Published OnlineFirst November 19, 2013; DOI: 10.1158/1940-6207.

CAPR-13-0264

Cancer
Prevention
Research Article Research

Folate Deficiency Induces Dysfunctional Long and

Short Telomeres; Both States Are Associated with
Hypomethylation and DNA Damage in Human
WIL2-NS Cells
Caroline F. Bull1,2, Graham Mayrhofer2, Nathan J. O'Callaghan1, Amy Y. Au3, Hilda A. Pickett3,4,
Grace Kah Mun Low5, Dimphy Zeegers5, M. Prakash Hande5,6, and Michael F. Fenech1

Abstract
The essential role of dietary micronutrients for genome stability is well documented, yet the effect of folate
deficiency or excess on telomeres is not known. Accordingly, human WIL2-NS cells were maintained in
medium containing 30, 300, or 3,000 nmol/L folic acid (FA) for 42 days to test the hypothesis that chronic
folate deficiency would cause telomere shortening and dysfunction. After 14 days, telomere length (TL) in
FA-deficient (30 nmol/L) cultures was 26% longer than that of 3,000 nmol/L FA cultures; however, this was
followed by rapid telomere attrition over the subsequent 28 days (P trend, P < 0.0001); both long and short
telomere status was positively correlated with biomarkers of chromosome instability (P
0.003) and
mitotic dysfunction (P ¼ 0.01), measured by the cytokinesis-block micronucleus cytome (CBMN-cyt) assay.
The early increase in TL was associated with FA-deficiency–induced global DNA hypomethylation (P ¼
0.05), with an effect size similar to that induced by the DNA methyltransferase inhibitor, 5-aza-20 -
deoxycytidine. Quantitative PCR analysis indicated a negative association between FA concentration and
uracil incorporation into telomeric DNA (r ¼ 0.47, P ¼ 0.1), suggesting a possible plausible mechanism for
uracil as a cause of folate deficiency–induced telomere dysfunction or deletion. Peptide nucleic acid-FISH
(PNA-FISH) analysis showed that FA deficiency resulted in 60% of micronuclei containing acentric terminal
fragments, an observation consistent with the 3-fold increase in terminal deletions (P ¼ 0.0001). Together,
these results demonstrate the impact of folate deficiency on biomarkers of telomere maintenance and
integrity, and provide evidence that dysfunctional long telomeres may be as important as critically short
telomeres as a cause of chromosomal instability. Cancer Prev Res; 7(1); 128–38. 2013 AACR.

Introduction
Functionally intact telomeres are critical for protecting
against degradation by nucleases, end-to-end fusions, and
structural rearrangement of chromosomes (1), each a
potential initiating factor for cancer and degenerative dis-
ease (1). Accelerated telomere shortening has been asso-
Authors' Affiliations: 1Nutritional Genomics Laboratory, CSIRO Animal,
Food and Health Sciences, Adelaide, South Australia; 2Discipline of Micro-
biology and Immunology, School of Molecular and Biomedical Sciences,
University of Adelaide, South Australia; 3Children's Medical Research
Institute, Westmead, New South Wales, Australia; 4Sydney Medical
School, University of Sydney, New South Wales, Australia; 5Department
of Physiology, Yong Loo Lin School of Medicine, National University of
Singapore; and 6Tembusu College, National University of Singapore,
Singapore
Note: Supplementary data for this article are available at Cancer Prevention
Research Online (http://cancerprevres.aacrjournals.org/).
Corresponding Authors: Caroline F. Bull, CSIRO Animal, Food and
Health Sciences, P.O. Box 10041, Adelaide BC, South Australia 5000,
Australia. Phone: 61-8-8303-8931; Fax: 61-8-8303-8899; E-mail:
caroline.bull@csiro.au; and Michael F. Fenech, michael.fenech@csiro.au
doi: 10.1158/1940-6207.CAPR-13-0264
2013 American Association for Cancer Research.
ciated with unhealthy diet and lifestyle (2, 3), exposure to
chronic stress (4), childhood violence (5), and negative
psychological states (6). Conversely, meditation, exercise,
and a healthy diet offer a protective effect against telomere
erosion (7, 8). The essential role of dietary factors in
maintenance of chromosome stability is well doc-
umented (9, 10); however, knowledge of the requirements
for individual micronutrients for maintenance of opti-
mal telomere length (TL) and functionality is extremely
limited.
Folate (vitamin B9) is increasingly consumed in the
supplemental (monoglutamated) form of folic acid (FA).
Moderate folate deficiency can cause chromosomal insta-
bility (CIN) comparable with that induced by carcino-
genic doses of ionizing radiotherapy in vitro (10). In its
role as a one carbon (methyl)-group donor, folate is
required for genomic stability via two distinct, yet inter-
dependent, biochemical pathways: (i) the synthesis of S-
adenosyl methionine (SAM), essential for maintaining
DNA cytosine methylation, epigenetic control of gene
expression, and structural integrity at regions of hyper-
methylated tandem repeats (centromeres and the subte-
lomere; refs. 10–13); and (ii) methylation of dUMP to

