PML is required for telomere stability in non-neoplastic human cells
PML is required for telomere stability in non-neoplastic human cells
M Marchesini 0 1 4
R Matocci 0
L Tasselli 0 1 5
V Cambiaghi 1
A Orleth 1
L Furia 1
C Marinelli 0 1
S Lombardi 1
G Sammarelli 2
F Aversa 2
S Minucci 1 3
M Faretta 1
PG Pelicci 1
F Grignani 0
0 General Pathology Section, Department of Experimental Medicine, University of Perugia , Perugia , Italy
1 Department of Experimental Oncology, European Institute of Oncology , Milan , Italy
2 Hematology and Bone Marrow Transplantation Unit, Department of Clinical and Experimental Medicine, University of Parma , Parma , Italy
3 Department of F Grignani, General Pathology Section, Department of Experimental Medicine , Piazza Lucio Severi, 1, Perugia 06132 Italy or Professor PG Pelicci , Department of Experimental Oncology, European Institute of Oncology , Via Giuseppe Ripamonti, 435, 20141 Milan , Italy
4 Present address: Department of Leukemia, MD Anderson Cancer Center, The University of Texas , Huston, TX , USA
5 Present address: Division of Endocrinology, Gerontology, and Metabolism, Department of Medicine, Stanford University School of Medicine , Stanford, CA , USA and Geriatric Research, Education, and Clinical Center, VA Palo Alto Health Care System , Palo Alto, CA , USA
Telomeres interact with numerous proteins, including components of the shelterin complex, whose alteration, similarly to proliferation-induced telomere shortening, initiates cellular senescence. In tumors, telomere length is maintained by Telomerase activity or by the Alternative Lengthening of Telomeres mechanism, whose hallmark is the telomeric localization of the promyelocytic leukemia (PML) protein. Whether PML contributes to telomeres maintenance in normal cells is unknown. We show that in normal human fibroblasts the PML protein associates with few telomeres, preferentially when they are damaged. Proliferation-induced telomere attrition or their damage due to alteration of the shelterin complex enhances the telomeric localization of PML, which is increased in human T-lymphocytes derived from patients genetically deficient in telomerase. In normal fibroblasts, PML depletion induces telomere damage, nuclear and chromosomal abnormalities, and senescence. Expression of the leukemia protein PML/RAR? in hematopoietic progenitors displaces PML from telomeres and induces telomere shortening in the bone marrow of pre-leukemic mice. Our work provides a novel view of the physiologic function of PML, which participates in telomeres surveillance in normal cells. Our data further imply that a diminished PML function may contribute to cell senescence, genomic instability, and tumorigenesis.
Chromosomal telomeres surveillance and repair mechanisms
continuously operate in proliferating cells to prevent the
activation of DNA damage signaling and the development of
chromosomal abnormalities. Telomeres structure is protected by a
number of proteins constituting the shelterin complex, whose
functions include the maintenance of the structure of telomeric
DNA loops and the inhibition of the activity of DNA repair
proteins, which are found associated with telomeres.1?3
The protein members of the shelterin complex dynamically
interact with telomeric RNAs and with other proteins involved in
telomere surveillance, including DNA damage response factors.4
The shelterin complex exerts a crucial function in the protection of
telomeric repeats, since the depletion of its protein members,
such as TRF2 and POT1, causes telomere uncapping and damage.
As a consequence telomeres associate with a number of proteins,
including 53BP1 and gamma-H2AX, constituting the telomere
dysfunction-induced focus (TIF), which are considered as markers
of telomere damage.5?7 However, the complex interplay between
the shelterin complex and the numerous proteins that engage in
transient or stable interactions with telomeric structures is only
Telomeres surveillance is critical for the regulation of cell life
span.8,9 Indeed, the replication potential of normal cells is limited
by a proliferation-dependent telomere attrition, which triggers cell
senescence upon excessive shortening of the telomeric DNA
repeats.10,11 Such telomeres attrition leads to progressive
modifications of the cell phenotype, which are linked to cellular
dysfunctions associated with human aging and age-related
disease.12?14 The maintenance of telomeric DNA repeats length
is physiologically necessary to prevent senescence in a few cell
types, including stem cells and germ cells. This function is
accomplished by the telomerase ribonucleoprotein complex,
whose reverse transcriptase component (TERT) elongates
telomeric repeats.15,16 Activation of telomerase occurs also in
normal T-lymphocytes when stimulated to proliferate by antigens
or lectins, allowing the expansion of an antigen reactive T-cell
population.1 Telomerase becomes abnormally activated in the
majority of tumor types, allowing indefinite proliferation of cancer
cells.17 However, 10?15% of tumors do not show telomerase
reactivation. In these tumors, the maintenance of telomeres
length is accomplished by a telomerase-independent mechanism,
referred to as Alternative Lengthening of Telomeres (ALT).18,19
Hallmarks of ALT are heterogeneity of telomere lengths, circular
telomeric DNA, and the association between telomeres and
the promyelocytic leukemia (PML) protein.20 This protein was
first discovered as the product of the PML gene, which fuses
with the RAR? gene in the t(15;17) chromosomal translocation.
This chromosomal abnormality causes the human acute
promyelocytic leukemia (APL), where a fusion PML/RAR? protein is
expressed.21?24 In normal cells, the PML protein aggregates
nuclear structures called PML nuclear bodies (PML-NBs), where it
interacts with multiple protein partners to accomplish a wide
variety of functions, including regulation of transcription and p53
activation.25,26 PML also participates in DNA damage response and
is overall regarded as a tumor suppressor.27?30 The interaction
between the PML protein and telomeres is clearly recognized in
ALT cells, where the PML protein is present within telomeric
bodies named ALT-associated PML nuclear bodies.20 However, the
specific role played by the PML protein in this context is still under
investigation. Another fundamental question is whether PML-NBs
exert a telomeric function in normal cells. Evidence for the
presence of the PML proteins at the telomeres of non-neoplastic
cells have been reported in human endothelial cells and mouse
embryonic stem cells, where the PML protein appears to be
relevant for telomeres stability.31,32 However, it is not clear
whether this localization is functionally significant nor if it has a
role upon proliferative telomeric attrition or damage. Another
open question is whether an altered function of the telomeric PML
could contribute to the pathogenesis of leukemia. The PML/RAR?
protein33 acts as a transcriptional repressor of RAR? and non-RAR?
target genes and disrupts the PML-NBs exerting a dominant
negative activity on their function.25,34 Whether the leukemogenic
function of the PML/RAR? protein involves an alteration of the
function of PML at telomeres is still unknown.
