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Cell Cycle
ISSN: 1538-4101 (Print) 1551-4005 (Online) Journal homepage: https://www.tandfonline.com/loi/kccy20
Cleavage of MCM2 licensing protein fosters
senescence in human keratinocytes
Hideki Harada, Hiroshi Nakagawa, Munenori Takaoka, James Lee, Meenhard
Herlyn, J. Allan Diehl & Anil K. Rustgi
To cite this article: Hideki Harada, Hiroshi Nakagawa, Munenori Takaoka, James Lee, Meenhard
Herlyn, J. Allan Diehl & Anil K. Rustgi (2008) Cleavage of MCM2 licensing protein fosters
senescence in human keratinocytes, Cell Cycle, 7:22, 3534-3538, DOI: 10.4161/cc.7.22.7043
To link to this article: https://doi.org/10.4161/cc.7.22.7043
Published online: 15 Nov 2008.
Submit your article to this journal
Article views: 68
Citing articles: 10 View citing articles
© 2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
[Cell Cycle 7:22, 3534-3538; 15 November 2008]; ©2008 Landes Bioscience
3534 Cell Cycle 2008; Vol. 7 Issue 22
In eukaryotic cells, MCM, the minichromosome maintenance
proteins, form a heterohexamer during G1 phase in the cell cycle
and constitute a DNA helicase activity at the onset of replication.
MCM proteins are downregulated and dissociated from chromatin
when cells exit the cell cycle. MCM proteins are upregulated
frequently in a variety of dysplastic and cancer cells. To delineate
the role of MCM in esophageal epithelial biology, we determined
the MCM family gene expression during cellular senescence,
immortalization, differentiation and apoptosis. All of the MCM2-7
proteins appeared to be downregulated in primary human esopha-
geal keratinocytes upon replicative senescence. Their expression
was restored by ectopic expression of a catalytic subunit of human
telomerase, resulting in immortalization. Interestingly, we found
a reciprocal induction of a novel MCM2-related protein fragment
upon cell growth inhibition associated with senescence, contact
inhibition or terminal differentiation, but not apoptosis. Epitope
mapping of this MCM2-related fragment suggested the lack of
amino- and carboxyl-terminal regions, including one of the puta-
tive nuclear localization signals and the ATPase domain, the MCM
box. The absence of multiple MCM2 transcripts implied a possible
posttranslational molecular cleavage in generation of the MCM2-
related fragment, and a potential functional role in the regulation
of the activity of the MCM protein complex.
Introduction
DNA replication occurs in a precise fashion during eukaryotic
cell division. This tight control is orchestrated by many regulatory
molecules, including members of the minichromosome mainte-
nance gene family, designated as MCM. Initially, MCM proteins are
recruited to sites of DNA replication and interact with each other,
forming the MCM2-7 complex during G1 phase.1 This complex has
helicase activity and facilitates DNA replication.2,3 Therefore, the
MCM proteins are essential for proliferating cells. In fact, MCM
proteins are upregulated frequently in a variety of dysplastic and
cancer cells.4-6
Normal human epithelial cells are limited in their prolifera-
tive capacity and eventually undergo differentiation, senescence or
apoptosis. In this context, deregulated cells have mechanisms to
suppress such processes, thereby resulting in unlimited cell prolifera-
tion, termed immortalization. Immortalization of human esophageal
epithelial cells can be achieved by the ectopic expression of human
telomerase, hTERT.7 Importantly, these cells maintain cell cycle
checkpoints such as p16INK4a/pRb and p14ARF/p53/p21WAF1.7
When oncogenic Ha-Ras is expressed ectopically in immortalized
human esophageal epithelial cells, these cells undergo senescence,
accompanied by upregulation of p16INK4a and hypophosphory-
lated pRb.8 To understand the molecular mechanisms underlying
constrained cellular growth arrest induced by senescence or differ-
entiation, we used normal human esophageal epithelial cells (EPC2)
and their derivative immortalized cells (EPC2-hTERT). We found
that MCM2 was cleaved in senescence, while cancer cells prevented
MCM2 from being cleaved. These results might indicate that the
cleavage of MCM2 plays a critical role in cellular growth arrest
induced by senescence or differentiation.
Results
A novel MCM2-related protein fragment is induced upon
replicative senescence through a post transcriptional mechanism.
