telomere length

TELOMERE LENGTH
− INHERITANCE PATTERN AND
ROLE AS A BIOMARKER
AKADEMISK AVHANDLING
som med vederbörligt tillstånd från Rektorsämbetet vid Umeå
Universitet för avläggande av filosofie doktorsexamen kommer att
offentligen försvaras i hörsal Betula, byggnad 6M, Norrlands
Universitetssjukhus,
fredagen den 10 oktober 2008, kl 09.00.
Avhandlingen kommer att försvaras på engelska.
Av
Katarina Nordfjäll
Institutionen för Medicinsk Biovetenskap, Patologi.
2008
Fakultetsopponent:
Professor Thomas von Zglinicki
Institute for Aging and Health
Telomere Length – Inheritance Pattern and Role as a Biomarker
Katarina Nordfjäll
Department of Medical Biosciences, Pathology, Umeå University,
SE-907 87 Umeå, Sweden.
New Series No. 1200
ISSN 0346-6612
ISBN 978-91-7264-620-9
ABSTRACT
Telomeres are repetitive TTAGGG structures ending each chromosome and thereby
protecting its integrity. Due to the end-replication problem, telomeres shorten with
each cell division. When reaching a critical telomere length (TL), the cells stop dividing
and enter replicative senescence. It has been speculated that telomeres might regulate
lifespan at the organism level but this hypothesis is controversial. However, telomeres
in human blood cells do shorten with increasing age.
Telomerase is an enzyme capable of lengthen telomeres. It consists of a
catalytic subunit, hTERT, and a RNA template, hTR. Telomerase is active in germ cells,
stem cells, activated lymphocytes and highly proliferating epithelial cells while no
activity is found in other somatic cells. One step in order to produce a tumour mass is
that cancer cells need to have a limitless replicative potential and this can be achieved
by activating telomerase. Most tumour cells express telomerase activity and hence, the
enzyme is an interesting target for cancer therapy.
Telomere length is in part inherited. Two separate family cohorts were
investigated to elucidate the inheritance pattern and a strong paternal inheritance was
observed. In the larger, multifamily cohort spanning up to four generations, a weak
correlation between the TL of the mother and the child was also found, as well as a
significant
correlation
between
grandparent-grandchild
pairs.
Interestingly,
the
heritable impact diminished with increasing age, indicating than non-heritable factors
might influence TL during life.
A functional T to C transition polymorphism in the hTERT promoter was
previously reported, showing that the
-1327
C/C genotype was correlated with shorter TL
compared to the alternative genotypes in healthy individuals and in coronary artery
disease patients. When investigating 226 myocardial infarction patients and 444
controls separately, no differences were observed regarding mean TL or increased
attrition rate between the different genotypes.
TL in blood cells is shown to be altered in patients with certain types of solid
tumours. In our breast cancer cohort, TL was a strong prognostic marker. Short
telomeres were associated with increased survival, especially in young patients and in
those with advanced tumours. It has been speculated that cancer patients might have
a faster telomere attrition rate than controls but this has not been experimentally
proven. Two blood samples from the same individual taken with 9-11 years interval
was investigated. Some were diagnosed with a malignancy after the second blood
draw. When comparing patients with controls, telomere attrition rate was not
correlated to future tumour development. About one third of the individuals elongated
their telomeres over a decade and the individual telomere attrition rate was telomere
length dependent, showing an inverse correlation to TL at a highly significant level.
This strongly suggests that the TL maintenance mechanism shown to provide
protection for short telomeres in vitro is important also in human cells in vivo.
Keywords:
Telomere length, peripheral blood, inheritance, breast cancer, survival,
polymorphism
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New Series No. 1200
ISSN 0346-6612
ISBN 978-91-7264-620-9
TELOMERE LENGTH
− INHERITANCE PATTERN AND
ROLE AS A BIOMARKER
Katarina Nordfjäll
From the Department of Medical Biosciences, Pathology.
Umeå University, Sweden
Umeå 2008
Cover
Photo: Andreas Gottvall
Models: Rickhard Nordfjäll, Marlene Nordfjäll and Axel Nordfjäll
Layout: Ida Holmgren
Copyright © 2008 by Katarina Nordfjäll
New Series No. 1200
ISSN 0346-6612
ISBN 987-91-7264-620-9
Printed by Print & Media, Umeå 2008
To my family
In my end is my beginning
- Stuart, Mary
SUMMARY
Telomere Length – Inheritance Pattern and Role as a Biomarker
STUDY POPULATIONS
AIM
RESULTS
Paper I
132 healthy individuals in
49 unrelated families were
analysed regarding
telomere length.
To investigate if
telomere length is
inherited and if so,
what the mode of
inheritance is.
Paper II
A multifamily cohort with
962 individuals spanning
over four generations
were investigated
regarding telomere length.
444 were included in
inheritance analysis.
To further investigate
telomere length
inheritance in different age groups and
over generations.
Paper III
226 cases with myocardial
infarction patients and 444
controls were included in
the study.
The different
T/C
hTERT promoter
genotypes were
analysed in cases
and controls to
investigate if the
polymorphism was a
functional one.
Paper IV
Telomere length in blood
cells from 265 newly
diagnosed breast cancer
patients and 300 controls
were measured.
To investigate if telomere length differ
between cases and
controls and if it
could be used as a
risk factor and
prognostic marker.
Telomere length was
measured in two blood
samples with 9-11 years in
between. 314 persons
were diagnosed with
cancer after the second
blood sample. 645
individuals were used as
controls.
To investigate
telomere length
attrition at the
individual level in a
longitudinal study
and correlate it to
future tumour
development.
-1327
Paper V
There was a significant
correlation between fatherson and father-daughter pairs
but not between mother-son
or mother-daughter pairs,
indicating a paternal
inheritance.
A stronger paternal than
maternal inheritance was
observed, and a grandparentgrandchild correlation was
also found. Moreover,
telomere length inheritance
seemed to diminish with age.
The polymorphism is not
-1327
functional since the
C/C
indivuduals did not display
significantly shorter telomeres
or faster attrition rate compared to the other genotypes.
There was no difference
between patients and
controls.
Patients with long blood
telomeres had a significantly
worse survival compared to
patients with shorter telomeres. Long telomeres were
also a risk factor for breast
cancer. The patients had in
general longer telomere
length compared to controls.
Telomere length is not
correlated to future tumour
development. However, it is
strongly correlated to the
telomere length at baseline,
indicating that the event of
telomere elongation and
attrition is telomere length
dependent.
TABLE OF CONTENTS
ABSTRACT.......................................................................................................... 6
ORIGINAL ARTICLES ......................................................................................... 7
ABBREVIATIONS................................................................................................ 8
POPULÄRVETENSKAPLIG SAMMANFATTNING ............................................. 9
INTRODUCTION ............................................................................................... 11
The end......................................................................................................... 11
Replicative senescence and immortality....................................................... 13
Telomerase ................................................................................................... 14
Components ............................................................................................. 14
Telomerase and telomere length regulation ................................................. 15
Telomere structure........................................................................................ 16
The shelterins ........................................................................................... 16
The TRF1 and TRF2 complex .................................................................. 17
ALT ............................................................................................................... 17
Telomere length measurements ................................................................... 18
Telomere length in normal individuals .......................................................... 19
Telomere length and heredity ....................................................................... 20
Telomeres and disease ................................................................................ 21
Constitutional genetic disorders ............................................................... 21
Telomeres and tumours............................................................................ 22
Telomerase and cancer therapy............................................................... 24
Non-malignant disorders .......................................................................... 25
AIMS .................................................................................................................. 26
MATERIAL AND METHODS ............................................................................. 27
Study populations ......................................................................................... 27
Telomere real time PCR ............................................................................... 29
TRAPeze....................................................................................................... 31
SNP analysis................................................................................................. 31
Statistical analyses ....................................................................................... 32
RESULTS AND DISCUSSION .......................................................................... 33
Telomere length inheritance ......................................................................... 33
The -1327T/C hTERT promoter polymorphism ............................................... 36
Telomere length as a biomarker in breast cancer ........................................ 37
Telomere attrition is telomere length dependent .......................................... 39
Strength and limitations of the thesis............................................................ 41
General discussion ....................................................................................... 42
CONCLUSIONS ................................................................................................ 44
ACKNOWLEDGEMENTS.................................................................................. 45
REFERENCE LIST ............................................................................................ 47
ABSTRACT
Telomeres are repetitive TTAGGG structures ending each chromosome and
thereby protecting its integrity. Due to the end-replication problem, telomeres
shorten with each cell division. When reaching a critical telomere length (TL),
the cells stop dividing and enter replicative senescence. It has been speculated
that telomeres might regulate lifespan at the organism level but this hypothesis
is controversial. However, telomeres in human blood cells do shorten with
increasing age.
Telomerase is an enzyme capable of lengthen telomeres. It consists of a
catalytic subunit, hTERT, and a RNA template, hTR. Telomerase is active in
germ cells, stem cells, activated lymphocytes and highly proliferating epithelial
cells while no activity is found in other somatic cells. One step in order to
produce a tumour mass is that cancer cells need to have a limitless replicative
potential and this can be achieved by activating telomerase. Most tumour cells
express telomerase activity and hence, the enzyme is an interesting target for
cancer therapy.
Telomere length is in part inherited. Two separate family cohorts were
investigated to elucidate the inheritance pattern and a strong paternal
inheritance was observed. In the larger, multifamily cohort spanning up to four
generations, a weak correlation between the TL of the mother and the child was
also found, as well as a significant correlation between grandparent-grandchild
pairs. Interestingly, the heritable impact diminished with increasing age,
indicating than non-heritable factors might influence TL during life.
A functional T to C transition polymorphism in the hTERT promoter was
previously reported, showing that the -1327C/C genotype was correlated with
shorter TL compared to the alternative genotypes in healthy individuals and in
coronary artery disease patients. When investigating 226 myocardial infarction
patients and 444 controls separately, no differences were observed regarding
mean TL or increased attrition rate between the different genotypes.
TL in blood cells is shown to be altered in patients with certain types of
solid tumours. In our breast cancer cohort, TL was a strong prognostic marker.
Short telomeres were associated with increased survival, especially in young
patients and in those with advanced tumours. It has been speculated that
cancer patients might have a faster telomere attrition rate than controls but this
has not been experimentally proven. Two blood samples from the same
individual taken with 9-11 years interval was investigated. Some were
diagnosed with a malignancy after the second blood draw. When comparing
patients with controls, telomere attrition rate was not correlated to future tumour
development. About one third of the individuals elongated their telomeres over a
decade and the individual telomere attrition rate was telomere length
dependent, showing an inverse correlation to TL at a highly significant level.
This strongly suggests that the TL maintenance mechanism shown to provide
protection for short telomeres in vitro is important also in human cells in vivo.
6
ORIGINAL ARTICLES
Paper I
Nordfjäll,K., Larefalk,Å., Lindgren,P., Holmberg,D., Roos,G. Telomere length
and heredity: Indications of paternal inheritance. (2005) Proc Natl Acad Sci U S
A, 102, 16374-16378.