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dTTP, forming thymidine for DNA synthesis and repair
(14, 15). When folate is limiting, thymidine residues in
DNA can be replaced by uracil, initiating a cycle of base
excision repair, increased frequency of abasic sites, and
heightened risk of double-strand breaks (DSB; ref. 16). In
addition, compelling evidence has shown that repair
responses can be restricted at telomeric DNA, suggesting
that damage may be prolonged (17–19). The telomere,
comprised of a repeating DNA hexamer (TTAGGGn), is
bound by proteins of the shelterin complex to form the
"telosome," key components of which are "telomere
repeat binding factors" (TRF1 and TRF2), the binding
kinetics for which are highly sensitive to substrate
changes (20, 21). It is plausible, therefore, that the thy-
midine-rich nature of telomeres makes them vulnerable
to uracil misincorporation, incomplete excision repair of
uracil, leading to abasic sites, unrepaired DNA strand
breaks, and compromised telosome formation when
folate is deficient. Accordingly, we hypothesized that
telomeres would shorten at an accelerated rate under
FA-deficient conditions, and tested this in an in vitro
human cell line model.
Materials and Methods
Design
An in vitro model of chronic FA deficiency was established
using human WIL2-NS cells. FA concentrations were deter-
mined based on pilot growth data, and on previous studies
indicating that DNA damage occurs in WIL2-NS cells at
20 and 200 nmol/L FA (22). Cells were cultured for 42
days in medium either deficient (30 nmol/L), replete (300
nmol/L), or supraphysiological (control, 3,000 nmol/L) for
FA. The "replete" concentration of 300 nmol/L is one which
would only be achieved in serum of individuals supple-
menting with several milligrams of FA daily, whereas the
"control" concentration of 3,000 nmol/L is not achievable
in vivo in humans. We do, however, live in an era in which
cells are increasingly taken out of our bodies, grown in
culture, and returned to the body (e.g., stem cell, bone
marrow, and skin graft transplantation). Such culture sys-
tems routinely use media with supraphysiological concen-
trations of FA and yet it is not known whether this is optimal
for DNA damage prevention. The human WIL2-NS (B-
lymphoblastoid) cell line was selected because folate defi-
ciency has been identified as an important risk factor for B-
cell lymphoma (23), and because its p53-deficient status
allowed observation of substantial genome damage events
without excessive cell death through apoptosis (24). Deter-
mination was made of TL by flow cytometry, biomarkers
of DNA damage and cytostasis by cytokinesis-block micro-
nucleus cytome (CBMN-cyt) assay, expression of human
telomerase reverse transcriptase [(hTERT); quantitative PCR
(qPCR)], uracil incorporation into telomeric DNA (qPCR),
and global methylation status (ELISA). In addition, both
metaphase and (induced) binucleated interphase cell pre-
parations were used for molecular cytogenetic analysis of
chromosome aberrations, loss of chromosome terminal
ends, the presence of interstitial telomeric DNA, and the
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Folate Deficiency Induces Dysfunctional Telomeres
in situ location of centromeric and telomeric DNA within
biomarkers of CIN.
Cell culture
WIL2-NS cells (American Type Culture Collection; CRL-
8155) were cultured for 7 days in complete RPMI 1640
medium (Sigma) before seeding in treatment medium at
day 0 of the experimental period. Cells were authenticated
at the time of delivery (2002) to confirm p53-negative
status, and in 2011 were confirmed as being karyotypically
consistent with previous reports (25). Duplicate 90 mL
cultures were maintained in 75 cm2 vented-cap culture
flasks (Becton Dickinson) at 37 C in a humidified atmo-
sphere with 5% CO2. Medium was replaced twice weekly.
Complete RPMI medium (control 3,000 nmol/L FA condi-
tion) was supplemented to contain 5% FBS (Thermo) and
1% penicillin/streptomycin (Sigma). L-Glutamine (1%;
Sigma) was added immediately before use. Medium con-
taining 30 or 300 nmol/L FA was prepared by dilution of
complete RPMI medium with an appropriate volume of
supplemented FA-free RPMI (Sigma). For 5-aza-20 -deoxy-
cytidine (5azadC) studies, filter sterilized 5azadC (Sigma)
stock solution (21.9 mmol/L in PBS) was added to RPMI
medium (3,000 nmol/L FA) to final concentrations of either
0.2 or 1.0 mmol/L 5azadC. Control (0 mmol/L 5azadC)
medium was prepared by adding equivalent volume of
vehicle (1 PBS).
Determination of TL
TL was measured in viable single cells at G0–G1 using the
flow cytometric method detailed previously (26). TL was
expressed relative to a (long telomere) control cell line,
1301 European Collection of Cell Cultures (ECACC). Telo-
mere-specific peptide nucleic acid-fluorescein isothiocyan-
ate (PNA-FITC) labeling was conducted using kit K5327
(Dako). Doublets, debris (dead and dying cells), and cells at
G2 were excluded based on cell cycle staining with propi-
dium iodide. The coefficient of variation of replicate mea-
surements was 13.5%.
CBMN-cyt assay
Chromosomal damage and cytostasis measures were
determined using the CBMN-cyt assay, using the standard
protocol detailed previously (27), with additional scoring
criteria for fused nuclei (FUS) morphologies as described
(9). See details in Supplementary Materials and Methods.
Measurement of uracil incorporation into telomeric
DNA by quantitative real-time PCR
The amount of uracil present in telomeric repeat
sequences was measured using a modification of the quan-
titative real-time PCR (qPCR) method used to measure TL,
incorporating additional digestion steps. For details see
Supplementary Materials and Methods.
Detailed methods are provided in Supplementary
Materials and Methods for cell growth, the CBMN-cyt
assay, binucleated cell preparations for PNA-fluorescence
in situ hybridisation (FISH), metaphase chromosome
Cancer Prev Res; 7(1) January 2014 129
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Bull et al.

preparations, FISH with PNA probes, immunoaffinity

purification-telomeric repeat amplification protocol (IP-
TRAP), C-circle assay, terminal restriction fragment (TRF)
analysis, DNA isolation and global DNA methylation,
RNA isolation and expression analysis of hTERT, mea-
surement of uracil incorporation into telomeric DNA by
qPCR, and statistical analyses.