We have previously carried out extensive characterization of EPC2
primary normal human esophageal epithelial cells.7 EPC2 cells
cease proliferation as they undergo replicative senescence by 44 PD
with an induction of p16INK4a protein and senescence-associated
β-galactosidase activity. Retrovirus-mediated stable transduction of a
catalytic subunit of telomerase (hTERT) in the presenescent EPC2
cells (42 PD) permitted the cells to reenter the cell cycle and resulted
in immortalization.7 The MCM family proteins are associated with
cell proliferation.9 In agreement, all of the examined MCM family
members were found downregulated as EPC2 cells underwent
senescence as documented by a reduced phosphorylation level of
pRB protein (Fig. 2A). By contrast, their expression was reversed
*Correspondence to: Anil K. Rustgi; 600 CRB; University of Pennsylvania; 415
Curie Blvd.; Philadelphia, Pennsylvania 19104 USA; Tel.: 215.898.0154; Fax:
215.573.5412; Email: anil2@mail.med.upenn.edu
Submitted: 08/29/08; Accepted: 09/18/08
Previously published online as a Cell Cycle E-publication:
http://www.landesbioscience.com/journals/cc/article/7043
Report
Cleavage of MCM2 licensing protein fosters senescence in human
keratinocytes
Hideki Harada,1,3,† Hiroshi Nakagawa,1,3,† Munenori Takaoka,1,3 James Lee,1,3 Meenhard Herlyn,4 J. Alan Diehl5,6 and
Anil K. Rustgi1-3,*
1Gastroenterology Division and Department of Medicine; 2Department of Genetics; 3Abramson Cancer Center; 4Wistar Institute; 5Department of Cancer Biology; 6Abramson
Family Cancer Research Institute; University of Pennsylvania; Philadelphia, Pennsylvania USA
†Co-first authors
Key words: MCM2, senescence, differentiation, esophageal keratinocytes
© 2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
MCM2 and cellular senescence
www.landesbioscience.com Cell Cycle 3535
upon hTERT transduction (Fig. 2A). Interestingly, a novel band was
induced reciprocally in senescing cells (Fig. 2A). This molecular mass
of approximately 55 kDa was detected by an anti-MCM2 antibody
raised against synthetic peptides corresponding to amino acid resi-
dues 131–150 of the MCM2 protein (Fig. 1), and thus designated
as a MCM2-related fragment. Since there is no known protein with
substantive similarity to this peptide sequence, we hypothesized that
the MCM2-related fragment may represent a novel splicing variant
or posttranslational cleavage of MCM2, and that its expression is
associated with the status of cell proliferation.
To determine a possible splicing variant, quantitative PCR was
performed by employing three independent sets of primers and a
TaqMan® probe, in which the MCM2 exon 3–4 boundary region is
specifically detected by the sequence encoding amino acids 131–150
of MCM2. Figure 2B demonstrates that all of the TaqMan® assays
detected a reduction in MCM2 mRNA expression upon cellular
senescence in parallel with the protein level. In addition, Northern
blotting with a full length MCM2 cDNA probe detected a single
transcript only throughout the PDs of EPC2 cells with or without
hTERT expression (data not shown), although the sensitivity of
detecting low copy mRNA species was not as great as that by PCR.
These observations argue against the idea that a unique splicing
variant is expressed upon cellular senescence.
MCM2-related protein fragment is also induced upon contact
inhibition and terminal differentiation, but not apoptosis. We next
tested whether or not the MCM2-related fragment may be induced
by cellular conditions other than senescence and detected by other
MCM2 antibodies raised against different epitopes within the
amino-terminal or carboxyl-terminal regions (Fig. 1). First, EPC2
cells were grown in monolayer culture until they reach an overly
confluent status. Contact inhibition was confirmed by decreased cell
proliferation and pRB phosphorylation (Fig. 3B). As shown in Figure
3A, the MCM2-related fragment was detected progressively by only
the anti-MCM2 antibody recognizing amino acid residues 131–150
in post confluent day 3 through day 11 while downregulation of the
full length MCM2 protein was detected consistently by all of the
antibodies in postconfluent cells (Fig. 3A). Thus, the MCM2-related
fragment was thought to lack both amino- and carboxyl-termini of
the full length MCM2 protein as denoted in Figure 1.
Such an induction of the MCM2-related fragment was observed
also in hTERT-immortalized EPC2 cells upon contact inhibition
(Fig. 3C and data not shown). Of note, the MCM2-related fragment
was expressed weakly in subconfluent EPC2 cells, where subpopula-
tion cells undergoes spontaneous senescence in primary culture (Figs.