In this paper, we characterize the telomeric localization and
function of the PML protein in non-neoplastic cells. We show that
PML/telomere associations increase upon proliferative telomere
attrition and telomeric damage. PML depletion causes telomeric
damage, genomic instability and cell senescence, indicating
a critical role for PML in telomeres surveillance. Moreover, the
PML/RAR? fusion protein displaces PML from telomeres and
induces telomeres shortening in human hematopoietic precursor
cells, identifying a novel contribution to APL leukemogenesis.
The PML protein localizes at telomeres in normal cells
We investigate the localization of PML at telomere in cell types
with different telomere maintaining mechanisms. To this end,
we performed telomeric fluorescence in situ hybridization (FISH)
analysis combined with immunofluorescence for PML (immuno-FISH)
in interphase ALT tumor cell lines (U2-OS, SK-LU-1 and
SA-OS-2), normal human fibroblasts (WI38, MRC-5), mouse
embryonic fibroblasts and telomerase-positive cancer cell lines
(HeLa and A549 and U937) (Figure 1a and Supplementary Figures
1a?c). 3D reconstructions shown in Supplementary Figure 2 and
Supplementary video 1 illustrate the standards for colocalization
scoring. As expected, in ALT cell lines we observed that 30?80%
of the PML-NBs colocalized with telomeres (Figure 1b and
Supplementary Figure 1). Normal human and mouse fibroblast cells
and telomerase-positive cancer cells also showed colocalization
between PML-NBs and telomeres, although at much lower
frequency than ALT cell lines (Figure 1b and Supplementary
Figure 1d). The percentage of PML-NBs at telomere in non-ALT
cells was small but consistent, ranging from 4 to 14% of total
PMLNBs number in normal fibroblasts and 22?35% in
telomerasepositive cells, independently from the number of PML-NBs per cell.
In the different cell lines, the number of PML-telomere colocalization
per cell ranged from 0.36 to 5.86 (Supplementary Figure 1d). Thus, a
fraction of the PML protein colocalizes with telomeres in
telomerasepositive cancer cells and in non-neoplastic cells, suggesting an
extended and physiologic function for PML at telomere.
PML association with telomeres increases upon telomeres attrition
We thereafter focused on non-neoplastic cells and asked whether
the PML protein may contribute to telomere surveillance during
specific phases of the cell cycle, since telomere replication is a
critical process, requiring accurate care.35 Considering the low
frequency of the telomeric localization of PML, we took advantage
of an automated fluorescence image analysis,36,37 allowing the
study of a large number of cells and their cell-cycle phase
(Figures 2c?f). MRC5 normal human fibroblasts were stained with
anti-PML, -TRF2 and -53BP1 antibodies, in association with DAPI
incorporation to detect DNA amount and Ethinyl-Deoxyuridine
(EdU) staining, which allows replication measurement (Figures 2a
and d). This procedure allowed (i) the detection of telomeres, as
identified by TRF2 signals, (ii) the detection of damaged telomeres
as damage induced foci (TIFs), identified by measuring
53BP1TRF2 overlap in the proximity ligation assay36,37 (PLA, Figure 2b);
(iii) the identification of the cell-cycle phase and (iv) the analysis of
the PML/telomeres and damaged telomeres colocalization. We
detected an average of 0.65 PML-telomere colocalizations per cell
(Figure 2c). The distribution of the number of PML-NBs and the
PML/TIF colocalizations, depending on the DNA content of the
cells (Figures 2d?f), did not show statistically significant
differences, indicating that the telomeric function of PML is not specific
for a cell-cycle phase. When we compared proliferating and late
passage (see Materials and methods) near senescent (NS) cells,8?10
Telomerase Positive Cells
the number of TIFs and PML-NBs increased in the latter (Figures 2g
and h),26 where most of the cells displayed at least one TIF
(Figure 2i). In these experiments, a fraction of the telomeric PML
colocalized with dysfunctional telomeres (TIFs), as 5% of
proliferating cells displayed PML/TIFs colocalization. Notably, this
percentage increased to 36% of the NS cells (Figure 2j).
To further investigate the link between senescence-associated
telomere attrition with and recruitment of the PML protein at
telomere, we analyzed proliferating and NS WI38 fibroblasts by
immuno-FISH, using anti-PML and anti-?H2AX antibodies staining
together with a PNA telomeric probe (Figure 3a). The number of
TIFs per cell, as identified by overlapping ?H2AX and telomeric
signals, increased in NS WI38 cells (Figure 3b). The number of
PML-NBs and the number of their colocalization with telomeres
and TIFs increased significantly (Figure 3b and Supplementary
Figure 3a and b). Moreover, the percentage of the total NBs
associated with telomeres (PML/Tel%) and with TIFs (PML/TIFs%)
was almost double in NS than in proliferating cells (Figure 3b).
Overall, these results indicate that physiologic,
proliferationinduced attrition triggers PML colocalization with telomeres.
We next asked whether the localization of PML at telomeres
could be specifically relevant in the context of telomere attrition
due to premature senescence. To this end, we studied IL-2
stimulated peripheral blood T-lymphocytes from a patient
carrying a germline mutation of the TERT gene, resulting in TERT
aploinsufficiency (Figures 3c and e). In immuno-FISH experiments,
the number of PML-NBs, identified in patient?s cells was similar to
that of age-matched control T-lymphocytes and remained
relatively stable during prolonged in vitro culture (Figure 3e).
However, the number of TIFs was higher in TERT mutated
T-lymphocytes than in controls and increased with in vitro culture
(Figure 3d), indicating augmented telomere attrition. Significantly,
the percentage of PML-NBs colocalizing with telomeres (PML/Tel)
was higher in TERT-deficient cells and further increased to more
than double than control cells upon cell proliferation in culture
(Figure 3d), suggesting a progressive recruitment of PML to
proliferation-induced dysfunctional telomeres.
Specific telomeric damage increases PML localization at telomeres
The above data suggested that PML may participate in damaged
telomeres surveillance. Thus, we measured PML/telomere
colocalization after the induction of telomere-specific damage. The
G-quadruplex ligand RHPS4 delocalizes POT1 from telomeres and
induces TIFs formation.38,39 We treated early passage WI38 normal
human fibroblasts with RHPS4 for 24 h and performed
immunoFISH staining (Figure 4a). In exponentially growing cells, the
treatment induced a significant increase in TIFs number
(Figure 4b). PML-NBs increased in number (Figure 4b) and were
recruited to telomeres, as shown by a threefold increase in the
number of PML/telomere colocalizations (Supplementary Figure 3c)
and by a 3.5-fold raise in the percentage of PML-NBs colocalizing
with telomeres (Figure 4b). Moreover, the number of
colocalizations between PML-NBs and TIFs markedly increased
(Supplementary Figure 3d) and the percentage of total PML-NBs
colocalizing with TIFs increased by tenfold, as compared with
control cells (Figure 4b).
