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Immortalization of normal human mammary epithelial
cells in two steps by direct targeting of senescence
barriers does not require gross genomic alterations
James C Garbea, Lukas Vrbabcd, Klara Sputovaa, Laura Fuchse, Petr Novakbd, Arthur R
Brothmane, Mark Jacksonf, Koei Ching, Mark A LaBargea, George Wattsb, Bernard W
Futscherbc & Martha R Stampferab
a Life Sciences Division; Lawrence Berkeley National Laboratory; Berkeley, CA USA
b Arizona Cancer Center; The University of Arizona; Tucson, AZ USA
c College of Pharmacy; Department of Pharmacology & Toxicology; The University of Arizona,
Tucson, AZ USA
d Biology Centre ASCR; v.v.i., Institute of Plant Molecular Biology; Ceske Budejovice, Czech
Republic
e Department of Pathology; The University of Arizona College of Medicine; Tucson, AZ USA
f Case Comprehensive Cancer Center; Case Western Reserve University; Cleveland, OH USA
g University of California San Francisco; San Francisco, CA USA
Accepted author version posted online: 29 Oct 2014.Published online: 14 Nov 2014.
To cite this article: James C Garbe, Lukas Vrba, Klara Sputova, Laura Fuchs, Petr Novak, Arthur R Brothman, Mark Jackson,
Koei Chin, Mark A LaBarge, George Watts, Bernard W Futscher & Martha R Stampfer (2014) Immortalization of normal human
mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations,
Cell Cycle, 13:21, 3423-3435, DOI: 10.4161/15384101.2014.954456
To link to this article: http://dx.doi.org/10.4161/15384101.2014.954456
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Immortalization of normal human mammary
epithelial cells in two steps by direct targeting
of senescence barriers does not require gross
genomic alterations
James C Garbe
1,
*, Lukas Vrba
2,3,4
, Klara Sputova
1
, Laura Fuchs
5
, Petr Novak
2,4
, Arthur R Brothman
5
, Mark Jackson
6
,
Koei Chin
7
, Mark A LaBarge
1
, George Watts
2
, Bernard W Futscher
2,3
, and Martha R Stampfer
1,2,
*
1
Life Sciences Division; Lawrence Berkeley National Laboratory; Berkeley, CA USA;
2
Arizona Cancer Center; The University of Arizona; Tucson, AZ USA;
3
College of Pharmacy;
Department of Pharmacology & Toxicology; The University of Arizona, Tucson, AZ USA;
4
Biology Centre ASCR; v.v.i., Institute of Plant Molecular Biology; Ceske Budejovice,
Czech Republic;
5
Department of Pathology; The University of Arizona College of Medicine; Tucson, AZ USA;
6
Case Comprehensive Cancer Center;
Case Western Reserve University; Cleveland, OH USA;
7
University of California San Francisco; San Francisco, CA USA
Keywords: carcinogenesis, c-Myc, genomic instability, human mammary epithelial cells, immortalization, p16INK4a,
senescence, telomerase
Abbreviations: BaP, benzo(a)pyrene; CT, cholera toxin; DDR, DNA damage response; DMR, differentially methylated regions;
HMEC, human mammary epithelial cells; OIS, oncogene-induced senescence; p, passage; p16sh, shRNA to p16
INK4A
;
PD, population doublings; RB, retinoblastoma protein; TTS, transcription start site; X, oxytocin.
Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known
about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model
systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-
clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence
barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16
INK4
;
replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction.
Thus, 2 pathologically relevant oncogenic agents are sufficient to immortally transform normal HMEC. The resultant
non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable
cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells
with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1)
telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for
telomerase reactivation and immortalization, as well as many additional “passenger”errors carried forward into
resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome
tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a
need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of
“passenger”genomic errors, should facilitate examination of telomerase regulation during human carcinoma
progression, and exploration of agents that could prevent immortalization.
Introduction
Acquisition of sufficient telomerase activity to maintain stable
telomere lengths is necessary for immortalization of most human
epithelial cells. In turn, immortalization appears essential for
development and progression of malignant human carcinomas.
1
Despite the crucial role of telomerase and immortalization in
human carcinogenesis, the mechanisms that control telomerase
expression, and the aberrations that allow telomerase reactivation
during malignant progression, remain poorly understood.
© James C Garbe, Lukas Vrba, Klara Sputova, Laura Fuchs, Petr Novak, Arthur R Brothman, Mark Jackson, Koei Chin, Mark A LaBarge, George Watts, Bernard W
Futscher, and Martha R Stampfer
*Correspondence to: Martha R Stampfer; Email: mrstampfer@lbl.gov; James Garbe; Email: JCGarbe@lbl.gov
Submitted: 08/07/2014; Accepted: 08/08/2014
http://dx.doi.org/10.4161/15384101.2014.954456
This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The
moral rights of the named author(s) have been asserted.
www.landesbioscience.com 3423Cell Cycle
Cell Cycle 13:21, 3423--3435; November 1, 2014; Published with license by Taylor & Francis Group, LLC
REPORT
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The lack of appropriate experimentally tractable model systems
of human cancer–associated telomerase reactivation and immor-
talization has contributed to this knowledge gap. Murine cells
have significant differences from human in regulation of telome-
rase, including less stringent telomerase repression.
2,3
Conse-
quently, telomerase activity is not limiting in most murine
carcinoma model systems. On the other hand, there is a paucity
of human epithelial cell immortalization models suitable for
experimental examination of telomerase reactivation during
carcinogenesis. Immortalization models employing transduc-
tion of ectopic hTERT, the catalytic subunit of telomerase,
preclude identifying the errors responsible for telomerase
reactivation during in vivo human carcinogenesis. Determin-
ing the errors responsible for driving cancer-associated
immortalization using in vivo human tissues is difficult due
to the many genomic errors and the genomic instability usu-
ally exhibited by carcinoma cells. Normal finite human epi-
thelial cells contain intact genomes; short telomeres and
widespread genomic instabilitycanfirstbeobservedinmany
pre-malignant lesions, such as DCIS in breast.
4,5
We have
postulated that genomic instability caused by the critically
shortened telomeres present in finite cells as they approach
replicative senescence may give rise to rare errors permissive
for telomerase reactivation, and underlie many of the passen-
ger errors seen in carcinomas.
1,6
Our previous studies have used pathologically relevant
agents to transform normal finite lifespan human mammary
epithelial cells (HMEC) to immortality.