Results and Discussion

Culture in folate-deficient medium is associated with
rapid fluctuations in TL
WIL2-NS cells were maintained in medium deficient (30
nmol/L), replete (300 nmol/L), or supraphysiological
(3,000 nmol/L; control) for FA. The doubling time of the
cells in culture varied considerably for each FA condition.
The number of viable cells at each time point, indicating rate
of cell division, was reduced in both the 30 and 300 nmol/L
FA cultures, compared with the 3,000 nmol/L condition
(Supplementary Fig. S1a). Cell viability and nuclear divi-
sion index (NDI) were significantly lower at every time
point in 30 nmol/L compared with that of 3,000 nmol/L
FA (P < 0.0001), whereas the frequency of necrotic cells
significantly increased at all time points in the 30 nmol/L
condition (P < 0.0001; Supplementary Fig. S1b–S1d).
Pilot TL data from short-term (21 days) cultures indicated
TL significantly increased in the short term (7–14 days)
under FA-deficient conditions, followed by a modest
decline between day 14 and day 21 (Supplementary Fig.
S2). To examine the longer term chronic effects of FA
deficiency, cells were then maintained in culture for 42
days. TL of samples at baseline, and weekly or fortnightly
thereafter, were compared by flow cytometry and results
expressed relative to the TL of reference cell line 1301. At day
14, TL in 30 nmol/L FA was 18.1  2.1 (mean  SD), 1.4-
fold that of baseline (12.7  3.4), followed by rapid
attrition (P trend, P < 0.0001). By day 42, TL had shortened
to 12.6  1.2, comparable with cells grown for the same
length of time in 3,000 nmol/L FA (13.5  2.7; Fig. 1A). TL
of control cultures remained stable throughout the 42-day
period (mean, 13.7  0.6). Mean area under the curves
(AUC TL) for the 30, 300, and 3,000 nmol/L FA conditions
was 638, 628, and 576, respectively. Despite the rapid TL
shortening beyond day 14 in the 30 nmol/L condition, a
negative correlation was recorded between [FA] and AUC
TL, over the 42 days (r ¼ 0.47; P ¼ 0.008). These are new
and intriguing observations, which do not support the
original hypothesis that FA deficiency is associated with
Figure 1. TL and biomarkers of CIN. A, TL in WIL2-NS cells cultured for
telomere shortening. 42 days in medium containing 30, 300, or 3,000 nmol/L FA. (Mean 
SD; N ¼ 10 at each time point for each FA concentration; n ¼ 20 for
TL is positively associated with biomarkers of CIN in day 0). B–F, frequency per 1,000 binucleated (BN) cells displaying
the short term, but negatively associated in the longer biomarkers of CIN following culture in medium containing 30,
term 300, or 3,000 nmol/L FA over 42 days; B, 1 micronuclei (MN); C, 1
The relationships between [FA], TL, and CIN have not nucleoplasmic bridges (NPB); D, 1 nuclear buds (NBud); E, "fused"
(FUS) nuclei; F, combined total frequency of BN cells (per 1,000
previously been examined. Biomarkers of CIN were BN cells) displaying any CIN biomarker (MN or NPB or NBud or FUS;
scored in binucleated cells using the CBMN-cyt assay, mean  SD; N ¼ 2). Data at each time point not sharing the same
specifically; micronuclei (MN; broken fragments or lag- superscript letter differ significantly (P
0.05), as measured by the
ging chromosomes), nucleoplasmic bridges (NPB; indic- Bonferroni post-test. Two-way ANOVA analyses shown (P value;
% variance [in parentheses]; Int, interaction of [FA] with time).
ative of dicentric chromosome (DC) formation due to

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Folate Deficiency Induces Dysfunctional Telomeres

Table 1. Correlations between biomarkers of CIN, concentration of FA in culture medium, and TL

DNA damage
(all markers
MN NPB NBud FUS combined) TL day 14 TL day 42
Log [FA] P 0.0005 0.02 0.7 0.07 0.01 0.0003 0.065
r 0.98 0.88 0.21 0.77 0.9 0.98 0.8
MN P 0.01 0.9 0.05 0.005 0.005 0.03
r 0.9 0.04 0.81 0.94 0.94 0.85
NPB P 0.8 0.003 0.0002 0.004 0.002
r 0.11 0.96 0.98 0.95 0.97
NBud P 0.58 0.77 0.02 0.64
r 0.29 0.15 0.9 0.24
FUS P 0.003 0.01 0.01
r 0.95 0.9 0.91
DNA damage P 0.0002 0.003
(all markers combined) r 0.98 0.95

NOTE: Shaded values indicate statistically significant associations (P < 0.05), r ¼ Pearson correlation coefficient. DNA damage
(all markers combined), frequency of binucleated cells displaying one or more MN, NPB, NBud, or FUS.
All data are calculated using duplicate values at day 42, except TL data, which use mean TL values for each condition at two key time
points: days 14 and 42.