To investigate the interaction of the PML protein with uncapped
telomeres, we depleted the TRF2 protein40 in early passage WI38
and MRC5 normal human fibroblasts by lentivirus-mediated short
hairpin RNA (shRNA) interference (TRF2 KD), using as a control a
vector carrying a shRNA directed against a luciferase sequence
(CTRL). After cell selection, TRF2 depletion was verified by western
blotting and immunofluorescence assays (Supplementary Figures
4a and b). In agreement with previous reports,5 WI38 and MRC5
human fibroblasts infected with a TRF2KD vector, but not with the
control vector, displayed a significant increase in TIFs and
PML-NBs (Figures 4c and d and Supplementary Figures 4c and
d). The number of PML colocalizations with telomeres and
specifically with TIFs increased markedly in both TRF2-depleted
WI38 and MRC5 cells, as compared with controls (Supplementary
Figure 4e and f). This increase is not a consequence of the
increased number of PML-NBs in TRF2-depleted cells, since, the
percentage of the total PML-NBs colocalizing with telomeres
raised markedly and the percentage of PML-NBs specifically
associated with TIFs increased more than 25- and 35-fold in WI38
and MRC5 cells, respectively (Figure 4d and Supplementary Figure
4d). Overall, these data show that telomere damage induced by
loss of components of the shelterin complex causes PML
association with altered telomeres.
telomere dysfunction was most likely the trigger for p53
activation. Taken together, these data show that PML contributes
to telomere integrity in normal cells and that its depletion causes
telomeric damage and the activation of premature senescence.
PML depletion leads to telomere dysfunction and senescence in
To unveil the role of the PML protein at telomere in normal cells,
we depleted its expression in early passage WI38 and MRC5 cells.
To this end, we infected and selected the cells using two PML
shRNA lentiviral vectors (PML KD1 and PML KD2) targeting exon 3
sequences common to all the PML isoforms (Figure 5a). Strikingly,
PML knockdown caused a dose-dependent decrease in cell
proliferation (Figure 5a), morphological changes consistent with
a senescent phenotype and an increased expression of the
senescence-related ?-galactosidase enzyme (Figures 5b and c).
Moreover, a senescence molecular pathway was activated, as
shown by an increased expression of the p53, p16INK4a and
p21WAF1 proteins in PML-depleted cells with respect to control
cells (Figure 5d). Significantly, PML depletion did not induce
senescence, proliferation arrest or reduced clonogenicity in the
telomerase-positive HeLa cell line (Supplementary Figure 5).
To investigate whether this phenotype was linked to the role of
PML at telomeres, we measured the generation of TIFs upon PML
depletion (Figure 5e). The data showed a significant increase in
TIFs formation in both WI38 and MRC5 PML-KD cells with respect
to control cells, indicating that PML depletion induces telomeric
damage (Figure 5f). Immuno-FISH experiments employing a PNA
telomeric probe, anti-?H2AX and anti-p53 antibodies, showed that
the number of p53-positive cells was markedly increased in
PML-KD MRC5 cells (Figures 5g and h). More than 90% of PML-KD
p53-positive cells were TIF positive (Figure 5i), indicating that
PML depletion is associated with chromosomal abnormalities and
The above data uncover the role of PML in maintaining telomere
integrity of normal human fibroblasts. To further investigate the
relevance for chromosomal stability of PML activity at telomeres,
we analyzed nuclear abnormalities in MRC5 cells after
siRNAmediated PML depletion. More than 20% of cells displayed
binucleation (Figures 6a and b). To emphasize defects in nuclear
division, we treated the cells with blebbistatin, a cytokinesis
inhibitor. DAPI stained nuclei showed several abnormalities,
including nucleoplasmic bridges (NPBs), micronuclei (MNs) and
nuclear buds (Buds) (Figures 6c and d), which are considered
hallmarks of genomic instability due to telomeric dysfunction.41
These abnormalities were scored in 450% of PML-depleted cells
(Figure 6d). To link these observations with telomere dysfunction,
we analyzed chromosome spreads of control and PML-KD MRC5
cells and performed telomeric FISH to investigate the status of
chromosome ends. Chromosomes of PML-KD MRC5 cells showed
a number of aberrations, including end-to-end fusion, circle
chromosomes and fragile telomeres (Figures 6e and f). These
abnormalities most likely derive from the telomeric dysfunction
associated with PML depletion. In addition, the metaphases
showed chromosomal breaks and fragments (Figure 6f), which
seemingly indicate the development of genomic instability.
Overall, PML depletion in normal human fibroblast cells is
associated with telomere dysfunction and chromosomal instability,
leading to senescence induction.
CTRL PMLKD1 PML KD2
CTRL PML KD1
CTRL PML KD1
The telomeric function of PML is hampered by the APL PML/RAR?
Leukemia cells in APL have short telomeres.42 Since the APL fusion
protein PML/RAR? disrupts PML-NBs,25 we investigated whether
an impairment of the telomeric function of PML could contribute
to the pathological activity of PML/RAR?. To this end, we infected
normal human CD34+ hematopoietic progenitor cells (HPCs) in
liquid culture with a retrovirus vector expressing PML/RAR? or a
control vector and analyzed the PML/telomere colocalization
(Figure 7a), using the criteria illustrated in Supplementary Figure 1.
In these conditions, normal HPCs showed at least one
PML/Telomere colocalization event in about 50% of the cells.
However, in PML/RAR? expressing HPCs colocalization was only
found in 15% of the cells (Figure 7b). Moreover, 50% of the control
cells had 410% of the PML signals colocalizing with telomeres
whereas this was observed in only 3% of the PML/RAR? cells
(Figure 7c). Overall, these data indicate that PML/RAR? expression
impairs PML association with telomeres. To investigate whether
this fact could relate with the telomere shortening of APL
blasts, we analyzed bone marrow cells derived from the
preleukemic stage of mice transplanted with PML/RAR? expressing
Lin ? hematopoietic progenitors.43 We found that telomeres
displayed a 20% average reduction in length (Figure 6d),
indicating that, before the development of leukemia, the
expression of PML/RAR? and displacement of PML from telomeres
correlate with an overall reduction of telomeres length.
CTRL PML KD1
DAPI PML CTRL PML KD1
This study shows that the PML protein may participate in
telomeric surveillance in non-neoplastic cells. The low levels of
telomeric damage generated by in vitro culture of early passage
normal human fibroblasts in exponential proliferation are
sufficient to induce the telomeric localization of PML.