Paper II
Nordfjäll,K*., Svenson,U*., Norrback,K.F., Adolfsson,R., Roos,G. The paternal
influence on telomere length is stronger than the maternal and the heritable
impact diminishes with age. Manuscript, submitted.
*Contributed equally.
Paper III
Nordfjäll,K., Osterman,P., Melander,O., Nilsson,P., Roos,G. hTERT -1327T/C
polymorphism is not associated with age-related telomere attrition in peripheral
blood. (2007) Biochem Biophys Res Commun., 358, 215-218.
Paper IV
Svenson,U*., Nordfjäll,K*., Stegmayr,B., Manjer,J., Nilsson,P., Tavelin,B.,
Henriksson,R., Lenner,P., Roos,G. Breast cancer survival is associated with
telomere length in peripheral blood cells. (2008) Cancer Res., 68, 3618-3623.
*Contributed equally.
Paper V
Nordfjäll,K., Svenson,U., Norrback,K.F., Adolfsson,R., Lenner,P., Roos,G.
Human blood cell telomere attrition rate is telomere length dependent but not
associated to tumour development. Manuscript, submitted.
The original papers were reprinted with kind permission from the publishers.
7
ABBREVIATIONS
AA
ALT
ANCOVA
APBs
bp
CAD
CI
Ct
CVD
DBS
DC
D-loop
ds
DZ
EBV
ER
FISH
HR
hTR or hTERC
hTERT
kb
M1/M2
MHC
MI
MNC
MONICA
MSP
MZ
NSHDS
OR
PCR
Q-FISH
RNP
RT-PCR
ss
STELA
TL
TPG
TRAP
TRF
VIP
8
aplastic anemia
alternative lengthening of telomeres
analysis of covariance
ALT-associated promyelocytic leukemia bodies
base pair
coronary artery disease
confidence interval
cycle threshold
cardiovascular disease
double stranded DNA break
dyskeratosis congenita
displacement loop
double stranded
dizygotic twins
Epstein Barr virus
estrogen receptor
fluorescence in situ hybridisation
hazard ratio
RNA component of telomerase
catalytic subunit of telomerase
kilo base pairs
mortality stage 1 and 2
major histocompatibility complex
myocardial infarction
mononuclear cells
Multinational Monitoring of Trends and Determinations
in Cardiovascular Disease
Mammography Screening Project
monozygotic twins
Northern Sweden Health and Disease Study
odds ratio
polymerase chain reaction
quantitative fluorescent in situ hybridisation
ribonucleoprotein
real time polymerase chain reaction
single stranded
single telomere length analysis
telomere length
total product generated
telomeric repeat amplification protocol
terminal restriction fragments
Västerbotten Intervention Project
POPULÄRVETENSKAPLIG SAMMANFATTNING
Kromosomerna är de enheter i cellen som innehåller arvsmassa eller DNA.
Längst ut på dessa finns en struktur som kallas telomer och som består av
många upprepade sekvenser av kvävebaserna TTAGGG. Liknande
telomersekvens återfinns hos alla däggdjur men också hos lägre stående djur
och växter. Att sekvensen bibehållits genom evolutionen tyder på att
telomererna har en central roll i cellen. Deras funktion är inte helt klarlagd men
de skyddar arvsmassan som helhet och stabiliserar kromosomerna. En cells
telomerer förkortas för varje celldelning på grund av att cellerna inte kan kopiera
den yttersta delen av DNA:et inför delningen. I och med detta fungerar
telomererna som en buffertzon så att inte DNA som kodar för viktiga gener ska
förloras. I en odlad cellkultur slutar cellerna att dela sig när telomererna blir
kritiskt korta. Telomerförkortningen kan ses hos människor genom att till
exempel studera telomerlängden i blod från unga respektive gamla individer.
Men det finns en stor skillnad i telomerlängd mellan olika personer så man kan
inte uttala sig om åldern på en människa genom att enbart studera telomererna.
Ibland dras parallellen mellan telomerlängd och livslängd. På populationsnivå
har det visat sig att kvinnor generellt har längre telomerer än män, och kvinnor
har ju också något längre medelöverlevnad. Att telomerlängden styr livslängden
stämmer möjligtvis på cellnivå men i en komplett organism är det många
samverkande faktorer som påverkar.
Ett specifikt enzym som har förmågan att förlänga telomererna upptäcktes
1985 och fick namnet telomeras. Efter fosterstadiet uttrycks enzymet bara i ett
fåtal av kroppens celler till exempel i könsceller, stamceller och aktiverade
immunceller. Dock kan produktionen av enzymet aktiveras i de flesta
cancerceller och på så vis bidra till att dessa celler kan genomgå ett obegränsat
antal celldelningar och ge upphov till en tumörmassa. Därför har telomerer och
telomeras studerats såväl inom cancerforskningen som ur ett
åldrandeperspektiv.
Det har sedan tidigare varit känt att telomerlängden i viss mån ärvs.
Genom att studera två olika familjematerial från Västerbotten och Norrbotten
har vi visat att telomerlängden i större utsträckning ärvs från fadern än från
modern. Det ärftliga mönstret är dock svårare att upptäcka hos äldre. Detta
skulle kunna tyda på att telomererna förändras olika hos enskilda individer
genom livet och att vi eventuellt kan påverka vår telomerlängd genom vårt sätt
att leva.
I den gen som kodar för telomeras finns en position där kvävebasen inte
är den samma hos alla människor. Det har tidigare observerats att individer
som har uppsättningen C/C, det vill säga en så kallad C/C-genotyp, har kortare
telomerer jämfört med andra genotyper hos friska personer såväl som hos
9
patienter med kranskärlssjukdom. När vi undersökte 226 hjärtinfarktspatienter
och 444 friska kontroller kunde vi dock inte verifiera detta påstående.
Undersökningar av telomerlängden i blodceller hos cancerpatienter har
visat att den kan skilja sig från telomerlängden hos friska. I en
bröstcancerkohort på 265 patienter kunde vi visa att telomerlängden fungerade
som en markör för sjukdomens prognos. Korta telomerer indikerade längre
överlevnad, speciellt hos yngre patienter och hos de med avancerad sjukdom.
Det har förekommit teorier om att cancerpatienter tappar telomerer snabbare
med ökad ålder jämfört med friska men ingen har experimentellt visat detta. I
Västerbottens biobank fanns tillgång till dubbla blodprover från samma individ
tagna med 9-11 års mellanrum. 343 av dessa drabbades av cancer och som
kontroller användes 444 individer som ej fått någon sådan diagnos. Vi kunde
inte visa någon skillnad i telomerförlust mellan cancerpatienter och kontroller i
blodceller. Dock kunde vi se en mycket stark positiv korrelation mellan graden
av telomerförlust och telomerlängden vid första provet som var taget. Det vill
säga att om man hade långa telomerer från början så var tapper per år större
jämfört med om man hade korta telomerer från start. Ungefär 1/3 av alla
individer förlängde faktiskt sina telomerer. Detta antyder att det finns en
mekanism som ser till att skydda korta telomerer.
10
INTRODUCTION
INTRODUCTION
Telomeres are the repetitive structures terminating each chromosome. Although
telomeres are comprised of DNA probably not coding for that many genes, this
does not diminish their importance. They are crucial for chromosome stability
and integrity and function as a cellular biological clock. Thus, the telomeres play
a central role in the biology of cancer and aging.
The end
The knowledge and research about telomeres started in the 1930s when two
independent geneticists, Barbara McClintock and Herman J. Müller working with
Zea mays and Drosophila melanogaster respectively, both suggested that
chromosome ends have special features providing chromosome stability. Müller
observed that chromosome ends were sealed by a “terminal gene” and he
coined the term telomere from the Greek words telos (end) and meros (part).1
McClintock showed that ends created by chromosome breaks fused and formed
dicentric chromosomes, but that broken ends could be restored by recreating
the telomere. The conclusion from her work was that telomeres play a
fundamental role in the integrity of the chromosomes, preventing bridge-fusionbreakage cycles which are catastrophic for cell survival.2
The end-replication problem, namely that linear DNA can not be fully
replicated, was suggested in the 1970s by James Watson3 and Alexey
Olovnikov.4 The DNA polymerase can only synthesise in 5’ to 3’ direction,
leaving the 5’ end of the DNA shorter with each cell cycle (figure 1). In the
"Theory of Marginotomy" by Olovnikov,5 it was proposed that this progressive
chromosome shortening might underlie the limited number of replications
observed in vitro for normal somatic cells, and that malignant cells must have
mechanisms to escape the end replication problem.6,7
The DNA sequence of the telomeres was revealed in 1978 by studying
the fresh water living, ciliated protozoa Tetrahymena thermophila.8 It consisted
of tandem TTGGGG repeats, a very similar sequence to the human variant,
TTAGGG, which was isolated from a human recombinant repetitive DNA library
ten years later.9 In fact, the telomeric sequence is well conserved among most
vertebrates, further indicating the crucial role of telomeres in cellular biology.10
When measured by Southern blot, the telomere lengths of human
11
INTRODUCTION
DNA polymerase
DNA ligase
Okazaki fragments
3’
5’
Leading strand
5’
3’
3’
5’
5’ 3’
Lagging strand
5’
3’
5’
3’
5’
3’
Figure 1. The end replication problem. The DNA polymerase can only synthesise in 5’ to 3’
direction. Therefore, an RNA template is needed for for lagging strand synthesis of discontinued
sets of Okazaki fragments which are heald to a complete strand by a ligase. Beyond the end there
is no DNA where the template can bind, and a gap at the lagging strand 5´ end are created, making
the telomeres shorter for each cell division.
chromosomes are about 8-12 kb but have a great interindividual variability for
all ages.11-15 They also vary among chromosomes, including homologous, from
the same cell.16-20 Adjacent to the telomere is the subtelomeric region
comprising DNA of variable lengths (1 kb-~300 kb) and of high sequence
similarity to the telomeres.21-24
In 1985, Elisabeth Blackburn and Carol Greider used synthetic telomere
primers to find an enzyme adding TTAGGG repeats in Tetrahymena cell free
extracts.25 The enzyme was called telomerase and was shown to have an RNA
component with a sequence complementing the telomeric repeats, acting as a
template for de novo telomere synthesis.26-28 Due to this feature, telomerase
can extend the number of replications a cell may undergo.
These central discoveries were the start of what is now a large research
field with main focus on cancer and aging. In recent years telomeres have been
suggested to be involved in cardiovascular diseases, diabetes, inflammatory
conditions and stress and age-related degenerative diseases as well.
