Am J Physiol Lung Cell Mol Physiol 285: L889–L898, 2003.
First published June 27, 2003; 10.1152/ajplung.00065.2003.
SPRR1B overexpression enhances entry of cells into the G0
phase of the cell cycle
Yohannes Tesfaigzi,1 Paul S. Wright,2 and Steven A. Belinsky1
1
Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87108;
and 2Aventis Pharmaceutical, Bridgewater, New Jersey 08870
Submitted 11 March 2003; accepted in final form 20 June 2003
squamous; quiescence; differentiation; adenocarcinomas;
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
THE SPRR GENE FAMILY maps to human chromosome 1q21,
clustered in a 170-kb region within the epidermal differentiation complex (15, 26, 27). The SPRR family
comprises 11 genes: SPRR1A, SPRR1B, seven SPRR2
genes, SPRR3, and the recently identified SPRR4 gene
(5, 6). This protein family is an important component of
the cornified cell envelope, a structure formed beneath
the plasma membrane of squamous differentiated cells
by extensive cross-linking of several proteins (34). Nu-
Address for reprint requests and other correspondence: Y. Tesfaigzi, Lovelace Respiratory Research Inst., 2425 Ridgecrest Dr., SE,
Albuquerque, NM 87108 (E-mail: [email protected]).
http://www.ajplung.org
merous studies have established a role of SPRR1B in
squamous differentiating cells in vivo (43, 45) and in
vitro (1, 21, 33, and reviewed in Ref. 40) and indicate
that SPRR proteins are believed to affect the strength
and flexibility of stratified squamous epithelia (7, 36).
Moreover, SPRR1 is expressed in squamous tumors of
the lung (44).
In addition to its established role in squamous cells,
expression of SPRR1 also occurs in nonsquamous tissues and cell lines. SPRR1 is detected in myoepithelial
cells and in smooth muscle cells of human head skin
(22), in the myoepithelium of eccrine sweat glands, and
in the muscle layer of blood vessels (23). Studies in cell
lines derived from squamous cell carcinomas that do
not stratify in low-calcium medium (45) led to the
conclusion that SPRR1 expression must be under a
different signal transduction pathway and may have a
role in processes not directly related to terminal differentiation (25).
Further indication of the function of SPRR1 in
nonsquamous cells can be found from studies in Chinese hamster ovary (CHO) cells, a nondifferentiating
cell line (11, 38). Expression of Gadd 33, the hamster
SPRR1B homolog, appears to be associated with certain stressful growth-inhibitory responses. In addition,
expression of SPRR1 mRNA is induced before CHO
cells enter the G0 phase of the cell cycle, suggesting
that its expression could be in response to growtharresting signals (38).
The purpose of the current study was to determine
the expression profile for the SPRR1B gene in adenocarcinomas from mice, rats, and humans and to investigate the effect of its overexpression on cell cycle
regulation in nonsquamous cells. Expression of
SPRR1B in adenocarcinomas was restricted to individual or clusters of cells. Constitutive overexpression of
SPRR1B induced a change in ploidy in adenocarcinoma
cell lines from several species, whereas transient, inducible expression of SPRR1B accelerated entry into
G0. These results demonstrate that SPRR1B may play
a role in the transition of cells to G0 and may disrupt
the normal progression to mitosis resulting in changes
in ploidy. These studies provide additional insight into
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1040-0605/03 $5.00 Copyright © 2003 the American Physiological Society
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Tesfaigzi, Yohannes, Paul S. Wright, and Steven A.
Belinsky. SPRR1B overexpression enhances entry of cells
into the G0 phase of the cell cycle. Am J Physiol Lung Cell
Mol Physiol 285: L889–L898, 2003. First published June 27,
2003; 10.1152/ajplung.00065.2003.—Many studies have established the role of SPRR1B during squamous differentiation of skin and respiratory epithelial cells. However, its role
in nonsquamous cells is largely unknown. We reported that
expression of SPRR1B in Chinese hamster ovary (CHO) cells
is increased as they enter the G0 phase of the cell cycle. The
purpose of this study was to further investigate the SPRR1B
expression pattern in nonsquamous tumors and to study its
role in these cells. Expression of SPRR1B was detected
by Northern blotting in a higher percentage of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced compared with
beryllium metal-induced rat lung adenocarcinomas. In situ
hybridizations confirmed that SPRR1B is expressed in individual or clusters of cells of nonsquamous cells from mouse,
rat, and human adenocarcinomas. The same pattern of expression was observed in adenocarcinomas formed in nude
mice from cell lines established from adenocarcinomas.