Telomerase-positive tumor cell lines also displayed telomeric
PML signals, overall extending the telomeric localization of PML
beyond the neoplastic ALT cells displaying ALT-associated PML
bodies.20 These data are in agreement with reported observations
obtained in human umbilical vein endothelial cells,
telomerasepositive cancer cell lines44 and mouse embryonic stem cells.32 The
telomeric localization of the PML protein in normal cells is a low
frequency phenomenon. We therefore used an automated
confocal microscopy method,36,37 which allowed us to precisely
quantify PML/telomere colocalization in a large number of cells,
while relating the findings to the cell-cycle phase. Telomere
replication is a critical step in the cell cycle. Capping proteins must
dissociate from telomeres making them accessible for DNA
replication factors and regulatory proteins, whereas DNA damage
protein is transiently recruited to telomeres during G2/M phase.45
However, we found that the association of PML-TIFs is not
cellcycle phase dependent suggesting that PML exerts its function
independently from telomeric DNA replication. In normal human
fibroblasts, PML interacts with telomere at a much lower
frequency than in ALT tumor cells. Most likely, this is due to the
more prominent telomere damage and instability in tumor cells
activating the ALT system. The abundance of telomeric PML
protein in ALT cells may suggests that PML is recruited to
damaged telomeres. Whether this hypothesis could hold true in
normal cells is not clearly established, although an activation of
the ALT pathway has been reported upon radiation induced
general DNA damage.46 We addressed this issue in diverse model
systems. First, we quantitatively analyzed telomeric PML in NS
CTRL PML/RAR? PML Tel
fibroblast cells. We found that PML associates with telomeres
undergoing attrition upon cell passage in vitro, specifically
colocalizing with damaged telomeres. Thus, PML interacts with
telomeres when they are physiologically shortened during cell
proliferation. An accelerated telomere shortening is seen in rare
syndromes caused by mutations in components of the telomerase
complex.47 Indeed, we found that T-lymphocytes derived from a
patient carrying a germline mutation of the TERT gene diffusely
displayed a higher number of TIFs, with respect to age matched
controls. TIFs increased further upon in vitro culture and the
percentage of PML-NBs co-localizing with telomeres was much
higher than in control cells, confirming an association of PML with
shortened telomeres. Thus, it can be speculated that PML may
participate in telomere surveillance and stability during cellular
senescence and, possibly, human aging.
We also asked whether PML might participate in the
surveillance on telomere damage induced by alterations in the
shelterin complex. We specifically induced telomere damage in
normal fibroblasts by treating the cells with the drug RHPS4,
which delocalizes POT1 from telomeres39 or by depleting the cells
of TRF2, an essential component of the shelterin complex.40 In
both cases, the association of PML with damaged telomeres
increased markedly. These data support the view that, in
nonneoplastic cells, PML comes into play when telomeres are
damaged by diverse causes, thus participating in physiologic
telomere homeostasis. The effects of PML depletion observed in
normal cells support this novel prospect on PML function.
An inadequate amount of PML induces the activation of a
senescence program, including an increased expression of p53,
p21 and p16, and undergo proliferation arrest. In these conditions,
we did not observe significant changes in cell death, suggesting
that senescence is the major mechanism activated by PML
depletion. Moreover, PML-depleted cells develop nuclear and
chromosomal abnormalities indicating that a defective PML
function is associated with chromosomal instability. A decreased
activity of PML on genomic DNA damage foci27,48 could contribute
to these aberrations. However, the observed chromosomal lesions
include lesions such as NPBs, MNs and nuclear buds, which
strongly suggest that telomere damage gives a major contribution
to this effect.
Overall, our data suggest that PML participates in physiologic
telomere surveillance. Its function may be specifically relevant
when the activity of telomerase is low, as in ALT cells,
TERTdeficient cells or in normal cells. In fact, PML depletion in
telomerase-positive tumor cells does not lead to proliferation
arrest or senescence (our unpublished results). PML may function
by participating in the surveillance of telomeric chromatin, since
the induction of an ALT phenotype was reported upon depletion
of the histone chaperone ASF1.49
The APL-specific PML/RAR? chimeric protein acts as an
abnormal chromatin regulator.50?52 Indeed this fusion protein
can be considered as a dominant negative PML mutant, since it
disrupts PML-NBs.25 We found that the telomeric localization of
PML is markedly impaired in normal HPCs expressing the PML/
RAR? protein. This finding may imply that the integrity of the
protein interactions of PML and its effects on sumoylation, which
are altered by the PML/RAR? protein, are required for PML
telomeric function. Interestingly, APL blasts have shortened
telomeres,42 suggesting that the decreased PML function may
impact on telomere stability. Pre-leukemic bone marrow cells
derived from mice transplanted with hematopoietic precursors
expressing PML/RAR? show a reduction in telomere length. Thus,
before the development of a full-blown leukemia phenotype,
PML/RAR? expression alone induces telomere shortening. This is
associated with the displacement of PML from telomeres,
suggesting that the dominant negative effect of PML/RAR? on
PML may impair telomere surveillance, contributing to the
pathogenesis of leukemia. APL typically displays a 15;17
chromosomal translocation. However, complex chromosomal
abnormalities are detected in about 30% of the cases.53,54 In the remaining
patients, the impaired telomeric surveillance may not translate in
complex chromosomal aberrations due to the rapid natural history
of this leukemia or the effective treatment.
Overall, our data identify a novel and important functional role
for PML in telomeres surveillance in normal cells and imply that a
diminished PML function may contribute to cell senescence,
genomic instability and tumorigenesis.
MATERIALS AND METHODS
Primary cells and cell lines
293 T packaging cells, HeLa cells, the lung carcinoma A549 and the
osteosarcoma U2OS cell lines and normal human WI38 fibroblasts were
maintained in Dulbecco?s Modified Eagle?s Medium supplemented with
10% fetal bovine serum (EuroClone, Pero, Italy). MRC-5 normal human
fibroblasts and the lung adenocarcinoma cell line SK-LU-1 were cultured in
Eagle's Minimum Essential Medium (EuroClone) with the above
supplementation, non-essential amino acids (0.1 mM) (EuroClone) and sodium
pyruvate (1 mM) (EuroClone). The osteosarcoma cell line Saos-2 was
cultured in McCoy?s Medium (Sigma-Aldrich, St Louis, MO, USA)
supplemented with 15% fetal bovine serum. Media were supplemented
with penicillin (100 units/ml) and streptomycin (100 ?g/ml, EuroClone). All
cells were cultured in 5% CO2 at 37 ?C. Cell lines were purchased from
American Type Culture Collection (ATCC) Manassas, VA, USA). Mouse
embryonic fibroblasts were isolated from in house animals, following
standard procedures, from embryos surgically removed at embryonic day
13.5 and cultured in Dulbecco?s Modified Eagle?s Medium supplemented
with antibiotics and 10% fetal bovine serum, in 9% CO2 at 37 ?C. Late
passage near senescent MRC5 and WI38 cells (NS) are defined as passages
30?34, corresponding to about 35?40 population doublings, when the
cells decrease their proliferation rate to about one half of early passage
cells. For colony assays 0.5 ? 103 HeLa cells were plated in triplicate in 6 cm
plates in growth medium. Ten days after plating, colony number was
evaluated. The cells were washed twice with PBS, fixed in 5%
formaldehyde, rinsed and stained for 5 min with 0.05% crystal violet.