3A and 4A).
Next, EPC2 and EPC2-hTERT cells were treated with calcium
chloride to induce terminal differentiation as indicated by induction
of involucrin (Fig. 4). Again, the full length MCM2 was down-
regulated and the MCM2-related fragment was induced, indicating
that the MCM2-related fragment can be induced upon cell growth
inhibition associated with cellular senescence, contact inhibition or
terminal differentiation. Finally, cells were exposed to actinomycin
D or gamma-irradiation. While apoptosis was induced within 12–24
hours as indicated by fragmentation of caspase 3, the MCM2-related
fragment was not detected (Fig. 5 and data not shown). Importantly,
the full length MCM2 level was not altered by apoptosis. These data
indicate that the MCM2 protein undergoes a molecular shift from
the full-length form to a short form upon chronic, but not acute,
induction of cell growth inhibition or cell cycle arrest.
MCM2 protein expression may be regulated through post-
translational modification. The above data suggested that the full
length MCM2 protein may be cleaved to generate the MCM2-
related fragment upon cell growth inhibition. However, the lack of
both amino- and carboxyl-terminal regions (Fig. 1) precluded us
from determining whether the MCM2-related fragment is derived
from the full-length form of MCM2 protein. Nonetheless, we
expressed ectopically an amino-terminally Flag-epitope tagged full-
length MCM2 protein in EPC2-hTERT cells. Flag-tagged MCM2
was expressed constitutively under the retroviral LTR promoter.
Interestingly, contact inhibition or terminal differentiation reduced
the expression of the Flag-tagged MCM2 (Figs. 3C and 4B),
suggesting that MCM2 protein may undergo degradation and/or
cleavage although the resulting putative MCM2-related fragment per
se could not be detected by an anti-Flag antibody.
Figure 1. Schematic representation of the MCM2 protein and a novel MCM2-related fragment. Horizontal bars indicate the primary structure of the 120
kDa MCM2 protein and putative molecular mass of approximately 55 kDa (MCM2-RF), sharing an epitope recognized by an antibody directed against
amino acids 131–150 of human MCM2. Failure of detection of the MCM2-RF by antibodies directed against peptides containing amino acids 1–50 or
amino acids 805–904 (see Figs. 3–5), indicated by horizontal arrow, predicted the molecular region of MCM2-RF. MCM2-RF is likely to contain one of
two putative nuclear-targeting sequences (NTS) and a Zn2+ finger motif, but not the MCM box, encompassing Walker A/B ATPase motifs, evolutionarily
conserved in the MCM2-7 family. For quantitative determination of MCM2 mRNA, TaqMan® Gene Expression Assays were carried out detecting transcripts
derived from exonic boundaries of the MCM2 gene, indicated by open vertical bars. Note that the TaqMan® assay detecting the exon 3–4 overlaps the
gene region encoding amino acids 131–150.
© 2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
MCM2 and cellular senescence
3536 Cell Cycle 2008; Vol. 7 Issue 22
Our data with MCM2 mRNA quantitation with three indepen-
dent probe sets (Fig. 2B) did not support the possibility of de novo
transcription of an alternative spliced form to account for the novel
MCM2-related fragment. Although post-translational cleavage of
MCM2 protein is suggested, neither the precise cleavage site(s) nor
the responsible proteases were identified in the present study. First,
in silico analysis using the peptide cutter program10 failed to identify
promising cleavage sites for MCM2 compatible with the predicted
cleavage pattern (Fig. 1) based upon the distribution of epitopes for
the antibodies we used. Second, the lack of either amino- or carboxyl
terminal region in the MCM2 related fragment hampered the use of
epitope-tagged protein to be expressed ectopically. In fact, amino-
terminally Flag-epitope tagged MCM2 only disappeared following
differentiation (Fig. 3C). Finally, a low protein yield from the senes-
cent or terminally differentiated cells limited affinity purification
with an antibody. Amongst the MCM family members, MCM3 has
been reported to undergo proteolytic cleavage mediated by Caspase-
3.11 Consistent with that notion, MCM2 remained uncleaved upon
caspase-3 activation (Fig. 5).
