiment showed, by the extent of the chromosomal damages, that the cultivated
cells have different reactions to ionizi ng radiation. It is evident that the radiotherapy used for
the cancer treatment induces genomic instability [21] not only on tumoral cells, but also on
normal adjacent cells, contributing to the secondary malignancies in long-time cancer
survivors [22]. Thus, the radiation bystander effect can lead to DNA damage, enhanced
mutagenesis, and ultimately oncogenic transformation in cells not directly exposed to
irradiation treatment [21, 23, 24, 25, 26, 27].
The inherited and induced chromosomal instability represents a fragile bridge between
genome integrity mechanisms and tumorigenesis [28], telomeres playing an important role in
both processes. FISH technique is an approp riate method for the investigation of the
relationship between telomeres, i rradiation effects and telomerase activity, such studies being
very important, both from theoretical point of view and from practical medicine.
In this study, the FISH analysis results were in agreement with other studies in which
the length variability at chromosomal ends or the lack of telomeric sequences at end-to-end
junctions was observed after beta irradiation [2 9, 1, 30]. However, the method used in this
study identified only the presence/absence of telomeric end, without providing information
about their length. The variation of repeat nu mber is important for the estimation of the
dysfunction extent at telomere level, so, in a future study, this aspect will be investigated.
In fact, many environmental factors, includi ng the exposure to radiation can accelerate
telomere shortening, increasing the risk for numerous cancers [31]. Telomerase is one of the
most important players in telomere maintenance, in the DNA repair process, particularly of
DSB, frequently observed after ionizing radiat ion exposure [19]. Several studies have
revealed that telomerase activity is almost absent in normal somatic cells. However, a low
level of telomerase activity has been discovered in mitotic active cells, including skin cell, lymphocytes, and endometrial cells. Also, stem cells express telomerase to maintain telomere
length throughout their life cycle, and, about 90% of cancer cells contain high levels of
telomerase activity [32, 33].
Because many studies reported an increased te lomerase activity in irradiated cells, [34,
35, 36, 37, 38, 39, 40], the response of hTERC and hTERT genes to beta rays was also
investigated by FISH. The hTERC gene amplification revealed by those six fluorescent
signals found apart from that recorded in polyploid cells or endomitosis suggests a possible
3q26 locus-specific amplification rather than a polisomy of chromosome 3. Similar situation
was reported in high-grade squamous intraepithelial neoplasia and invasive carcinoma, where
the presence of extra copies of the hTERC gene is relatively common [41].
Another amplification phenomenon was noticed for hTERT gene after beta irradiation,
confirming once more the telomerase activity in the cell surviving post irradiation. This
amplification was also reported on human lung and breast cancer [42, 43].
For better understanding of the relationship between telomere damage and telomerase
activity in normal cell exposed to ionizing radiation, the hTERC and hTERT genes must be
further investigated using other methods such as : telomere repeat amplification protocol, real
time PCR or monochrome multiplex quantitative PCR [44, 45, 46].

Conclusion

Classic cytogenetics and FISH technique proved to be inexpensive, rapid and reliable
methods for the initial evaluation of telomeres and telomerase activity in lymphocyte cells
before and after irradiation. This type of analysis could enter in clinical practice as a screening

POJOGA (USURELU) MARIA DANIELA, MANAILA ELENA, CONSTANTIN NICOLETA,
DUTA CORNESCU GEORGIANA, CIMPONERIU DANUT, SIMON-GRUITA ALEXANDRA

8610 Romanian Biotechnological Letters, Vol. 18, No. 5, 2013 of radiotherapy effects in patients with differe nt cancers, especially because little is known
about the damages produced by beta radiations on the cells adjacent to the malignant tumors.

Acknowledgements
In memoriam Prof. Dr. Lucian Gavrila.

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