6-9
However, immor-
talization was clonal with multiple genomic errors present in
immortalized lines,
1
and the alterations specifically responsi-
ble for immortalization were not fully identified. The spo-
radic nature of the immortalization events has prevented
examining the immortalization process as it occurs. We there-
fore sought to define a reproducible protocol, using agents
that might recapitulate molecular alterations occurring during
in vivo breast cancer progression, which could achieve non-
clonal transformation of normal HMEC to immortality.
Design of this protocol was based on our model of
the tumor-suppressive senescence barriers normal HMEC
need to bypass or overcome to attain immortality and malig-
nancy
6,10
(see Fig. 1A). Further, we wanted to determine
whether direct targeting of senescence barriers could generate
immortal lines lacking gross genomic errors. Cultured
HMEC can encounter at least 3 distinct tumor-suppressive
senescence barriers.
6,10,11
A first barrier, stasis, is stress-associ-
ated and mediated by the retinoblastoma protein (RB).
HMEC at stasis express elevated levels of the cyclin-depen-
dent kinase inhibitor CDKN2A/p16
INK4A
(p16), and do not
show genomic instability or critically short telomeres.
10,12,13
A second barrier, replicative senescence, is a consequence of
critically shortened telomeres from ongoing replication in the
absence of sufficient telomerase, and is associated with telo-
mere dysfunction, genomic instability, and a DNA damage
response (DDR).
5,6,13,14
When functional p53 is present, this
barrier has been called agonescence; cell populations remain
mostly viable. If p53 function is abrogated, cells enter crisis
and eventually die.
6
Overcoming the third barrier, oncogene-
induced senescence (OIS), is associated with acquiring telo-
merase activity and immortalization; thus a single additional
oncogene can confer malignancy-associated properties once a
cell is immortally transformed.
11,15
By exposing normal pre-stasis HMEC to different culture
conditions and oncogenic agents, we have generated numerous
post-stasis and immortal HMEC with distinct phenotypes.
HMEC grown in our original MM medium ceased growth at
stasis after »15-30 population doublings (PD)(Fig. 1B, upper
panel), but rare clonal outgrowths emerged after primary cul-
tures were exposed to the chemical carcinogen benzo(a)pyrene
(BaP), generating the BaP post-stasis populations (originally
termed Extended Life).
7,16
BaP post-stasis cultures examined
lacked p16 expression, due to gene mutation or promoter
silencing,
12,17,18
and grew an additional 10–40 PD before ago-
nescence. Rare immortal lines have emerged from BaP post-sta-
sis populations at the telomere dysfunction barrier. Pre-stasis
HMEC grown in serum-free MCDB170 medium showed
more limited proliferative potential, with a rapid rise in p16
expression leading to stasis by »10–20 PD; cells at stasis exhib-
ited abundant stress fibers.
12,19
MCDB170 induces rare post-
stasis cells, called post-selection, with silenced p16 as well as
many other differentially methylated regions (DMR).
12,18
Post-
selection post-stasis HMEC proliferate for an additional 30-70
PD before the population ceases growth at agonescence.
Immortal lines were produced by transducing BaP and post-
selection post-stasis HMEC with the breast cancer-associated
oncogenes c-MYC and/or ZNF217.
1,8
In those studies, trans-
duced c-MYC, a transactivator of hTERT, did not by itself
immortalize post-selection post-stasis HMEC; however, when
c-MYC was later transduced into the BaP post-stasis culture
184Aa, uniform immortalization was observed. Consequently,
we tested the hypothesis that exposure to highly stressful (i.e.,
rapid p16-inducing) culture environments such as growth in
serum-free MCDB170 produced post-stasis populations refrac-
tory to c-MYC induction of telomerase, whereas post-stasis cells
thathadnotexperiencedhighstresscouldbeimmortalizedby
c-MYC.
In the current studies, additional, independently derived
BaP post-stasis cultures also showed induction of telomerase
activity and uniform immortalization following c-MYC trans-
duction. However, these BaP-exposed p16(-) cells harbor
BaP-induced small genomic and epigenomic errors (
18,20
;
Severson et al. in prep). We therefore generated and exam-
ined the effect of c-MYC transduction on HMEC populations
made post-stasis by transduction of shRNA to p16 (p16sh)
into unstressed pre-stasis cells. In addition to trying to
achieve reproducible non-clonal immortalization, we wanted
to examine whether direct targeting of the stasis and replica-
tive senescence barriers could produce immortalized lines
without gross genomic changes. We report that transduction
of p16sh to bypass stasis, followed by transduced c-MYC to
induce hTERT, efficiently immortalized pre-stasis HMEC
populations grown in low stress-inducing media. Resultant
immortalized lines possessed a normal karyotype at early
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passages, and none to few genomic copy number changes at
higher passages. The failure of c-MYC to immortalize the
p16(-) post-selection post-stasis HMEC was not due to dif-
ferences in the hTERT gene locus DNA methylation state, or
repressive (H3K27me3) or permissive (H3K4me3) histone
modifications. These data indicate that just 2 oncogenic
agents are sufficient to immortally transform unstressed nor-
mal HMEC, and support our hypothesis that the genomic
instability commonly present in human carcinomas may not
be required per se for transformation, but is needed to gener-
ate errors that can overcome tumor suppressive barriers.
Results
Immortalization of HMEC by p16sh and c-MYC
Figure 1A illustrates our model of the senescence barriers
encountered by cultured primary HMEC and Fig. 1B shows
the derivation and nomenclature of the finite and immortal-
ized HMEC described in this study, and the agents employed
to promote bypass or overcoming of the senescence barriers.
In 10 independent experiments, c-MYC transduction of the
post-selection post-stasis HMEC produced only one instance
of clonal immortalization, generating the 184SMY1 line
Figure 1. HMEC model system.
(A) Schematic representation of cul-
tured HMEC tumor-suppressive
senescence barriers. Thick black bars
represent the proliferation barriers
of stasis and replicative senescence.
Orange bolts represent genomic
and/or epigenomic errors allowing
these barriers to be bypassed or
overcome. Red arrows indicate cru-
cial changes occurring prior to a bar-
rier. (B) Derivation of isogenic HMEC
from specimens 184, 48R, and 240L
at different stages of transformation
ranging from normal pre-stasis to
malignant. Cells were grown in
media varying in stress induction,
measured by increased p16 expres-
sion (left column), and exposed to
various oncogenic agents (red). The
distinct types of post-stasis HMEC
are shown in the middle column;
nomenclature for types is based on
agent used for immortalization (e.g.,
BaP; p16sh) or historical naming
(e.g., post-selection 19). Transduced
finite cultures are indicated by speci-
men number and batch (e.g., 184F,
184D, 184B) followed by a “-“and
the agent transduced (e.g., -p16sh);
the BaP post-stasis nomenclature is
based on original publications, and
includes specimen number and
batch (e.g., 184A, 184B, 184C) 7, 16.