12
INTRODUCTION
Replicative senescence and immortality
In the 1960s, Leonard Hayflick observed that cells have a limited number of cell
divisions and this phenomenon was called “The Hayflick limit”.6,7,29 Although the
cells had stopped dividing he noticed that they continued their metabolism. In
1990 Harley et al. showed for the first time that telomeres shorten with
increasing population doublings in cultured human fibroblasts. The telomeres
shortened with about 50-100 bp with each cell cycle, and after 50-70 population
doublings they stoped dividing.12,30 The telomere attrition with age was found to
be true also in vivo.12,13,31,32
Human cells in culture enter replicative senescence at a critical telomere
length,33 also called mortality stage 1 (M1),34 with a phenotype showing cell
cycle arrest, low metabolic rate and β-galactosidase expression.35 When the
two tumour suppressor genes p53 and pRb are inactivated by viral
oncoproteins, the cells can escape M1 and further divide about 20 doublings
until the cells reach crisis or M2.36,37 At this stage, the telomeres are extremely
short (~1,5 kb) and massive cell death and chromosome instability are
observed.38 A rare fraction of the cells can bypass this checkpoint as well and
become immortalised usually by activating telomerase.39 Telomere length are
then stabilised and the frequency of dicentric chromosomes decreases.40 The
final evidence that telomere maintenance is crucial for immortalisation came
from experiments showing that overexpression of hTERT (human Telomerase
Reverse Transcriptase), the catalytic subunit of telomerase, results in
prevention of senescence induction.41-43 Senescence can probably be induced
by diverse signals, for example DNA damage checkpoints and by loss of the 3’
overhang.44-48 However, hTERT does not induce cell transformation but can
perhaps alter gene expression.49-52 In fact, transformation can occur in the
absence of telomerase.53
Telomerase is active during embryogenesis but is silenced in most
somatic cells.54 Activity can also be observed in stem cells, for example in the
hematopoietic system55 and in the basal crypts of the intestine,56 as well as in
activated lymphocytes57,58 (figure 2).
13
INTRODUCTION
Telomere length
Germ cells
15 kb
Somatic cells
Activated lymphocytes
Stem cells
Highly proliferating epithelial cells
5-7 kb
Telomerase
activation
2-4 kb
Cell divisions
Senescence
”Crisis”
Figure 2. Telomeres shorten with increasing population doublings in somatic cells until a critical
length is reached. The cells can then enter senescence or activate telomerase in order to maintain
the telomeres and become immortalised. Telomerase activity is constitutively expressed in germ
cells and transient expression is observed in stem cells, activated lymphocytes, and highly
proliferating epithelial cells. (Adapted from Harley 2002)
Telomerase
Components
Telomerase is a ribonucleoprotein (RNP) complex with reverse transcriptase
activity. It consists of hTERT,59,60 an RNA component, hTR or hTERC,61 and a
number of other proteins involved in the RNP complex. The enzyme can only
bind to and act on the 3’ end of the telomeres. The RNA component consists of
11 nucleotides (CUAACCCUAAC) complementing the human telomeric
sequence (figure 3). hTERT is only detectable in tissues/cells with telomerase
activity while hTR is present in telomerase negative tissues/cells as well.62,63
Since hTERT expression correlates with telomerase activity, it is believed that
hTERT is the key determinant of the enzyme level, even if hTR is also coupled
to telomerase activity.64 Recent papers indicate that two molecules each of
hTERT, hTR, and the dyskerin protein form the active telomerase complex. The
ATPases pontin and reptin are able to interact with both hTERT and dyskerin
and are shown to be required for assembly of the RNP.65,66
14
INTRODUCTION
Telomerase
Telomere
hTR
C A AUCCCA AUC
GT T A
G
hTERT
G
G
Figure 3. Telomerase is an enzyme with reverse transcriptase activity able to elongate the
chromosomes by the novo syntesis of telomeric repeats. The component hTR function as a RNA
template so that the catalytic subunit hTERT can add the correct nucleotides to the 3’ end.
Telomerase and telomere length regulation
The hTERT gene is 35 kb long and consists of 16 exons and 15 introns. It is a
GC-rich region and contains binding sites for various proteins.67-69 The
transcription of telomerase can be regulated by diverse transcription factors for
example estrogen, since the hTERT promoter carries a palindromic estrogenresponsive element, or c-Myc.70,71,72 hTERT can also undergo alternative
splicing and subcellular localisation. The 14-3-3 proteins can interact with
hTERT and thereby induce its accumulation in the nucleus.73 The localisation
within the nucleus however, seem to vary between cell cycle stages. In time for
replication, telomerase is sequestered from nucleolar sites into the
nucleoplasm.74 During S-phase, hTR and hTERT have been shown to be
recruited to the telomeres.75 Therefore, telomerase is believed to act in this
stage of the cell cycle. It has also been shown that telomerase preferentially
expand the shortest telomeres.76-78 However, high telomerase activity does not
always correspond to longer telomere length. Human cell lines show a large
variation in telomere length and the ones with shortest telomeres do not
express the lowest telomerase activity. This indicates that telomerase do not
regulate telomere length alone. The regulation is most likely executed by
complex mechanisms involving telomere associated proteins. It is believed that
telomeres can exist in an open, uncapped state allowing elongation by
telomerase, or in a closed, capped formation representing a telomerase nonextendible state.76,79
15
INTRODUCTION
Telomere structure
The telomere structure is complex and one of its functions is to protect the ends
from being recognised as double strand breaks (DSB). The last 50-200 bp of
the telomere consists of a single stranded (ss) G-rich 3’ overhang.80 The 3’ end
seems to terminate randomly, but a high fraction of the 5’ strand ends with
CCAATC-5', suggesting that a nuclease with high specificity processes the C
strand.81 The double stranded (ds) part of the telomere is invaded by the 3’ end,
thereby creating ring structures of a duplex T-loop and a smaller, ss
displacement loop (D-loop).82,83 These can form into more complex secondary
DNA conformations such as intermolecular G-quadruplexes which could simply
sequester the telomere end from being observed as a DNA break.84,85 However,
data suggest that telomeres are recognised as DNA damage during the G2 cell
cycle phase and that homologous recombination factors are important for
restoration of the protective structure.86
It has since long been assumed that telomeres are transcriptionally silent
due to their heterochromatic structure, but two recent papers have shown that
mammalian telomeres can be transcribed into RNA, so called TelRNAs or
TERRA. These could be important for telomere integrity.87,88 The
heterochromatic structure is enriched in epigenetic marks and one of its
characteristics is the ability to silence nearby genes. Telomeres have been
shown to silence genes in the subtelomeric region by telomere positioning
effects.89 Alteration of these marks correlates with the loss of telomere length
control, and in the opposite way, if telomeres are shortened to a critical length
epigenetic marks will be changed.90-92 Because of this, epigenetic status might
be crucial in telomere length regulation.
The shelterins
A variety of proteins are associated with the telomeres and six of them,
collectively referred to as the shelterin complex (previously also telosome93), are
telomere specific.79 These are TRF1, TRF2, TIN2, Rap1, POT1 and TPP1 (or
PTOP).94,95 TRF1, TRF2 (telomeric repeat binding factors) and POT1
(protection of telomeres 1) binds directly to the TTAGGG repeats. Together with
the three other shelterin subunits they allow cells to distinguish the telomeres
from DNA damage. Many of the human telomere associated proteins have
closely related proteins or homologues with similar function in prokaryotic cells.
Experiments using these model systems have often resulted in fundamental
16
INTRODUCTION
theories regarding telomere length regulation, proven lately in human cells as
well. However, these studies are not addressed in this thesis.
The TRF1 and TRF2 complex
TRF1 show great specificity for the ds telomeric repeats and the total amount of
TRF1 is correlated to the telomere length.96-101 Even though telomerase activity
is not altered, overexpression leads to telomere shortening until a new setting is
achieved while inhibition will result in telomere lengthening. TRF1 does not
modulate telomere length in telomerase negative cells but has been shown to
inhibit telomerase in a cis-acting manner via a protein-counting machinery
regulating telomere length via feedback mechanisms.102-104
TIN2 (TRF1-interacting nuclear factor 2) has a central role in keeping the
core telomere proteins together by its binding to three of the shelterins (TPP1,
TRF1, TRF2). POT1 can bind to the ss 3’ overhang but in association with
TRF1 it can also localise to the ds part.100,105 It is believed to inhibit the binding
of telomerase.100,106,107 TPP1 is a protein associated with both the TRF1/TIN2
complex and with POT1. It enhances POT1 affinity to telomeric ss DNA and
with telomerase, bridging the enzyme to the shelterins.
TRF2 plays a role in telomere length regulation and preservations of the
telomeres. When inhibiting TRF2, the telomeres are recognised as DSB and
apoptosis, via activation of the p53/ATM kinase pathway, or senescence is
observed.108 One explanation to why loss of TRF2 leads to rapid cell death is
that it can bend and remodel telomeres into forming T-loops.82,83 Rap1
(repressor/activator protein) is a TRF2 binding partner unable to bind to the
telomere on its own. Knockdown will lead to telomere elongation and thus,
Rap1 is suggested to act in a negative telomere length manner.109,110 Recently
TRF2 has, when in combination with telomerase depletion, been shown to act
as an oncogene in vivo.111 Upregulation of TRF2 has also been observed in
several tumours.112-114 Interestingly, inhibition of TRF2 could therefore be
beneficial in cancer therapy.115,116
ALT
Some mammalian cell lines can maintain their telomeres without telomerase.117These cells show very long and heterogeneous telomeres, sometimes >50
kb. This mechanism is named alternative lengthening of telomeres (ALT).120
119
17
INTRODUCTION
About 10 % of human tumours do not express telomerase activity, instead some
of them use ALT.121 A larger fraction of tumours with mesenchymal,
(osteosarcomas, liposarcomas) or neuroepithelial (astrocytomas) origin extend
their telomeres by ALT compared to other tumours.122,123
The mechanism for telomere elongation by ALT is recombination events,
but the precise process is not fully elucidated.124,125 A hallmark for ALT-cells, not
present in telomerase positive cells, is the ALT-associated promyelocytic
leukaemia bodies (APBs) involved in DNA damage response.126 They are found
in about 5% of the ALT-cells but are not essential for the ALT phenotype.127,128
Their role has not been demonstrated conclusively but they could sequester
linear DNA away from proteins recognising DSB and are suggested to be the
site of ALT activity.124
Telomere length measurement
A variety of methods for measuring telomeres have been developed. The
golden standard has for long been Southern blotting, where mean terminal
restriction fragments (TRF) are analysed. The DNA used must be of good
quality and not degraded since restriction enzymes are used in the assay for
fragmentation of the DNA before it is separated on an agarose gel, transferred
to a nitrocellulose membrane and hybridised to a for example radioactively
labelled telomere specific probe. The method requires a lot of DNA (~3-10 µg),
the subtelomeric region is included in the analysis and it can not detect very
small differences in telomere length.129 Nor is it an ideal method for measuring
long telomeres, such as in the ALT-cells. For this, pulse field electrophoresis
can be used. An alternative to Southern blot is the Slot blot method where the
telomere DNA content is calculated expressed as the percentage of average
chemiluminescent signal compared to a reference DNA. This method requires
only small amounts of DNA (~20 ng). However, it is not suitable to monitor small
changes in telomere length either.130
The FISH technique can be used to visualise telomeres and quantify
telomere length by measuring the fluorescence signal (Q-FISH). Peptic nucleic
acid probe usage instead of standard oligonucleotides stabilises the method
and by performing Flow-FISH, non-dividing cells can be investigated.9,17,131
FISH can also be combined with immunostaining.9,17,132 This allows examination
of paraffin embedded tissues and identification of different cell types.