fusion of dysfunctional or critically short telomeres, or
misrepair of DNA breaks), nuclear buds (NBud; gene
amplification; ref. 27), and fused nuclei (FUS; suggesting
failed chromatid separation and/or dysfunctional meta-
phase/anaphase transition; ref. 9). FA deficiency induced
highly significant increases in the frequency of all bio-
markers over 42 days (P < 0.0001–P ¼ 0.0009; Fig. 1B–F).
Consistent with previous (shorter term) findings, micro-
nuclei, NPBs, and FUS were positively correlated, yet fre-
quencies of each were strongly, negatively associated with
[FA] (Table 1; refs. 9, 10). Relationships between TL and
CIN biomarkers at day14 (the point at which the longest TL
was observed) were positive and significant. By day 42, this
had reversed, with correlations between CIN and TL being
strongly negative (Table 1).
These data show that FA deficiency induces both shorter
and longer telomeres, and that both states are strongly
associated with CIN, indicating telomere dysfunction
regardless of length.
Possible mechanisms underlying the increase in TL
Questions that arise from the above findings are the
following: (i) What mechanisms are responsible for the
increase in TL in folate-deficient cultures? and (ii) Does a
causal link exist between these and the evidence of CIN?
Telomere shortening is a pivotal event early in the patho-
genesis of cancer; however, in established cancers telome-
rase activity (TA) can induce longer TL than that of adjacent
normal tissue (28). An alternative TA-independent, recom-
bination-driven telomere maintenance mechanism (TMM),
called the alternative lengthening of telomeres (ALT), has
been identified in approximately 10% of cancers (29, 30).
Hypomethylation of the subtelomeric promoter for "telo-
meric repeat-containing RNA" (TERRA) may influence the
ALT phenotype, directly or indirectly (11, 31, 32). Accord-
ingly, we considered whether the increased TL and CIN in
WIL2-NS cells from FA-deficient cultures may be due to ALT-
associated recombination, the latter potentially inducing
formation of DC containing interstitial telomeric DNA,
initiating telomere amplification via breakage–fusion–
bridge (BFB) cycling.
To elucidate the mechanism of telomere elongation
induced by FA deficiency, we explored (i) intrachromoso-
mal (interstitial) telomeric DNA to determine if amplifica-
tion may have occurred as a result of BFB cycling; (ii) the
ALT or telomerase status of WIL2-NS cells; (iii) whether FA
deficiency reduced global DNA methylation, and the asso-
ciation between DNA methylation and TL; and (iv) the
impact of FA deficiency on expression of hTERT, the active
subunit of telomerase.
BFB cycling is not responsible for amplification of
telomeric DNA
Numerous studies report increased incidence of ana-
phase bridges when TL and/or integrity is compromised
(33–36); however, to our knowledge, evidence of telomeric
DNA amplification through this mechanism has not been
reported. DC formation is a known initiator of BFB cycling,
gene amplification, and altered gene dosage (34, 36). We
speculated that random breakage of DCs formed by fusion
of dysfunctional telomeres may result in daughter cells
receiving uncapped (fusigenic) chromosomes; one contain-
ing telomere sequences of both fused chromosomes (inter-
stitially), and the other receiving a reduced telomere content

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Bull et al.

Figure 2. Investigations into mechanisms underlying transient telomere elongation in low FA culture conditions. A, proposed model for telomeric DNA
amplification via BFB cycling, wherein compromised (uncapped or broken) telomere ends fuse to form a dicentric chromosome containing intrachromosomal
telomeric DNA. Red squares, telomeric DNA; green circles, centromeres. B, southern (TRF) analysis of (day 0, untreated) WIL2-NS cells (reference cell lines;
 þ þ þ  þ
HeLa, ALT /TA , VA13, ALT ). C, detection of C-circles (CC) shows WIL2-NS cells to be CC-negative compared with ALT (IIICF/c) and ALT /TA (HeLa)
reference cells. D, WIL2-NS cells are positive (þ) for TA, determined using the IP-TRAP assay. Sizes of molecular weight markers are indicated to the
þ þ 
left of B and D. Buffer, buffer-only negative control; HCT116, telomerase-positive (TA ) reference cell line; GM847, ALT /TA reference cell line. E and F, global
methylation status of WIL2-NS cells cultured for 14 days and 42 days (E) in medium containing 30 or 3,000 nmol/L FA (a, P ¼ 0.046; b, P ¼ 0.06); and for 4 days
(F) in complete medium (CM; 3,000 nmol/L FA) containing 0, 0.2, or 1.0 mmol/L 5azadC (points not sharing the same letter differ significantly; P < 0.05).
%5-meC; percentage of cytosine residues methylated at carbon-5, as a percentage of total DNA; mean  SD; N ¼ 3. G, TL of WIL2-NS cells cultured
for 4 days in CM containing 0, 0.2, or 1.0 mmol/L 5azadC. Mean  SD; N ¼ 20 at day 0, n ¼ 10 at day 4 (points not sharing the same letter differ significantly:
P < 0.05).

