# Analysis of Gene Expression in Normal and Cancer Cells Exposed toγ-Radiation

Authors: M. Ahmad Chaudhry
Journal: Journal of Biomedicine and Biotechnology
 (2008-02-05)

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## Abstract

The expression of many genes is modulated after exposure to ionizing radiation. Identification of specific genes may allow the determination of pathways important in radiation responses. We previously identified modulation of the expression of several genes in response to ionizing radiation treatment. In the present study, we monitored the expression of RGS1, CC3, THBS1, vWF, MADH7, and a novel gene encoding a secreted protein in irradiated Jurkat, TK6, HeLa, and HFL1 cells. The RGS1 is involved in G-protein signaling pathway, CC3 belongs to the complement system, THBS1 is a component of the extracellular matrix, vWF takes part in blood coagulation, and MADH7 is a member of the TGF-β signal transduction pathway. Our objective was to find similarities and differences in the expression of these genes in ionizing radiation-exposed diverse cell types. RGS1 was downregulated in Jurkat cells but was upregulated in TK6 and HFL1 cells. The expression of CC3 was repressed in Jurkat and HFL1 cells but was induced in TK6 and HeLa cells. THBS1 was downregulated in irradiated TK6 and HFL1 cells. vWF was induced in radiation-exposed HeLa cells, but its expression was downregulated in Jurkat cells. The expression of MADH7 was induced in all the cell types examined. These results indicate cell specific modulation of gene expression and suggest the involvement of different pathways in cellular response to radiation treatment in different cells.


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## Body

## 1. Introduction

Ionizing radiation (IR) is known to
potentially interfere with cellular functions at all levels of cell
organization. The path from
irradiation of the cells to the induction of biological effects comprises
several complex steps provoking alterations to a variety of cellular
components. The radiation-induced cellular effects include sister-chromatid
exchanges, chromosome aberrations, apoptosis, micronucleation, transformation,
mutations, and gene expression alterations. The critical DNA lesions generated by IR
are double-strand breaks, which are repaired by homologous recombination and
nonhomologous end joining. Depending on the structure of broken DNA ends, some
of the single-strand breaks may be repaired by long-patch base excision repair (BER) pathway. Oxidative DNA
damage is taken care of by short-patch BER [1]. The accuracy of DNA repair depends on the
complexity of the DNA lesion [2] and on the fidelity of the DNA repair
machinery itself.The
cellular response to IR is mediated via genes that control complex regulatory
pathways. In response to IR exposure, several signal transduction pathways
(e.g., ERK1/2, JNK, p38, and ATM) and transcription factors (e.g., AP1, NFκB,
GADD153, and p53) are activated [3]. The ionizing
radiation-induced stress response is very complex and involves altered
expression of many genes. Ionizing
radiation-induced alterations in the expression of many genes have been
reported. In addition to classical radiation responsive genes, new studies have
discovered the involvement of other genes as well. Apo1, nuclease sensitive
element binding protein 1, syntaxin (a membrane integrated protein involved in
exocytosis), cyclin G1, hNOP56, paraoxonase (hydrolyze oxidized lipids), and
glutathione peroxidase were overexpressed after irradiation [4]. Activating transcription
factor 3 (ATF3) was induced after X-irradiation [5]. IR exposure results in the induction of clusterin (associated with apoptosis) gene expression [6]. CXC chemokines and other secretory products (secretogranin II, thrombospondin
type I domain containing 2, amphiregulin, and interleukin-6) have shown to be
modulated by IR [7]. The hematopoietic zinc finger protein, Hzf, was shown to be
upregulated in response to genotoxic stress [8]. The isolation and characterization of a novel
ionizing radiation-induced gene, apoptosis enhancing nuclease (AEN) has
been described. AEN protein acts as DNase and enhances apoptosis following
irradiation [9]. The modulation of connexin43
(associated with gap junctions) expression has been shown after exposure to IR [10]. The expression of chromatin
assembly factor 1 (CAF-1) and proliferating cell nuclear antigen (PCNA)
is dramatically induced as a result of DNA damage by the radiomimetic drug
bleocin (a bleomycin compound) or by IR [11]. The expression of inducible
nitric oxide synthase (iNOS) in X-ray irradiated mouse skin at both the
mRNA and protein levels was recently published [12]. The expression and
activation of MMP -2, -3, -9, -14 are induced in rat colon after abdominal
X-irradiation [13].Various
studies have examined the radiation-induced gene expression profiles in diverse
cell types. Microarray approach has identified genes regulated in response to IR
[14–18]. The
differential expression of apoptosis-related BAX and BCL-XL [19] and a dose-response relationship for the induction of CDKN1A,
MDM2, GADD45A, ATF3, and BAX has been reported [20]. Early gene expression profile in mouse brain after exposure to IR identified
change in the expression of JAK3, DFFB, NSEP1, and  TERF1 genes [21]. IR was shown to enhance the
expression of the nonsteroidal anti-inflammatory drug-activated gene (NAG1)
[22]. Microarray analysis of the
transcriptional response to single or multiple doses of IR has also been
reported [23]. Based on microarray studies,
we previously identified the induction of complement component 3 (CC3),
von Willebrand factor (vWF), mothers against decapentaplegic homolog 7
(MADH7), regulator of G-protein signaling 1 (RGS1),
thrombospondin 1 (THBS1), and a novel gene encoding a secreted protein (Sec
Pro) genes in irradiated cells. In the present study, we examined the
expression of CC3, vWF, MADH7, RGS1, THBS1, and  Sec Pro genes in irradiated normal and tumor cells of
various types. Our aim was to investigate variations in the response of these
genes in diverse cell types and to identify any genes that are universally
induced after exposure to ionizing radiation.