Squamous epithelial cells, or keratinocytes, are distinguished
by their proliferation-differentiation gradient and renewal during
homeostasis and tissue regeneration. Such cells slough that is trig-
gered by apoptosis and senescence prior to renewal. While the
expression of MCMs is decreased, if not lost, during differentiation
in somatic cells,12 it is unclear to what extent changes in their struc-
tures and functions impact upon differentiation. We have discovered
In aggregate, the MCM2 protein may give rise to a putative
cleaved form designated as a MCM2-related fragment, lacking both
amino- and carboxyl-terminal regions as well as the MCM box of
MCM2 (Fig. 1) upon cellular senescence, contact inhibition or
terminal differentiation, but not apoptosis.
Discussion
MCM2-7 proteins are related to each other and form a complex
for DNA synthesis initiation. The MCM complex binds origins of
DNA replication during the late M/early G1 phases of the cell cycle.
Through the action of S phase protein kinases, the MCM complexes
unwind the double-stranded DNA at the origin, with subsequent
recruitment of DNA polymerases and initiation of DNA synthesis.
They then disengage from replication origins, so DNA replication
cannot be reinitiated. MCMs are targets of S phase checkpoints.
That being said, in premalignant and malignant conditions, the
MCMs are often expressed abnormally, and have been advocated
as serving as diagnostic biomarkers to distinguish between normal
cells and malignant cells, and in cancers themselves as potential
therapeutic targets. In fact, we have observed marked upregulation
of MCM2 mRNA and its 120-kDa protein product in primary
esophageal tumor tissues as well as actively proliferating esophageal
cell lines (Fig. 2 and unpublished observations). Nevertheless, by
virtue of MCM dysregulation, chromosomal aberrations accrue
during tumorigenesis; loss of MCM proteins triggers DNA damage
and genomic instability.
Figure 2. Reciprocal expression of MCM2 protein and the MCM2 fragment with senescence and immortalization of primary human esophageal keratino-
cytes. (A) Western blotting demonstrates progressive downregulation of examined MCM2-7 family proteins, including the 120 kDa MCM2 (solid arrow)
in EPC2 cells undergoing replicative senescence by 42 population doublings (PD) while they were reinduced variably upon immortalization of EPC2 by
hTERT. By contrast, an MCM2 fragment (open arrow) was reciprocally induced upon senescence and suppressed by immortalization when Western blot
was carried out with an MCM2 antibody directed against amino acids 131–150 of MCM (Fig. 1). Cellular senescence was accompanied by a reduction
in the phosphorylation level of RB protein. MCM2, but not the MCM2 fragment, was detected in TE3 esophageal cancer cells, an esophageal squamous
cell carcinoma cell line. β-actin was used as a loading control. Note that protein yield from presenescent cells (42PD) was low and only 1 μg of protein
was loaded while other lanes were loaded with 10 μg of protein. Nonetheless, the MCM2 fragment was detectable at 42PD. (B) MCM2 mRNA level was
determined by TaqMan® Gene Expression Assays at indicated population doublings using a set of sequence specific primers and a probe detecting different
exonic junctions of the MCM2 gene transcript as described in Figure 1.
© 2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
MCM2 and cellular senescence
www.landesbioscience.com Cell Cycle 3537
instructions. The MCM2 mRNA was determined by TaqMan® Gene
Expression Assays (Applied Biosystems) using three independent
sets of primers and the probes (Hs00170472_m1 for exons 2–3,
Hs01091568_g1 for exons 3–4 and Hs01091564_m1 for exons
13–14), targeting different exon boundaries of the MCM2 transcript
(MN_004526.2). SYBR green reagent (Applied Biosystems) was
used to quantitate mRNA for GAPDH as an internal control. All
PCR reactions were performed in triplicate. The relative expression
level of MCM2 mRNA was calculated by normalizing to GAPDH
that while all of the MCM family members were
markedly downregulated during cellular senescence
(Fig. 2) and differentiation (data not shown), there
is the emergence of a unique fragmented MCM2
protein that may arise due to cleavage, and lacks both
the amino- and carboxyl domains as well as the MCM
box. It is conceivable that this fragment is responsible
for preventing DNA replication during periods of
prolonged or terminal differentiation, and/or during
senescence. In a recent study, Komamura-Kohno Y, et
al., analyzed the MCM2 protein biochemically through
partial digestion with trypsin.13 They demonsrated
that MCM2 fragments derived from the C-terminal
region inhibit DNA helicase activity through their
ability to bind to ssDNA. By contrast, two fragments
(148–441 and 442–676) from the central region were
responsible chiefly for the interaction between MCM2
and MCM4.13 Thus, the MCM2 fragment identified
in our study may prevent assembly of the MCM2-7
complex. It is also quite possible that fragments of
MCM2 might confer different effects in comparison
to full-length MCM2, depending upon subcellular
localization based upon solubility, or lack of it, as has
been reported in mouse ovarian oocytes,14 implying
there may be posttranslational modification of MCM2
in association with different functional properties.