Visible colonies were counted. The experiments were repeated at least
three times. Mean and s.d. were calculated among the experiments.
RHPS4 and blebbistatin
The G-quadruplex ligand RHPS438,39 was added directly to the culture
medium at the concentration of 1 ?M for 24 h to induce specific telomeric
DNA damage in human fibroblasts. Blebbistatin 20 ?M (Sigma-Aldrich) was
added to the culture medium for 24 h to block cytokinesis. All experiments
were performed at least three times.
Lentiviral vectors constructs and siRNA for RNA interference
The following oligonucleotides were used to generate shRNAs for PML,
TRF2 and a control luciferase sequence (Luc). (Eurofins MWG Operon,
PMLKD1: For 5?-TGACCTCAGCTCTTGCATCATTCAAGAGATGATGCAAGAGCT
GAGGTCTTTTTTC3?; Rev 5?-TCGAGAAAAAAGACCTCAGCTCTTGCATCATCTCTTG
AATGATGCAAGAGCTGAGGTCA-3?; PMLKD2: For 5?-TGGGACCCTATTGACGTTG
ATTCAAGAGATCAACGTCAATAGGGTCCCTTTTTTC-3?; Rev 5?-TCGAGAAAAAAG
GGACCCTATTGACGTTGATCTCTTGAATCAACGTCAATAGGGTCCCA-3?; LUC: For
-3?; Rev 5?-TCGAGAAAAAACTTACGCTGAGTACTTCGATCTCTTGAATCGAAGTAC
TCAGCGTAAGA-3?; TRF2 KD: For 5?-TGAGGATGAACTGTTTCAAGTTCAAGAGAC
TTGAAACAGTTCATCCTCTTTTTTC-3?; Rev 5?-TCGAGAAAAAAGAGGATGAACTG
Oligonucleotides were annealed and cloned into pSIcoR vectors.55
Lentiviral particles were generated in 293 T cells by standard methods and
used for infection.56 Infected cells were selected with puromycin for 3 days
and selection was confirmed by the death of puromycin-treated
Oligonucleotide sequences used for siRNA experiments were the
following. Luciferase (Luc): 5?-CTTACGCTGAGTACTTCGA-3?; PMLKD1:
5?-GACCTCAGCTCTTGCATCA-3?; PML KD2: 5?-GGGACCCTATTGACGTTGA-3?.
SiRNA was transfected at 20 nM using Lipofectamine RNAi MAX kit
(Invitrogen, Carlsbad, CA, USA) following the manufacturer protocol.
Growth curves and ?-galactoside assays
Primary fibroblasts were seeded in 6-well plates, in triplicate. Cells were
counted at 3, 6, 8 and 10 days after plating for growth curves evaluation. For
?-galactoside staining, the cells were washed in PBS (EuroClone), fixed with
4% paraformaldehyde and incubated overnight at 37 ?C in staining buffer
(1 mg/ml 5-bromo-4-chloro-3-indolyl ?-D- galactoside (X-Gal), 5 mM
potassium ferrocyanide, 40 mM citric acid, 150 mM NaCl2, 2 mM MgCl2 and sodium
phosphate pH 6.0). At the end of incubation, cells were washed with PBS
and analyzed.57 All the experiments were performed at least three times.
Immunofluorescence and microscopy
Cells were grown directly on glass coverslips, fixed with 4%
paraformaldehyde, permeabilized in PBS containing 0.1% Triton X-100 (Sigma-Aldrich) and
blocked 1 h in blocking solution (PBS 2% BSA). The cells were then stained
with a primary antibody for 1 h at room temperature and counterstained with
a secondary antibody. Cell nuclei were stained with
4?,6-diamidino-2phenylindole (DAPI). Images were captured with a CCD (charge-coupled
device) camera (Hamamatsu Bs/W CCD Camera CJ895, Hamamatsu Photonics
Italia, Milano, Italy), using a wide field (Olympus BX61, Olympus Italia, Segrate,
Italy) or a confocal (Leica TCS SP2 AOBS, Leica Microsystem, Milano, Italy)
microscope. Supplementary Figure 2 and Supplementary Video 1 show the
criteria for colocalization scoring. In situ 53BP1-Trf2 PLA was performed as
described.36,37 Coverslips for detection of cell-cycle distribution by wide field
high-resolution cytometry were stained with DAPI (10 ?M for 30 min) and
mounted in a Mowiol containing mounting media. DNA counterstain for
confocal analysis was performed by incubating cells with Chromomycin A3
(10 ?M in PBS/70 mM MgCl2) or Draq5 and mounting coverslips in a DABCO
containing glycerol-based mounting medium to preserve cell 3D structure.
Primary antibodies used were anti-PML (H238, sc-5621, Santa Cruz
Biotechnology, Dallas, TX, USA) (PGM3, sc-100410, Santa Cruz Biotechnology),
anti-?H2AX (Ser139) (Upstate 05-636, Millipore, Billerica, MA, USA), anti-TRF2
(clone 4A794, Millipore) rabbit anti-53BP1 (ab36823, Abcam, Cambridge, UK)
and anti-p53 (DO-1, sc-126, Santa Cruz Biotechnology). Secondary antibodies
used were CY3 donkey anti-Rabbit/Mouse, CY5 donkey anti-Rabbit/Mouse
(Jackson Immunoresearch, West Grove, PA, USA), Alexa488 donkey
antiRabbit/Mouse (Alexa) and Pacific Orange anti-Rabbit/Mouse (Jackson
Image cytometry analysis
The image cytometry experiments for simultaneous detection of cell-cycle
distribution, DNA replication, PML and telomeres content and spatial
localization were performed as described.36,37 Automated wide field and
confocal microscopes acquired images were analyzed by an ad-hoc image
analysis software developed in the ImageJ macro language. Since the
frequency of the detected events was particularly low in the exponential
growth phase, we increased the throughput of the confocal analysis
employing wide field microscopy to measure up to 15 000 cells. To increase
efficiency in the high-resolution detection of damaged telomeres, we
employed PLA between TRF2 and 53BP1 protein (see Immunofluorescence).