## 2. Materials and Methods

### 2.1. Cell Culture

Human normal lung fibroblast cell line HFL1 was
purchased from American Type Culture Collection (ATCC) (Manassas, Va, USA).
These cells were cultured as a monolayer in Ham's F12 K medium with 2 mM
L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate (Invitrogen,
Carlsbad, Calif, USA) supplemented with 10% fetal bovine serum (Invitrogen) and
antibiotics (50 units/mL penicillin and 50μg/mL streptomycin). Cells were maintained at a
density of 2 × 105 cells per mL after trypsin
treatment of adherent cells. HeLa cells (kindly provided by Dr. Markus Thali)
were cultured at 37°C
in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) with 10% fetal calf serum,
1% penicillin, and streptomycin. Subconfluent cultures were trypsinized in 0.25% trypsin,
0.03% EDTA solution to obtain a density of 2 × 105cells per mL, and grown as a monolayer.The TK6 human B lymphoblast cells (kindly provided by
Dr. Susan Wallace) were grown in suspension in RPMI 1640 medium
(Cellgro/Mediatech Inc.,
Herndon, Va, USA) supplemented with 10% fetal calf serum (Invitrogen, Grand
Island, NY, USA) at 37°C in 5% CO2 and 100% humidity. Cell densities were maintained at 1–10×105  cells/mL. Lymphoblast acute
T cell leukemia cell line Jurkat (kindly provided by Dr. Markus Thali)
was grown in suspension in RPMI 1640 medium supplemented with 2 mM L-glutamine adjusted
to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM
sodium pyruvate. The cell culture medium was supplemented with 10% fetal
bovine serum. Cultures were maintained in suspension at a cell concentration
between 1 × 105 and 1 × 106   viable cells/mL and the cell
density was not allowed to exceed 106 cells/mL.


### 2.2. Ionizing Radiation Treatment

Exponentially
growing cells were irradiated with 2 Gy ofγ-radiation using a 137Cs
irradiator (Nordion International ISO 1000, Model B) at dose rate of 0.12 Gy/s
at the Red Cross facility (Burlington, Vt, USA). The control cells did not
receive any radiation and were mock irradiated. The irradiated and control
cells were incubated at 37°C for various periods of times before isolating
total RNA.


