To unravel the molecular changes that underlie RTT, several mouse models with
different MECP2 mutations were generated. Mecp2-null male (Mecp2
) and female
) mice generated via cre/lox recombination have no apparent phenotype until
around 6 weeks. There follows a period of rapid regression resulting in reduced
spontaneous movement, uncoordinated gait, irregular breathing, hind limb clasping and
tremors. Rapid progression of symptoms leads to death at 8 weeks of age (Guy et al., 2001;
Chen et al., 2001). Detailed brain examination revealed that the brains of Mecp2 null mice
are smaller in size and weight than brains of wild type littermates, but have no detectable
structural abnormalities, except for smaller, more densely packed neurons (Chen et al.,
2001). In addition, the olfactory neurons of Mecp2 null mice demonstrate a transient delay
in differentiation, and abnormalities of axonal targeting, suggesting that Mecp2 mediates a
crucial function in the final stages of neuronal development (Matarazzo et al., 2004).
Recently, Pelka et al. (2006) reported a null mice with XO background (Mecp2
similar phenotypes with male Mecp2
mice. This finding indicates that the Y-
chromosome has no effect on the phenotypic manifestation in Mecp2 null mice (Pelka et al ., 2006).
Shahbazian et al. (2002) reported another RTT mouse model generated with
insertion of a stop codon in the Mecp2 gene at nucleotide position corresponding to amino
acid 309. This mutation results in a truncated protein with the MBD, TRD, and NLS
domain and a lack of the C-terminal region, which is predicted to have similar effects of
p.Arg294X mutation observed in RTT patients. Mecp2
mice display no initial
phenotype until 6 weeks of age, and then they develop progressive neurological
phenotypes, including motor dysfunction, forepaw stereotypies, hypoactivity, tremor,
seizures, kyphosis, social behavior abnormalities, decreased diurnal activity, increased
anxiety-related behavior, and learning and memory deficits, reminiscent of the clinical
picture in human girls with RTT. Female mice heterozygous for the truncation display
milder and more variable features. In vivo, the truncated protein maintains normal
chromatin localization, but histone H3 is hyperacetylated in the brain, indicating abnormal
chromatin architecture (Shahbazian et al., 2002; Moretti et al., 2005).
Collins et al. (2004) has developed a mouse model that transgenically over-expressed
MECP2 under the endogenous human promoter. Initially, MECP2 Tg
increased synaptic plasticity, with enhancement in motor and contextual learning abilities.
However, at 20 weeks of age, these mice developed seizures, hypoactivity and spasticity
with several other progressive neurological abnormalities.
The conditional inactivation of MeCP2 in only post-mitotic neurons of the forebrain
caused delayed onset of symptoms similar to those shown by Mecp2 knockout mice. This
finding indicates that Mecp2 plays an essential role in post-mitotic neurons (Chen et al.,
2001). Additionally, the expression of Mecp2 in only post-mitotic neurons of Mecp2-null
mice was shown to be sufficient to restore normal neurological function. This finding
indicates that Mecp2 deficiency in peripheral tissues does not significantly influence
disease manifestations and suggests that Mecp2 plays no essential role in the early stages
of brain development (Luikenhuis et al., 2004).
1.2.10. MeCP2 Target Genes and Their Relevance with Disease
Although biochemical evidence suggested that MeCP2 functions as a global
repressor of gene expression, transcriptional profiling failed to identify profound changes
of gene expression in the brain of Mecp2 knockout mice (Tudor et al., 2002). Using the
candidate gene approach or CGH analysis of samples from both human and mouse tissues,
only a few number of putative MeCP2 targets that might be relevant to the pathogenesis of
RTT was identified (Figure 1.7).