To evaluate cell-cycle distribution, cells were classified according to the
DNA and EdU content (G1: 2 N EdU negative; G2: 4 N EdU negative;
S phase: EdU positive). Spots were detected by first applying a Laplace of
Gaussian filter on background-subtracted images. Colocalization between
targeted spots (PLA (damaged telomeres) and PML-NBs) was considered by
calculating mutual distances of the fluorescence barycenter ranging between
100 (colocalization) and 400 nm (less than sum of the spots radius) (Figure 2b).
Immuno-FISH staining and telomere FISH. Cultured cells were fixed in 4%
paraformaldehyde for 30? and 100 mM glycine for 20? at room temperature.
The cells were then permeabilized in 0.5% Triton X-100/0.5% saponin.
Primary and secondary antibody staining was performed as described above
and followed by an additional fixation with 4% paraformaldehyde and
100 mM glycine. Telomere-FISH was carried out using the Telomere PNA FISH
Kit/Cy3 (DAKO, Glostrup, Denmark), according to the manufacturer protocol.
Briefly, slides were immersed in Tris-buffered Saline and permeabilized with
pretreatment solution. DNA was denatured by heating 5 min at 80 ?C in
hybridization solution (50% formamide, 20% dextran sulfate in ? 2 SSC )
containing a Cy3-conjugated PNA probe specific for telomeric repeats. After
the annealing step at RT in the dark, the slides were washed with
Trisbuffered Saline, counterstained with DAPI and mounted in Mowiol. Images
were collected as described above and ImageJ software (W. Rasband, NIH)
was used to process and analyze images. Experiments performed with this
technique shown in the manuscript are representative of three experiments.
As described in figure legends, statistics were carried out scoring nuclei
50?100/condition in every triplicate experiment.
Binucleation assay for abnormal nuclear morphology analysis
MRC-5 cells were grown on cover slips and transfected with 20 nM siRNA
targeting PML. Twelve hours later, cells were treated with 20 ?M
Blebbistatin (Sigma-Aldrich) for 24 h to block cytokinesis, fixed in 4%
paraformaldehyde and stained for Immunofluorescence as described
above. The analysis of abnormal nuclear morphologies was performed on
binucleated cells using described criteria.58 An NPB was considered to be
the narrow/wide chromatin segment connecting two cell nuclei, MNs were
morphologically identical to, but smaller than the cell nucleus, and round
and oval protrusions of the nuclear membrane, connected to the cell
nucleus, were classified as buds.59 The experiments were performed at
least three times. In every experiment 50 binucleated cells were analyzed.
Metaphase spreads were obtained from MRC-5 PML knockdown cells
treated with 0.1 ?g/ml colcemid (Gibco, Carlsbad, CA, USA) for 2 h. Cells
were trypsinized, washed in PBS and incubated in hypotonic solution
(75 mM KCl) for 30 min at 37 ?C. The cells were then fixed with methanol/
glacial acetic acid (3:1 vol/vol). For telomeric FISH, the cells were either
spun onto coverslips via a cytospin centrifuge or spread across a slide
presoaked in 2% TWEEN detergent (Sigma-Aldrich) and dried. Staining was
performed with a specific telomeric PNA probe as above. Fifty metaphases
were analyzed in each of three experiments.
Western blotting was performed as described previously.60 The primary
antibodies used were anti-PML (H238, sc-5621, Santa Cruz Biotechnology),
anti-TRF2 (clone 4A794, Millipore), anti-p53 (DO-1, sc-126, Santa Cruz
Biotechnology), anti-p21 (C-19, sc-397, Santa Cruz Biotechnology), anti-p16
(H-156, sc-759, Santa Cruz Biotechnology), anti-vinculin or anti-actin (Santa
Cruz Biotechnology) were used to normalize protein content of samples.
Images shown in the figures are representative of at least three experiments.
Isolation of primary T-lymphocytes
Peripheral blood mononuclear cells were obtained after informed consent
from a patient carrying a germline X456Y mutation in the TERT gene and
from an age-matched control donor. The cells were isolated by
centrifugation on Ficoll?Hypaque (Pharmacia, Uppsala, Sweden) and cultured in
RPMI-1640 supplemented with 10% FCS and penicillin/streptomycin
antibiotics (EuroClone). Monocytes were depleted by plastic adherence
and T-lymphocytes were activated for 16 h by 5 mg/ml
phytohaemoagglutinin A (Pharmacia) and then stimulated with interleukin-2 (500 U/ml)
(Pharmacia). Immuno-FISH experiments were performed on cytospin slides.
Where indicated data represented as mean ? s.d. were derived from at least three
independent experiments. Statistical significance between means was assessed
by Student?s t-test where a P-value of o0.05 was considered as significant.
Human CD34+ hematopoietic progenitors purification and
CD34+ cells were purified, cultured in vitro and transduced with a control or
a PML/RAR? cDNA containing retroviral vector, as previously described,61
but using a pBABE-Puro retrovirus vector backbone. Twenty-four hours after
the infection, the cells were selected in puromycin for 48 h. Immuno-FISH
experiments on CD34+ cells were performed as described above.
Mice transplantation and bone marrow analysis
The procedures for bone marrow isolation, transduction and
transplantation were performed as described,43 using 129/Sv inbred mice (The
Jackson Laboratory, Bar Harbor, MA, USA).
Telomere length measurement assays
The average length of telomere repeats was evaluated by flow FISH62 using
a telomere-specific FITC labeled probe according to the manufacturer
instructions (DAKO). Samples were then analyzed by flow cytometry
(FACScan, BD Biosciences, Milano, Italy) and relative telomere length was
determined by comparing isolated test cells with a control cell line (1301;
sub-line of the Epstain-Barr Virus genome negative T-cell leukemia line
CONFLICT OF INTEREST
The authors declare no conflict of interest.
This work was supported by AIRC IG grant to FG, and SM, and by a grant from the
Fondazione Cassa di Risparmio di Perugia to FG. We thank Lucilla Luzi and Alessandro
Brozzi (European Institute of Oncology, Milan) for their help in statistical analysis and
Annamaria Biroccio (Istituto Nazionale Tumori Regina Elena, Roma) for kindly
providing us the RHPS4 compound. We are grateful to Simona Colla (MD Anderson
Cancer Center) for encouragement and critical reading of the manuscript, to Giulio
Draetta and Luigi Nezi (MD Anderson Cancer Center) for their encouragement and
support. This work was supported by AIRC IG grant to FG, and SM, and by a grant
from the Fondazione Cassa di Risparmio di Perugia to FG.