### 2.3. RNA Isolation and cDNA Synthesis

Total RNA from cells was
isolated by using Trizol reagent (Invitrogen) according to the
protocol provided by the supplier. The concentration of samples was determined
by measuring the OD
at 260 nm wavelength in
a nanodrop spectrophotometer. The quality of the RNA was assessed on
Bioanalyzer 2100 (Agilent) with a nanochip. 10μg of total RNA was converted to cDNA
with Superscript II reverse transcriptase (Invitrogen) and an oligo-(dT)12–18 primer (Invitrogen) according to the manufacturer’s recommendations. 1 μL of the cDNA was used as
a template for amplification in the PCR.


### 2.4. Relative Quantitative RT-PCR

Relative quantitative
RT-PCR was performed using a commercially available kit (Ambion Inc., Calif, USA).
This procedure modulates the amplification
efficiency of a PCR template without affecting the performance of other targets
in a multiplex PCR. The multiplex RT-PCR usesβ-actin as an endogenous standard
combined with Ambion’s Competimer Technology. The
actin competimers are modified at their 3′ ends to block extension by DNA
polymerase. By mixing actin primers with actin competimers, the PCR
amplification efficiency of actin can be reduced without the primers becoming limited
and without loss of relative quantification. The actin primer pair and actin
competimer are supplied at a concentration of 5 μM. The β-actin primer: competimer mixtures were prepared as three different
ratios of 1 : 9,
2 : 8, and 3 : 7, and were examined for multiplex PCR with gene specific primers
to establish the optimum concentration. A ratio of 2 : 8 actin primers to
competimers is appropriate for most genes. A ratio of 1 : 9 is required for rare
messages, and a ratio of 3 : 7 works best for relatively abundant messages. A primer: competimer ratio of
2 : 8 was found to be optimum for all the genes amplified in a multiplex format
in this study. The
PCR was set up using first-strand cDNA, gene specific primers, and β-actin primer: competimer mixtures and Platinum Taq DNA polymerase (Invitrogen) according
to manufacturer’s recommendations. All the PCR primers used in this
study were designed with the Primer 3 software and were synthesized and
purified by Operon Biotechnologies. The sequence of all the primers is shown
in Table 1. The PCR
amplification products were electrophoresed in 1% agarose gels stained with
ethidium bromide (0.5 μg/mL). The gel images were captured with a Kodak
digital imaging system and quantified with NIH Image software. The resultant data was
plotted to show the alterations in the expression of these genes after exposure
to ionizing radiation.Table 1
RT-PCR primers for various genes.
GeneAccessionLeft primerRight primerProduct sizeG-protein signaling 1 (RGSI)NM_002922TGCCAGGAATGTTCTTCTCTCTATTAGCCTGCAGGTCATTTAGA580Von Willebrand factor (vWF)NM_000552.2GTCCGAGGCTGAGTTTGAAGTAACGATCTCGTCCCTTTGC500Mothers against the decapentaplegic homolog 7 (MADH7)NM_005904.1CCAACTGCAGACTGTCCAGAGTCGAAAGCCTTGATGGAGA450The complement component 3 (CC3)NM_000064.1GGAAAAGGAGGATGGAAAGCACCCAAAGACAACCATGCTC497Thrombospondin 1 (THBS1)NM_003246.2GGAGACAACAGCGTGTTTGAGCCAGGTCTCTGGTGAAGAC500Secreted protein (Sec Pro)BF440021.1GCTTTTCACCGAGGAGGAGTGCAGGAGATTCAGCACCTA393