Some PCR based methods for telomere length determination have also
been developed. Single telomere length analysis (STELA) is a method used for
18
INTRODUCTION
measuring telomere length on individual chromosomes where the primers for
the X and Y chromosomes have been best evaluated. It is highly telomere
specific but probably include the subtelomereic region.19 Amplification of
telomeres by PCR was long thought to be impossible due to their repetitive
structure. Richard Cawthon desiged primers with flapping 3’ ends, unable to
hybridise to each other and give primer-dimer products. By this, he succeeded
in using real time PCR for the analysis of telomere length.133 A great advantage
of this method is that only 10-20 ng of DNA are required. Mean telomere length
is measured, but a small part of the subtelomeric region can theoretically also
be included in the PCR-product if the region contains binding sites for the
telomere primers. The method is sensitive and can detect small changes in
telomere length.
Telomere length in normal individuals
From epidemiological studies it is well established that telomere length declines
with age, but there is a large interindividual variation both regarding telomere
loss134 and telomere length. Human blood cells shorten with approximately 1480 bp per year.135-139 Newborns shorten telomeres rapidly the first two years of
their lifes140,141 and there might be a plateau around 30-50 years of age where
telomere length is more or less unaffected.142 Telomere length of different
tissues in human fetuses as well as in newborns and older individuals seems to
correlate.14,143,144
Human peripheral blood contains a lot of different cell types which all
origin from a hematopoietic stem cell able to self-renew in the bone marrow
(figure 4). Telomere length might differ depending on cell type. Telomeres in Bcells have been shown to be about 15% longer and have a slower attrition rate
compared to T-cells. Moreover, memory B-cells seem to have longer telomeres
than naïve B-cells and an elongation was observed when inducing B-cell
differentiation into plasma cells.137 In the germinal centres, naïve B cells have
shorter telomeres compared to centroblasts which in turn have shorter telomere
length than centrocytes. The centroblasts and centrocytes express telomerase
activity while naïve and memory B-cells do not.145 It has also been shown that
granulocytes
have
longer
telomere
length
than
lymphocytes.141
19
INTRODUCTION
Hematopoietic stem cell
Multipotent progenitor
Lymphoid oligopotent
progenitor
Myeloid oligopotent
progenitor
Basophil
NK-cell
Eosinophil
T-cell
B-cell
Erythrocyte
Megakaryocyte
Monocyte
Neutrophil
Granulocytes
Platelet
Macrophage
Figure 4. Schematic simplified drawing of the hematopoietic system where one stem cell able to
self-renew gives rise to all peripheral blood cells.
It has been speculated that telomere length might regulate human lifespan
since long telomeres have been associated with increased survival.146,147
However, not in the oldest old.148-151 Women have a longer lifespan that men,
and women have in some papers been shown to actually exhibit longer
telomeres and/or shorten them at a lower rate than men.152-155 From this it can
be speculated that telomere length may act as a biomarker for age-related
conditions, where both the length per se and the attrition rate could be of
importance.
Telomere length and heredity
Twin studies and mapping experiments have documented that telomere length
is 36-84% inherited but the mode of inheritance and the genes involved are
unknown.141,153,156-158 An X-linked inheritance pattern was initially observed154
and a positive correlation was found when investigating mother-offspring
telomere length at partus.159 But a later publication have shown a stronger
paternal than maternal heredity regarding telomere length.15 Telomere length of
the children has been positively correlated to the age of the father at
conception, giving further support to the paternal inheritance theory.15,146,155,160
20
INTRODUCTION
Moreover, the paternal lifespan was associated with the daughters’ telomere
length when investigating an Amish population.15 Interesting is also the fact that
the telomere length in sperm actually increases with age.12,146,161 The telomere
length in the zygote might well influence telomere length in blood cells later in
life.
From a study showing equally strong telomere length inheritance between
monozygotic (MZ) and dizygotic twin (DZ) pairs, it was hypothesised that the
highly significant correlation depended mainly on shared environmental factors.
Otherwise there would be a stronger inheritance in MZ compared to DZ.162
However, other twin studies found that identical homologous telomeres from
aged MZ twins showed significantly less differences in relative telomere length
compared to the two alleles within one individual. From this it was concluded
that epigenetic/environmental effects on relative telomere length are relatively
minor during life.163,164 These results are conflictive.
Telomeres and diseases
Constitutional genetic disorders
Some genetic disorders demonstrate premature aging and involve telomeres
and telomere associated proteins. One such condition is dyskeratosis congenita
(DC) characterised by a classical triad of abnormal skin pigmentation, nail
dystrophy and leukoplakia.165 Other symptoms can also be observed where the
most severe ones are bone marrow failure, predisposition to malignancies and
pulmonary complications.166 The genetic traits are either X-linked, autosomal
dominant or recessive. It is caused by mutations in the gene encoding for
dyskerin (DKC1),167 or more rarely in hTR168,169 or hTERT genes.170 Cell lines
from patients with X-linked DC show reduced levels of hTR and shorter
telomere length compared to non-affected individuals. The shorter telomere
length in patients have also been found in vivo.171,172 Many patients with DC,
and other individuals with mutations in hTERT and hTR, suffer from idiopathic
pulmonary fibrosis. It is a fatal disease characterised by chronic lung fibrosis
and abnormal pulmonary gas exchange.173
hTR mutations have also been found in patients with inherited aplastic
anemia (AA) but not in the acquired form which is the most common.174
However, patients with acquired AA show shorter telomere length and have
higher telomerase activity compared to controls, even though they do not reach
21
INTRODUCTION
as high telomerase levels as the chronic form.175,176 Fanconi's anemia is an
autosomal recessive disorder causing bone marrow failure (aplastic anemia)
and abnormalities of inner organs and skeleton. The patients have an increased
risk of developing leukemia. They also have short telomeres, an increased
telomere attrition rate and express high telomerase activity.177 One possible
explanation to why the telomeres are affected in these diseases is the fact that
hematopoietic progenitor cells must proliferate very rapidly to resist the bone
marrow failure, but direct effects on the telomere machinery has also been
described.
Hutchinson-Gilford progeria syndrome is an extremely rare disorder
showing early aging as a symptom. It is caused by a truncated Lamin A gene
(called progerin), a key component of the nuclear lamina, but occurs
sporadically and is therefore not familiar but still congenital.178 Fibroblasts from
Hutchinson-Gilford patients show shorter telomere length compared to healthy
individuals.12
Werner and Blooms syndromes are two disorders caused by mutations in
the RecQ helicases WRN179 and BLM.180 These proteins interact with at least
three of the shelterin proteins namely TRF1, TRF2 and POT1. Werner
syndrome is an autosomal recessive inherited disorder showing premature
aging symptoms like hoariness, skin alterations, osteoporosis, cataracts,
atherosclerosis and cancer. Blooms syndrome patients do also show signs of
premature aging, predisposal to cancer and short telomere length.181
Ataxia telangiectasia is a rare autosomal recessive inherited disease
presenting itself with radiosensitivity, immunodeficiency, sterility and a
predisposition for mainly hematopoietic malignancies. A mutation in the ATM
kinase protein, which has a central role in DNA damage signalling introduced by
telomere dysfunction, is the cause of the disease.44,182,183 NBS1 is an ATM
substrate that when mutated gives rise to Nijmegen breakage syndrome. These
patients have neurological defects but also show predisposition for the
development of malignancies. The NBS1 protein forms a multimeric complex
with RAD50/MRE11 and recruits them to the site of double strand beaks.184
Telomeres and tumours
One of the hallmarks of cancer cells is genomic instability promoting stepwise
accumulation of genetic mutations in both oncogenes and tumour suppressor
genes. The process from premalignant lesions to metastatic tumours is slow
and probably starts years before diagnosis.185 There are a few essential
mechanisms, present in almost all tumour types, which are responsible for and
22
INTRODUCTION
dictate tumour growth. These are self-sufficiency in growth signals, insensitivity
to growth-inhibitory signals, evasion of apoptosis, sustained angiogenesis,
tissue invasion/metastasis and limitless replicative potential.186 Since telomeres
function as the “biological clock” of a cell, they can in part control tumour growth
by limiting the replicative potential. Exactly how telomeres, telomerase and their
associated proteins function and change during tumour development is not fully
elucidated and the pattern is probably not the same in all tumour types.
However, when the telomeres are lost a bridge-fusion-breakage cycle during
replication starts. Sister chromatids fuse at their ends and form a bridge during
anaphase which breaks when the two centromers are pulling in the opposite
direction. The bridge will not break at the same location as the end fusion,
leaving one chromosome with a repeat and the other one with a deletion. This
scenario will be repeated next round of replication leading to more and more
chromosome instability. These broken chromosomes can be healed again by de
novo addition of telomeric repeat by telomerase.187
Telomerase activity is found in about 85-90% of human tumours and has
been shown to be a prognostic marker in various malignancies.188,189
Interestingly, telomerase and hTERT do have extratelomeric functions
preventing cell death.190-192 Therefore, telomerase has a dual role in cell survival
(figure 5).
The role of telomere length as a diagnostic and prognostic marker has not
been as widely studied as the role of telomerase. Telomere length in tumour
lesions is often shorter than in the corresponding normal tissue.193 In fact, really
short telomeres, called T-stumps, have been found in human cancer cell
lines.194 Telomere length has also been associated with decreased survival of
patients with blood malignancies195-199 as well as patients with some solid
tumours.200 Interestingly, telomere length in blood cells from cancer patients
with certain types of solid tumours appears to be shorter compared to healthy
controls and associated with survival, indicating a systemic tumour effect.201-203
Emerging evidences that cancer stem cells are responsible for tumour
development and maintenance have been presented within the last years.204
The first evidence of this was demonstrated in leukemic cells.205 A debate
whether the proposed cancer stem cells arise from stem cells, progenitors, or
more mature cells is ongoing but a key feature is their self-renewal capacity. If
the hypothesis about cancer stem cells is correct, they are clearly an interesting
cancer therapy target, for example by inhibition of telomerase to limit their
proliferative capacity.206 The problem is to find them since there is no good
single marker for cancer stem cells available so far.207
23
INTRODUCTION
Telomerase
activity
Cell growth
Tissue protection
Tumour development
Age-related diseases
↑ Tissue fitness
↑ Life span
Cancer
Figure 5. Telomerase has a dual role in survival. (Adapted from Geserick et al. 2006)
Telomerase and cancer therapy
Since almost all human tumours express telomerase activity, inhibition of the
enzyme has been suggested as a general cancer therapy. Down regulation of
telomerase activity can be completed by introducing a dominant negative
hTERT in immortalised cell lines. The telomere length then shortens and the
cells eventually die.208 Because of the fact that the cells must divide a number
of times before the telomere length is affected, inhibiting telomerase can have a
delayed therapeutic effect. However, most malignant cells show shorter
telomeres than normal cells.11,32 Three main approaches for targeting
telomerase have been described and substances from all of them are under
clinical trials (table 1).206
24
INTRODUCTION
Table 1. Main cancer therapy approaches for targeting telomerase
Gene
therapy
Immunotherapy
Suicide gene
therapy
A vector targeting telomerase is introduced. The vector
encodes an enzyme that, when activated by a pro-drug, will kill
206,209
cells that are telomerase positive.
