OXIDATIVE STRESS INDUCED ALTERATIONS OF GENE
EXPRESSION IN HUMAN FIBROBLASTS HARBORING A8344G
MITOCHONDRIAL DNA MUTATION
Y.S. Ma1, C.Y. Lu1, Y.C. Chen1, C.Y. Liu1, T. Yang2, Y.H. Wei1
1
Department of Biochemistry and Center for Cellular and Molecular Biology,
National Yang-Ming University, Taipei, Taiwan 112, 2CAG Sector of Agilent
Technologies, Taiwan, Ltd., Taipei, Taiwan 105
ABSTRACT
The effect on gene expression of MERRF (myoclonic epilepsy with ragged-red
fibers)-associated A8344G mutation of mtDNA was investigated by Agilent’s human
1 cDNA microarray and RT-PCR. The activation of transcriptional responses, such as
the induction of inflammation-related proteins, matrix metalloproteases, and markers
of oxidative stress were observed in the patient’s fibroblasts. By contrast, the gene
expression of cytoskeleton-related proteins, extracellular matrix proteins, and
translation-related proteins were all down-regulated. We also found that the mRNA
and activity levels of Mn-SOD, but not catalase and glutathione peroxidase, were
significantly increased in skin fibroblasts of the MERRF patient. The intracellular
level of hydrogen peroxide was increased in skin fibroblasts of the MERRF patient as
compared to those of her family members and age-matched controls. As a result,
ROS may not be efficiently removed and in turn elicit an elevation of oxidative stress
in skin fibroblasts of the MERRF patient. Our data suggest that the alterations of
gene expression may represent the response of the MERRF patient’s fibroblasts to the
increased oxidative stress. Taken together, the changes in gene expression imply that
inflammation, disruption of mitochondrial reticulum together with degradation of
muscle proteins and oxidative damage to tissues are all involved in the
pathophysiology of MERRF syndrome.
AIM OF STUDY
To unravel the genetic and epigenetic factors involved in the phenotypic
heterogeneity of skin fibroblasts harboring the A8344G mtDNA mutation.
MATERIALS AND METHODS
Fig. 1. The pedigree of the MERRF family examined in this study. (A). The
patient (IV-1) was a fifteen-year-old female with MERRF syndrome but her younger
sister (IV-2) and brother (IV-3) were healthy at the time their skin samples were
collected. (B). The proportion of mutant mtDNA of each established skin fibroblasts
was determined by PCR-RFLP and analyzed by an Agilent 2100 Bioanalyzer (Lu et al.
Clin. Chim. Acta 318: 97-105, 2002). By introducing a mismatched primer, only the
PCR products amplified from mutant mtDNA as a template were recognized by Nae I
to produce two short restriction fragments of 197 and 26 bp. B, H, and S represent
DNA samples purified from whole blood cells, hair follicle cells, and skin biopsy,
respectively.
Fig. 2. Analysis of differentially expressed genes between the skin fibroblasts
from a MERRF patient and an age-matched healthy subject by a cDNA
microarray system. Gene expression profiles of these fibroblasts were analyzed by
using Agilent’s Human 1 cDNA Microarray Kits (Agilent Technologies, Ltd., CA)
that include 12,814 human cDNA clones on an array. The cDNA probes will be
made from 20 g of purified total RNA with SuperScript II RT (Life Technologies,
Inc., Bethesda, MD) to incorporate the Cy3 or Cy5 dye (NEN). Following
purification with the QIAquick spin columns, the purified labeled cDNA probes were
concentrated under vacuum in a rotary desiccator until dry. For each microarray to
be hybridized, a Cy3-/Cy5-labeled cDNA sample was suspended in deposition
hybridization buffer and mixed well with deposition control targets and Cot-1 DNA.
After denaturation at 98°C for 2 min, the cDNA probe was transferred to microarray
slide, which was then placed onto the base of a hybridization chamber and incubated
at 65ºC overnight. The slides was washed sequentially in 2 X SSC, 0.01% SDS; 0.5
X SSC, 0.01% SDS and finally in 0.06 X SSC before subjecting to scan for image
analysis using the Agilent's dual-laser Microarray Scanner (G2565AA) combined
with Feature Extraction Data Analysis Software (Agilent Technologies, Ltd.).
Fig. 3. A comparison between microarray and RT-PCR analysis for several
differentially expressed genes between the fibroblasts from normal subjects and
MERRF Chen patient. Total RNA were isolated from skin fibroblasts of MERRF
patients and age-matched healthy subjects. The cDNA from each of the RNA
samples was analyzed by RT-PCR with each gene specific primer pair and quantitated
by an Agilent 2100 bioanalyzer. The expression levels of these genes were measured
in each run using GAPDH as an internal standard. The results were shown in Table
3.Abbreviation: IL 8, Interleukin 8; CCIS, Complement Component 1s; CC3,
Complement Component 3; IFIT1, Interferon Induced Transmembrane Protein 1; IFIP,
Interferon, -inducible Protein; IFIP27, Interferon, -inducible Protein 27; IFIPTI,
Interferon-induced Protein with Tetratricopeptide repeats 1.
Table 1. Clinical features of patients with MERRF syndrome.
Patient
IV-1
M1
M2
Age / Sex
Age of onset
Myoclonic epilepsy
15/F
12
＋＋
18/F
11
＋
14/F
10
－
Muscle atrophy
＋＋＋
＋
＋
Cerebellar dysfunction: ataxia
Mental retardation
Ragged-red fibers
MtDNA mutation in fibroblasts
A8344G mutation
＋＋
＋
＋
＋
－
＋
＋
＋
＋
＋
＋
＋
84
86
55
Proportion of the mutant mtDNA (%)
IV-1is the patient of the pedigree examined in Figure 1. M1 and M2 are the two
unrelated MERRF patients.