## 2.1. Cell Culture

Human normal lung fibroblast cell line HFL1 was
purchased from American Type Culture Collection (ATCC) (Manassas, Va, USA).
These cells were cultured as a monolayer in Ham's F12 K medium with 2 mM
L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate (Invitrogen,
Carlsbad, Calif, USA) supplemented with 10% fetal bovine serum (Invitrogen) and
antibiotics (50 units/mL penicillin and 50μg/mL streptomycin). Cells were maintained at a
density of 2 × 105 cells per mL after trypsin
treatment of adherent cells. HeLa cells (kindly provided by Dr. Markus Thali)
were cultured at 37°C
in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) with 10% fetal calf serum,
1% penicillin, and streptomycin. Subconfluent cultures were trypsinized in 0.25% trypsin,
0.03% EDTA solution to obtain a density of 2 × 105cells per mL, and grown as a monolayer.The TK6 human B lymphoblast cells (kindly provided by
Dr. Susan Wallace) were grown in suspension in RPMI 1640 medium
(Cellgro/Mediatech Inc.,
Herndon, Va, USA) supplemented with 10% fetal calf serum (Invitrogen, Grand
Island, NY, USA) at 37°C in 5% CO2 and 100% humidity. Cell densities were maintained at 1–10×105  cells/mL. Lymphoblast acute
T cell leukemia cell line Jurkat (kindly provided by Dr. Markus Thali)
was grown in suspension in RPMI 1640 medium supplemented with 2 mM L-glutamine adjusted
to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM
sodium pyruvate. The cell culture medium was supplemented with 10% fetal
bovine serum. Cultures were maintained in suspension at a cell concentration
between 1 × 105 and 1 × 106   viable cells/mL and the cell
density was not allowed to exceed 106 cells/mL.


## 2.2. Ionizing Radiation Treatment

Exponentially
growing cells were irradiated with 2 Gy ofγ-radiation using a 137Cs
irradiator (Nordion International ISO 1000, Model B) at dose rate of 0.12 Gy/s
at the Red Cross facility (Burlington, Vt, USA). The control cells did not
receive any radiation and were mock irradiated. The irradiated and control
cells were incubated at 37°C for various periods of times before isolating
total RNA.


## 2.3. RNA Isolation and cDNA Synthesis

Total RNA from cells was
isolated by using Trizol reagent (Invitrogen) according to the
protocol provided by the supplier. The concentration of samples was determined
by measuring the OD
at 260 nm wavelength in
a nanodrop spectrophotometer. The quality of the RNA was assessed on
Bioanalyzer 2100 (Agilent) with a nanochip. 10μg of total RNA was converted to cDNA
with Superscript II reverse transcriptase (Invitrogen) and an oligo-(dT)12–18 primer (Invitrogen) according to the manufacturer’s recommendations. 1 μL of the cDNA was used as
a template for amplification in the PCR.


## 2.4. Relative Quantitative RT-PCR

Relative quantitative
RT-PCR was performed using a commercially available kit (Ambion Inc., Calif, USA).
This procedure modulates the amplification
efficiency of a PCR template without affecting the performance of other targets
in a multiplex PCR. The multiplex RT-PCR usesβ-actin as an endogenous standard
combined with Ambion’s Competimer Technology. The
actin competimers are modified at their 3′ ends to block extension by DNA
polymerase. By mixing actin primers with actin competimers, the PCR
amplification efficiency of actin can be reduced without the primers becoming limited
and without loss of relative quantification. The actin primer pair and actin
competimer are supplied at a concentration of 5 μM. The β-actin primer: competimer mixtures were prepared as three different
ratios of 1 : 9,
2 : 8, and 3 : 7, and were examined for multiplex PCR with gene specific primers
to establish the optimum concentration. A ratio of 2 : 8 actin primers to
competimers is appropriate for most genes. A ratio of 1 : 9 is required for rare
messages, and a ratio of 3 : 7 works best for relatively abundant messages. A primer: competimer ratio of
2 : 8 was found to be optimum for all the genes amplified in a multiplex format
in this study. The
PCR was set up using first-strand cDNA, gene specific primers, and β-actin primer: competimer mixtures and Platinum Taq DNA polymerase (Invitrogen) according
to manufacturer’s recommendations. All the PCR primers used in this
study were designed with the Primer 3 software and were synthesized and
purified by Operon Biotechnologies. The sequence of all the primers is shown
in Table 1. The PCR
amplification products were electrophoresed in 1% agarose gels stained with
ethidium bromide (0.5 μg/mL). The gel images were captured with a Kodak
digital imaging system and quantified with NIH Image software. The resultant data was
plotted to show the alterations in the expression of these genes after exposure
to ionizing radiation.Table 1
RT-PCR primers for various genes.
GeneAccessionLeft primerRight primerProduct sizeG-protein signaling 1 (RGSI)NM_002922TGCCAGGAATGTTCTTCTCTCTATTAGCCTGCAGGTCATTTAGA580Von Willebrand factor (vWF)NM_000552.2GTCCGAGGCTGAGTTTGAAGTAACGATCTCGTCCCTTTGC500Mothers against the decapentaplegic homolog 7 (MADH7)NM_005904.1CCAACTGCAGACTGTCCAGAGTCGAAAGCCTTGATGGAGA450The complement component 3 (CC3)NM_000064.1GGAAAAGGAGGATGGAAAGCACCCAAAGACAACCATGCTC497Thrombospondin 1 (THBS1)NM_003246.2GGAGACAACAGCGTGTTTGAGCCAGGTCTCTGGTGAAGAC500Secreted protein (Sec Pro)BF440021.1GCTTTTCACCGAGGAGGAGTGCAGGAGATTCAGCACCTA393


