METHYLMETABOLOMIC APPROACH OF GENOMIC INSTABILITY LINKED WITH DNA HYPOMETHYLATION BIOMARKERS

1
METHYLMETABOLOMIC APPROACH OF GENOMIC INSTABILITY
LINKED WITH DNA HYPOMETHYLATION BIOMARKERS I
Turcu2, M Matei3, G Anton4, N Belc5, D Duta5, LG
Radu6, C Albu6, A Botezatu4, C Posea7 and N
Cucu1 1. University of Bucharest, Faculty of
Biology, Dept of Genetics-MICROGEN-Epigenetics
laboratory, Romania 2. Ana Aslan International
Academy of Anti_Aging, Bucharest, Romania 3. CF2
Hospital Bucharest, Department of Assisted
Reproduction, Genetics Laboratory, Romania 4.
Stefan Nicolau National Institute of Virology,
Bucharest, Romania 5. National Institute of
Bioresources, Bucharest, Romania 6. National
Institute of Biology, Bucharest, Romania, 7. Dept
of Hemathology, Universitary Emergency Hospital
Bucharest, Romania
Abstract One carbon (methyl group) metabolism is
actually considered one very important cell
pathway linking the environment and the genome
stability with a dramatic impact on our health
status. Epigenetic biomarkers such as the DNA and
histone methylation and their associated
chromatin covalent modifications (histone
acetylation/deacetylation) are proposed to be
integrated with the already wellknown metabolites
S-adenosylmethionine (SAM) and its product,
S-adenosylhomocysteine (SAH) when an
investigation of the genome stability is
performed. An experimental model is proposed to
study the relation between the DNA
hypomethylation induced by aging process and
certain pathogenetic states (cancer and diabetes)
as well as by certain disease events and
treatments for the young individuals. In our
study a cohort of six - elderly (60 plus years),
adult (30-45 years), and young (18-30 years)
groups, has been approached, as follows one
young group with controlled sportive life, one
young group with uncontrolled lifestyle, two
adult groups with and without health problems,
and two elderly groups, with vs. without
significant pathology. DNA methylation dynamics
is similar both during aging and cancerogenesis
process showing concomitant global DNA hypo- and
local DNA hypermethylation. Both biomarkers have
been proved in tumors collected from breast
cancer patients through methylation specific PCR
method in local ER alpha and RAR beta promoters
and HPLC method, respectively, for the general
DNA methylation level. As the median age of this
group of individuals was 50 year, we approached
the biomarker study also using lymphocyte culture
comparatively with the young and aged groups with
and without health problems, proving similar
methylation profiles. The study comprises a
genetic (karyotyping) approach in lymphocyte
cultures, which showed again similarities
regarding the genome instability in terms of
frequent premature chromatides dissociations
(PCDs) involving chromosome 1 and 16. Also,
another epigenetic marker, the ratio of methyl
donor SAM and its methylation reaction product,
SAH was used to prove the similarity between the
aging and cancerogenesis process. The mthfr
(methylenetetrahydrofolate) gene plymorphism is
proposed for proving no alteration in diet folate
assimilation. Using specific epigenetic drugs,
such as Trichostatin A and 5Aza cyditidine, as
well as the Gerovital, a product containing
procaine, were approached in order to demonstrate
the involvement of the heterochromatin remodeling
through DNA methylation in satellite 2 DNA and
HDAC in pericentromeric region during both
developmental processes. The study envisaged the
importance of the epigenetic biomarkers when new
therapeutical or nutrition personalized
strategies have to be approached.
Introduction Specific biochemical
modifications of chromatin, such as global DNA
demethylation and HDAC activity alteration, are
considered hallmarks of the aging as well as
cancerogenesis process, which affect primarily
the chromosome architecture and hence its
function during cell division. Pericentromeric
region of the chromosomes contains repetitive,
satellite 2 DNA, in interaction with specific
centromeric proteins (CENP) and nonhistone
proteins, HP1. The DNA hypomethylation processes
are frequently targeted towards this DNA region.