The first gene shown to be repressed by MeCP2 was brain-derived neurotrophic
factor (BDNF), encoding a protein that has essential functions for neuronal plasticity,
learning and memory (Chen et al., 2003). In basal conditions, BDNF expression is
repressed by MeCP2 bound to its promoter; upon membrane depolarization,
phosphorylated MeCP2 dissociates from the promoter and BDNF expression is induced by
binding of CREB (Moretti et al., 2005 and 2006). Conditional deletion of Bdnf in
postmitotic neurons of mice mimicked some of the phenotypes observed in Mecp2 null
mice, including hind limb clasping, reduced brain weight, and reduced olfactory and
hippocampal neuronal sizes. However, Chang and colleagues reported that BDNF protein
levels are decreased rather than increased in brains of symptomatic Mecp2
Bdnf is known to be upregulated in response to neuronal activity, the reduced cortical
activity in Mecp2 null mice is expected to negatively affect Bdnf expression, hence
masking the expected upregulation that would normally result from loss of repression in
resting cortical neurons that lack MeCP2 (Chang et al., 2006). Consistent with this data,
forebrain-specific deletion of Bdnf in Mecp2
mice resulted in earlier onset of locomotor
dysfunction and reduced lifespan, while forebrain- specific overexpression of Bdnf in these
mice improved locomotor function and extended their lifespan (Chang et al., 2006).
Horike et al. (2005) reported the loss of imprinting of a maternally expressed gene,
distal-less homeobox 5 (DLX5), in both Mecp2-null mice and in lymphoblastoid cell lines
obtained from RTT patients. MeCP2 was shown to be essential for the formation of a silent
chromatin structure at the Dlx5 locus by histone methylation and through the formation of
a chromatin loop. Dlx5 regulates GABA neurotransmission and osteogenesis; therefore,
alterations in Dlx5 expression can account for epilepsy, osteoporosis and somatic
hypoevolutism observed in RTT girls.
MeCP2 was shown to affect the expression pattern of UBE3A located in PWS/AS
imprinted region. UBE3A encodes the ubiquitin ligase E3A and imprinted only in the
brain. Mutations in the maternal copy of the gene account for about 10 per cent of
Angelman Syndrome (AS) cases. UBE3A mRNA and protein levels are slightly reduced in
human and mouse MeCP2-deficient brains due to the overexpression of anti sense UBE3A (Makedonski et al., 2005). Since maternal mutations in UBE3A (or repression of the
maternal allele) give rise to AS, it is speculated that deregulation of UBE3A expression
that results from MeCP2 loss of function might contribute to the clinical manifestations of
Rett syndrome, such as mental retardation, seizures, muscular hypotonia and acquired
microcephaly, that are common to both conditions (Makedonski et al., 2005).
Chip analyses revealed that MeCP2 binds to promoter region of the corticotropin-
releasing hormone (CRH) gene, glucocorticoid-inducible genes, serum glucocorticoid-
inducible kinase 1 (Sgk1) and FK506-binding protein 5 (Fkbp5) in wild type brain. It was
shown that these genes are upregulated in Mecp2 null mice (Bale and Vale, 2004; Nuber et al ., 2005). Since the genes Sgk1, Fkbp5, and Crh are involved in regulation of behavioral
and physiological responses to stress it may be suggested that at least some RTT symptoms
arise from the disruption of MeCP2 regulation on stress-responsive genes (Nuber et al.,
Figure 1.7. MeCP2 target genes and their relevance with the disease. Loss of MeCP2
affects the expression pattern of specific genes: BDNF, DLX5, Sgk1, Fkbp5 and antisense
UBE3A (Mari et al., 2005).
1.3. Breast Cancer 1.3.1. Breast Tissue
The breast, being an apocrine gland, is composed of glandular, fatty, and fibrous
tissues positioned over the pectoral muscles of the chest wall and attached to the chest wall
by fibrous strands called Cooper’s ligaments. A layer of fatty tissue surrounds the breast
glands and extends throughout the breast. The fatty tissue gives the breast a soft
consistency. The glandular tissues of the breast house the lobules (milk producing glands at
the ends of the lobes) and the ducts (transporting milk from the milk glands to the nipple).
Toward the nipple, each duct widens to form a sac (ampulla) (Figure 1.8). During lactation,
the bulbs on the ends of the lobules produce milk. Once milk is produced, it is transferred
through the ducts to the nipple.