## 3. Results

Exponentially growing TK6, Jurkat, HeLa, and HFL1
cells were irradiated with 2 Gy ofγ-radiation and after incubation at 37°C
for 4-, 8-, 12-, and 24-hour, total RNA was isolated and converted to cDNA. We
examined the differential gene expression of six genes coding for CC3, vWF, MADH7,
RGS1, THBS1, and a “Sec Pro” in Jurkat, TK6, HeLa, and HFL1 cells. We employed relative
quantitative RT-PCR to examine differential gene expression. The relative
quantitative RT-PCR in a multiplex format was done by using actin/modified
actin primers (competimers) in combination with gene-specific primers.
Competimers are specially modified primers of the same sequence as the normal
β-actin primers that cannot be extended. By adjusting the ratio of β-actin
competimers to normal β-actin mRNA primers, we were able to attenuate the
signal for β-actin mRNA to the level of rare messages. In separate experiments,
all cDNAs corresponding
to the genes under investigation were amplified with 1 : 9, 2 : 8, and 3 : 7 ratios of actin primers to
actin competimers in a multiplex format (data not shown). The ratio of 2 : 8
resulted in optimum amplification of all the genes and actin cDNAs.We first monitored the radiation-induced expression of RGS1, CC3,
THBS1, vWF, MADH7, and Sec Pro genes in Jurkat cells (Figure1). The overall
expression of RGS and CC3 was lower in these cells as
compared to the expression levels of THBS1, vWF, MADH7, and Sec Pro. The
expression of RGS1 and CC3 remained unaltered up to 12 hours (Figure 1) but was
downregulated at 24-hour time point. A similar downregulation of vWF at the 24-hour
time point was also seen. The most prominent gene induction was seen for MADH7,
which was induced after 4 hours of radiation exposure of Jurkat cells and
remained upregulated up to 24 hours (Figure 1).Ionizing
radiation-induced gene expression in Jurkat cells. (a) The expression
ratios of various genes at 4-, 8-, 12-, and 24-hour time points compared to
unirradiated sham control. (b) Agarose gel images of relative quantative
RT-PCR for various genes. In all cases, the upper gel band represents gene-specific amplification and the lower
gel band indicates the relative amplification ofβ-actin internal standards.
(a)(b)The expression of variousgenes in irradiated
TK6 cells at various time points is shown in Figure2. RGS1, CC3, and Sec Pro were all induced after radiation treatment. These three
genes were induced at the 4-hour time point and remained upregulated till 24
hours. THBS1 showed two peeks of gene downregulation, one at 4 hours and
the other at 12 hours. An opposite effect was seen for the expression of MADH7 gene where an upregulation was seen at 4 and 12 hours after irradiation while
the expression of this gene returned to normal levels at 8 and 24 hours. The
overall expression of vWF was lower as compared to all the other genes
and did not appear to be altered after radiation treatment (Figure 2).Figure 2
The gene expression in ionizing radiation treated TK6 cells.
The expression ratios for various genes at 4, 8, 12, and 24 hours after radiation exposure were
determined and compared to the control mock irradiated cells.Figure3 shows the gene expression pattern in HeLa
cells after irradiation. The CC3 gene was induced after irradiation and
its expression level peeked at 12 hours. After this time point, a decline in
the expression of this gene was observed. A similar pattern of gene induction
was seen for vWF where the expression of this gene reached the maximum
level at 12 hours before showing a decline at the 24-hour time point. In case
of Sec Pro, the expression was the highest after at 8 hours after
irradiation. An interesting pattern of gene expression was seen for  MADH7,
where an upregulation was seen at 4-hour postirradiation and then a dramatic
gene repression was seen at all the other time points, suggesting a complete
shut down of this gene.Figure 3
The expression of RGS1, CC3, THBS1, vWF, MADH7, and Sec Pro at various time points in irradiated HeLa cells.Finally, we examined the gene expression in
irradiated HFL1 cells (Figure4).  RGS1 was induced in these cells after
8 hours of radiation exposure. The expression level of CC3 did not
change up to 8-hour postirradiation and then started to get downregulated. THBS1 gene was first repressed and later returned to normal levels at 12-hour time
point. MADH7 was upregulated at 8 hours, and its expression level
continued to be increased until 24 hours. vWF and  Sec Pro were
expressed at reduced levels in irradiated HFL1 cells.Figure 4
The expression of RGS1, CC3, THBS1, vWF, MADH7,
and Sec Pro at 4-, 8-, 12-, and 24-hour time points in irradiated HFL1
cells.