Consequently, the chromatin conformation in this
region may be dramatically changed, carrying on
altered DNA-protein and protein-protein
interactions, due to specific conditions aging
process, pathological condition, chemotherapy,
methyl depleted diet and DNMT3b mutation (lake in
the model ICF syndrome). The effects on cell
division may include premature chromatides
divisions (PCD) and frequent aneuploidies as
determinants of genome instability. Such
interesting phenomena are also encountered when
karyotyping healthy young/adult people which
received specific treatments, or karyotyping
women lymphocytes prelevated during menopause or
hormonal treatment, and very importantly, when
karyotyping cancer patients. The experimentally
5azaC or TSA induced PCDs in lymphocyte culture
from young people plede for the role of
epigenetic factors such as DNA demethylation and
HDAC inhibition in the genome instability
characteristic to aging and pathological
conditions. Also, the newly emerged metabolomic
domain is focusing on a major biochemical pathway
concerning the one carbon (-CH3), methyl group
addition and depletion from DNA and histones,
with interesting effects upon the genome
stability and chromosomal morphology. The SAM
regeneration from its product, SAH, represents
one of the central reaction involving folates and
B (12 and 6) vitamins and specific cathions (Mg
and Zn). Therefore, the SAM/SAH ratio and further
the Hcys level may be good candidate markers for
the general health status linked with chromatin
methylation processes. In this concern, the
polymorphism of mthfr gene was considered as one
major genetic factor involved in methyl
metabolome linked with the SAH to SAM
regeneration. Our goal was the study of the DNA
hypomethylation effects on chromosomal stability
during cell division in aging and cancerogenesis
process, considering also the methyl group
metabolism factors. This is the first step in our
research approach of the epigenetic control of
the genome stability during aging and cancer
processes and the pathological conditions linked
with this critical human developmental stage. The
further step envisages the focus on specific
heterochromatin features which define the
centromere and pericentromere stability through
specific histone covalent modifications,
DNA-histone and histone-nonhistone interactions.
Materials and methods Genomic DNA methylation
level, cell division patterns and methyl
metabolomic aspects have been studied in six
elderly (60 plus years), adult (30-45 years) and
young (18-30 years) groups, as follows one young
group with controlled sportive life, one young
group with uncontrolled lifestyle, two adult
groups with (cancer) and without health problems
and two elderly groups, with (diabates, CVD,
cancer) vs. without significant pathology. Usual
lymphocyte culture induction, chromosomal spreads
obtaining and karyotyping methods have been
performed. For the treatment of lymphocyte
cultures 6x10(-6)M 5azaC, and 1x10(7)M TSA were
used. For DNA extraction a Promega/wizard DNA
extraction kit was used. The isoschizomer pair
MspI and HpaII restriction enzymes has been used
for DNA methylation sensible restriction in order
to estimate the global DNA methylation level.
Another approach for this estimation was HPLC
analysis of the relative 5-methyldeoxycytidine
(5mdC) content it was done after hydrolysis of
DNA with nucleases P1 and alkaline phosphatase.
Also, the methyl group metabolites, SAM (methyl
donor) and its product SAH have been estimated by
HPLC in individual blood samples. Genetic
analyses by PCR with specific primers and
restriction enzyme have been performed for mthfr
polymorphism estimation.
Results and discussions Global DNA
hypomethylation and local (ER alpha and RAR beta)
DNA hypermethylation have been studied in tumor
tissues of cancer patients. The same marks
(ERalpha promoter hypermethylation and global DNA
hypomethylation) have been also approached in
blood lymphocytes as the mean age of the cancer
patients group was 58 and presented the same
epigenetic features. Our approach of the
lymphocyte cultures obtained from the two groups
of blood donators (a total cohort of 200
persons) (i) aged people (without major healthy
problems) and (ii) young healthy people,
comprised the karyotype and epigenetic (HPLC and
MspI / HpaII restriction) analyses. These
analyses have been performed also with a third
group of samples comprising treated cell cultures
(from young persons) with DNA inhibitor
(5azacytidine, 5azaC) and HDAC inhibitor
(trichostatin acid, TSA) as epigenetic
agents. In our study the untreated cell
cultures obtained from the elderly group
presented a specific karyotype, characterized by
frequent premature chromatides dissociations(PCDs)
and aneuploidy, as compared with the cultures
obtained from the young, untreated group (Fig.1
and 2). Further treatment of lymphocyte culture
obtained from young group with epigenetic agents
revealed the same frequent PCDs as in untreated
elderly lymphocytes (Fig.3). A link between the
DNA demethylation processes in the
pericentromeric region stability contributing to
PCDs is suggested. Moreover, similar PCD
phenomena have been observed in the chromosome
preparations obtained from adult-young groups
having severe healthy (cancer) problems or in
specific treatment conditions (antibiotics,
hormonal treatments in young women) (Fig.4,6) ,
demonstrating the same genomic instability.