Figure 1.8. A schematic diagram of a normal female breast (www.cancer.org/docroot/
1.3.2. Breast Cancer Risk
Breast cancer is the most common malignancy among women in industrialized
countries and diagnosed in 1 x 10
women in the world each year. The highest age-adjusted
incidence rate is reported for North America, being 86.3 per 100,000 women per year,
while the lowest rate, reported in China, is only 11.8. The average incidence is 63.2 for
more developed countries and 23.1 for the less developed regions.
Breast carcinomas originate from the epithelial cells lining the ducts or lobules,
therefore classified as ductal or lobular carcinomas. Ductal carcinoma is the most common
type of breast cancer, accounting for 85 to 90 per cent of the cases. Lobular carcinoma
occurs in 10 to 12 per cent of the cases (Feig SA, 2000). Ductal breast malignancies are
divided into two categories, pre-invasive and invasive. Pre-invasive ductal cancer is called
ductal carcinoma in situ (DCIS). It is a very early stage of breast cancer and is the result of
proliferation of the ductal luminal cells which fills the lumen but do not enter the basement
membrane and the surrounding stroma. It accounts for 5 per cent of all breast carcinomas.
Invasive (infiltrating) ductal carcinoma (IDC) is the most aggressive type with capability
of metastasis. Invasive lobular carcinomas (ILC) only account for about 10 per cent of all
breast cancers and they tend to be somewhat less aggressive than IDC. Unlike IDC, it is
now believed that lobular carcinoma in situ (LCIS) is not a precursor of invasive lobular
carcinoma. The confusion exists because LCIS, while it has the word carcinoma in its
name, does not behave like a cancerous condition. LCIS does not grow, form masses,
transform into invasive cancer, or metastasize. Therefore, it does not represent a true
The molecular mechanisms underlying the development of breast cancer are not
completely understood. However, it is generally believed that the initiation of breast cancer
is a consequence of cumulative genetic damages leading to genetic alterations that result in
activation of proto-oncogenes and inactivation of tumor suppressor genes. These in turn
are followed by uncontrolled cellular proliferation and/or aberrant programmed cell death,
or apoptosis. Also, the role of reactive oxygen species (ROS) has been related to the
etiology of cancer, as they are known to be mutagenic, and therefore capable of tumor
The risk of developing breast cancer is increased if a family history of the disease is
present. The epidemiological studies have shown that 12 per cent of women with breast
cancer had one and one per cent had two or more affected relatives. Therefore the genesis
of most breast cancers can not be explained by heritage. Age and the duration of exposure
to endogenous or exogenous steroid hormone levels are suggested as the best defined risk
factors for breast cancer. Breast cancer is uncommon among women younger than 30 years
of age but the incidence increases sharply with age. The rate of increase in breast cancer
incidence continues throughout life but slows somewhat between ages 45 and 50 years.
This finding strongly suggests the involvement of reproductive hormones in breast cancer
etiology, because non-hormone-dependent cancers do not exhibit this change in slope of
the incidence curve around the time of menopause (Pike et al., 1993). Several reproductive
factors that alter estrogen status affect the risk of breast cancer: early age at menarche and
late age at menopause are associated with increased risk of breast cancer. After
menopause, adipose tissue is the major source of estrogen, and obese postmenopausal
women have both higher levels of endogenous estrogen and a higher risk of breast cancer
(Harris et al., 1992; Huang et al., 1997). Postmenopausal hormone use increases the breast
cancer risk depending on the duration of use and whether estrogen alone or estrogen in
combination with progestin is taken (Ross et al., 2000).
The age might be the driving force for the accumulation of mutational load due to the
reactive oxygen species, telomere dysfunction, and increased epigenetic gene silencing.
Exposure to growth factors like estrogen increases the likelihood of occurrence of these
changes in breast epithelial stem cells as well as the propagation of these changes by
enabling the cells to divide.
Hereditary (familial) form of the breast cancer represents 5-10 per cent of all cases.