## 4. Discussion

IR
activates both pro- and antiproliferative signal transduction pathways, the
balance of which determines cell fate. The initiating and amplifying mechanisms
involved in the activation are poorly understood. The cellular response to IR is mediated via
genes that control complex regulatory pathways such as cell cycle progression,
apoptosis, or DNA repair. The relative contribution of changes in the
expression of these genes on signaling pathways is unknown. The mechanisms for
the survival of cells after IR exposure may involve the induction of DNA
repair, immediate early, cytokine, and growth factor genes. Cells respond to IR
with the activation of specific early and later response genes. The products of
early response genes may be responsible for regulating downstream genes that
are important in the adaptation of cells and tissues to radiation-induced
stress.We
investigated the radiation-induced gene expression in Jurkat, TK6, HeLa, and
HFL1 cell lines. Jurkat cells are p53 negative and TK6 cells are p53 positive.
Both of these cells have lymphoblast origin and are grown as suspension
cultures. On the other hand, HeLa (epithelial origin), and HFL1 (fibroblast
origin) cell lines are grown as adherent cultures. HeLa cells are p53 negative
while HFL1 cells are p53 positive. The examination of radiation-induced gene
expression in a variety of cell lines differing in p53 status or growth
conditions could provide information as signature pathways operating in all
cell types.A striking
observation was the induction of MADH7 in all the cell lines exposed to
ionizing radiation. MADH7 (also known as Smad7) belongs to the category
of Smad proteins. These proteins are signaling transducers downstream from TGF-β receptors [24]. Smad7 belongs to the
inhibitory Smads category of TGF-β signal transduction pathway and
prevents TGF-β signaling. Expression of TGF-β after radiation exposure is
well known [25]. It has been suggested that TGF-β is the master switch cytokine, which,
once activated after radiation, promotes a cascade of cellular
events. Escape from TGF-β-induced inhibition of growth and
proliferation may contribute to tumorigenesis. Our data suggests that MADH7 is
upregulated in all the cells examined in this study regardless of their p53
status.vWF was
induced in radiation-exposed HeLa cells (Figure3) but its expression was
downregulated in Jurkat cells (Figure 1). However, vWF is a glycoprotein
involved in blood coagulation. Increased amounts of vWF in blood plasma are
indicative of damaged endothelium. Ionizing irradiation damage to the
vasculature results in an increase in procoagulant activity of endothelial
cells, including elevated vWF secretion [26]. The induction of vWF in brain after IR exposure has been reported [27].RGS1 was
downregulated in Jurkat cells (Figure1) but was upregulated in TK6 (Figure 2)
and HFL1 cells (Figure 4). G-protein-coupled receptors (GPCRs) play a central
role in the regulation of cell communication and encompass a wide range of functions that
includes the rhodopsin-like GPCRs, the secretin-like GPCRs, the cAMP receptors,
the pheromone receptors, and the metabotropic glutamate receptor subfamilies as
well as nuclear receptors. A recent in vivo study examined the
effect of acute and fractionated low-dose radiation and sex differences in the
expression of the GPCRs in the murine muscle tissue [28]. Regulators of G-protein
signaling (RGS) play a critical role in GPCR signaling in mammalian cells and
could be involved in radiation-induced signal transduction pathways.The expression