(Fig. 2A). To test the hypothesis that BFB cycling was
responsible for telomere amplification, metaphase prepara-
tions from cells grown in control (3,000 nmol/L) or defi-
cient (30 nmol/L) FA for 0, 7, 14, 21, 28, or 35 days were
probed using PNA-FISH for the presence of interstitial
telomeric DNA (i.e., telomeric sequences at nonterminal
locations). Analysis showed no interstitial telomeric DNA
in these metaphases (n ¼ 40 metaphases per condition, per
time point; data not shown), thus refuting this hypothesis.
WIL2-NS cells are ALT-negative and telomerase-
positive
ALTþ cells are characterized by highly heterogeneous TL
distribution (potentially ranging from <3 kb to >50 kb), the
presence of ALT-associated promyelocytic leukemia bodies
(APB), and C-circles (29, 30). C-circles are a specific, quan-
tifiable marker of ALT (29, 30). To test whether WIL2-NS
cells are ALTþ, we examined untreated (day 0) cells first by
Southern blot to assess the degree of TL heterogeneity in
TRF, and second for the presence of C-circles by the C-circle
assay (30). TRF analysis showed a comparable spread of TL
relative to HeLa cells (ALT/TAþ), and smaller, less hetero-
geneous fragments than those from VA13 cells (ALTþ/
TA; Fig. 2B). WIL2-NS cells were then shown to be negative
for C-circles relative to ALTþ IIICF/c reference cells (Fig. 2C).
Together, these results suggest that WIL2-NS are ALT.
The IP-TRAP assay showed WIL2-NS are positive for TA
(Fig. 2D), consistent with an ALT phenotype (29), and
suggesting that the increase in TL observed at days 7 and 14
in the FA-deficient cultures may be mediated by telomerase.

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FA deficiency results in global DNA hypomethylation
The relationship between DNA methylation status and TL
is currently unclear. Telomeres lack CpG sites, the substrate
for DNA methyltransferase (DNMT) enzymes, and, as a
consequence, are unmethylated. In contrast, the subtelo-
mere is normally heavily methylated, reduction in which
(in gene knockout studies) has been associated with
dramatically elongated telomeres, APBs, and telomeric
sister chromatid exchange (TSCE; refs. 11, 32). Subtelo-
meric and pericentromeric DNA hypomethylation was
also associated with increased TL and TSCE in a panel
of neoplastic cells (12), suggesting a plausible association
between global and subtelomeric methylation status, and
telomere dysfunction.
The relationship between folate, DNA methylation, and
TL has not previously been reported. We hypothesized that
under FA-deficient conditions, TL and global DNA methyl-
ation status would be negatively associated. An ELISA-based
assay was used to determine the percentage of 5-methyl-
cytosine (%5me-C) in cells cultured in 30 nmol/L and 3,000
nmol/L FA at day 14 and day 42. Results showed global
DNA methylation was lower in the 30 nmol/L compared
with 3,000 nmol/L FA cultures, at both time points (Fig.
2E); 16% of variance was attributable to [FA] (ANOVA, P ¼
0.03), 43% to time (P ¼ 0.005), and 11% to their interac-
tion (P ¼ 0.15). Methylation status and [FA] were positively
associated at day 14 (r ¼ 0.8, P ¼ 0.05) and day 42 (r ¼ 0.8,
P ¼ 0.06). In agreement with our hypothesis, at day 14, the
point at which TL was longest in the 30 nmol/L FA culture,
TL was negatively and significantly associated with global
methylation status (r ¼ 0.8, P ¼ 0.05).
Global hypomethylation is associated with increased
TL
To examine the impact of hypomethylation on TL, without
the potentially confounding effect of reduced intracellular
thymidine through FA deprivation, the protocol was repeat-
ed using WIL2-NS in complete medium (3,000 nmol/L FA)
containing DNMT inhibitor, 5azadC. To minimize cytotoxic
effects, the lowest possible (bioefficacious) concentrations
(0.2 and 1.0 mmol/L) were used (37). Treatment could only
be maintained for 4 days before cells became nonviable;
however, as TL increased in cells in 30 nmol/L FA within 7
days, 4 days exposure to 5azadC was deemed sufficient to
induce a comparable effect.
We verified that methylation status of WIL2-NS exposed to
0.2 and 1.0 mmol/L 5azadC was significantly lower than
those from control (0 mmol/L) cultures; 21% of variance
attributable to [5azadC] (ANOVA, P ¼ 0.05; Fig. 2F). The
correlation between [5azadC] and DNA methylation at day 4
was significant and negative (r ¼ 0.8, P ¼ 0.01).
At day 4, TL was 29% and 50% greater, in the 0.2 and 1.0
mmol/L 5azadC, respectively, than in untreated controls
(Fig. 2G). [5azadC] explained 27% of TL variance (P <
0.0001), 39% due to time (P < 0.0001) and 27% due to
their interaction (P < 0.0001). A significant, negative cor-
relation was recorded between global DNA methylation
and TL in these samples (r ¼ 0.75, P ¼ 0.02).
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Folate Deficiency Induces Dysfunctional Telomeres
Relative to 3,000 nmol/L cultures, cells in 30 nmol/L FA
had 74% less DNA methylation at day 14, and 26% longer
TL. Similarly, the 30% reduction in methylation in the 0.2
mmol/L 5azadC culture had 30% longer TL, whereas the
355% reduction in methylation in the 1.0 mmol/L condition
was associated with 50% longer TL, relative to control
cultures. From these data, we concluded that DNA hypo-
methylation is a plausible candidate as the mechanism
underlying increased TL observed after 7 to 14 days of
suboptimal FA.
Expression of hTERT is unaffected by FA deficiency
TA is dependent upon presence of the active subunit,
hTERT, which is regulated at the transcriptional level
(38). Animal models show that diets deficient for methyl-
donors (including folate) increased DNMT expression,
resulting in CpG island hypermethylation, in certain
tissues (39), an effect that may plausibly impact hTERT
expression. Accordingly, we determined whether hTERT
expression was altered in WIL2-NS under FA-deficient
conditions by quantifying RNA transcripts in cells
following 14, 28, 35, and 42 days in 30, 300, or 3,000
nmol/L FA culture. qPCR results, expressed relative to
baseline (day 0), normalized against housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
showed no significant differences, with only 5.2% of
variance attributable to [FA] (P ¼ 0.77), and 18.2% of
variance due to time (P ¼ 0.75; data not shown).
Although telomerase is active in WIL2-NS, it seems that
neither FA deficiency per se, nor an associated change in
DNA methylation status, impacts transcription of hTERT,
thus increased TA is unlikely to have contributed to the
longer TL observed.
Mechanisms for accelerated telomere loss
Uracil incorporation into genomic DNA under folate-
deficient culture conditions is well documented (14), but
whether uracil is incorporated into telomeres has not pre-
viously been examined. A functional telosome, key com-
ponents of which are capping proteins TRF1 and TRF2,
inhibits nonhomologous end joining and end fusions (40).
The high specificity of capping proteins for their substrate
was demonstrated in an elegant study showing substitution
of a single telomeric guanine with 8-oxo-guanine (8oxoG)
resulted in 50% reduction in TRF1/2 binding, an abasic
site within the telomere reduced binding 3-fold, and a
lesion within each hexamer repeat reduced bound TRF1
and TRF2 to barely detectable levels (21). Accordingly, we
hypothesized that uracil in telomeric DNA may impact
telosome integrity in a similar manner. Furthermore, uracil
glycosylase (UDG), part of the base excision repair (BER)
mechanism, removes uracil from genomic DNA, resulting
in a transient abasic site intermediate (15, 16). In low FA
(low thymidine) conditions, uracil can be reincorporated at
the "gap-filling" step, leading to futile cycling of BER and
abasic site generation, potentially compromising TRF1/2
binding, and increasing the risk of DSB (15, 16). Recent
findings indicate, however, that telomeric DNA damage is
Cancer Prev Res; 7(1) January 2014 133
 Published OnlineFirst November 19, 2013; DOI: 10.1158/1940-6207.CAPR-13-0264