The cytogenetic results have been confirmed by
the molecular and biochemical measurement of the
global DNA methylation level (Fig.7,8, Table 1)
and also by the SAM/SAH ratio estimations (Table
2). Not only the DNA hypomethylation has been
detected in elderly blood but also in treated
lymphocyte culture from the young individuals.
The ratio value presented a decreased level with
age and pathological conditions. The estimated
mthfr polymorphism proved no implication of
mutant MTHFR in DNA hypomethylation and SAH
accumulation, for the studied cohort. Global
genomic DNA hypomethylation linked with genomic
instability is recognized as a hallmark of aging
processes and of certain pathological conditions,
such as cancer. The extensive research of the
molecular basis of the centromere instability
linked with PCD and aneuploidy and chromosomal
rearrangements pointed towards an interesting
syndrome (ICF, immunodeficiency, centromeric
region instability, facial anomalies) as a model
to investigate the DNA hypomethylation effects.
This is a pathological result of the DNMT3b
mutation, linked with severe DNA 2 satellite
demethylation that determined the pericentromere
instability linked chromosomal rearrangements
between certain chromosomes (1-16) containing
such repetitive DNA sequences (Fig.5).
Intriguingly,however, this syndrome is not linked
with cancer. Heterochromatin is the major
chromatin structure of centromere/pericentromere
regions whose stability is of crucial importance
for the cell division processes, and hence the
proper cohesion and segregation of the
chromatides in chromosomes . Pericentric
heterochromatin is found juxtaposed to centromere
one and remains condensed throughout the cell
cycle, but has to be flexible to external signals
and endogenous ones, linked especially with
replication. Not only DNA methylation marks
through satellite DNA 5-methylcytosine residues
but also other methylation process at the level
of pericentric heterochromatin have to be
seriously considered when studying the genome
architecture protective factors which assure the
proper chromosome functions (such as methylation
of histones in specific lysine residues).
Certain researcher groups (T.Jenuwein,
L.Almouzni) points to a crucial role of histone
underacetylation within pericentric
heterochromatin region and for their association
with heterochromatin protein 1 (HP1) in order
that centromere function to be properly
performed. They point also towards a magic mark
of the heterochromatin domains, encoded by
histone methylation at H3lys9 (H3K9), more
specifically its SET domain. This is a
heterochromatin subdomains (comprising minor and
major satellite DNA) marks for specific
interaction with centromeric proteins (as CENP A)
and, respectively, HP1. Moreover, the recently
discovered miRNA implications in the
heterochromatin structure and gene expression
control oriented us towards further new
exploration directions for the molecular details
of the centromere ctructures in order to explain
their instability induced by the epigenetic
factors during our experimental model of aging
process and its related pathologies. The study
is aiming at finding molecular aging hallmarks an
demonstrating the need for a personalized
approach of the aged people when a correct
diagnosis and therapeutic strategy have to be
applied.
