Replicative cellular senescence is a phenomenon of
irreversible growth arrest triggered by the accumulation
of a discrete number of cell divisions. In vivo studies
have implicated cellular senescence as an important
tumor suppression mechanism in a variety of human
and mouse tissues [1,2]. Cellular senescence has also
been linked with aging and age related pathology .
Telomere shortening was the first described cause of
senescence , but many other triggers have since been
documented, including oncogene activation, a variety of
genotoxic insults, and oxidative as well as other yet
poorly understood stresses
mechanism is the presence of unrepaired or persistent
DNA double-strand breaks (DSB), which arise from
telomere dysfunction or other genotoxic insults, and
[5,6]. One central
signal through the DNA damage response (DDR)
pathway to activate the p53 tumor suppressor, leading
to the upregulation of the cyclin-dependent kinase
(CDK) inhibitor p21 and cell cycle arrest .
The second pathway of considerable importance is
governed by the pRb tumor suppressor, which is
maintained in its active state by the upregulation of the
p16 CDK inhibitor [8,9]. The DDR can signal to p16
through mechanisms such as the activation of the p38
MAPK pathway, but the regulation of p16 is not well
understood, and likely involves components that are
independent of genotoxic stress [10,11]. For example,
while the expression of telomerase elongates telomeres
and hence prevents their dysfunction and activation of
the p53-p21 pathway, immortalization of some
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How to measure RNA expression in rare senescent cells expressing
any specific protein such as p16Ink4a
Jessie C. Jeyapalan and John M. Sedivy
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA
Key words: Cellular senescence, flow cytometry, immunostaining, RNA purification, transcriptome profiling
Received: 9/15/12; Accepted: 2/23/13; Published: 2/25/13
Correspondence to: John M. Sedivy, PhD; E‐mail: firstname.lastname@example.org
Copyright: © Jeyapalan and Sedivy. This is an open‐access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Abstract: Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of
formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a
phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The
underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous
and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with
telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting
method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative
analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to
5x107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde
crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery
of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered
RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR,
hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression
profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high,
indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented
in this communication should be of interest to many investigators working in diverse model systems.
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fibroblast strains and most epithelial cell types requires
the additional silencing of p16 [12-14].
We previously documented that when normal human
diploid fibroblasts (HDF) approaching replicative
senescence were monitored at the single cell level by
immunofluorescence microscopy, p21 and p16 were
initially upregulated in different cells . While this
suggested the possibility that p21 and p16 were
upregulated in response to different triggers, fully
senescent cells expressed high levels of both p21 and
p16, and expression of hTERT in presenescent cells was
sufficient to generate immortalized clones. We report
here the unexpected finding that HDF cultures fully
immortalized with hTERT continue to generate
senescent, p16-positive cells at an appreciable
frequency, with no evidence of DDR.
These observations indicate that presenescent and
senescent cultures are heterogeneous mixtures of cells
with different characteristics and fates [15-17]. This is
certainly expected to be the case in vivo, where
senescent cells are typically found at low frequencies
within tissues [18-20], and underscores the need for
single-cell techniques to molecularly analyze these rare
pools of cells. While laser capture microdissection has
been used with some success, these methods are
compromised by the poor quality of the recovered
RNA, and in the case of senescence, widely dispersed
cells. Flow cytometry has important advantages,
including the ability to recover substantial numbers of
cells, but has mostly been used with antibodies directed
at cell surface antigens. Given that p16 is an
intracellular antigen, we have developed and report here
a preparative method
crosslinking, followed by crosslink reversal for the
recovery of RNA. We can routinely obtain >106 cells,
the recovered RNA is of adequate quality for accurate
qPCR, microarray and next generation sequencing, and
the method should be adaptable to studying many
different cellular processes in addition to senescence.
Spontaneous and DDR-independent upregulation of
p16 in hTERT-immortalized HDF
We reported previously that p21 and p16 were
upregulated in different cells as cultures of fetal lung
HDF approached replicative exhaustion . Cells
singly positive for either p21 or p16 were senescent, as
verified by staining for the senescence-associated β-
galactosidase marker  and absence of BrdU
incorporation . Ectopic expression of telomerase in
early passage cells prevented telomere dysfunction and
based on formaldehyde
activation of the p53-p21 pathway , and readily
yielded immortalized clones, some of which have been
extensively propagated . We were thus surprised to
find that cultures of telomerase-immortalized HDF
(designated LF1/TERT, Methods) contained significant
proportions (15-20%) of p16-positive cells (Figure 1).
We previously showed that in non-immortalized
cultures, p16-positive cells are continuously generated
at low levels even at early passage, that this process
increases with passage, and is independent of telomere
dysfunction and p53-p21 pathway signaling .
Apparently, this process continues unabated after
immortalization with telomerase.
Further elucidating this telomere-independent, p16-pRb
pathway regulated senescence process would be of
considerable interest. We first investigated, using
single-cell immunofluorescence analysis, the correlation
between upregulation of p16 and the DDR (Figure 2
A,B). Using 53BP1 as a sensitive readout of DSB, we
found that in exponentially growing LF1/TERT
cultures, 53BP1 foci and p16 upregulation occurred
mostly in different cells, with only a very small fraction
(2%) of double-positive cells. This suggests that p16 is
upregulated independently of DDR signaling. The low
frequency of DSB (approximately 10% 53BP1-positive
cells) is caused by oxidative stress due to atmospheric
oxygen, and cannot be eliminated even by culture under
physiological (2.5%) oxygen tension (Methods).
The regulation of p16 expression has been of
considerable interest. Using immunoblot or qPCR
analyses the levels of p16 are seen to rise gradually as
Figure 1. Expression of p16 at the single cell level
measured by immunostaining with a p16 antibody
followed by immunohistochemical detection. (A) Non‐
immortalized HDF (LF1) at early passage. (B) LF1 HDF passaged
into senescence. (C) LF1 cells immortalized with telomerase
(LF1/TERT) under conditions of exponential proliferation.
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