SPRR1B expression was downregulated in the cell lines
derived from adenocarcinoma when cells were enriched in G0
at low confluence. Tetraploidy was induced in CHO, mouse,
and human tumor cell lines by stably overexpressing
SPRR1B, whereas control cells showed no change in ploidy.
Inducible expression of this protein for shorter periods using
the ecdyson system did not affect growth rate or the ploidy of
CHO cells but accelerated entry into G0/G1 compared with
controls. These findings indicate that SPRR1B is likely coupled primarily to signals responsible for withdrawal from the
proliferative state rather than the final stages of cellular
quiescence and that its overexpression for prolonged periods
may disrupt the normal progression of mitosis.
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the very poorly understood mechanisms involved in the
transition of cells from the cell cycle to the G0 phase.
MATERIALS AND METHODS
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Tumor generation and histopathology. Lung tumors were
induced in A/J mice (6–8 wk old; Jackson Laboratory, Bar
Harbor, ME) by intraperitoneal injection of a single dose (100
mg/kg body wt) of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as described earlier (3). Mice were killed 42 wk
after treatment. Lung adenocarcinomas in male Fischer (F)
344/N were induced by injections with NNK at a dose of 50
mg/kg body wt, three times a week for 20 wk and were killed
over a 100-wk period as described (4). Adenocarcinomas were
induced in 12-wk-old F344/N rats by a single nose-only exposure to a 980 mg/m3 beryllium (Be) metal aerosol for 40
min as described (28). Adenocarcinomas were produced in
athymic nude mice by injecting the cell lines I033, CL25, and
NCI-H596 subcutaneously in both flanks of the back and
waiting for 2–3 wk. The Lovelace Respiratory Research Institute is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.
Lung lesions and tumor specimens observed at necropsy
were trimmed of normal tissue and fixed in 10% neutral
buffered formalin. Small portions (0.2–0.5 mg) of large adenocarcinomas (1 cm) were also snap-frozen in liquid nitrogen
and stored at ⫺80°C for RNA isolation. Fixed lung tumors or
lung tissues fixed in neutral buffered formalin were embedded in paraffin for the preparation of tissue sections (5-␮m
thick), for staining with hematoxylin and eosin, and for in
situ hybridization. Histological diagnosis of lung lesions was
made from hematoxylin and eosin-stained tissue sections.
Adenocarcinomas of the human lung were obtained from the
Cooperative Human Tissue Network. Several board-certified
pathologists at our Institute confirmed these tumors as adenocarcinomas.
In situ hybridization. In vitro transcription of the coding
region of SPRR1B cDNA cloned in Bluescript SKII⫹ (Stratagene, La Jolla, CA) was performed using Riboprobe Gemini II
core system (Promega, Madison, WI). T3 and T7 RNA polymerases were used to generate the sense and antisense cRNA
probes, respectively. [33P]UTP (2,064 Ci/mmol; DuPontNEN, Boston, MA) was used to label the probes. In situ
hybridizations was performed as described by Simmons et al.
(32) with some modifications in the posthybridization washes
to decrease background. Briefly, sections were deparaffinized, hydrated in decreasing ethanol solutions, postfixed in
4% paraformaldehyde, digested with proteinase K (20 ␮g/ml)
for 15 min, refixed in 4% paraformaldehyde, treated with
0.25% acetic anhydride, and dehydrated. The hybridization
mixture contained 50% deionized formamide, 0.3 M sodium
chloride, 20 mM Tris 䡠 HCl (pH 8.0), 10% dextran sulfate, 0.5
mg/ml yeast RNA, 5 mM Na2EDTA, 10 mM sodium phosphate, and 20 mM dithiothreitol (DTT), plus 2 ⫻ 105 cpm/␮l
of either the sense or antisense cRNA probes. Tissue sections
were hybridized for 16 h at 55°C, then washed as follows: 1)
5⫻ SSC, 10 mM DTT, 30 min, 55°C; 2) 50% formamide, 2⫻
SSC, 10 mM DTT, 30 min, 65°C; 3) RNase A (20 ␮g/ml in 0.5
M sodium chloride, 10 mM Tris 䡠 HCl, pH 8.0, 5 mM EDTA),
30 min, 37°C; 4) repeat step 3 wash (minus RNase); 5) repeat
step 2 wash; 6) 2⫻ SSC, 15 min, 22°C; and 7) 0.1⫻ SSC, 15
min, 22°C. Slides were dehydrated, and autoradiography was
performed using Kodak NTB-2 emulsion. Sections hybridized
with SPRR1B probes (antisense and sense) were exposed for
14–21 days depending on signal intensities as estimated
from exposures to X-ray films at 4°C, then developed in
Kodak D19. Slides were lightly stained with toluidine blue
(0.02%, 30 s). Photomicrographs of the sections were taken
with an Olympus BH-2 microscope.