New immortalized lines described in
this paper are outlined in the right
columns; nomenclature is based on
the oncogenic agents employed
(e.g., p16s for p16sh, MY for c-MYC,
TERT). Numbers in parentheses
before the barriers indicate how
many time there was clonal or non-
clonal escape from that barrier out
of how many experiments per-
formed (e.g., c-MYC-transduced pre-
stasis HMEC were cultured to stasis
4 times; in 3 experiments there was
clonal escape from stasis leading to
3 clonally immortalized lines).
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(Fig. 1B middle panel). Figure 2A shows the growth of post-
selection 184B following transduction with c-MYC or control
vector; net growth ceases at replicative senescence at passage
(p) 15 in both conditions. Telomerase activity was examined
using the TRAP assay in cells from this experiment, as well
as one using the 184S post-selection batch, which ceases net
growth at 22 p. In both cases, no significant TRAP activity
could be detected in either control or c-MYC transduced pop-
ulations, consistent with the failure to immortalize. Similar
results were seen using post-selection post-stasis HMEC from
another specimen, 48RS (Fig. S1A).
In contrast, c-MYC transduction into the BaP post-stasis cul-
ture, 184Aa, produced continuous cell growth with increasing
TRAP activity (Fig. 2B). Similar results were seen in 5
independent experiments, generating the non-clonally immortal-
ized lines 184AaMY1–5 (Fig. 1B upper panel). While both these
post-
stasis types lack p16 expression, this was due to mutation in
184Aa and promoter silencing in the post-selection HMEC.
12,17
We therefore tested the effect of c-MYC transduction in 2 addi-
tional independent BaP post-stasis cultures that exhibited p16
promoter silencing, 184Be and 184Ce.
12,18
Both populations
showed continuous growth and increasing TRAP activity follow-
ing c-MYC transduction, generating the non-clonally immortal-
ized lines 184BeMY and 184CeMY (Figs. 1B upper panel, and
2B). These data indicate that these 2 different types of p16(-)
post-stasis HMEC, BaP and post-selection, differ significantly in
response to c-MYC transduction.
Figure 2. Effect of c-MYC on post-stasis HMEC growth and TRAP activity. (A) Post-stasis post-selection 184B HMEC grown in MCDB170 were transduced
with a c-MYC containing retrovirus (LXSN, red) or empty vector control at 7p (blue). Cultures ceased net growth at agonescence (15p). Post-selection
184S HMEC were transduced with c-MYC or control at 15p; net growth ceased at 22p (not shown). No significantly increased TRAP activity was seen fol-
lowing c-MYC transduction in either experiment. (B) BaP post-stasis 184Aa, 184Be, and 184Ce HMEC grown in MCDB170 were transduced with a c-MYC
containing retrovirus (LXSN/ BH2), red) or empty vector (blue) at the indicated passages. Control cells ceased net growth at agonescence while c-MYC-
transduced populations maintained proliferation indefinitely, associated with increased TRAP activity. The continuous exponential growth following
c-MYC transduction reflects the visually observed non-clonal immortalization; growth was maintained throughout the dish with no areas of clonal
growth. Proliferating control cultures of 184Ce expressed low TRAP activity. (C) Schematic representation of protocol to directly target senescence bar-
riers to achieve non-clonal immortalization. (D) Pre-stasis 184D and 240L HMEC grown in M87ACCTCX were transduced at 3p with a p16sh-expressing
retrovirus (MSCV, blue) or empty vector (black). At 4p cultures §p16sh were transduced with c-MYC (BH2)(red Cp16sh; purple -p16sh). c-MYC-transduced
p16sh post-stasis HMEC maintained active growth indefinitely, associated with increased TRAP activity. The continuous exponential growth following c-
myc transduction of the 4p p16sh-post-stasis populations reflects the observed non-clonal immortalization. Cells transduced with p16sh alone bypassed
stasis and ceased net growth at agonescence, with rare clonal immortalization at agonescence. Cells transduced with c-MYC alone ceased growth at sta-
sis, with rare clonal escape from stasis leading to immortalized lines. Control cultures transduced with empty vectors ceased growth at stasis. In some
TRAP assays, heat-treated controls (C) were run next to unheated (-) samples. Positive TRAP control samples are indicted by “C”(E).
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We then examined the effect of transduced c-MYC on HMEC
made post-stasis by direct knockdown of p16 using p16sh
(Figs. 1B lower panel, 2C, 2D). The non-clonal p16sh post-stasis
populations would not harbor the BaP-induced errors present in
the clonal BaP post-stasis cultures, and do not contain the exten-
sive DMR present in the post-selection post-stasis HMEC.
18
These studies used pre-stasis HMEC grown in media formula-
tions (M87A or M85) that delay the onset of p16 expression and
support up to »60 PD.
10
Early passage pre-stasis HMEC from
specimen 240 L and 2 batches from specimen 184 were trans-
duced with p16sh-containing or control retrovirus, followed by
c-MYC or control transduction at the next passage (Figs. 2C and D;
Fig. S1B). At these early passages, <10% of the population
expressed p16 protein.
10
Control cultures ceased growth at stasis,
and p16sh-transduced cultures ceased growth at replicative senes-
cence, with rare exceptions. p16sh post-stasis cultures that
received c-MYC showed uniform continuous growth and TRAP
activity, generating the non-clonal immortal lines 184Dp16sMY,
184Fp16sMY and 240Lp16sMY (Fig. 1B lower panel). These
studies indicate that the ability of c-MYC to induce rapid uniform
immortalization in p16(-) post-stasis HMEC is not dependent
upon pre-existing genomic errors.