Materials and Methods
Cell culture, retroviral vectors and retroviral trans-
duction. Normal human esophageal epithelial cells
(EPC2) and their hTERT-immortalized derivative
(EPC2-hTERT) were described previously.7 A full-
length human MCM2 cDNA was generated by reverse
transcription-polymerase chain reaction (RT-PCR)
using total RNA extracted from EPC2 cells as a
template. The PCR product, tagged with BamHI and
XhoI sites at the 5' and 3' ends, respectively, was
cloned into the pCMV FLAG-tagging mammalian
expression vector (Stratagene). For stable expression,
FLAG-tagged human MCM2 coding sequence was
further subcloned into the pBabe puromycin-resistant
retrovirus vector.15 Retrovirus production and infec-
tion were carried out as described previously.7,16
In brief, retroviral expression vectors were trans-
fected into Phoenix-Ampho packaging cells and the
virus-containing culture medium supernatants were
collected 48 and 72 hours post-transfection. EPC2-
hTERT cells were infected with the virus in a 6-well
plate (0.5 x 105 cells/well), followed by drug selection with
0.5 μg/mL of puromycin for 5 days.
RNA extraction, cDNA preparation and real-time PCR. Briefly,
total RNA was isolated from cells with the RNeasy Mini Kit (Qiagen,
Inc., Valencia, CA). DNaseI treatment was done on column and
cDNA was synthesized with the SuperscriptTM First Strand Synthesis
System (Invitrogen, Carlsbad, CA). Real-time PCR was performed
using the ABI PRISM® 7000 Sequence Detection System (Applied
Biosystems, Foster City, CA), according to the manufacturer’s
Figure 3. Contact inhibition of human primary esophageal keratinocytes is associated with
reduction in the full-length MCM2 protein and induction of the MCM2 fragment through a
posttranscriptional mechanism. (A) Western blotting using anti-MCM2 antibodies recogniz-
ing different epitopes as depicted in Figure 1 detects changes in expression of full-length
MCM2 (solid arrow) and an MCM2-related fragment (open arrow) in EPC2 cells harvested
at various cell densities. In (B), contact inhibition was documented by a reduction in cell pro-
liferation. 3H-thymidine incorporation was adjusted by the amount of protein from the whole
cell lysate used for Western blotting to detect the hyper-phosphorylated form of RB protein
shown in the insert. In (A and B), days elapsed after 100% confluent status are indicated in
the parentheses. (C) EPC2-hTERT cells stably transduced with amino-terminally Flag-epitope
tagged MCM2 protein (Flag-MCM2) or a control empty vector (puro) were harvested at
90% cell density or 3 days after reaching 100% confluency and subjected to Western blot-
ting with the indicated antibody. Note that anti-Flag antibody detected a reduced level of
exogenously expressed epitope-tagged full-length MCM2 protein but not MCM2 protein that
lacks the amino-terminal sequence (Fig. 1). β-actin was used as a loading control.
© 2008 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
MCM2 and cellular senescence
3538 Cell Cycle 2008; Vol. 7 Issue 22
Figure 5. Caspase-3 activation does not affect MCM2 protein. EPC2-hTERT
cells were treated with or without 2 μg/ml actinomycin D (Act. D) for 5 hours
(A) or exposed to indicated amount of γ-ray for 24 hours (B) and subjected
to Western blotting to detect MCM2 (solid arrow), full-size (open triangle) or
fragmented (solid triangle) forms of caspase-3 (Casp.-3), and Phospho-Chk2
(P-Chk2). β-actin was used as a loading control.
6. Laskey R. The Croonian Lecture 2001 hunting the antisocial cancer cell: MCM proteins
and their exploitation. Philos Trans R Soc Lond B Biol Sci 2005; 360:1119-32.
7. Harada H, Nakagawa H, Oyama K, Takaoka M, Andl CD, Jacobmeier B, von Werder A,
Enders GH, Opitz OG, Rustgi AK. Telomerase induces immortalization of human esopha-
geal keratinocytes without p16INK4a inactivation. Mol Cancer Res 2003; 1:729-38.