Bull et al.

not repaired (18, 19), resulting in a persistent damage
response (phosphorylated (g)-H2AX and/or ATM) at
lesions, prolonged cell cycle arrest, and potentially replica-
tive senescence.
To examine the factors underlying the telomere attrition
observed in extended 30 nmol/L FA cultures, we tested (i)
the relationship between [FA] and uracil within telomeres,
(ii) the presence of telomeric DNA in biomarkers of DNA
damage in cytokinesis-blocked binucleated cells, and (iii)
"loss of telomere" events in metaphase preparations.
Uracil incorporation into telomeric DNA is negatively
associated with [FA] in medium
Uracil content of telomeric DNA was measured in UDG-
digested and -undigested DNA using an adaptation of a
qPCR method developed in this laboratory to detect abnor-
mal bases by digesting DNA with lesion-specific enzymes
(41). Results showed that, following 42 days of culture in
either 30 nmol/L or 300 nmol/L FA, the amount of uracil
per kb of telomeric DNA was 2.9- and 3.4-fold greater,
respectively than at baseline, whereas no uracil was detect-
able in telomeres of cells from 3,000 nmol/L FA cultures
(n ¼ 4, P ¼ 0.3). The slightly greater amount of uracil
observed in the 300 nmol/L culture (compared with 30
nmol/L) is consistent with previous findings in genomic
DNA, an effect possibly due to the different rates of DNA
synthesis and cell division between the midrange and
lowest [FA], thus influencing the rate of uracil incorporation
(14). As predicted, [FA] and uracil content in telomeric DNA
were negatively associated (r ¼ 0.47, P ¼ 0.1) after 42 days
in culture.
FA deficiency is associated with DSBs, loss of terminal
telomeric fragments, and mitotic dysfunction
To examine the in situ location of telomeric and centro-
meric DNA in FA-deficient cultures, dual fluorochrome
PNA-FISH was conducted on cytokinesis-blocked binucle-
ated cells cultured in 30 nmol/L FA for 7, 14, 21, 28, or 35
days. Of 151 micronuclei scored (day 7, n ¼ 37; day 14, n ¼
21; day 21, n ¼ 41; day 28, n ¼ 31; day 35, n ¼ 21), 38%
contained centromeric DNA (Cþ) and 79% contained
telomeric DNA (Tþ; Fig. 3A–C). Consistent with previous
data from 9-day FA-deficient cultures (42), only 25% of the
53 micronuclei containing 2 T signals were also Cþ

Figure 3. Representative PNA-FISH images of CIN biomarkers in cytokinesis-blocked binucleated (BN) WIL2-NS cells cultured in medium containing
30 nmol/L FA. Black and white images indicate 4',6-diamidino-2-phenylindole (DAPI) staining of DNA (white), with arrows indicating the location of CIN
þ
biomarkers. Corresponding color images show DAPI-stained DNA (blue), the location of 5-carboxy-fluorescein (FAM)-labeled centromeric probe (green; C )
þ
and/or Cy3-labeled telomeric probe (red; T ), visualized using PNA-FISH. A–C, BN cells after 7, 21, and 28 days in culture, respectively, each
þ þ þ þ  þ
containing a single C T micronucleus, the micronucleus in B contains 2C and 4T signals, and C also contains a single C T NPB. D (28 days culture),
   þ
E and F (35 days cultures), BN cells containing 1 or 2 NPB. All are C T , except for the left NPB in F which is C T . G, H, and I, fused (FUS) cells after
þ þ
(G, 28 days), (H and I, 35 days) in culture. Dashed lines indicate nuclear boundaries of fusion regions (FR) for scoring purposes. In each case, FR are C T .
þ þ þ þ þ þ þ
G, contains 4C , 1T , the FR of (H) contain 3T , 2C , and FR of (I) contain 1T , 1C . The large size of C signals suggests close proximity of more
than one hybridization site (indicated by arrows in H and I); magnification, 630.