Cell culture and flow cytometry. The mouse cell lines CL25
and I033 were derived from adenocarcinomas (35). NCIH596, an adenosquamous carcinoma cell line; SK-MES-1, a
squamous carcinoma cell line; and CHO cells were purchased
from the American Type Culture Collection. I033, CL25, and
CHO cells were grown in Ham’s F-12 (Life Technologies,
Grand Island, NY) supplemented with 10% fetal bovine serum. NCI-H596 was grown in Ham’s F-12 supplemented with
10% fetal bovine serum and 5 ␮g/ml insulin (Collaborative
Research Products), 5 ␮g/ml transferrin (Collaborative Research Products), 12.5 ng/ml EGF (Collaborative Research
Products), 20 ng/ml cholera toxin (Sigma, St. Louis, MO), 1.8
nM hydrocortisone (Collaborative Research Products), and
25 ␮g/ml gentamicin (Sigma). Cells were grown to the desired
stage of confluence, harvested by trypsinizing, washed twice
in Dulbecco’s phosphate-buffered saline (DBPS; Life Technologies), and stored as pellets at ⫺80°C for further analysis.
DNA content was analyzed by flow cytometry as described
(41). Briefly, after being harvested, cells were fixed in ice-cold
70% methanol in H2O, washed in DPBS, and incubated in 2.5
␮g/ml propidium iodide (Sigma) and 100 ␮g/ml RNase A
(Sigma) in DPBS for 1 h.
RNA isolation and Northern blot analysis. Total RNA was
isolated from the adenocarcinomas using the TRIzol reagent
according to the manufacturer’s protocol (Life Technologies).
RNA was isolated from cultured cells using the Nonidet P-40
lysis method as described (39). Twenty micrograms of total
RNA from each tumor sample were electrophoresed on 1%
agarose formaldehyde gels, transferred to Hybond N membranes (Amersham, Arlington Heights, IL), and cross-linked
to the membrane by irradiation from an ultraviolet Stratalinker (Stratagene). Probes (cDNAs to SPRR1B, cdc2, and
GAPDH) were 32P-labeled by random priming using rediprime DNA labeling system (Amersham). After hybridization, membranes were subjected to autoradiography using
Hyperfilm-MP (Amersham). The intensity of autoradiographic bands was quantified via densitometry using a
Fluor-S Max Imager and Quantity One software (Bio-Rad,
Hercules, CA). The band intensities were normalized with
the corresponding band intensities for GAPDH mRNA.
Construction of the pREPSPRR1B vector and the
pINDSPRR1B muristerone A-inducible vector. The open
reading frame of SPRR1B cDNA (44) was cloned into the
BamHI site of pREP10 (Invitrogen, San Diego, CA) by standard protocols (31). The orientation of the cDNA clone was
verified by sequence analysis.
SPRR1B cDNA was cloned unidirectionally into the EcoRV
and HindIII restriction sites of the pIND plasmid (Invitrogen) in the sense position related to the promoter, generating
the pINDSPRR1 expression vector. The cDNA sequence was
verified by sequence analysis. The ecdyson-inducible expression system utilizes a heterodimer of the ecdysone receptor
(VgEcR) and the retinoid X receptor that binds a hybrid
ecdysone response element in the presence of the synthetic
analog of ecdysone muristerone A (29). Binding of the heterodimer to the modified ecdysone response element, present
in the minimal promoter in the pIND vector, activates transcription. EcRCHO cells that were stably transfected with
VgEcR were purchased (Invitrogen).
Transfections. Cells were transfected by Lipofectin reagent
(Life Technologies) according to the manufacturer’s protocol.
In brief, CHO cells were collected by low-speed centrifugation
and washed twice with serum-free medium. The cells were
resuspended in serum-free medium, and 1 ⫻ 105 cells were
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RESULTS
Expression of SPRR1B in adenocarcinomas. Expression of SPRR1B was assessed in adenocarcinomas induced in mice and rats by the tobacco carcinogen NNK
and also in rats by Be metal. A 550-nt mRNA product
was detected that corresponds to SPRR1B (20).