Oncolytic viral
therapy
An oncolytic adenovirus, able to kill cancer cells, is introduced
206,210
and the viral replication is driven by the hTERT promoter.
Dendritic cell
presentation
Peptide
vaccine
Small
molecular
inhibitors
hTERT peptides are presented by the major histocompatibility
complex (MHC) to the immune system, and the cytotoxic Tcells can provide protective immunity against tumours.
Dendritic cells, which are the most efficient antigen-presenting
cells, can be used. Also a mixture of MHC class II peptides
derived from the active site of telomerase can be injected to
206,211-213
give an immune response.
Small molecule oligonucleotides function as telomerase template antagonists
thereby inhibiting telomerase action. G-quadraplexes have also been tested as
well as reverse transcriptase inhibitors and other small interacting
214,215
molecules.
Non-malignant disorders
In recent years, telomere length and its implication for non-malignant disorders
has been investigated and the largest field of this type of telomere research is
cardiovascular diseases (CVD). In the beginning of this century, short telomeres
were observed in patients with various types of heart conditions (myocardial
infarction, coronary heart disease and heart failure).216-220 However, conflicting
results have been published.221 Numerous data on CVD risk factors and
telomere length have also been published. Contradictions in the literature make
it difficult to reach consensus on this topic, but new data indicate that lifestyle
rather than single blood tests could affect telomere length.222-224 It has also been
discussed whether the inflammatory, and oxidative stress components in
atherosclerosis could affect telomere length, causing attrition in patients via
increased proliferation or by direct effect on the telomeres. Both processes have
been shown to shorten telomere length.218,225-227
Psychological stress, depression and other mood disorders have been
associated with short telomeres228-231 as well as patients with
osteoporosis,232,233 rheumatoid arthritis,234 and individuals having low socioeconomic status.235 This new research field on telomeres is growing.
25
AIMS
AIMS
General aim
The research on telomeres and telomerase is expanding beyond the cancer
field. This thesis aims to explore telomere length as a general biomarker
effecting, or perhaps more likely, being affected by processes involved in
cancer, normal aging and other disorders such as cardiovascular disease.
Telomere length is suggested to be inherited and if short telomeres are
predisposing for certain diseases, it is interesting to further elucidate telomere
heredity and its mode of inheritance.
Specific aims
Paper I & II
To investigate if telomere length in blood cells is inherited and if so, what the
mode of inheritance is and if the degree of inheritance is diminished by nonheritable factors affecting telomere length during life.
Paper III
To test a previously published difference regarding telomere length and
telomere attrition rate between the diverse -1327T/C hTERT genotypes in both
controls and myocardial infarction patients.
Paper IV
To evaluate blood telomere length at diagnosis as a possible biomarker for
breast cancer risk and survival.
Paper V
To investigate individual telomere loss with age in blood, and examine whether
the magnitude of yearly loss is associated with future tumour formation and
dependent on telomere length at baseline.
26
MATERIAL & METHODS
MATERIAL AND METHODS
Study populations
All studies are approved by ethical committees and the participating individuals
have given informed consent.
Paper I
132 healthy individuals in 49 families, living in northern Sweden were included
in this study. After blood draw, peripheral mononuclear cells (MNC) (monocytes
and lymphocytes) were collected and divided in two parts. One part was frozen
in DMSO and the other part was EBV (Epstein Barr virus) transformed after
which viable lymphoblastoid cells were DMSO frozen. 27 daughters and 22
sons, age 32-42 years (mean 37), and their parents (41 mothers and 42 fathers,
age 52-86 years) were investigated regarding telomere length in both the
transformed and non-transformed samples.
Paper II
Telomere length was investigated on a subset of a multigenerational family
cohort (2-5 generations) where the oldest individuals from the county of
Västerbotten and their relatives were invited to a research project, originally
aimed at studying genetic and environmental factors influencing heredity of
personality traits, upbringing, general health and longevity. Whole blood was
available from 962 individuals in 68 families (445 men and 517 women) with an
age-span of 0-102 years. DNA was extracted and telomere length was
measured on all of them. The inheritance pattern was investigated by examining
duos and trios created from drawn pedigrees, totally 444 individuals. DNA was
not available from all individuals in the pedigrees. Duos included one parent and
his or her child (n = 207, age 13-102 years) while trios included both parents
and one of their children (n = 10, age 12-91 years). Trios instead of duos were
selected telomere length data was available on both parents. Only one duo or
trio from each pedigree contained a parent born within the family (i.e. was not
in-laws), and the oldest parent and child were exclusively included in the
analysis to avoid selection bias and confounding factors. The remaining duos
27
MATERIAL & METHODS
included in-law parents. Using the same criteria, duos (n = 79, age: 13-101) and
trios (n = 3, age: 15-91) were created for grandparents and grandchildren (n =
85).
Paper III
226 cases of myocardial infarction (MI) patients (155 men and 71 women) and
444 controls (306 men and 138 women) were selected from a part of the Malmö
Cancer and Diet Study. Cases and controls were age, and gender matched so
that the age-span was 48-68 years (mean 61) for both groups. Blood was drawn
and DNA extracted from MNC preparations.
Paper IV
All samples were collected as buffy coats (all leucocytes) from breast cancer
patients referred to the Oncology Clinic at Umeå University Hospital in
Västerbotten County, Sweden, within three month after morphological
diagnosis. 265 patients, not diagnosed with any previous cancer form and
untreated, except for surgical removal of their tumour, were included in the
analysis. As controls, 300 women from the MONICA study in Northern Sweden
(“Multinational Monitoring of Trends and Determinations in Cardiovascular
Disease”) and 146 women from the Malmö Cancer and Diet Study were
selected (also used in Paper III). We consider both cases and controls
representative for the general population of Sweden. The age range was 29-75
years for controls (median 55), and 29-86 years for cases (median 57).
Data regarding tumour size, nodal status and estrogen receptor (ER)
expression was collected from the clinical charts at the Oncology Clinic. Since
two different techniques were used to detect ER status in the cohort, four
patients had to be excluded from the statistical analysis regarding ER since they
were classified as “uncertain”. The information of the cause of death was
obtained from clinical charts and death certificates, with the last follow up in
May 2007.
.
Paper V
Individuals from the Västerbotten Intervention Project (VIP), launched 1985 in
the County of Västerbotten, the MONICA cohort and the local Mammography
Screening Project (MSP) are all included in the North Sweden Health and
Disease Study (NSHDS). All residents are invited in the year of their
28
MATERIAL & METHODS
40th, 50th, or 60th birthday for an volunteer interview and a blood draw for the
VIP. Between 1985 and 1996, residents were also invited at the age of 30.
Blood has been drawn with anticoagulants, separated into plasma, erythrocytes
and buffy coat and stored at -80°C in small aliquots. In the NSHDS collection
we identified >7000 individuals who had donated blood samples with about 10
years interval (9-11 years) and of these, 343 persons had obtained a cancer
diagnosis after the second blood sample (time from sample 2 to diagnosis: 0-11
years, mean 2.7). In the cohort with two repeated samples, 686 age, and
gender matched controls were also selected. The age-span was 30-61 years for
sample 1 and 40-70 years for sample 2. Cancer cases were identified through
record linkages with the regional Cancer Register. In the statistical analyses
314 of the cases and 645 controls (totally 1918 samples) were included due to
non-sufficient amounts of buffy coat for DNA extraction or unsuccessful real
time PCR.
To study possible family patterns regarding TL attrition, 13 families from
the multifamily cohort in paper II was utilised. In these families it was possible to
selected 10 or more related individuals, i.e. no in-laws, in at least three
generations.
Telomere real time PCR
Telomere length was measured by real time PCR first described by Richard
Cawthon in 2002.133 β2-globin was used as a housekeeping gene to normalise
the DNA loading. DNA from each individual and from a reference cell line
(CCRF-CEM) was denatured at 95°C, chilled on ice, centrifuged at 730 g and
tested in triplicates using 96 well plates except for in paper V where 384 well
plates were used. To test the efficiency of the PCR reaction, a standard curve
ranging from 0.3 to 5 ng/μl of DNA was produced in every plate using the cell
line CCRF-CEM. Telomere single copy gene (T/S) values were calculated by
the formula 2-ΔCt where ΔCt = Cttelomere - Ctβ2-globin. Relative T/S values were
determined by dividing sample T/S values with the T/S value of the reference
DNA. Mean inter-assay coefficiency of variance (CV) for this method was 3.96%
in our laboratory when a separate series of blood samples was analysed twice
by two separate investigators.
29
MATERIAL & METHODS
Paper I
In paper I the primers Tel1a and Tel2a were used for telomere amplification and
primers Hbg1 and Hbg2 for β2-globin amplification (table 2). All samples were
diluted to 1.75 ng/µl in TE buffer and tested in triplicates using 35 ng DNA/well
in a total reaction volume of 50 µl. Final concentrations of reagents were 1.25
units of AmpliTaq Gold DNA polymerase (Applied Biosystems), 150 nM 6-ROX
(Molecular Probes), 0.2x SYBR Green 1 (Roche Diagnostics), 1% DMSO, 0.2
mM of each dNTP (MBI Fermentas, Amherst, NY), 5 mM DTT, 15 mM Tris-HCl
(pH 8.0), 50 mM KCl, and 2 mM MgCl2. PCR amplification was performed in a
PRISM 7000 sequence detection system (Applied Biosystems). Cycling
conditions for the telomere amplification comprised an initial denaturation step
at 95 degrees for 10 minutes followed by 35 cycles of 95 degrees for 15
seconds and 54 degrees for 120 seconds. The same denaturation step
initialised the β2-globin amplification but was followed by 40 cycles of 95
degrees for 15 seconds and 58 degrees for 60 seconds. SDS v1.1 software
(Applied Biosystems) was used for construction of a standard curve and cycle
threshold (Ct) values.
Table 2. Primers for telomere real time PCR
Name
Tel1a
Tel2a
Hbg1
Hbg2
Tel1b
Tel2b
Hbg3
Hbg4
Sequence 5´ to 3´
GGT TTT TGA GGG TGA GGG TGA GGG TGA GGG TGA GGG T
TCC CGA CTA TCC CTA TCC CTT CCC TAT CCC TAT CCC TA
GCT TCT GAC ACA ACT GTG TTC ACT AGC
CAC CAA CTT CAT CCA CGT TCA CC
CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT
GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT
TGT GCT GGC CCA TCA CTT TG
ACC AGC CAC CAC TTT CTG ATA GG
Final conc.