Karyotype analysis
Global DNA hypomethylation
Methyl group metabolome
Scheme of one carbon (-CH3 group) metabolome
(after L Sharp and J Little, Polymorphisms in
Genes Involved in Folate Metabolism and
Colorectal Neoplasia in Am J Epidemiol
2004159423443)
Fig.5. Hypothetical model of the mechanism
involved in radial chromosome formation (after
Reik et al,1999 )
Table 1. Variation of the global DNA methylation
level estimated with the two major groups- the
young and aged groups
Fig. 6. Diagram of PCS percent estimated with the
three experimental models treated and untreated
young people, untreated aged group
DNA hypermethylation
Breast cancer patients-tumor tissue

DIONA PROJECT- Romanian National Research
Program The main purpose of DIONA is to
address nutritional issues that affect the ageing
process in the hope of improving the health and
quality of life of the ageing population. A
target study group of around 1200 elderly
persons, will be selected through
socio-demographical screening on different
elderly population groups and the care needs of
the elderly under study will be assessed in
accordance with their dependency degree as
institutionalized or at home and their actually
nutritional status and needs. Metabolomic
approach of nutrigenomics in elderly under study
will be applied with focus on the impact of the
methyl donor bioactive compounds. Based on
the results about nutritional needs of the
elderly, scientific and technical improvements
and more efficient methods will be developed in
order to produce the best food products. Three
new foods will be designed, adapting them to the
traditional recipes of the different European
countries. Bioresources containing proper
bioactive compounds for a personalized diet
specific for healthy ageing and age-related
pathology as preventive/curative diets will be
selected and will be included in new food
recipes. Formulation and development of food in
easy opening containers, mainly plastic
containers, with different grades of grinding for
old people with mastication problems will be
considered. The food products will be
elaborated at pilot plant scale and will be
clinically tested and, then the proper ones will
be transferred to the industry for production.
In addition, the results will be efficiently
disseminated to the industry, researchers,
consumers by creating tailored training and
detailed instructions as well as to policy
makers for an establishment and alignment of food
strategy related to health/nutrition European
claims.
RAR beta
Elderly-ER alpha-blood
ER alpha
Conclusions The results pointed out to a
pronounced global DNA hypomethylation concomitant
with local DNA hypermethylation (ER alpha) in the
healthy elderly as well as in cancer affected
adult persons similar epigenetic aspects have
been observed by methyl metabolome analyses,
which indicated a decrease in methyl donor level
in these individuals. Such results explained the
altered cell division pattern characterized by
frequent PCDs and even a marked genome
instability proved by the radial chromosomal
morphology. These features have been identified
also in the young subjects with cancer or
antiviral or hormonal therapy as well as in adult
cancer bearing patients, when compared with the
young sportive group. However, the involvement of
the epigenetic factors associated with the
pericentromeric heterochromatin structure has
been proved by obtaining similar genome
instability features when treatment of the young
group lymphocyte culture with DNA methylase and
HDAC inhibitors have been performed. Epigenetic
factors such as DNA hypomethylation, accompanied
by HDAC inhibition, Lsh gene alteration and
histone H3K9 methylation in the pericentromeric
region containing repetitive satellite DNA may
result in alterations of the heterochromatin
structure which is of crucial importance for the
correct cohesin, separase and ciclins regulation
activity of the chromatides cohesion.
Karyotyping lymphocytes from different
individuals grouped as function of age, health
conditions and specific treatments revealed
interesting preliminary results which correlated
the global DNA hypomethylation and premature
chromatide separation processes.Treatment of
lymphocyte cultures from the young healthy donors
with the DNA demethylating agent and the HDAC
inhibitor resulted in similar PCDs frequency, as
compared with the percentage observed with
normal elderly karyotype, demonstrating the
relevance of the two epigenetic factors in
maintaining the genome stability through the
centromere-pericentromere heterochromatin
structure. The molecular Msp/Hpa restriction
profiles and HPLC determinations proved the
decrease in global DNA methylation level with
aging. PCR screening, performed in order to
ensure that the mthfr mutations had no
implications in this study, proved that the
methyl group metabolism was not influenced by
this factor. By the other hand, this specific
feature of elderly methyl group metabolome may be
monitored by diets and supplements which may
provide proper elements (folates, vitamins B12
and B6, Mg and Zn cathions and other secondary
methyl donors such as betaine and choline) for
maintenance of a normal genomic stability in
aging conditions and perhaps improving the
genomic structure in cancer. This study is
curently continued by targeting other molecular
actors such as H3K9 and K27 di and trimethylated,
LSH demethylase, and also the study of certain
HDAC inhibitors (SAHA, butyrate etc) upon the
demethylation in promoter ER alpha and RAR beta
and their gene expression reactivation. These
studies are concomitant with other epigenetic
therapies alternatives, such as known procaine
DNA demethylating agent.
Breast cancer patients -ER alpha- blood
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