134 Cancer Prev Res; 7(1) January 2014 Cancer Prevention Research

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 Published OnlineFirst November 19, 2013; DOI: 10.1158/1940-6207.CAPR-13-0264

Folate Deficiency Induces Dysfunctional Telomeres

A C D E F
3,000 nmol/L
30 nmol/L
3.5
G H I
3.0
LOT (Relative to day 0)

2.5

2.0 J K L
1.5

1.0

0.5

0.0 M N
0 7 14 21 28 35
Day
B

O P

Day 14 Day 21 Day 28 Day 35

Figure 4. Chromosome aberrations in cells cultured in FA-deficient conditions. A, Loss of telomere (LOT) events in WIL2-NS metaphases cultured for 35 days
in medium containing 30 or 3,000 nmol/L FA. [Mean  SD, n ¼ 2, 20 metaphase spreads scored per duplicate, a total of 40 per condition per time point. Data
not sharing the same superscript letter at each time point differ significantly, measured by the Bonferroni post-test (P
0.05)]. B–P, PNA-FISH–stained partial
metaphase spreads from WIL2-NS cultured in 30 or 3,000 nmol/L FA; DAPI-stained DNA (blue), FAM-labeled centromeric DNA (green), Cy3-labeled
telomeric DNA (red). B, representative images of LOT events from 30 nmol/L FA cultures (yellow arrows, single LOT event; white arrows, double LOT event).
C–P, chromosome aberrations (yellow arrows, centromeres). C, a dicentric chromosome (DC) observed after 21 days in 3,000 nmol/L FA. (D–P are all from
30 nmol/L FA cultures); (D, E; day 14) ring chromosomes formed by end fusion (pink arrow) within the same chromosome; (F, day 21) DC; (G and I, day 14) DC;
(H, day 14) q-arm fusion (pink arrow); (J, K, day 28) DC; (L–P, day 35) DC. Image M also contains an acentric fragment (pink arrow), representing a
fusion between two broken chromosome ends; magnification, 630.

(indicating a lagging chromatid or chromosome), suggest-
ing that the majority of Tþ micronuclei represent acentric
fragments arising from DSB. Of 21 NPBs scored after days 7
(n ¼ 6), 14 (n ¼ 1), 21 (n ¼ 2), 28 (n ¼ 8), or 35 (n ¼ 4) in 30
nmol/L FA (Fig. 3D–F), 38% were Tþ, suggesting fusion
between dysfunctional or compromised telomeres, 43%
were Cþ, and 24% contained both (CþTþ), which together
suggest mitotic disruption (9). The greatest proportion
(43%) was for CT, suggesting that these NPBs arose from
fusion between uncapped chromosomes, or DSB misrepair
of breaks at nontelomeric chromosome regions. Cells dis-
playing FUS morphologies (n ¼ 196) were scored following
days 7 (n ¼ 37), 14 (n ¼ 21), 21 (n ¼ 40), 28 (n ¼ 30), or 35
(n ¼ 21) in 30 nmol/L FA (Fig. 3G–I). The majority (78%) of
fusion regions were Cþ, and/or Tþ (74%), with 61% exhi-
biting both (CþTþ) and only 8% displaying neither (CT).
These results suggest that telomere end fusions or failure of
sister chromatid separation could cause FUS morphologies
supporting our previous observations (9).
FA deficiency causes telomere loss and chromosome
aberrations
"Loss of telomere" (LOT) events were scored in meta-
phase spreads from cells cultured in 30 or 3,000 nmol/L
FA for days 0, 7, 14, 21, 28, or 35 (20 metaphases/slide,
n ¼ 2; Fig. 4A and B). Data were expressed relative to that of
baseline cultures (set arbitrarily at 1.0). Confirming indica-
tions from the above data, metaphases from 30 nmol/L FA
cultures had an approximately 3-fold increase in LOT from
day 14 to day 35, compared with cells maintained in 3,000
nmol/L FA. Of the variance in LOT, 43% was attributable to
[FA] (P ¼ 0.0001), 22% to time (P ¼ 0.04), and 18% to their
interaction (P ¼ 0.08). At day 35, LOT was negatively
correlated with log[FA] (r ¼ 0.96, P ¼ 0.03), and positively
correlated with binucleated cells containing 1 micronuclei
(r ¼ 0.96, P ¼ 0.04).
These same spreads were analyzed for aberrations (DC,
acentric fragments, double minutes, chromosome or chro-
matid breaks, ring chromosomes, q or p arm fusions). The

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Research.
 Published OnlineFirst November 19, 2013; DOI: 10.1158/1940-6207.CAPR-13-0264

Bull et al.