BRCA1, BRCA2, p53, ATM, CHECK2, and PTEN are the major breast cancer
susceptibility genes (Marcus et al., 1996; Miki et al., 1994; Stratton and Wooster, 1995;
Hill et al., 1997, Bell et al 1999, Cantor et al 2001). BRCA1, BRCA2 and ATM genes
maintain genomic stability and involved in repair of double-strand breaks. BRCA1 and
BRCA2 play also role in transcription and cell cycle control acting as a tumor suppressor
gene (Venkitaraman, 2002). p53 is known to be involved in cell cycle regulation, DNA
damage repair, apoptosis and inhibition of angiogenesis. Therefore, loss of functional
protein eliminates the growth arrest in response to DNA damage and allows the replication
of mutated DNA. CHECK2, a G2 check point kinase, is involved in the repair of DNA
breaks. PTEN is a lipid phosphatase that was identified as a candidate tumor suppressor
gene. It is suggested to have an inhibitory role on PKB/Akt that is required for cell growth
and survival (Downward J, 1998). It is also found to inhibit integrin-mediated cell
migration thus preventing metastasis (Tamura et al., 1998).
1.3.3. Breast Carcinogenesis
Breast cancer progression is a multi-step process encompassing progressive changes
from normal, to hyperplasia with and without atypia, carcinoma in situ, invasive
carcinoma, and metastasis (Figure 1.9) ( Simpson et al., 2005).
Figure 1.9. Simplified multi-step model of breast cancer progression based on
morphological features (Simpson et al., 2005).
It has been suggested that a cell has to acquire six features to become malignant (IDC
or ILC): (1) limitless replicative potential, (2) self-sufficiency in growth signals, (3)
insensitivity to growth-inhibitory signals, (4) evasion of programmed cell death, (5)
sustained angiogenesis and (6) tissue invasion and metastasis (Hanahan and Weinberg,
2000). This process requires complex series of stochastic genetic events including gene
amplifications, gene deletions, point mutations, loss of heterozygosity, chromosomal
rearrangements, and overall aneuploidy. Some of the observed genetic lesions are loss of
16q, 11q, 14q, 8p, 13q, gain of 17q, 8q, 5p, and amplifications on 17q12, 17q22–24, 6q22,
8q22, 11q13, and 20q13. Besides the genetic alterations, epigenetic alterations are among
the most common molecular alterations in human neoplasia (Baylin and Herman, 2000;
Jones, 1996; Jones and Laird, 1999). Hypermethylation and global hypomethylation of
more than 25 genes have been correlated with breast carcinogenesis. Abnormal
methylation of each gene enables cells to acquire new capabilities needed for
tumorigenesis (Figure 1.10 and Table 1.1).
Figure 1.10. View of breast carcinogenesis from a DNA methylation standpoint
(Widschwendter and Jones, 2002).
Table 1.1. The list of methylated genes in breast cancer (Widschwendter et al., 2002).
Table 1.1. The list of methylated genes in breast cancer (continued).
1.3.4. DNA Methylation and Genetic Instability
The genomic instability is the common feature of all cancer types and DNA
methylation might be responsible for these chromosomal instabilities. Methylation leads to
instability in several ways. First, 5meCs serve as sites of transition mutations by the
hydrolytic deamination of 5meC to thymine. For example, such mutations frequently occur
in the tumor suppressor genes p53, Rb, and c-H-ras-1 (Magewu and Jones, 1994; Ghazi et al ., 1990). Secondly, epigenetic inactivation of certain critical genes in cancer by promoter
methylation may predispose to genetic instability (Herman and Baylin, 2000). For instance,
methylation of MLH1, a gene involved in mismatch repair, precedes the MIN + phenotype
in sporadic colon, gastric and endometrial cancers (Esteller et al. 1999). Furthermore, there
is a striking correlation between mismatch repair, genetic instability and methylation
capacity in colon cancer cell models (Lengauer et al., 1997, 1998). In addition, promoter
CpG island methylation and resulting inactivation of the detoxifying π-class glutathione S-
transferase (GST) can lead to accumulation of oxygen radicals and subsequent DNA
damage (Lee et al., 1994, Henderson et al., 1998, Matsui et al., 2000). A p53-inducible
gene, 14–3–3σ, is methylated and inactivated in many breast cancers. Loss of its
expression may also facilitate the accumulation of genetic damages (Ferguson et al., 2000).