270 nM
900 nM
400 nM
400 nM
100 nM
900 nM
400 nM
400 nM
Paper II, III and IV
Sample and reference DNA were diluted to 1.75 ng/µl in TE buffer containing E.
coli DNA (Sigma Aldrich) to stabilise the PCR reaction. The primers Tel1b and
Tel2b were used for telomere amplifications and Hbg3 and Hbg4 for control
gene amplification (table 2). Final concentrations of both the telomere and β2globin PCR mix were 50 mM KCl, 10 mM Tris-HCl (pH 8.0), 0.2 mM of each
dNTP, 150 nM Rox (Molecular Probes), 0.2x Sybr Green (Roche Diagnostics
GmBH), 1% DMSO and 1.25 U AmpliTaq Gold DNA Polymerase (Applied
Biosystems). Final concentrations specific for the telomere and β2-globin mix
were 1.7 mM MgCl2, 2.5 mM DTT and 2 mM MgCl2, 5mM DTT, respectively.
30
MATERIAL & METHODS
PCR amplification was performed in a ABI Prism 9700 sequence detection
system (Applied Biosystems). Cycling conditions for the telomere amplification
comprised an initial denaturation step at 95 degrees for 10 minutes followed by
25 cycles of 95 degrees for 15 seconds and 54 degrees for 1 min. The same
denaturation step initialised the β2-globin amplification but was followed by 35
cycles of 95 degrees for 15 seconds and 56 degrees for 60 seconds. SDS v2.1
(Applied Biosystems) was used for the creation of a standard curve and
calculation of Ct values.
Paper V
Primer sequences and final concentrations were as in Paper II, III and IV but
17.5 µl DNA/well was used in a 384 well plate with a total reaction volume of 20
µl.
TRAPeze
Telomerase activity was measured by the TRAPeze method (TRAPeze
Telomerase Detection Kit, Oncor Inc., Gaithersburg, MD) according to
guidelines given by the supplier. Briefly, protein extracts were prepared and an
RNAse inhibitor was added to the lysis buffer to preserve the endogenous RNA.
The telomerase in the extract was then allowed to work on an added
radioactively labelled non-telomeric oligonucleotide (T/S) forward primer, and in
the second step the extension products were amplified by adding the reverse
primer to a PCR reaction. The PCR products were visualised on a
polyacrylamide gel and quantified by phosphimager. The relative telomerase
activity level was expressed as units of TPG (Total Product Generated).
SNP analysis
An hTERT TaqMan SNP genotyping assay was bought from Applied
Biosystems (No. C_1839086_10, Foster City, CA, USA). For PCR the
GeneAmp 9700 system (Applied Biosystems) was used and PCR-plates were
read (allelic discrimination) on the ABI Prism 7900 instrument. Assay and PCR
31
MATERIAL & METHODS
conditions were according to the supplemented protocol except that the PCR
was run on 10 ng DNA in a 10 µl reaction volume. The SDS v2.1 software
(Applied Biosystems) was used when calling genotypes. 7% of the samples
were rerun to check for genotype call accuracy, showing 100% consistency.
Statistical analyses
Statistical Package for the Social Sciences (SPSS) was used for statistical
analyses. In paper II and IV, telomere length values were converted into their
corresponding natural logarithm to achieve a normal distribution, so that tests
requiring normality could be performed. Pearson partial correlation with age,
and gender adjustments was used in paper I, II, IV and V while Pearson
correlation was performed in paper I and Spearman rank correlation coefficient
in paper III. Analysis of covariance (ANCOVA) with age, and/or genderadjustment was performed in paper III, IV and V for adjusted comparisons
between groups and for 95% confidence interval (CI) calculations. The chi
square test was also used in paper III for group comparison. Odds ratios (OR)
for breast cancer risk were calculated by binary logistic regression and hazard
ratios (HR) were obtained by multivariate Cox regression analysis in paper IV.
In the same paper and in paper V, Kaplan-Meier with the log-rank test was used
for survival analyses. In paper V, the MLwiN (Centre for Multilevel Modelling,
University of Bristol), a software for multilevel analysis, was used to test for
parallelism between families. 95% CI for correlation coefficients and
significance of the difference between two correlation coefficients were
calculated
using
VassarStats
(Lowry
1998-2008),
available
at
http://faculty.vassar.edu/lowry/VassarStats.html.
32
RESULTS AND DISCUSSION
RESULTS AND DISCUSSION
Telomere length inheritance
In paper I, a family material from northern Sweden consisting of “trios” (mother,
father and their child) and “duos” (any parent and his/her child) was investigated
regarding the mode of telomere length inheritance. This was further analysed in
Paper II on a larger, multifamily cohort spanning up to four generations. It was
previously shown that telomere length is, at least in part, inherited141,153,156-159
and an X-linked inheritance has been suggested.154 A significant motheroffspring telomere length correlation in newborns has also been found.159
In Paper I, telomere length declined by age (r = -0.356, p < 0.0001, n =
132), with a loss of 21 bp per year (women 16 bp, men 25 bp) after
recalculating the relative T/S results to Southern blot data using a transforming
formula.196 This corresponds well to previously published results using Southern
Blot154,155 or real time PCR150 for telomere length measurements. The
inheritance pattern was explored by examining the interfamilial relationships
regarding telomere length. A positive correlation between father-child pairs was
observed but not between mother-child pairs. When stratifying the children by
gender, a positive correlation was still observed for both father-daughter and
father-son pairs. This was not found on the maternal side. A correlation test
between spouses was performed as a negative control showing no significance
as expected. Age adjustments of all correlations did not change the statistical
outcome (table 3).
Table 3. Interfamiliar relationships Paper I
n
Non-adjusted
r
p
Age-adjusted
r
p
father-children
father-son
father-daughter
42
20
22
0.569
0.558
0.578
<0.001
0.011
0.005
0.564
0.540
0.603
<0.001
0.021
0.005
mother-children
mother-son
mother-daughter
41
18
23
0.361
0.141
0.161
0.146
0.577
0.463
0.159
0.149
0.163
0.335
0.582
0.482
spouses
35
0.219
0.207
0.107
0.559
33
RESULTS AND DISCUSSION
The results from Paper I could be confirmed in Paper II. Again, the father-child
correlation was highly significant independent of offspring gender. The mothers’
telomere length did not correlate significantly with offspring telomere length as a
whole group. However it did to the daughters’ telomere length but not to the
sons’. There was a significant correlation between grandparents-grandchildren
illustrating that the heritability of telomere length can be observed over two
generations (table 4). The sample size did not allow further subdivisions.
Tabel 4. Interfamiliar relationships Paper II
father-children
father-son
father-daughter
n
98
51
47
Age-adjusted
r
p
0.454
<0.001
0.465
<0.001
0.474
<0.001
mother-children
mother-son
mother-daughter
129
57
72
0.148
0.080
0.297
0.098
0.561
0.013
grandparents-grandchildren
85
0.272
0.013
Since the cohort studied in Paper II showed a great age-span, heritability in
different age-groups between parent and child, separated according to the age
of the parent at blood draw, could be calculated by R squared correlations
shown in percentages (table 5). This data illustrate that both paternal and
maternal telomere length inheritance diminishes with age probably due to nonheritable factors modulating telomere length during life. A number of
environmental and lifestyle factors are shown to do that.218,222,224,236 Chromatin
modifications important for telomere dynamics occur throughout life.237 It can
not be ruled out that lifestyle can influence such epigenetic modifications.
Table 5. R2 correlations in different age-groups
father-child
mother-child
34
Parents
<50 years
49 %
12 %
Parents
≥50 - < 70 years
29 %
7.5 %
Parents
≥70 years
4.9 %
0.03%
RESULTS AND DISCUSSION
From paper I and II it can be concluded that telomere length is inherited from
both the father and the mother, but that the paternal influence is far stronger
than the maternal. The non-significant correlation to the mother in Paper I is
likely due to the lack of power caused by small sample size. These results were
also confirmed in an Amish population.15 The age of the father at conception
seems to be associated to the telomere length of the offspring.15,146,155,160 This
provides indirect support to the paternal inheritance theory. In Paper II we found
a positive correlation between the age of the parents at the birth of the child and
offspring telomere length but it did not reach significance (father: r = 0.160, p =
0.117, n = 98. mother: r = 0.138, p = 0.120, n = 129). Moreover, telomere length
in sperm actually elongates with increasing age.12,146,161 Since an X-linked
inheritance also has been suggested, inconsistence in the literature exists.154
We were very cautious when selecting duos and trios. If these constellations
were created with more than one parent born within the family, a potential
lineage related confounder could be introduced. The selection process is not
fully stated in previous publications. The genes involved in the complex
inheritance of telomere length have not been elucidated. Imprinting has been
suggested as a possible mechanism but no experimental studies have been
done.15,238
Since a part of the MNC preparations in Paper I was EBV transformed
and cultured for about one month before DNA extraction, it was possible to
compare telomere length in non-cultivated cells with cultivated cells from the
same individual. There was a significant correlation between the two samples (r
= 0.220, p = 0.012) and the majority of the cell lines had elongated their
telomeres during cultivation. However, part of the elongation might be an
artefact since a selection of B-cells occurs by EBV transfection and these cells
have longer telomeres than T-cells and monocytes.137 The initial MNC telomere
length was inversely correlated to change in telomere length during cultivation
of the same cell cohort with a high statistical significance (r = -0.246, p = 0.005),
indicating that telomerase preferentially acts on cells with short telomeres. This
feature has been suggested previously.76,77,239 Telomerase activity was
measured in 13 of the cell cultures showing a variation from 0 to 97 TPG. The
enzyme activity did not significantly correlate with the telomere length (r =
0.425, p = 0.147), however the sample size was very limited.
35
RESULTS AND DISCUSSION
The -1327T/C hTERT promoter polymorphism
The transcription of hTERT has been widely studied but less is known about the
impact of genetic variations. A functional T to C polymorphism has previously
been found 1327 bp upstream the hTERT promoter starting site. Shorter
telomere length was found in peripheral blood from Japanese individuals not
carrying the -1327T-allele compared to the T carriers and the shortest telomeres
were found in the -1327C/C group.240 Moreover, the -1327C/C genotype was
overrepresented in coronary artery disease (CAD) patients and this group
showed shorter telomeres compared to the other CAD patients.241
To test this finding, telomere length and the -1327T/C polymorphism were
investigated in 226 patients with MI and 444 controls from southern Sweden.
However, the former results could not be reproduced. Neither in patients nor in
controls did the individuals carrying the -1327C/C polymorphism show
significantly shorter telomeres after age adjustments compared to the other
genotypes (p = 0.339 and p = 0.794, respectively). This was also true after
telomere length comparison between the -1327C/C group and a merged group
consisting of the -1327T/C and -1327T/T genotypes (p = 0.551). The -1327C/C
genotype was not overrepresented in the patient cohort compared to the control
cohort (p = 0.897).