of CC3 was repressed after exposure to IR in Jurkat cells (Figure1) and
HFL1 cells (Figure 4). On the contrary, this gene was induced in TK6 and HeLa
cells (Figures 2 and 3, resp.). The complement system has long been
regarded as consisting of proinflammatory proteins circulating in the peripheral blood and
tissue fluids. The complement system is an important mediator of natural
and acquired immunity. It consists of approximately 30 proteins that can
exhibit catalytic activity, function as regulators, or act as cellular surface receptors [29]. These components normally circulate in inactive forms and
are activated by the classical, alternative, or lectin pathways. CC3 plays a
central role in all three activation pathways where it exerts several important
biological roles. Different cell groups of immune system give different responses
in individuals exposed to long-term ionizing radiation. Recently, the effect of
occupational exposure to low levels of IR were investigated in selected indices
of cellular and humoral immunity in radiology workers and were found to be
weaker in radiation workers [30].The expression
of THBS1 was found to be downregulated in irradiated TK6 (Figure2) and
HFL1 cells (Figure 4). THBS1, a matricellular glycoprotein, is a
component of the extracellular matrix predominantly during active or subacute
processes [31]. THBS1 is released by
platelets during the formation of a hemostatic plug. In addition, macrophages,
monocytes, fibroblasts, vascular smooth muscle cells, tumor cells, and
endothelial cells also secrete THBS1 or their function is modulated by THBS. THBS1 is also an endogenous angiogenesis inhibitor. Our studies are
consistent with those of others showing THBS1 to be downregulated by ultraviolet
B light [32] and by IR [7].Many
studies have reported the modulation of gene expression in IR-treated
cells.  The results of such studies are
mixed and perhaps are not surprising, given the complexity of the response to
damage and the multitude of factors that contribute to the diversity of
cellular sensitivity. Additional difficulties to interpret data involve
variations in growth conditions of cells under investigation and a variety of IR
doses and quality employed. The ionizing radiation-induced gene expression
could be cell-type-specific. The expression of connexin43 after exposure to IR has shown to vary in
different cell lines [10]. The variation in the response has not been fully
explained and may be related to intrinsic biologic capability of the cells. The
investigation of the regulation of the gene expression in multiple cells of
various origins exposed to IR is necessary for an understanding of the
generality of gene expression response.The
identification of radiation responsive genes in various cell types will permit
an understanding of the molecular mechanisms underlying radiation-induced
physiological responses and an ability to predict the radiation susceptibility
of normal tissues in radiotherapy patients. The ability to detect altered gene
expression has prompted a search for molecular markers that could predict tumor
response to therapy. Advances in the knowledge of how cells respond to IR will
provide opportunities for the development of new approaches that selectively
enhance radiotherapy of tumors. Additionally, the availability of biomarkers to
monitor potentially exposed individuals after a radiological accident would be
extremely valuable. The investigation of gene expression after exposure to IR could serve as a potential molecular
marker for such biodosimetry.



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*Source: 541678-2008-03-18.xml*