Almost all pre-stasis HMEC receiving c-MYC alone ceased
growth at stasis; however clonal outgrowths of rare cells that
escaped stasis by unknown means produced clonal immortal lines
(184DMY3, 184FMY2, 240LMY; Fig. 1B lower panel) with
increased TRAP activity following the passages where most cells
stopped at stasis (Fig. 2D). For example, pre-stasis 184D-myc
initially stopped growth by 8–10 p. However, a culture reiniti-
ated from 5 p frozen stocks exhibited 1-2 clonal outgrowths per
dish at 9 p, against a background of senescing cells; these colonies
maintained growth, generating the 184DMY3 line. We presume
that once this population became post-stasis, the transduced c-
MYC could immortalize it similar to the effect of c-MYC on the
BaP and p16sh post-stasis populations. Although we have never
observed spontaneous immortalization at the telomere dysfunc-
tion barrier in unperturbed post-selection post-stasis HMEC
(cells that had experienced high pre-stasis culture stress), rare
clonal outgrowths in a background of senescent cells were seen at
this barrier in some p16sh post-stasis cultures (cells that had
bypassed stasis prior to p16 elevation). These colonies main-
tained growth, generating the clonal immortal lines 184Fp16s
and 240Lp16s (Fig. 1B lower panel). The immortalization-
producing error in 184Fp16s must have occurred after 9 p, since
re-initiation of frozen 9 p 184F-p16sh stock did not yield an
immortal line (Fig. S1B). We hypothesize that the difference in
spontaneous immortalization in the post-selection vs p16sh post-
stasis HMEC, during the period of genomic instability, is related
to the need for multiple errors for telomerase reactivation in the
post-selection cells compared to the ability of just one error, such
as transduced c-MYC, to immortalize the p16sh post-stasis
HMEC. Altogether, these data suggest that a prior exposure to
high culture stress may invoke alterations preventing c-MYC
induction of hTERT in post-stasis populations.
Exposure of pre-stasis HMEC to high culture stress also influ-
enced the ability of hTERT to produce efficient immortalization.
Previous studies indicated that hTERT could not immortalize
pre-stasis HMEC grown in high stress media such as
MCDB170/MEGM,
21
and yielded only one p16(-) clonal line
(184FTERT) when transduced into 3 p HMEC grown in mod-
erate stress MM medium.
22
In contrast, hTERT transduced into
3 p HMEC grown in low stress M87A efficiently immortalized
the population, with no growth slowdown at the stasis barrier
(184DTERT, Figs. 1B lower panel; Fig. S1C). As expected,
given hTERT’s ability to immortalize post-selection post-stasis
HMEC,
21,22
transduction of hTERT into the p16sh post-stasis
cells 240L-p16sh also produced efficient immortalization
(240Lp16sTERT; Fig. S1D), with continuous growth similar to
that seen following c-MYC transduction of 240L-p16sh
(Fig. 1D).
We previously reported that proliferative pre-stasis HMEC
grown in MM exhibit low levels of TRAP activity at 4 p.
23
Pre-stasis HMEC from specimen 184 grown in M85/M87A also
show low TRAP activity at early passages, but activity is not
detectable when the cells approach stasis (Fig. 2D;Fig. S1E).
Transduction with p16sh appeared to slightly increase TRAP
activity compared to controls, with levels reduced by agonescence
(Fig. 2D;Fig. S1B). The low TRAP activity in unperturbed
240L was increased by p16sh transduction, while the immortal-
ized clonal line 240Lp16s emerged from replicative senescence
with robust TRAP activity (Fig. 2D). c-MYC alone transiently
increased TRAP activity in proliferative pre-stasis populations,
with further increased activity seen in immortalized lines
(184DMY3, 240LMY).
The effect of transduced p16sh in reducing p16 protein
expression is shown by Western analysis in Fig. S2A for the finite
and immortal cultures, and by immunochemistry for post-stasis
184D-p16sh and immortal 184DMY3 (Fig. S2C). Higher pas-
sage pre-stasis 184D and 240LB express significant p16; trans-
duction of p16sh reduced most but not all p16 expression in
both the p16sh post-stasis HMEC, and the immortal lines
derived from them. p16 protein was seen in 2 of the MYC-alone
transduced clonal immortal lines; the high expression in
240LMY suggests that an error elsewhere in the RB pathway
enabled the cell giving rise to that line to overcome stasis, while
the mixed p16 expression and 2 distinct morphologies present in
184DMY3 suggests it consists of 2 distinct clones, one of which
retains p16 expression. HMEC lines containing transduced c-
MYC showed variably increased MYC expression levels compared
to normal pre-stasis HMEC (Fig. S2B). Of note, MYC levels in
c-MYC-transduced normal pre-stasis HMEC were not signifi-
cantly elevated, but were increased in abnormal post-selection
184B-myc, which did not immortalize. As has been suggested for
cancer cells,
24
dysregulation of MYC, as well as increased expres-
sion, may play a role in carcinogenesis, and in some circumstan-
ces, low level deregulated c-MYC may be more efficient at
oncogenesis than overexpressed c-MYC.
25,26
Altogether, these data indicate that post-selection post-stasis
HMEC are refractory to c-MYC-induced telomerase induction
and immortalization, while other p16(-) post-stasis types are
readily immortalized by c-MYC, and are more vulnerable to
immortalization from errors generated during telomere
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dysfunction. Most significantly, the data show that normal
HMEC can be efficiently immortalized with endogenous telome-
rase reactivation by just 2 pathologically relevant oncogenic
agents, p16sh and c-MYC.
Genomic profiles of immortally transformed HMEC lines
The studies described above have produced at least 12 new
non-hTERT immortalized HMEC lines (Figure 1B, outlined in
right columns). To examine the role of genomic errors in their
generation, lines were assayed for karyotype and/or genome copy
number by (a)array CGH. Karyotype at early passages following
immortalization was determined for the non-clonally immortal-
ized lines 184AaMY1 (17 p), 184BeMY (11 p), 184CeMY
(12 p), 184Fp16sMY (16 p), 184Dp16sMY (16 p), and
240Lp16sMY (16 p)(Table 1,Figure 3A). aCGH was performed
on these, and additional clonal lines, at higher passages (Fig. 3B;
Fig. S3A). Clonal lines exhibited numerous copy number
changes, consistent with a need to generate genomic errors to
overcome stasis in the MYC-alone lines, and replicative senes-
cence in the p16sh-alone lines. Some genomic errors, e.g., 1 q
and 20 q amplification, are commonly seen in breast cancer.
27
The karyotype of all 3 p16sh-MYC-derived lines, and one of
the 3 BaP-MYC lines (184CeMY), showed no abnormalities at
early passage. At higher passages, 1-2 copy-number changes were
observed in 184Dp16sMY (30p) and 240Lp16sMY (25 p). Both
contained small deletions in the p16 locus on 9p21 that would
not be obvious by karyology (Figure S3B), and a subpopulation
of 240Lp16sMY showed a 1q amplification. MYC-induced
genomic instability
24
and/or retroviral-induced insertional muta-
genesis
9
could have produced a 1q error conferring preferential
growth to a 240Lp16sMY cell. The origin of the 9p deletion in
lines that had received both p16sh and c-MYC is currently
unknown. The gross genomic errors in 184AaMY1 and
184BeMY are likely due to these post-stasis cultures being trans-
duced by c-MYC close to the point of agonescence (Fig. 2B),
when the populations would already contain cells with genomic
errors due to telomere dysfunction,
13
as these errors are not pres-
ent in earlier passages of 184Aa or 184Be.