The MI patients carrying the C/C polymorphism did not show a faster
telomere attrition rate with age compared to the other patients (-0.157, p =
0.249). An elongation was rather indicated in the C/C controls (r = 0.221, p =
0.021). None of the other genotypes, of either patients or controls, showed any
significant age related changes in telomere length (Controls: -1327T/C: r = -0.047,
p = 0.471, -1327T/T; r = -0.067, p = 0.505. Patients: -1327T/C: r = -0.037, p = 0.707,
-1327
T/T; r = -0.183, p = 0.154). Taken together, the C/C group do not show
shorter telomere length or faster telomere attrition rate in controls or MI patients.
One explanation to the discrepancy between Japanese and Swedish
individuals regarding the -1327T/C polymorphism is that gene expression can
differ depending on ethnicity.242 Therefore, the conclusion that the
polymorphism is not functional can only be drawn on Caucasians from this
study. Another possibility is that the polymorphisms co-segregate with genes
having different impact in diverse ethnicities.
36
RESULTS AND DISCUSSION
Telomere length as a biomarker in breast cancer
Breast cancer is the most frequently diagnosed malignant tumour in women of
the western world, affecting about 10%.243 Is has recently been shown that
telomere length in peripheral blood is shorter in patients with certain types of
solid tumours compared to controls, indicating that a simple blood draw can
give useful information on the disease process and for early diagnostics,
perhaps years before tumour development.201-203,243 However, in a recent study
on breast cancer, no difference in telomere length in peripheral blood between
patients and controls was shown.244
In our cohort both controls and patients experienced shortened telomere
length with age (n = 446, r = -0.241, p < 0.001 and n = 265, r = -0.307, p <
0.001, respectively). Two different control cohorts were merged to match the
age distribution of the patients. They did not differ regarding telomere length
after age adjustments justifying the merge (p = 0.219). We observed that the
patients actually had longer blood telomeres compared to controls after age
adjustments. This was true also after subdivision into age groups (table 6).
Table 6. Telomere length in controls and breast cancer patients subdivided into
different age groups
n
All ages
446
<45 years
158
45-54 years
63
55-64 years
160
>64 years
65
Controls
telomere length
(95%CI)
0.65
(0.63-0.67)
0.71
(0.68-0.74)
0.65
(0.61-0.70)
0.64
(0.61-0.67)
0.56
(0.52-0.61)
n
265
32
83
87
63
Cases
telomere length
(95%CI)
0.74
(0.71-0.77)
0.80
(0.73-0.88)
0.75
(0.71-0.80)
0.72
(0.68-0.76)
0.69
(0.64-0.75)
p
<0.001
0.018
0.001
<0.001
<0.001
The conflicting findings in our study compared to Barwell et al.244 can depend on
the method used for telomere length measurements since we used real time
PCR and they used Southern blot. A recent publication has indicated that very
short telomeres exist, so called T-stumps. These might not be detected by
Southern blot but probably by the PCR since it is a more sensitive method.194
To investigate whether telomere length could function as a risk marker for
breast cancer, odds ratios (OR) between quartiles divided according to telomere
37
RESULTS AND DISCUSSION
length among controls were calculated. The risk enhanced with increasing
telomere length (Ptrend < 0.001), with a maximal OR of 5.17 (95% CI: 3.09-8.64).
Moreover, telomere length in blood was also a prognostic factor for cancer
specific death since the patients with the shortest telomeres had an increased
survival compared to the longest telomere group (p = 0.006, subdivision
according to median telomere length among patients). Telomere length was a
stronger prognostic factor in patients <50 years (p = 0.024) compared to
patients ≥50 years (p = 0.095). Unpublished results from renal cell carcinoma
give support for this finding since the patients with long blood telomeres had a
worse prognosis compared to controls. However, this could not be observed in
the actual tumour tissue or normal kidney tissue from the same patients.245
Information on tumour size and nodal status, two well established
prognostic markers for breast cancer, was available on the patients. As
expected, large tumours and node positive patients had a poorer outcome. The
patient cohort was dichotomised using median tumour size as a cut off (0.16
mm). In patients with large tumours, individuals with short telomeres showed a
significantly increased survival compared to the ones with long telomeres (p =
0.006) but interestingly only in patients <50 years (p = 0.006). No correlation
was found on patients ≥50 years (p = 0.226) or on patients with small tumours
(p = 0.529, all ages). Node positivity together with long telomeres was
associated with worse prognosis for all ages (p = 0.001) as well as for women
<50 years (p = 0.039) and ≥50 years (p = 0.008). In node negative patients,
telomere length did not predict prognosis (p = 0.444, all ages). This indicates
that patients with short blood telomeres at diagnosis have a better prognosis,
especially in young individuals. This finding was further established with
multivariate Cox regression including age, nodal status, tumour size and blood
telomere length. The model indicates that short telomere length is actually an
independent, positive prognostic marker for breast cancer survival (HR 2.92,
95% CI: 1.33-6.39, p = 0.007).
ER status was investigated in 200 of the 265 patients. 78% was ER+ and
showed slightly longer telomeres compared to the ER- group (ER+: 0.73, 95%
CI: 0.70-0.75. ER-: 0.69, 95% CI: 0.65-0.74) but it did not reach statistical
significance (p = 0.191). ER+ patients had longer telomeres compared to the
controls (p < 0.001) while a borderline significance was calculated between ERand controls (p = 0.055). ER+ was not associated to better survival in our
patient cohort (p = 0.909). In both ER+ and ER- patients however, short
telomere length was correlated with increased survival (p = 0.005 and p =
0.025, respectively).
Telomere length has previously been shown to be shorter in tumour
lesions than in the normal corresponding tissue despite the fact that those
tumours to a great extent express telomerase.193 Tumour formation is likely to
start in cells with short telomeres, partly since they are genetically unstable.
38
RESULTS AND DISCUSSION
They also undergo more cell divisions by which the risk for mutations increases.
This is probably also the case in our cohort. Unfortunately, telomere length in
the tumour tissue could not be investigated. There are several possible
explanations to the discrepancy between telomere length in the tumours and
blood. Hematopoietic cells can, when activated by the immune system, express
telomerase and this is a feature separating them from endothelial cells. Brother
of the regulator of imprinted sites (BORIS) is a protein activated in various
cancers that can inhibit CCCTC-binding factor (CTCF).246 CTCF is a chromatin
insulator protein known to down regulate hTERT, and epigenetic alterations are
suggested to modulate telomere length.247,248 Since high levels of BORIS have
been detected in blood from breast cancer patients this might well provide an
explanation to the long telomeres found in the patient cohort compared to
controls. Several interleukins capable of activating telomerase249-253 have been
found in serum from breast cancer patients, providing further explanations to
why the patients display longer telomeres.254 Breast cancer is a hormone
related cancer and estrogen is a risk factor for tumour development.255 In
addition estrogen possess the ability to up regulate telomerase via estrogen
responsive elements on the hTERT promoter, or by posttranscriptional
regulation of hTERT via Akt-dependent phosphorylation.72,256 Moreover,
telomere shortening is accelerated by oxidative stress and estrogen have antioxidative capacity.236,255
We believe that the long telomeres shown in beast cancer patients are
due to the tumour, not that they are born with longer telomeres and hence have
a greater risk for tumour development. However, this can not be excluded from
this type of cohort. For this, longitudinal studies are required. The hypothesis
that telomere length in peripheral blood can give information about risk, and
perhaps more important prognosis, is very interesting and further studies are
needed to elucidate this suggestion.
Telomere attrition is telomere length dependent
The question whether short telomere length or increased attrition with age in
peripheral blood can predict future tumour development has been raised many
times but no experimental data has yet proven the theory. In order to explore
this we investigated telomere length in individuals having donated two blood
samples with 9-11 years in between. 314 individuals of the cohort developed
malignant tumours after the second blood draw while 645 individuals, still free
from tumours, were used as controls. Telomere length in blood cells did not
39
RESULTS AND DISCUSSION
differ between patients and controls after age adjustments in sample 1 or 2,
indicating that telomere length is not a good predictor of later tumour
development early or close to the diagnosis (p = 0.263 and p = 0.770,
respectively).
Another speculation is that cancer patients loose telomeres at a higher
rate than non-affected individuals, and that this accelerated telomere loss could
occur years before diagnosis. This could not be proven in our cohort since we
found similar yearly change in telomere length for both patients and controls (p
= 0.446). The majority of all individuals lost telomeres with age but 34% actually
increased their telomeres between sample 1 and 2 which were taken with 9-11
years interval. A large variation in individual telomere attrition rate has also
been shown earlier.134 Previous studies have indicated that telomerase
preferentially elongates the shortest telomeres,76,127,239 and when telomere
length in sample 1 was plotted as a function of yearly telomere loss, a very
strong correlation was observed (r = -0.752, p < 0.001, n = 959). This highly
significant data was found for both controls and patients (r = -0.730, p < 0.001,
n = 645 and r = -0.788, p < 0.001, n = 314, respectively). Therefore, a telomere
length maintenance mechanism is suggested providing protection for short
telomeres in vivo. Studies have indicated that telomere length, at least in the
middle-aged, can predict life span.146-151,257 Our data argue against this finding
and also against the hypothetical possibility to predict later tumour development
by measuring telomere length in blood years before diagnosis.
In a separate multifamily cohort, 13 families were selected to investigate
telomere attrition and initial telomere length on the familial level. They were
chosen since DNA was available from more than 10 individuals spanning over 3
generations in each family. The 13 family specific regression lines between age
and telomere length significantly differed from each other (p = 0.009). When the
slope from each family was plotted as a function of the corresponding intercept,
a significant correlation was observed (r = -0.691, p < 0.001). Hence, this data
demonstrate that the telomere loss was higher within families with longer
telomeres.
In conclusion, comparable data were obtained regarding the relationship
between telomere length and telomere length maintenance analysing individual
telomere loss and individual families in two separate sample cohorts. Human
cells in vivo seem to have a telomere maintenance system giving priority to the
shortest ones indicating that the frequency of telomere elongation and attrition
is telomere length dependent. It might be possible to modulate this mechanism
by life style and environmental factors as recently indicated.218,222,224,235
40
RESULTS AND DISCUSSION
Strength and limitations of this thesis
All five investigations included in this thesis are epidemiological studies based
on rather large cohorts. All cohorts have weaknesses, especially when initially
collected for other purposes than telomere research, but also strengths. In
general, we have good presumptions in Sweden to perform epidemiological
research since we can run different register against each other to find
previously unknown connections. This was useful in Paper IV when performing
survival analysis on breast cancer patients. The patient cohort was rather small
making it impossible to include more factors of importance, for example ER
status, in the multiple regression model. The different techniques used for
analysing ER status was also a drawback perhaps influencing the somewhat
unexpected results that ER+ patients did not have a better prognosis compared
to ER-. In order to have an equal age-span among controls and patients we had
to use two different control cohorts comprising different blood cells (buffy coat
compared to granulocyte preparations) which might have diverse telomere
lengths. However, this does not affect the most important finding of the study
namely that telomere length is a prognostic marker for breast cancer. In this
study it would also have been very interesting to investigate telomere length
both in blood and in the actual tumour tissue. The tumours have been
embedded in paraffin but unfortunately, the telomere PCR method does not
work on such extracted DNA. This will certainly be a challenge for future
optimisation of the method since every pathological clinic has plenty of such
samples saved. Another challenge will be to find a good, commercial, reference
sample so that results can be directly compared between different laboratories.