RESULTS
Table 2. Global view of transcriptional alterations in patient with MEERF
syndrome compared with normal subject.
According to the extraction result created by Feature Extraction software,
non-uniform features flagged by the algorithm of software were manually eliminated
to increase the reliability of the data. The genes that differentially expressed by 
1.5 fold between the skin fibroblasts established from the patient with MERRF
syndrome and normal subject were picked up and grouped the genes according to
their classification.
Table 3. The activities of free radical scavenging enzyme Mn-SOD and
catalase and the relative levels of superoxide anions and hydrogen peroxide in
the skin fibroblasts from the MERRF patient and her family members.
Subject
IV-1
III-10
IV-2
IV-3
Activities of free radical scavenging enzyme (mole/mg/mg)
Mn-SOD
22732*
14826
12529
13141
Catalase
9.50.7
9.32.4
5.50.6*
11.23.0
Contents of superoxide anions and hydrogen peroxide
Superoxide anions
Hydrogen
peroxide
8112
9627
12135
9038
150 9*
8514
18349*
54 7*
CAT activity was determined by monitoring the rate of decomposition of H 2O2 by the
decrease in the absorbance at 240 nm (Methods Enzymol 105:121-126, 1985). The
total SOD activity was assayed by monitoring nitroblue tetrazolium (NBT) reduction
according to Spitz and Oberley (J. Cell. Physiol. 175:359-369, 1998). Western blot
for Mn-SOD and CAT in each sample were also carried out and similar results were
observed. The contents of superoxide anion and hydrogen peroxide were relative
intensities of hydroethidine (HE) and 2’, 7’-dichlorofluorescin diacetate (DCF),
respectively, as compared with normal subjects with no mtDNA mutation and
measured by staining with HE and DCF and then analyzed by flow cytometry. All
the data in this table were obtained by three independent experiments and are
expressed as mean  SD.
*Significantly different from normal subjects, p < 0.01.
between three and six population doubling time.
The cell lines studied were
(A)
Intracellular pro-MMP-1
Extracellular pro-MMP-1
GAPDH
IV-1
Relative activity
(fold to normal control)
(B)
III-10
IV-2
20
IV-3
N
Active MMP-1
Total MMP-1
15
10
5
0
IV-1
III-10
IV-2
IV-3
N
Fig. 4. Relative level of secreted MMP-1 in skin fibroblasts from the MERRF
patient and her family members.
(A) Western blot of intracellular and
extracellular pro-MMP-1. (B) Relative activity of MMP-1. The acticvity of
MMP-1 in cell culture medium were measured by Fluorokine E Active MMP-1 kit (R
& D Systems). This kit allowed us to measure the levels of both endogenous active
MMP-1 in cell culture medium based on the fact that MMP-1 can be activated by
p-aminophenylmercuric acetate (APMA) in the assay mixture.
Fig. 5. The abnormality of the distribution of mitochondria in the skin
fibroblasts established from the MERRF patient. The fibroblasts were stained
with Mito Tracker Red. Compared with extended and clustered mitochondria of a
normal subject, the skin fibroblasts of the MERRF patient showed small and isolated
mitochondria are revealed by a confocal microscopy. (A) and (B) represent skin
fibroblasts of MERRF patient (IV-1) harboring A8344G point mutation in mtDNA; (C)
and (D) indicate skin fibroblasts of a normal subject.
DISCUSSION
The differentially expressed genes between the skin fibroblasts from a MERRF
patient and an age-matched healthy subject were analyzed by a cDNA microarray
system developed by Agilent Technologies (Figs. 1-2 and Tables 1-2). To diminish
the systematic error, the data were obtained by duplicate experiments and dye swap.
Based on the excellent performance of Agilent bioanalyzer on nucleic acid analysis as
described recently (Lu et al. Clin. Chim. Acta, 318: 97-105, 2002), we performed
RT-PCR on these target genes and quantitated by the Agilent bioanalyzer. We found
that the genes of several cytoskeleton-related proteins like actin, integrin, and tubulin
were repressed and matrix metalloproteinase (MMPs) gene families were
up-regulateded in the fibroblasts from the MERRF patient as compared with the
controls. By staining the skin fibroblasts with Mito Tracker Red, extended and
clustered mitochondria could be observed in the fibroblasts derived from normal
subject but the skin fibroblasts of the MERRF patient showed small and isolated
(disconnected) mitochondria scattered in the cytoplasm in a rather random manner
(Fig. 5). The matrix-degrading enzymes MMPs, which are activated in tissue
remodeling and repair during development and inflammation, as well as many
interferon-induced proteins were up-regulated. These results could explain the
muscle wasting and weakness-related symptoms in most patients with MERRF
syndrome. Moreover, we observed up-regulated transcription of manganese
superoxide dismutase (Mn-SOD), which correlates with our previous findings that the
protein content and enzyme activity of the enzyme were increased in the MERRF
fibroblasts. Based on the cellular and clinical observations, we suggest that the
communication between nuclear DNA and mitochondrial genome may play a very
important role in the pathogenesis of the MERRF syndrome. Further analysis of
differential gene expression in specific signaling and metabolic pathways will be
performed by a conjugated database and literature integrated software.