20
In summary, by targeting the stasis and telomere dysfunc-
tion barriers with p16sh and c-MYC respectively, we could
Table 1. Karyology of non-clonally immortalized lines at early passage
Cell line, passage
Karyotype and Aberrations
[# cells examined]
184Fp16sMY, 16p 46,XX normal diploid [10]
184Dp16sMY, 16p 46,XX normal diploid [12]
240Lp16sMY, 16p 46,XX normal diploid [11]
184AaMY1, 17p 46,XX normal diploid [14]
47,XX,Ci(1)(q10) [6]
184BeMY, 11p 45,X,add(X)(q28),-4,der(5)t(5;15)(q11.2;q11.2),
der(12)t(5;12)(q11.2;q24.3),-15,Cmar [cp16]
184CeMY, 12p 46,XX normal diploid [10]
Figure 3. Genomic analysis of newly developed lines from 184D and 240L. (A) Representative karyograms of newly derived immortalized lines at early
passages; non-clonal 184Dp16sMY is show as an example of a normal karyotype: 46,XX. Individual abnormalities in 184AaMY1: 47,XX,Ci(1)(q10), and
184BeMY: 46,X,add(X)(q28),-4,der(5)t(5;15)(q11.2;q11.2),der(12)t(5;12)(q11.2;q24.3),¡15,C2mar, are shown by arrows. (B) aCGH analysis of lines at the
indicated passage level using an Agilent human genome microarray with 44,000 probes per array.
3428 Volume 13 Issue 21Cell Cycle
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transform normal finite lifespan pre-stasis HMEC to immor-
tality in the absence of gross genomic changes. These data
are consistent with our hypothesis that cancer-associated
genomic changes are needed to overcome tumor suppressive
barriers and gain malignant properties, but gross genomic
changes per se are not inherently necessary for cancer-associ-
ated immortalization.
Epigenetic state of the
hTERT promoter in the
cultured HMEC
The above data showed that
c-MYC can induce telomerase
activity and immortalization in
p16(-) BaP and p16sh post-sta-
sis, but not post-selection post-
stasis HMEC. One possible basis
for this difference could be dis-
tinct hTERT chromatin states
that affect accessibility of c-
MYC, an hTERT transactivator.
To evaluate this possibility and
to gain better understand of HMEC telomerase regulation, the
hTERT gene locus was examined for DNA methylation and
permissive (H3K4me3) or repressive (H3K27me3) histone
modifications using 5-methylcytosine and chromatin immuno-
precipitations (ChIP) coupled to custom tiling microarray
hybridization. Post-stasis BaP, post-selection, and p16sh cultures
were examined along with other HMEC with different levels of
Figure 4. Epigenetic analysis of
the hTERT gene promoter.
(A) Shows the tiling microarray
data from the TERT promoter
region displayed as a heatmap,
with blue indicating high enrich-
ment of particular epigenetic
mark and yellow indicating no
enrichment. This region includes
the areas bound by H3K4me3 and
transcription factors including c-
MYC according to online data
(http://genome.ucsc.edu). Upper
and middle sections of the heat-
map show permissive H3K4me3
and repressive H3K27me3 histone
marks, respectively; the bottom
section shows DNA methylation
data. Two regions (UP and TSS)
indicated by brown bars at the
bottom were analyzed for DNA
methylationathigherresolution
by MassARRAY analysis. The small
black rectangles above the heat-
map indicate positions of individ-
ual microarray probes. The vertical
bars below the heatmap indicate
positions of individual CpG dinu-
cleotides. The CpG island is
marked in green. The 5’part of
the hTERT gene is in blue. The
genomic coordinates at the top
are hg18. (B and C) MassARRAY
analysis data for regions UP and
TSS indicated in (A). The data are
presented as a heatmap with
methylated CpG units in blue and
unmethylated CpG units in yellow.
www.landesbioscience.com 3429Cell Cycle
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TRAP activity, ranging from normal pre-stasis 184D (low activ-
ity, Fig. 2D;Fig. S1C), isogenic 184 mammary fibroblasts (no
activity, not shown), immortal 184A1 (moderate activity;
Fig. S1C), and several breast tumor lines. The DNA methylation
microarray results for the »6kb region that brackets the hTERT
transcriptional start site are shown in Figure 4A, lower panel.
The TERT locus was extensively methylated in all samples ana-
lyzed, with no differences detected or correlated to the level of
basal or MYC-inducible TRAP activity. To increase resolution
and sensitivity of the DNA methylation analysis, 2 regions were
analyzed in greater detail using MassARRAY (Figs. 4B, C). One
region that extended from 400 bp upstream to 200 bp down-
stream of the transcription start site (TSS) was unmethylated in
the pre-stasis, post-stasis, and in vitro immortalized HMEC
assayed, but partially methylated in some breast cancer cell lines.
A second region located 850 to 1400 bp upstream of the TSS
was extensively DNA methylated in all HMEC cultures, with
lower levels in 2 of the 4 cancer cell lines. Therefore, there was
no obvious correlation between DNA methylation state and
TRAP activity among the cell types analyzed.
The unmethylated region immediately surrounding the
TSS of the hTERT gene suggests a state permissive to tran-
scription, so the absence of TRAP activity in some of these
cultures might be due to other epigenetic marks. Using ChIP
linked microarray, we analyzed the HMEC for 2 histone
modifications at the hTERT gene region - H3K27me3, a pol-
ycomb-mediated repressive modification,
28
and H3K4me3, a
permissive modification present on all active and even some
inactive promoters.
29
Figure 4A (middle panel) shows that all
the cultures have repressive H3K27me3 near the hTERT pro-
moter, with no detectable correlation to TRAP activity. Sur-
prisingly, the permissive H3K4me3 mark was not detected in
the hTERT promoter region in any of the analyzed samples
(Fig. 4A, top panel), including the in vitro immortalized and
cancer lines, known to possess sufficient telomerase activity to
maintain stable telomeres. The genomic region displayed in
Figure 4 includes the area occupied by H3K4me3 in TERT-
expressing human embryonic stem cells according to the
online data (http://neomorph.salk.edu/human_methylome/)
and we detected the permissive H3K4me3 at the GAPDH
promoter (Fig. S4) and other active genes covered by the
microarray.
Overall, the data show that the epigenetic states of the
hTERT locus in the analyzed HMEC samples, with respect
to DNA methylation, H3K4me3, and H3K27me3, are indis-
tinguishable from one another and therefore do not appear
to play a role in the differential response of post-stasis types
to c-MYC transduction.