SPRR1B was detected at varying levels in 8 of 13 (61%)
and 9 of 22 (41%) NNK-induced mouse and rat lung
tumors, respectively (Fig. 1, A and B). In contrast, only
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Fig. 1. SPRR1B expression in 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone (NNK)- and Be-induced adenocarcinomas. RNA was isolated from individual tumors, and 20 ␮g of total RNA were analyzed
by Northern blot analysis as described in MATERIALS AND METHODS. A:
RNA was isolated from 13 NNK-induced tumors obtained from 5
different mice, and 8 tumors showed mRNA transcript for SPRR1B.
B: 4 of 12 NNK-induced adenocarcinomas from different rats showed
mRNA transcript for SPRR1B. C: 2 of 15 Be-induced tumors showed
mRNA transcript for SPRR1B.
2 of 15 (13%) Be-induced lung adenocarcinomas
showed SPRR1B expression. (Fig. 1C).
Previous studies have shown that NNK-induced
lung adenocarcinomas in AJ mice originate from type
II cells and do not contain any squamous differentiated
cells (3). However, in rats the spectra of neoplasms
induced by NNK are classified as papillary adenocarcinoma (60%), squamous cell carcinoma (25%), and as
solid or mixed carcinomas (15%) (4). To confirm that
the expression of SPRR1B seen in mice and rats was
localized to nonsquamous epithelial cells, we performed in situ hybridization. Interestingly, the expression seen by Northern analysis was not correlated with
homogenous expression throughout the tumor; rather
individual or clusters of cells demonstrated intense
reactivity to the antisense cRNA probe (Fig. 2A). This
pattern was also observed in preneoplastic hyperplasias (Fig. 2B). The sense cRNA probe showed no signal.
Detailed observation of these cells at higher magnification verified that SPRR1B-expressing cells within
the hyperplasias and adenocarcinomas were not squamous in morphology.
Expression of SPRR1 was also determined in human
adenocarcinomas only by in situ hybridization, because
most human adenocarcinomas contain abundant inflammatory cells, stromal cells, and connective tissue.
SPRR1 mRNA was detected in localized areas of only
two of 12 tumors analyzed (17%), and these cells also
showed no squamous morphology (Fig. 2C). The areas
that showed hybridization were smaller than that seen
in mouse and rat lung tumors and may have remained
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plated onto 30-mm dishes. After 24 h, cells were washed with
serum-free medium, and the Lipofectin reagent containing
the plasmid DNA was added to the cells in a volume of 1 ml.
After 2 h of incubation at 37°C, 1 ml of medium supplemented with 4% serum was added; cells continued to incubate for another 24 h, and medium was changed to F-12 with
10% serum. After a 48-h recovery period, selection for pREPtransfected cells was started by adding 50 ␮g/ml hygromycin
(Boehringer Mannheim, Indianapolis, IN) to the culture medium. Clonal cell populations from pINDSPRR1B-transfected EcRCHO cells were selected using zeocin (100 ␮g/ml)
and neomycin (300 ␮g/ml) antibiotics for plasmid selection.
These cell populations were always maintained in the respective selection medium.
Western blot analysis. Preparation and specificity of the
affinity-purified antibodies to peptides corresponding to the
23 amino acids of the COOH-terminal region (C23) (PKVPE
PCPSP VIPAPA QQKTKQK) and to the 29 amino acids of the
NH2-terminal region (minus methionine) (N29) (SSQQQ
KQPCT PPPQP QQQQV KQPCQ PPPQ) of SPRR1B have
been described (37).
Western blot analysis was carried out essentially as described (42). Briefly, protein extracts were subjected to 12.5%
SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes
(Millipore, Bedford, MA) using a miniblot apparatus (BioRad, Richmond, CA). After staining the blot with Ponceau S
(Sigma), the molecular weight lane was removed for reference, and the blot was incubated for 2 h in 5% nonfat dry milk
in Tris-buffered saline (TBS) (pH 7.4). The blot was then
incubated overnight at 4°C with the respective antibody
diluted in 5% nonfat dry milk. The C23 and N29 antibodies
were used at 1:1,000 and 1:500 dilutions, respectively. After
extensive washes with TBS and TBS containing 0.05%
Tween 20 (Sigma), membranes were incubated for 1 h at
room temperature with a secondary peroxidase-conjugated
anti-rabbit antibody (Jackson ImmunoResearch Laboratories). After extensive washes, the blots were developed with
the enhanced chemiluminescence kit (Amersham) as described by the manufacturers.