1 Akbar AN , Vukmanovic-Stejic M. Telomerase in T lymphocytes: use it and lose it? J Immunol 2007 ; 178 : 6689 - 6694 .
2 Bailey SM , Meyne J , Chen DJ , Kurimasa A , Li GC , Lehnert BE et al. DNA doublestrand break repair proteins are required to cap the ends of mammalian chromosomes . Proc Nati Acad Sci USA 1999 ; 96 : 14899 - 14904 .
3 Nandakumar J , Cech TR . Finding the end: recruitment of telomerase to telomeres . Nat Rev Mol Cell Biol 2013 ; 14 : 69 - 82 .
4 d'Adda di Fagagna F , Reaper PM , Clay-Farrace L , Fiegler H , Carr P , Von Zglinicki T et al. A DNA damage checkpoint response in telomere-initiated senescence . Nature 2003 ; 426 : 194 - 198 .
5 Takai H , Smogorzewska A , de Lange T. DNA damage foci at dysfunctional telomeres . Curr Biol 2003 ; 13 : 1549 - 1556 .
6 Stagno D'Alcontres M , Mendez-Bermudez A , Foxon JL , Royle NJ , Salomoni P . Lack of TRF2 in ALT cells causes PML-dependent p53 activation and loss of telomeric DNA . J Cell Biol 2007 ; 179 : 855 - 867 .
7 Karlseder J , Smogorzewska A. de Lange T. Senescence induced by altered telomere state, not telomere loss . Science 2002 ; 295 : 2446 - 2449 .
8 Hayflick L , Moorhead PS . The serial cultivation of human diploid cell strains . Exp Cell Res 1961 ; 25 : 585 - 621 .
9 Harley CB , Futcher AB , Greider CW . Telomeres shorten during ageing of human fibroblasts . Nature 1990 ; 345 : 458 - 460 .
10 Allsopp RC , Vaziri H , Patterson C , Goldstein S , Younglai EV , Futcher AB et al. Telomere length predicts replicative capacity of human fibroblasts . Proc Nati Acad Sci USA 1992 ; 89 : 10114 - 10118 .
11 Kuilman T , Michaloglou C , Mooi WJ , Peeper DS . The essence of senescence . Genes Dev 2010 ; 24 : 2463 - 2479 .
12 Armanios M. Telomeres and age-related disease: how telomere biology informs clinical paradigms . J Clin Invest 2013 ; 123 : 996 - 1002 .
13 Newgard CB , Sharpless NE . Coming of age: molecular drivers of aging and therapeutic opportunities . J Clin Invest 2013 ; 123 : 946 - 950 .
14 van Deursen JM. The role of senescent cells in ageing . Nature 2014 ; 509 : 439 - 446 .
15 Greider CW , Blackburn EH . Identification of a specific telomere terminal transferase activity in Tetrahymena extracts . Cell 1985 ; 43 : 405 - 413 .
16 Flores I , Benetti R , Blasco MA . Telomerase regulation and stem cell behaviour . Curr Opin Cell Biol 2006 ; 18 : 254 - 260 .
17 Kim NW , Piatyszek MA , Prowse KR , Harley CB , West MD , Ho PL et al. Specific association of human telomerase activity with immortal cells and cancer . Science 1994 ; 266 : 2011 - 2015 .
18 Bryan TM , Englezou A , Dalla-Pozza L , Dunham MA , Reddel RR . Evidence for an alternative mechanism for maintaining telomere length in human tumors and tumor-derived cell lines . Nat Med 1997 ; 3 : 1271 - 1274 .
19 Cesare AJ , Reddel RR . Alternative lengthening of telomeres: models, mechanisms and implications . Nat Rev Genet 2010 ; 11 : 319 - 330 .
20 Yeager TR , Neumann AA , Englezou A , Huschtscha LI , Noble JR , Reddel RR . Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body . Cancer Res 1999 ; 59 : 4175 - 4179 .
21 Pandolfi PP, Grignani F , Alcalay M , Mencarelli A , Biondi A , LoCoco F et al. Structure and origin of the acute promyelocytic leukemia myl/RAR alpha cDNA and characterization of its retinoid-binding and transactivation properties . Oncogene 1991 ; 6 : 1285 - 1292 .
22 de The H , Lavau C , Marchio A , Chomienne C , Degos L , Dejean A . The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR . Cell 1991 ; 66 : 675 - 684 .
23 Kakizuka A , Miller Jr WH , Umesono K , Warrell Jr RP , Frankel SR , Murty VV et al. Chromosomal translocation t( 15 ; 17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor , PML. Cell 1991 ; 66 : 663 - 674 .
24 Goddard AD , Borrow J , Freemont PS , Solomon E. Characterization of a zinc finger gene disrupted by the t(15;17) in acute promyelocytic leukemia . Science 1991 ; 254 : 1371 - 1374 .
25 de The H , Le Bras M , Lallemand-Breitenbach V . The cell biology of disease: Acute promyelocytic leukemia, arsenic, and PML bodies . J Cell Biol 2012 ; 198 : 11 - 21 .
26 Pearson M , Pelicci PG . PML interaction with p53 and its role in apoptosis and replicative senescence . Oncogene 2001 ; 20 : 7250 - 7256 .
27 Carbone R , Pearson M , Minucci S , Pelicci PG . PML NBs associate with the hMre11 complex and p53 at sites of irradiation induced DNA damage . Oncogene 2002 ; 21 : 1633 - 1640 .
28 Dellaire G , Ching RW , Ahmed K , Jalali F , Tse KC , Bristow RG et al. Promyelocytic leukemia nuclear bodies behave as DNA damage sensors whose response to DNA double-strand breaks is regulated by NBS1 and the kinases ATM, Chk2, and ATR . J Cell Biol 2006 ; 175 : 55 - 66 .
29 Salomoni P , Pandolfi PP. The role of PML in tumor suppression . Cell 2002 ; 108 : 165 - 170 .
30 Mazza M , Pelicci PG . Is PML a tumor suppressor? Front Oncol 2013 ; 3 : 174 .
31 Slatter TL , Tan X , Yuen YC , Gunningham S , Ma SS , Daly E et al. The alternative lengthening of telomeres pathway may operate in non-neoplastic human cells . J Phatol 2012 ; 226 : 509 - 518 .
32 Chang FT , McGhie JD , Chan FL , Tang MC , Anderson MA , Mann JR et al. PML bodies provide an important platform for the maintenance of telomeric chromatin integrity in embryonic stem cells . Nucleic Acids Res 2013 ; 41 : 4447 - 4458 .
33 de The H , Chomienne C , Lanotte M , Degos L , Dejean A . The t( 15 ;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor alpha gene to a novel transcribed locus . Nature 1990 ; 347 : 558 - 561 .
34 Melnick A , Licht JD . Deconstructing a disease: RARalpha, its fusion partners, and their roles in the pathogenesis of acute promyelocytic leukemia . Blood 1999 ; 93 : 3167 - 3215 .