Characterization of immortally transformed HMEC lines
The newly developed lines were characterized for lineage
markers by FACS and immunofluorescence, and for AIG. Most
of the lines did not display the malignancy-associated property of
AIG (Fig. 1B); the one exception, 184FMY2, has other proper-
ties associated with more aggressive breast cancer cells (see
below).
FACS analyses using the cell surface markers CD227 (Muc-1)
and CD10 (Calla) can distinguish CD227C/CD10- luminal
from CD227-/CD10Cmyoepithelial lineages in normal pre-
stasis HMEC (Fig. S5A). While normal pre-stasis 240L HMEC
exhibit distinct luminal and myoepithelial populations, all the
cell lines exhibited a predominantly basal/myoepithelial-like phe-
notype, showing expression of CD10, along with minor to signif-
icant expression of CD227 (Figs. S5B and C). All lines examined
showed expression of the basal-associated intermediate filament
protein keratin 14 and little or no expression of luminal-
associated keratin 19 (Fig. S6).
Antibodies recognizing the surface antigens CD44 and CD24
have been widely used in the putative identification of carcinoma
cells with tumor-initiating properties.
30,31
Normal pre-stasis
240 L HMEC are predominantly CD44
hi
/CD24
hi
, with a small
CD44
lo
/CD24
hi
subpopulation. Almost all the lines exhibited
co-expression of CD44 and CD24 at varying levels in all cells,
but some had separate subpopulations with increased CD44 and
decreased CD24, e.g., 184CeMY and 240Lp16sMY. Interest-
ingly, the 184FMY2 cell line with AIG exhibited a very promi-
nent CD44
hi
/CD24
low
population and evidence of EMT (Vrba,
Garbe, Stampfer, Futscher unpublished), but no tumor-forming
ability when injected subcutaneously in immune-compromised
mice (data not shown). In general, these immortalized lines
derived from young reduction mammoplasty specimens dis-
played basal-like phenotypes compared to the heterogeneous
composition of their normal pre-stasis populations.
Discussion
Immortalization of normal cultured HMEC using agents
associated with breast cancer pathogenesis in vivo has been diffi-
cult to achieve. We report here that reproducible non-clonal
immortalization was attained by targeting 2 tumor suppressive
senescence barriers, stasis and replicative senescence, and that
resultant immortalized lines exhibit normal karyotypes at early
passage. Our prior studies have indicated that stasis is enforced in
cultured HMEC by elevated p16 levels maintaining RB in an
active state. Unlike some other human epithelial cell types, e.g.,
keratinocytes,
32
p53-dependent p21 is not upregulated in cul-
tured HMEC at stasis;
10,12,13
consequently, transduction of
shRNA to p16 can be sufficient to bypass stasis. Overcoming the
telomere dysfunction barrier at replicative senescence requires, at
minimum, sufficient levels of telomerase activity to maintain sta-
ble telomere lengths. Transduction of c-MYC could induce telo-
merase activity and immortalization in some, but not all types of
p16(-) post-stasis HMEC. These results demonstrate that bypass-
ing these 2 barriers is sufficient to transform normal finite
HMEC to immortality; genomic instability and gross genomic
errors are not required. The data also validate our model of the
functionally and molecularly distinct tumor suppressive senes-
cence barriers encountered by cultured HMEC: stasis, a stress-
associated arrest independent of telomere length and extent of
replication, and replicative senescence due to ongoing replication
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in the absence of sufficient telomerase producing critically short
telomeres and telomere dysfunction.
6,10
Expression of sufficient telomerase activity is crucial for
human carcinoma progression. Almost all human breast cancer
cell lines and tissues have detectable telomerase;
33,34
the ALT
method for telomere maintenance is very rare.
35
The presence of
short telomeres and genomic instability in most DCIS, as well as
in pre-malignant lesions from other human organ systems, indi-
cates that these lesions did not develop from cells expressing suffi-
cient telomerase for telomere maintenance.
4,5,36,37
While
malignancy requires immortality to support ongoing tumor cell
proliferation, telomerase can also provide significant additional
malignancy-promoting properties.
38
Telomerase reactivation has
been associated with gaining resistance to OIS,
11,15,39
and expres-
sion of hTERT can confer resistance to TGF-b growth inhibi-
tion
22
and affect other signaling pathways.
38,40
Given the
importance of telomerase and immortalization for human carci-
nogenesis, it is surprising that so little is known about the regula-
tion of hTERT as normal cells transform to cancer. The lack of
appropriate experimentally tractable model systems has contrib-
uted to this knowledge gap. Unlike humans, small short-lived
animals such as mice do not exert stringent repression of telome-
rase activity in adult cells, which can spontaneously immortalize
in culture.
2,3
Comparison of the human and mouse TERT gene
shows significant differences in regulatory regions.
41
The impor-
tance of telomerase in murine carcinogenesis has been demon-
strated using animals engineered to lack telomerase activity,
42
however such models do not address the mechanisms that allow
endogenous hTERT to become reactivated during human carci-
nogenesis. There has also been a lack of human epithelial cell sys-
tems that model immortalization as it might occur during in vivo
tumorigenesis. The use of ectopic hTERT to achieve immortali-
zation precludes study of the factors that regulate endogenous
hTERT in vivo, while viral oncogenes such as HPVE6E7 or
SV40T are not etiologic agents for most human carcinomas,
including breast, and have many characterized and uncharacter-
ized effects.
We have employed reduction mammoplasty-derived primary
HMEC grown under different culture conditions and exposed to
a number of oncogenic agents, to generate cell types that may
represent the different stages and heterogeneity of in vivo malig-
nant progression.
6-9,11
Prior studies revealed divergence in trans-
formation pathways at the earliest stage, becoming post-stasis.
Post-selection post-stasis HMEC exhibited »200 DMR, most of
which are also found in breast cancer cells, compared to »10 in
BaP and »5 in p16sh post-stasis HMEC.
18
Of note, it has been
suggested that post-selection post-stasis HMEC (also referred to
as vHMEC,
43
and sold commercially as “normal” primary
HMEC (Lonza CC-2551; Life Technologies A10565)) may be
on a pathway to metaplastic cancer.
44
Here we show an
additional difference among post-stasis types: the inability of
post-stasis post-selection HMEC to become immortalized by
transduced c-MYC. While the molecular processes underlying
this difference remain unknown, we note an association with
prior exposure to culture stress. Post-selection HMEC overcame
stasis following growth in medium that rapidly induces p16,
whereas p16sh post-stasis HMEC bypassed stasis prior to p16
induction. The distinct properties of the post-selection HMEC
may result from their prior experience of p16-inducing stresses.