8. Takaoka M, Harada H, Deramaudt TB, Oyama K, Andl CD, Johnstone CN, Rhoades B,
Enders GH, Opitz OG, Nakagawa H. Ha-Ras(G12V) induces senescence in primary and
immortalized human esophageal keratinocytes with p53 dysfunction. Oncogene 2004;
23:6760-8.
9. Stoeber K, Tlsty TD, Happerfield L, Thomas GA, Romanov S, Bobrow L, Williams ED,
Williams GH. DNA replication licensing and human cell proliferation. J Cell Sci 2001;
114:2027-41.
10. Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A. ExPASy: The
proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 2003;
31:3784-8.
11. Schwab BL, Leist M, Knippers R, Nicotera P. Selective proteolysis of the nuclear replication
factor MCM3 in apoptosis. Exp Cell Res 1998; 238:415-21.
12. Barkley LR, Hong HK, Kingsbury SR, James M, Stoeber K, Williams GH. Cdc6 is a rate-
limiting factor for proliferative capacity during HL60 cell differentiation. Exp Cell Res
2007; 313:3789-99.
13. Komamura-Kohno Y, Tanaka R, Omori A, Kohno T, Ishimi Y. Biochemical characterization
of fragmented human MCM2. Febs J 2008; 275:727-38.
14. Swiech L, Kisiel K, Czolowska R, Zientarski M, Borsuk E. Accumulation and dynamics of
proteins of the MCM family during mouse oogenesis and the first embryonic cell cycle. Int
J Dev Biol 2007; 51:283-95.
15. Morgenstern JP, Land H. Advanced mammalian gene transfer: high titre retroviral vectors
with multiple drug selection markers and a complementary helper-free packaging cell line.
Nucleic Acids Res 1990; 18:3587-96.
16. Andl CD, Mizushima T, Nakagawa H, Oyama K, Harada H, Chruma K, Herlyn M, Rustgi AK.
Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggrega-
tion in esophageal keratinocytes in vitro and in vivo. J Biol Chem 2003; 278:1824-30.
mRNA expression level. Data were analyzed using ABI PRISM®
7000 sequence detection system software (Applied Biosystems).
Immunoprecipitation and western blot analysis. 10 μg protein
from whole-cell extracts was separated by SDS-PAGE and transferred
to Immobilon-P membranes (Millipore). The membranes were incu-
bated with anti-MCM2 (A300-191A) (amino acid residues 1–50)
(Bethyl laboratories, Montgomery, TX), anti-MCM2 (559542)
(amino acid residues 131–150) (BD Pharmingen), anti-MCM2
(clone 6A8) (amino acid residues 805–904) (Novus Biologicals,
Littleton, CO), anti-MCM3 (BD Pharmingen), anti-MCM4 (BD
Pharmingen), anti-MCM5 (BD Pharmingen), anti-MCM6 (BD
Pharmingen), anti-MCM7 (BD Pharmingen), anti-FLAG M2
(Sigma), AC-74 against β-actin (Sigma), and DMA1A + DM1B
against tubulin (Neo Markers). Staining was detected by using
ECL Plus (Amersham Pharmacia biotech). The FLAG-tagged
proteins were immunoprecipitated using FLAG Tagged Protein
Immunoprecipitation Kit (Sigma).
Acknowledgements
This research is supported by NCI grant PO1-CA098101 (M.H.,
J.A. H.N, A.K.R), the NIH/NIDDK Center for Molecular Studies
in Digestive and Liver Diseases P30-DK50306, the Morphology
Core, Molecular Biology Core and Cell Culture Core facilities, and
NIH grant R01-DK077005 (H.N.).
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Figure 4. Terminal differentiation in primary human esophageal keratinocytes
is associated with a reciprocal expression of MCM2 and the MCM2 frag-
ment. Subconfluent EPC2 cells (A) and EPC2-hTERT cells stably expressing
Flag-epitope tagged MCM2 (B) were treated for up to 72 hours with 1.8 mM
CaCl2 to induce terminal differentiation which was documented by Western
blotting for involucrin. Indicated anti-MCM2 antibodies and anti-Flag anti-
body detected full size MCM2 (solid arrow) or a MCM2 fragment (open
arrow). β-actin was used as a loading control.