Figure 5. Possible pathways by which folate insufficiency could impact TL, telomere integrity, and chromosomal stability. SAM, methyl carrier/donor
S-adenosyl methionine; ALT, the telomerase-independent (alternative) mechanism for maintenance of TL; MRN complex, DNA repair complex associated
with ALT, consisting of Mre11, Rad50, and Nbs1.

presence of DC was of particular interest, as they represent
end fusions from telomere loss or dysfunction, or DSB
misrepair. Only one aberration, a DC at day 21, was
observed in 3,000 nmol/L FA spreads (Fig. 4C). In contrast,
21 aberrations were recorded from 30 nmol/L FA cultures,
10 of which were DC (2 at day 14, 1 at day 21, 2 at day 28, 5
at day 35; Fig. 4D–P). No aberrations were recorded at day 0
or day 7, from 30 or 3,000 nmol/L FA cultures.
Together, these data show long-term FA deficiency is
associated with micronuclei containing acentric fragments
and lagging chromosomes, terminal chromosome loss,
fused chromosomes (including DC), apparent dysfunction-
al chromatid separation, and uracil in telomeric DNA. These
findings are consistent with those that may be expected
from uracil incorporation into genomic and telomeric
DNA, and support our original hypothesis that FA deficien-
cy could cause accelerated telomere erosion.
Conclusions
The novel data herein demonstrate that FA deficiency
compromises telomere homeostasis, evidenced by substan-
tial fluctuations in TL, increased chromosome fusions
(NPBs and DCs), and terminal deletions. These effects
coincide with global DNA hypomethylation, increased
presence of uracil in telomeres, and increased CIN. We
propose a model whereby FA deficiency results in short-
term net gain in telomere content arising from hypomethy-
lation, reduced capping and enhanced access by telomerase
to its substrate. In the longer term, however, as telomere
maintenance and DNA repair mechanisms become over-
whelmed (due to increasing uracil incorporation, BER pro-
cesses, abasic sites, and DSB), homeostatic balance is lost
resulting in increasing telomere attrition with time. Fur-
thermore, we provide evidence that the rapid telomere loss
observed beyond day 14 in the 30 nmol/L FA condition
coincides with an increase in terminal deletions, leaving
exposed chromosome ends to which telomerase can no
longer bind, and is thus unable to add telomeric repeats. A
diagrammatic summary of proposed pathways underlying
FA-deficiency–induced telomeric and genomic instability is
provided in Fig. 5.
Increased TL under hypomethylating conditions has only
been reported previously in mouse knockout models
(11, 32), or in cell lines exposed to cancer drugs such as
5azadC (12). The present data, however, show telomere
lengthening and DNA hypomethylation induced by FA
deficiency to a similar extent as that caused by 5azadC. As
epigenetic modifications are strongly associated with cancer
initiation, these findings potentially have wide-reaching
implications for disease risk minimization in populations
exposed to deficiency of folate, vitamins required for folate
metabolism (such as vitamin B12), or essential factors in

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 Published OnlineFirst November 19, 2013; DOI: 10.1158/1940-6207.CAPR-13-0264
one-carbon (methyl) metabolism (e.g., choline; ref. 43).
Until testing has been undertaken in nontransformed cells,
however, these data can only be considered directly relevant
to p53-negative cells with active telomerase, and can not be
extrapolated to primary lymphocytes. As such, these obser-
vations are relevant to cancer cells, and highlight how
periods of FA deficiency may compromise TL homeostasis
and functional integrity. We also can not exclude the pos-
sibility that some of the effects we observed are specific to
FA, as opposed to methyltetrahydrofolate, a form of the
cofactor to which lymphoid cells would also be exposed in
vivo. Furthermore, senescence-induced inflammation has
been associated with enhanced cancer promotion (44), an
effect likely to be increased when telomeres are damaged
and/or become dysfunctional. Together, these mechanisms
may provide plausible bases for recent prospective epide-
miologic studies showing positive associations between TL
and cancer risk (28).
Together, these results demonstrate the impact of methyl
insufficiency on telomere homeostasis and provide evi-
dence that dysfunctional long telomeres may be as impor-
tant as critically short telomeres as a cause of chromosomal
instability in p53-negative cells.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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Folate Deficiency Induces Dysfunctional Telomeres
Authors' Contributions
Conception and design: C.F. Bull, G. Mayrhofer, M.F. Fenech
Development of methodology: C.F. Bull, N.J. O’Callaghan, M.F. Fenech
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): C.F. Bull, A.Y. Au, H.A. Pickett, M.P. Hande
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Writing, review, and/or revision of the manuscript: C.F. Bull, G. Mayr-
hofer, N.J. O’Callaghan, G.K.M. Low, D. Zeegers, M.P. Hande, M.F. Fenech
Study supervision: C.F. Bull, G. Mayrhofer, M.F. Fenech
Acknowledgments
The authors thank M. Hor for technical assistance for the 5azadC work,
and S. Mitchell [the Commonwealth Scientific and Industrial Research
Organisation (CSIRO), Sydney, Australia] and V. Dhillon (CSIRO, Adelaide,
Australia) for generating preliminary methylation status data for these
samples.
Grant Support
C.F. Bull was supported by an Australian Postgraduate Award (APA)
Scholarship through the University of Adelaide, CSIRO’s Preventative Health
Flagship, and a Science Industry Endowment Fund (SIEF) John Stocker
Postdoctoral Research Fellowship.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received July 14, 2013; revised October 30, 2013; accepted November 1,
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Cancer Prevention Research
 Published OnlineFirst November 19, 2013; DOI: 10.1158/1940-6207.CAPR-13-0264

Folate Deficiency Induces Dysfunctional Long and Short

Telomeres; Both States Are Associated with Hypomethylation and
DNA Damage in Human WIL2-NS Cells
Caroline F. Bull, Graham Mayrhofer, Nathan J. O'Callaghan, et al.

Cancer Prev Res 2014;7:128-138. Published OnlineFirst November 19, 2013.

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