Current studies are addressing the hypothesis that mechanical
stressors may influence telomerase expression. Functionally, our
results suggest that neither post-selection HMEC, nor pre-stasis
HMEC cultured in MCDB170-type media, would be suitable
substrates for the immortalization protocol presented here.
The molecular phenotype of cancer cells likely varies depend-
ing upon initial target cell as well as the specific errors that pro-
mote transformation. Progenitor cell types have been suggested
to be the initial target in some situations.
45-47
Our M87A/85
media support proliferation of pre-stasis HMEC with progenitor
lineage markers, and allow robust proliferation prior to p16 upre-
gulation.
10,48
Such lower stress/p16-inducing conditions may be
reflective of early stage carcinogenesis in vivo, if unstressed pro-
genitor cells are initial targets.
Our results support the hypothesis that genomic errors are
needed to overcome tumor suppressive barriers, but instability
and aneuploidy per se may not be required for transformation.
6,10
While all our clonally derived lines exhibit multiple genomic alter-
ations,
1,8,9
non-clonal lines without gross genomic errors could be
generated by directly targeting the 2 main barriers to immortality,
stasis and replicative senescence. Most human carcinomas contain
many genomic changes, however, only a small number of these
are estimated to play a driving role in carcinogenesis.
49
Several
hypotheses have addressed the causes of genomic instability and
aneuploidy in carcinomas, including mutator phenotype,
50
DNA
damage,
51
and altered genomic copy number models.
52,53
We,
and others, have proposed that the inherent genomic instability
during telomere dysfunction at replicative senescence may be
responsible for initiating most of the genomic errors seen in pri-
mary breast cancers.
4,6,10,54,55
This instability will render most
cells non-proliferative or dead, but rare cells that generate errors
allowing telomerase reactivation may immortalize, carrying with
them all the other errors accumulated to that point. Conse-
quently, genomic instability in pre-malignant cells may be the
source of many of the “passenger” mutations present in carcino-
mas, as well as of “driver” mutations that influence prognosis. If
bridge-fusion-breakage cycles have begun, immortalized cells will
maintain some ongoing instability.
9
This hypothesis is consistent
with DCIS cells possessing short telomeres, genomic instability,
and many breast cancer-associated properties, including specific
genomic errors and aggressiveness,
56-59
as well as detection of telo-
merase activity in some DCIS tissues. Further, our results suggest
that once a cell acquires the errors that allow stasis bypass, and
then maintains proliferation to telomere dysfunction, no external
agents may be needed to support rare progression to immortality.
Although gross genomic changes were not required for immortali-
zation of post-stasis HMEC by transduced c-MYC,epigenetic
changes might be needed: changes have been observed associated
with immortalization, even in non-clonally immortalized lines
with no gross karyotypic abnormalities (
18
and unpublished). Our
genomically normal non-clonal immortalized lines lack malig-
nancy-associated properties; however, we and others have seen
that these OIS-resistant populations can be readily further
www.landesbioscience.com 3431Cell Cycle
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transformed to AIG and/or tumorigenicity by transduction of
individual oncogenes.
1,11,60
Genomic analysis of non-clonal lines
malignantly transformed at early passage will be needed to deter-
mine whether a malignant phenotype can be achieved without
gross genomic errors.
Our DNA methylation and histone modification analysis of
the TERT locus provides an overview of the hTERT epigenetic
state in normal to malignant cells, with varying expression of tel-
omerase activity, from one organ system. We did not find any
changes in DNA methylation or histone modification state that
could explain the distinct responses to transduced c-MYC by
post-selection post-stasis HMEC compared to the BaP and
p16sh post-stasis types. Overall, we did not find a correlation
between DNA methylation or histone modification and TRAP
activity in all the HMEC examined. Specifically, the CpG-rich
region that immediately surrounds the TERT TSS is DNA
unmethylated in pre-stasis, post-stasis, and TRAP(C) immortal
HMEC cultures. These results using isogenic HMEC indicate
that the lack of DNA methylation in this region may be permis-
sive for, but is not by itself indicative of telomerase activity.
61
This DNA methylation state is similar to what is seen in TERT-
expressing human embryonic stem cells (hESC) or induced
pluripotent stem cells (http://neomorph.salk.edu/human_
methylome/). Outside of the TERT TSS region, the rest of the
TERT promoter is densely DNA methylated in most of the
examined HMEC, consistent with previous reports for human
cancer cells,
61,62
as is the large CpG island that extends from the
promoter to approximately 5 kb into the gene itself, similar to
hESC and iPSC (http://neomorph.salk.edu/human_methylome/
). Our histone modification analysis did not detect the
H3K4me3 mark at the TERT promoter/TSS in HMEC with
and without telomerase activity. The polycomb-specific
H3K27me3 mark was detected both upstream and downstream
of the TSS region, but similar to DNA methylation, the
H3K27me3 levels decreased near the TSS. These results are in
contrast to hESC cells, where the TERT promoter exists in a
bivalent state, occupied by both H3K4me3 and H3K27me3
(http://neomorph.salk.edu/human_methylome/). Altogether,
these analyses highlight some unusual qualities of the hTERT
locus, in addition to the absence of any obvious epigenetic regula-
tion correlated with TRAP activity. The absence of permissive
H3K4me3 mark and the presence of 2 distinct repressive epige-
netic marks at the HMEC TERT promoter suggests it exists in a
repressed or inactive chromatin state, regardless of TRAP activity
or finite vs immortal status. This type of redundant chromatin
repression may reflect human cells general need, as part of tumor
suppression, to limit TERT induction to prevent sustained aber-
rant overexpression and cell immortalization. Further support of
this possibility is the presence of very high DNA methylation lev-
els in the unusually large CpG island at the 5’end of the hTERT
gene, a structure usually associated with transcriptional repression
and heterochromatic state. Additionally, since TERT expression
is usually very low and dynamic, being predominant during
S-phase, at a given moment promoters permissive for transcrip-
tion may be present only in a small proportion of the cells, mak-
ing it difficult to detect active chromatin.
The process of telomerase reactivation during human carcino-
genesis may present a valuable target for clinical intervention.
While breast cancers are known to be heterogeneous, both among
and within a given tumor, the requirement for immortalization is
common to almost all human carcinomas. Further, unlike the
signaling pathways involved in cell growth and survival, there are
no commonly used alternative pathways to telomerase reactiva-
tion during HMEC immortalization, thus decreasing the possi-
bility for emergence of therapeutic resistance. However,
development of potential therapeutics has been limited by the
lack of information on the mechanisms underlying human epi-
thelial cell immortalization, and by the absence of a significant
immortalization barrier in murine carcinogenesis, precluding
usage of murine models for testing pharmacologic interventions
in immortalization. The reproducible immortalization of
HMEC in the absence of “passenger” errors that is achievable
with our system can facilitate further examination of the mecha-
nisms involved in hTERT regulation during carcinogenesis. Bet-
ter understanding of hTERT regulation may offer new clinical
opportunities that involve not just targeting telomerase activity
but the reactivation process itself.