Apart from regional hypermethylation of some critical tumor suppressor genes, genome-
wide hypomethylation is an important feature in cancer and can also contribute to genetic
instability (Schmutte and Fishel, 1999).
Genomic integrity, senescence, and evasion of programmed cell death (apoptosis) are
thought to be important barriers to the development of malignant lesions. DNA repair
proteins have emerged as the key regulators among the multitude of players involved in
cell cycle control and apoptosis by inducing apoptosis in stressed or abnormal cells,
thereby protecting the organism from cancer development.
1.3.5. DNA Repair System
DNA repair enzymes maintain the integrity of the genetic code by removing
damaged DNA segments and minimizing replication errors. DNA damage may be a
consequence of normal cellular function (e.g. replication errors, oxidative metabolism,
reactive metabolites of hormone synthesis) or of environmental factors such as radiation
(UV) or xenobiotic chemicals (Mohrenweiser and Jones, 1998). Cells with damaged DNA
may be removed by apoptosis, or programmed cell death. If DNA adducts escape cellular
repair mechanisms and persist, they may lead to miscoding, resulting in permanent
mutations. If a permanent mutation occurs in a critical region of an oncogene or tumor
suppressor gene, it can lead to activation of the oncogene or deactivation of the tumor
suppressor gene. Multiple events of this type lead to aberrant cells with loss of normal
growth control and ultimately to cancer.
Multiple and complementary DNA repair systems have evolved to protect the
genome against the detrimental effects of DNA lesions. DNA repair may take place by one
of several pathways, depending on the type of damage, including nucleotide excision repair
(NER), base excision repair (BER), mismatch repair, or recombinatorial repair. Nucleotide
excision repair (NER) enzymes are responsible for removing ‘bulky’ DNA damage that
distorts the DNA helix such as UV photoproducts (thymine dimers), chemically induced
intra-strand crosslinks and bulky chemical adducts (polycyclic aromatic hydrocarbons and
aromatic amines). Two distinct sub-pathways are present in the mammalian NER system:
i.e., global genome NER (GG-NER) that operates genome wide, and transcription-coupled
NER (TC-NER) that is specialized to eliminate transcription-blocking lesions on the DNA
strand of active genes (Figure 1.11). The NER pathway includes several steps: (i) DNA
damage recognition; (ii) assembly of repair factors; (iii) incision of damaged DNA; (iv)
repair synthesis to fill gapped DNA; and (v) DNA ligation. DNA damage is recognized by
the Xeroderma Pigmentosum Group C (XPC)–hHR23B complex, followed by recruitment
of the transcription factor IIH (TFIIH) complex of proteins. The TFIIH complex is
composed of nine subunits, including XPD and XPB (de Boer and Hoeijmakers, 2000).
TFIIH unwinds the DNA duplex around the damaged site. Next, XPG binds to the TFIIH
complex and DNA, followed by recruitment of the XPF–ERCC1 complex. XPG and XPF–
ERCC1 produce dual incisions of 30 and 50 nucleotides at damaged site. After release of
the damaged DNA strand, the gap is filled by repair synthesis and ligation (Bradsher et al.,
The in vitro and in vivo experiments suggest that XPC-hHR23B complex is the initial
component for detecting DNA damage and plays at least four roles in DNA damage
recognition. First, XPC-hHR23B can discriminate between DNA distortions and the
Watson-Crick structure. Second, XPC-hHR23B may be the first NER factor to respond to
DNA damage in GGR. Third, XPC-hHR23B’s presence at sites of DNA damage can
induce a further bending of the DNA, which may enhance the binding of other downstream
NER proteins to the site of DNA lesions (Sugasawa et al., 1998). Finally, XPC-hHR23B
has a critical role in the recruitment of TFIIH, which is known to promote the opening of
the DNA helix in the vicinity of the lesion, presumably to assist in the assembly of
subsequent NER factors for further processing of the lesion (Schaeffer et al., 1993; Feaver
et al ., 1993; Drapkin et al., 1994).