35 Gunes C , Rudolph KL . The role of telomeres in stem cells and cancer . Cell 2013 ; 152 : 390 - 393 .
36 Furia L , Pelicci PG , Faretta M. A computational platform for robotized fluorescence microscopy (I): high-content image-based cell-cycle analysis . Cytometry A 2013 ; 83 : 333 - 343 .
37 Furia L , Pelicci PG , Faretta M. A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high-content high-resolution multiparameter image-cytometry . Cytometry A 2013 ; 83 : 344 - 355 .
38 Leonetti C , Amodei S , D'Angelo C , Rizzo A , Benassi B , Antonelli A et al. Biological activity of the G-quadruplex ligand RHPS4 (3 , 11 -difluoro- 6 , 8 , 13 -trimethyl-8Hquino [4 , 3 ,2 -kl]acridinium methosulfate) is associated with telomere capping alteration . Mol Pharmacol 2004 ; 66 : 1138 - 1146 .
39 Salvati E , Leonetti C , Rizzo A , Scarsella M , Mottolese M , Galati R et al. Telomere damage induced by the G-quadruplex ligand RHPS4 has an antitumor effect . J Clin Invest 2007 ; 117 : 3236 - 3247 .
40 van Steensel B , Smogorzewska A , de Lange T. TRF2 protects human telomeres from end-to-end fusions . Cell 1998 ; 92 : 401 - 413 .
41 Fenech M , Crott JW . Micronuclei nucleoplasmic bridges and nuclear buds induced in folic acid deficient human lymphocytes-evidence for breakagefusion-bridge cycles in the cytokinesis-block micronucleus assay . Mutat Res 2002 ; 504 : 131 - 136 .
42 Ghaffari SH , Shayan-Asl N , Jamialahmadi AH , Alimoghaddam K , Ghavamzadeh A . Telomerase activity and telomere length in patients with acute promyelocytic leukemia: indicative of proliferative activity, disease progression, and overall survival . Ann Oncol 2008 ; 19 : 1927 - 1934 .
43 Minucci S , Monestiroli S , Giavara S , Ronzoni S , Marchesi F , Insinga A et al. PML-RAR induces promyelocytic leukemias with high efficiency following retroviral gene transfer into purified murine hematopoietic progenitors . Blood 2002 ; 100 : 2989 - 2995 .
44 Slatter T , Gifford-Garner J , Wiles A , Tan X , Chen YJ , MacFarlane M et al. Pilocytic astrocytomas have telomere-associated promyelocytic leukemia bodies without alternatively lengthened telomeres . Am J Pathol 2010 ; 177 : 2694 - 2700 .
45 Verdun RE , Crabbe L , Haggblom C , Karlseder J . Functional human telomeres are recognized as DNA damage in G2 of the cell cycle . Mol Cell 2005 ; 20 : 551 - 561 .
46 Berardinelli F , Antoccia A , Cherubini R , De Nadal V , Gerardi S , Cirrone GA et al. Transient activation of the ALT pathway in human primary fibroblasts exposed to high-LET radiation . Radiat Res 2010 ; 174 : 539 - 549 .
47 Armanios M , Blackburn EH . The telomere syndromes . Nat Rev Genet 2012 ; 13 : 693 - 704 .
48 Dellaire G , Bazett-Jones DP . PML nuclear bodies: dynamic sensors of DNA damage and cellular stress . Bioessays 2004 ; 9 : 963 - 977 .
49 O 'Sullivan RJ , Arnoult N , Lackner DH , Oganesian L , Haggblom C , Corpet A et al. Rapid induction of alternative lengthening of telomeres by depletion of the histone chaperone ASF1 . Nat Struct Mol Biol 2014 ; 21 : 167 - 174 .
50 Grignani F , De Matteis S , Nervi C , Tomassoni L , Gelmetti V , Cioce M et al. Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia . Nature 1998 ; 391 : 815 - 818 .
51 Lin RJ , Nagy L , Inoue S , Shao W , Miller Jr WH , Evans RM . Role of the histone deacetylase complex in acute promyelocytic leukaemia . Nature 1998 ; 391 : 811 - 814 .
52 He LZ , Guidez F , Tribioli C , Peruzzi D , Ruthardt M , Zelent A et al. Distinct interactions of PML-RARalpha and PLZF-RARalpha with co-repressors determine differential responses to RA in APL . Nat Genet 1998 ; 18 : 126 - 135 .
53 Hiorns LR , Swansbury GJ , Mehta J , Min T , Dainton MG , Treleaven J et al. Additional chromosome abnormalities confer worse prognosis in acute promyelocytic leukaemia . Br J Haematol 1997 ; 96 : 314 - 321 .
54 Pantic M , Novak A , Marisavljevic D , Djordjevic V , Elezovic I , Vidovic A et al. Additional chromosome aberrations in acute promyelocytic leukemia: characteristics and prognostic influence . Med Oncol 2000 ; 17 : 307 - 313 .
55 Ventura A , Meissner A , Dillon CP , McManus M , Sharp PA , Van Parijs L et al. Cre-loxregulated conditional RNA interference from transgenes . Proc Natl Acad Sci USA 2004 ; 101 : 10380 - 10385 .
56 Vian L , Di Carlo M , Pelosi E , Fazi F , Santoro S , Cerio AM et al. Transcriptional finetuning of microRNA-223 levels directs lineage choice of human hematopoietic progenitors . Cell Death Differ 2014 ; 21 : 290 - 301 .
57 Dimri GP , Lee X , Basile G , Acosta M , Scott G , Roskelley C et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo . Proc Nati Acad Sci USA 1995 ; 92 : 9363 - 9367 .
58 Fenech M. The in vitro micronucleus technique . Mutat Res 2000 ; 455 : 81 - 95 .
59 Gisselsson D , Bjork J , Hoglund M , Mertens F , Dal Cin P , Akerman M et al. Abnormal nuclear shape in solid tumors reflects mitotic instability . Am J Pathol 2001 ; 158 : 199 - 206 .
60 Racanicchi S , Maccherani C , Liberatore C , Billi M , Gelmetti V , Panigada M et al. Targeting fusion protein/corepressor contact restores differentiation response in leukemia cells . EMBO J 2005 ; 24 : 1232 - 1242 .
61 Grignani F , Valtieri M , Gabbianelli M , Gelmetti V , Botta R , Luchetti L et al. PML/RAR alpha fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype . Blood 2000 ; 96 : 1531 - 1537 .
62 Rufer N , Dragowska W , Thornbury G , Roosnek E , Lansdorp PM . Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry . Nat Biotechnol 1998 ; 16 : 743 - 747 .