In Paper I a material from a very limited part of Sweden was investigated
where not so much net migration has occurred until today’s generation. The
genes are well conserved over generations which usually is an advantage when
studying inheritance. Blood cells were available from both non-cultivated cells
frozen directly in DMSO and cultivated cells. This gave an exclusive opportunity
to observe what happened with telomere length during the first passages of
cultivation. Since the cells were EBV transformed the analysis could have been
biased by the fact that a selection for B-cells occurred and these cells show
longer telomere length compared to other blood cells. The study groups for the
interfamilial relationship were rather small, probably causing the lack of some
correlation between mother and children.
The cohort investigated in paper II is unique because it is large and spans
over four generations, providing a great age-span which gives the opportunity to
subdivide the individuals in groups according to age and still having sufficient
numbers to perform powerful statistics. By this, we could carefully select the
41
RESULTS AND DISCUSSION
persons included in the inheritance analyses, limiting the possibility to introduce
confounding factors. This has not been done by others. We observed less
inheritance (weaker correlations) in older individuals indicating that
environmental and epigenetic factors during life could perhaps alter telomere
attrition rate. It would have been interesting to further investigate these possible
factors but no such data were available.
Longitudinal studies are the most powerful ones within epidemiology. In
northern Sweden we are fortuned with a large biobank continuously collecting
samples. This made study number V possible. The drawback with the study
was that each cancer group was rather small, making some statistical
calculations impossible. However, the main questions of the study, namely if
telomere length could predict future development and whether telomere attrition
was correlated to telomere length at baseline, could well be answered.
General discussion
Telomere research in humans has in recent years expanded from mainly being
focused on cancer and aging to also include cardiovascular diseases, diabetes,
psychology, dementia, rheumatology and more. Telomere length has been
shown to be altered in all these conditions, implying that common mechanisms
for these diseases act to disrupt a general, basic telomere length homeostasis
machinery probably directed by the shelterins and other telomere associated
proteins. Exactly what the mechanisms are is not fully elucidated but failure of
immunosurveillance is a potential candidate since it appears to be a risk factor
for many diseases, including cancer, and involved in normal aging.258 Likely, the
mechanisms can also be influenced by environmental, genetic and epigenetic
factors thereby indirectly affecting telomere length.
Some believe that short telomeres at birth predispose for several of these
diseases and malignancies but there is no experimental data showing such a
scenario. Our results do not support this theory. It is well established in vitro that
short telomeres are associated with an unstable genome which increases the
risk of mutations and eventually malignant transformation. This probably
requires extremely short telomeres perhaps never reached generally in vivo, at
least not at the mean telomere length level measured by the common
techniques used. However, it seems to be the shortest telomere in a cell that
triggers DNA damage checkpoints and senescence,194,239 but measuring single
telomeres in large cohorts would be very time consuming.
The fact that telomere length in blood has been found to be altered in
patients with certain types solid tumours201-203,243 indicates that the tumours,
42
RESULTS AND DISCUSSION
when they arise and not many years earlier, are able to interfere with the
telomere lengthening machinery. Telomere length in blood could therefore
provide an interesting prognostic marker for cancer. The PCR method is a good
and fast tool for screening through many samples and could therefore be used
in the clinic. The problem is that there is such a large distribution range in
telomere length among individuals of the same age, making it difficult to set a
pathological threshold for telomere length.
Since much evidence exists that telomere length is inherited, short
telomeres would then also affect future generations. Today, it is not clear if
telomere length inheritance is of importance. There are emerging proofs
pointing towards a much stronger paternal than maternal inheritance but why
this is the case is hard to answer. It has been observed that telomere length in
sperm increases by age, probably due to constitutive telomerase activity present
in adult testis.12,146,161 Women however, are born with all the oocytes they will
ever have. Imprinting is another plausible mechanism but this is yet to be
proven experimentally. It is interesting that paternal age at conception is
positively correlated to the children’s telomere length.15,146,155,160 This might
therefore be one of the contributors to interindividual telomere length variations.
Today, the parental age at conception is increasing and it remains to find out
whether telomere length of the general population then will increase and what
possible impact this will have considering the disease discussion above.
The mystery of aging and the question whether eternal life is possible
have puzzled mankind for centuries. Today, we tend to look upon aging as a
disease and not a natural phase of life. Telomere length has been hypothesised
to be able to predict future lifespan. This is a tempting scenario not the least
since telomere length seems to be longer in women than in men and that
women have a longer mean lifespan. However, aging in an organism is likely a
very complex process with many contributing factors and it is perhaps therefore
naive to believe that the net result of all of them can be interpreted by
investigating telomere length. The step from being a biomarker to a determinant
of life span is perhaps too big.
43
CONCLUSIONS
CONCLUSIONS
•
By investigation of two separate family cohorts, telomere length in
peripheral blood was found to be inherited. When performing correlation
analyses between parent and offspring telomere length, a stronger paternal
than maternal inheritance pattern was found. A correlation was also
observed between grandparents and grandchildren.
•
The telomere length heredity decreased with age, showing weaker telomere
length correlation at older ages compared to younger parents and children.
This indicates that life style, and environmental factors or an endogenous
telomere length maintenance mechanism probably modulate telomere
length during life, diminishing the hereditary component.
•
The -1327T/C polymorphism upstream the hTERT promoter starting site is not
a functional one as previously indicated. There was no difference in
telomere length after age adjustments between the possible genotypes
when investigating healthy individuals or myocardial infarction patients.
None of the genotypes were overrepresented in the patient group. A
possible telomere length elongation was observed with age in the -1327C/C
group. The other groups did not differ regarding telomere loss with age.
•
Breast cancer patients were found to have longer telomeres in peripheral
blood compared to controls after age adjustments. Having long telomeres
actually increased the risk of developing breast cancer. Even more
interesting was the fact that telomere length could predict outcome, showing
increased survival of patients belonging to the group with short telomeres.
This was especially pronounced in young patients and in those with
advanced disease.
•
Telomere length or telomere attrition rate in peripheral blood could not
predict future tumour development early or close to diagnosis. Further,
cancer patients do not show accentuated telomere shortening with age
before diagnosis compared to controls.
•
Initial telomere length was highly correlated to telomere loss with age
indicating a telomere maintenance mechanism giving priority to the shortest
telomeres.
44
ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
The warmest thanks to Göran Roos, my supervisor and the one who introduced me into
the fascinating world of research. Thank you for giving me the possibility to combine my
PhD studies with medical school, not all supervisors would accept such an arrangement.
You have given me great support, opportunites to participate in conferences and always
believed in my work (well, you have actually accused me for mixing the samples up a
couple of times when experiments showed unexpected results but…). Östersund’s
hospital seems to be a nice place to work at but I will miss the lab a lot, that is for sure!
A huge hug to all PhD students in the lab! Sofie, thank you for teaching me a lot of the
methods we use. You also took good care of me when I first came to the group as a
summer student. Much of my knowledge about telomeres I have learned from you!
Ulrika, you were the undergraduate student who now has become a coauthor, PhD
student and friend. You have given me invaluable assistance and contributed a lot to this
thesis! Pawel, you did a great job optimising the Tel-PCR. It has been extremely useful!
Magnus, you have really taken part in the discussion of other peoples work and always
come up with useful ideas, which shows great knowledge beyond your own projects.
Ravi, you brought some nice Indian influences to the group and did the right thing
coming to work with us.
Ulla-Britt, you have been the book of reference regarding lab methods. I have always
been able to ask you for help and no task is too big for you. Pia, you came back to the
lab with new ideas although you are an old lab member. Thank you for running
polymorphisms for me! Inger, thank you for answering all my thousand PCR questions,
teaching me how to design primers and helping me with sequencing! Bodil, thank you
for cultivating cells and buying the newspaper almost every morning! Eleonor, you
taught me how to extract DNA, the most basic lab work which I have had good use of!
I have been fortuned to have two second supervisors. Dan Holmberg, thank you for
good collaborations on Paper I which ended up as a nice first publication. Magnus
Hultdin, what would I have done without your help when mass media wanted interviews
and Göran was far away in China!?
Thank you Birgitta Stegmayr and Mats Eliasson, principle investigators of the
MONICA project. It has been very nice performing research with you. Even if two of our
common papers are not included in this thesis, they make a nice contribution to my
publication list! Thanks also to Åsa Ågren and Thore Johansson at the Medical
Biobank for helping me collect samples from the freezers and for DNA extraction.
45
ACKNOWLEDGEMENTS
Thank you to Per Lenner and Roger Henriksson at the Oncology Clinic for providing
DNA, clinical data and help with manuscript writing.
Thank you to the colleagues in Malmö: Peter Nilsson, Olle Melander and Jonas
Manjer. You have provided us with excellent DNA samples and been a great support
writing and commenting on manuscripts.
Thank you to KF Norrback, Rolf Adolfsson and Annelie Nordin from the Department
of Clinical Sciences, Psychiatry for good collaboration. It is very nice when your previous
results can be repeated, don’t you think?
You can not have too many statisticians up your sleeve when performing epidemiological
studies. Thank you Petter Lindgren, Hans Stenlund and Björn Tavelin!
Nice colleagues are a nessesity for successful work. Therefore, thanks to all of you in the
“fika” and lunch gang!
Thank you Terry, Åsa and most of all Karin for all administrative help!
Tack till alla mina vänner, gamla som nya. Speciellt till Daniel, Marcus, Micke, Isabelle,
Sara, Sonia och Åsa, alla medlemmar av Biomedicingänget. Vi har haft så mycket skoj
ihop under alla dessa år, andra helgen i mars kommer alltid att vara bokad exklusivt för
er! Tack också till Anna och Fia från Läkarprogrammet. Ni har varit med mig i med-, och
motgång och alltid funnits till hands när det har behövts som mest!
Mamma och Pappa! Ni har verkligen ställt upp till 100% på allt jag tagit mig för. Skjutsat
till träningar och matcher, hämtat mig mitt i natten när kylan varit för sträng för klänning
och tunna strumpbyxor, ordnat kalas, flyttstädat mm. Och hörrni, visst är Umeå nära men
det är ju inte så långt till Östersund heller! Tack också till världen bästa storebror
Rickhard som jag alltid sett upp till och beundrat. Du har bildat världens mysigaste familj
med Marlene och lille Axel. Tack inte minst för att ni tre gör er så bra på framsidan av
denna avhandling!
Tack Andreas för det mesta och bästa här i livet! Du har varit så tålmodig och hjälpsam
alla kvällar och helger jag suttit framför datorn och jobbat febrilt. I vinter väntar bara
skidåkning, myspys och utflykter!
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