Material and Methods
Cell culture
Finite lifespan HMEC from specimens 184, 240L, and 48R
were obtained from reduction mammoplasty tissue of women
aged 21, 19, and 16 respectively. Pre-stasis 184 (batch D), 240 L
(batch B), 48R (batch T) HMEC were grown in M87A supple-
mented with 0.5 ng/ml cholera toxin (CT), and 0.1 nM oxyto-
cin (X) (Bachem); pre-stasis 184 (batch F) were grown in
M85CCT, as described.
10
Post-selection post-stasis HMEC 184
(batch B, agonescence at »passage (p) 15; batch S, agonescence
at »22 p), and 48R batch S, agonescence at »22 p, as well as
BaP post-stasis 184Aa, 184Be, and 184Ce HMEC (agonescence
at »16 p, 10 p, 15 p respectively) were grown in serum-free
MCDB170 medium (commercially available versions MEGM,
Lonza, or M171, Life Technologies) plus supplements.
19
Total
PD level was calculated as described.
10
Anchorage-independent
growth (AIG) was assayed as described
9
using 1.5% methylcellu-
lose solution made up in M87ACCTCX. Details on the deriva-
tion and culture of these HMEC can be found at http://hmec.
lbl.gov. Research was conducted under LBNL Human Subjects
Committee IRB protocols 259H001 and 108H004.
Retroviral transduction
The p16 shRNA vector (MSCV) was obtained from Greg
Hannon Narita,
63
Four different c-Myc vectors were used:
LXSN for 184B, 184S, 184Aa, 184F; pBabe–hygro (BH2) for
184Be, 184Ce, 184D, 240LB; LNCX2-MYC-ires-GFP for
48RS;
60
Myc:ER for 184S, 184B.
64
The hTERT vector pBabe-
hygro-TERT was obtained from Bob Weinberg.
65
The p16-con-
taining construct was pLenti-p16-neo vector, plasmid 22260,
Addgene.Retroviral stocks were generated, supernatants collected
3432 Volume 13 Issue 21Cell Cycle
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in MCDB170 medium containing 0.1% bovine serum albumin
or M87A medium, and infections performed as described.
9
TRAP assays
Telomerase activity assays employed the TRAPeze Telomerase
detection kit (Millipore) using 0.2 mg of protein extract per reac-
tion. Reaction products were separated on a 10% polyacrylamide
gel and visualized using a Storm 860 imaging system (Molecular
Dynamics).
DNA isolation
Genomic DNA was extracted using the DNeasy Blood and
Tissue Kit (Qiagen) according to manufacturer protocol and
quantified spectrophotometrically.
Comparative genomic hybridizations (CGH) and karyology
CGH was performed at the Genomics Shared Service of the
Arizona Cancer Center using the Agilent human genome CGH
microarray with 44,000 probes per array, and analyzed using
Bioconductor in an R environment.
66
Low passage isogenic pre-
stasis HMEC were used as a reference. CGH for the 184F lines
was performed as described.
5
Karyology was performed as
described.
67
Epigenetic analysis of the hTERT gene
Methyl cytosine DNA immunoprecipitation (MeDIP), chro-
matin immunoprecipitation (ChIP), sample labeling and micro-
array hybridization were performed as described.
68
Microarray
data were analyzed in R
66
as described
68
(GEO Accession number
GSE48504). DNA methylation analysis by MassARRAY was
performed as described.
18
Primer sequences are listed in
Table S1; oligonucleotides were obtained from Integrated DNA
Technologies.
Western and ELISA analysis
Protein lysates for p16 were collected and processed as
described
23
and 50 mg samples were resolved on a 4–12% Novex
Bis/Tris gel (Invitrogen). Protein lysates for c-MYC were pre-
pared using cell extraction buffer (Invitrogen cat# FNN0011)
with protease inhibitors (Sigma cat# P2714). For detection of c-
MYC by western blot, 25 mg of extracts were separated on a 4–
12% Criterion TGX gel (Biorad). Separated proteins were trans-
ferred to Immobilon PVDF membrane (Millipore) and blocked
in PBS 0.05% Tween20 with 1% nonfat milk for 1 hour. Bind-
ing of mAb Y69 to c-MYC (Abcam) and mAb G175–405 to p16
(BD Biosciences) was detected by chemiluminescence using the
VersaDoc MP imaging system and quantified using Quanity-
One software (Biorad). The total c-MYC ELISA assay
(Invitrogen cat# KH02041) was performed following man-
ufacturer’s directions.
Immunohistochemistry and immunofluorescence
Immunohistochemical analysis for p16 was performed as
described using the JC8
22
or MAB G175-405 antibody (BD Bio-
science). Immunofluorescence was performed as described
23
using anti-K14 (1:500, Thermo, polyclonal) and anti-K19
(1:500, Sigma, clone A53-B/A2). Cells were counterstained with
DAPI (Sigma) and imaged with an epifluorescence Axioplan
microscope (Carl Zeiss).
FACS
Cells were trypsinized and resuspended in ice-cold M87A
media. Cells were stained for surface antigens using anti-CD227-
FITC (Becton Dickinson, clone HMPV), anti-CD10-PE or
–APC (BioLegend, clone HI10a), anti-CD24-Alexa488 (Biole-
gend, clone ML5), or anti-CD44-PE (BioLegend, clone IM7).
Results were obtained on a FACS Calibur (Becton Dickenson)
analysis platform as described.
48
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Gerri Levine, Batul Merchant, and Annie Pang for
technical assistance.
Funding
This work was supported by DOD BCRP BC060444 (JCG,
MRS), NIH CA24844 (JCG, MRS), NIH NIA R00AG033176
and R01AG040081 (JCG, KS, MAL), SWEHSC NIEHS
ES06694 and NIH CA23074 (LV, PN, GW, BWF), Margaret
E. and Fenton L. Maynard Endowment for Breast Cancer
Research (BWF), RVO:60077344 (PN), University of Arizona
Cytogenomics Laboratory (LF, AB), ACS RSG CCG 122517
(MJ), and the Office of Energy Research, Office of Health and
Biological Research, US. Department of Energy under Contract
No. DE-AC02-05CH11231. (JCG, KS, MAL, MRS).
Supplemental Materials
Supplemental materials for this article can be found on the pub-
lisher’s website.
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