Immunohistochemistry. After deparaffinization of tissues
with xylene, slides were hydrated in graded ethanol and
deionized water. Endogenous peroxidase activity was blocked
by incubating the sections in 0.1% hydrogen peroxide for 15
min. Slides were then rinsed in PBS (pH 7.4) and incubated
in 0.5% saponin for 30 min at room temperature to unmask
the SPRR1 protein. After blocking in 5% normal goat serum
in PBS, slides were incubated for 1 h at 37°C with the C23
SPRR1 antibody. SPRR1 immunoreaction was detected using
the Vectastain rabbit ABC kit and the peroxidase substrate
diaminobenzidine (Vector Laboratories, Burlingame, CA) according to the manufacturer’s directions.
Statistical analysis. Grouped results were expressed as
means ⫾ SE, and differences between groups were assessed
for significance by Student’s t-test. A P value of ⬍0.05 was
considered to indicate statistical significance.
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Fig. 2. SPRR1B expression in lung adenocarcinomas
from rats and humans. Tissue sections from NNK-,
Be-induced adenocarcinomas and human adenocarcinomas were hybridized with antisense (AS) and sense
(S) SPRR1B cRNA probes in situ. SPRR1B was detected in NNK-induced mouse and rat adenocarcinomas (A), preneoplastic hyperplasias (B), and in human
adenocarcinomas (C) using the AS cRNA probe. Arrows
show areas of intense hybridization. The S cRNA probe
showed no hybridization. D: tissue sections from mouse
adenocarcinomas were also stained with the C23
SPRR1 antibody (Ab), and this immunoreaction could
be inhibited when the antibody was preincubated with
the antigenic peptide (Pep).
undetected if RNA was isolated from the entire tumor
and analyzed by Northern blotting. Furthermore,
when the mouse adenocarcinomas were immunostained with the C23 SPRR1 antibody, individual or
clusters of cells were positive for this protein (Fig. 2C).
We demonstrated immunospecificity for the SPRR1
antibody by inhibiting the immunoreaction with the
antigenic peptide (Fig. 2D).
AJP-Lung Cell Mol Physiol • VOL
Expression of SPRR1B in adenocarcinoma cell lines
and their derived tumors. Cell lines established from
NNK-induced mouse adenocarcinomas (35) were analyzed for their ability to express SPRR1B. Both I033
and CL25 cell lines showed extensive expression of
SPRR1B by Northern blot analyses (Fig. 3A). When
injected into nude mice, I033 resulted in a papillary
adenocarcinoma, which highly resembles the morphol-
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ogy of the primary tumor, and CL25 produced a solid
adenocarcinoma. Injection of a human adenocarcinoma
cell line (NCI-H596) that did not express SPRR1B in
culture (Fig. 3A) also produced adenocarcinomas in
nude mice. In situ hybridization showed that SPRR1
mRNA expression in tumors derived from the mouse
cell lines, I033 (Fig. 3B), and the human cell line
NCI-H596 (Fig. 3C) was restricted to individual or
clusters of cells as observed in the primary tumors.
SPRR1B expression during entry into G0. Our previous studies with CHO cells had shown that SPRR1 is
increased 10-fold just before cells enter the G0 phase
and is downregulated after cells cease growing (38). To
investigate whether the regulation of SPRR1B is similar in an adenocarcinoma-derived cell line, we enriched I033 cells in G0/G1 by low-serum medium and
analyzed SPRR1B expression. Densitometric analysis
of SPRR1B normalized to the corresponding GAPDH
mRNA levels showed that at 48 h after the switch to
low-serum medium, SPRR1B mRNA levels were decreased twofold compared with levels at 24 h (Fig. 4A).
The mRNA levels of cdc2p34, a regulator of the G2
phase of the cell cycle and known to be downregulated
in G0 (10), were also decreased by ⬃40% at 48 h when
AJP-Lung Cell Mol Physiol • VOL
normalized by the corresponding GAPDH mRNA. Sodium butyrate has previously been shown to cause cell
cycle arrest in the G0/G1 phase (8, 14). Densitometric
analysis of SPRR1B normalized to the corresponding
GAPDH mRNA levels showed a twofold decrease in
cells treated with 5 mM sodium butyrate compared
with cells treated with nothing as control or with 2 mM
sodium butyrate for 48 h (Fig. 4B). Normalized to the
corresponding GAPDH mRNA levels, cdc2p34 mRNA
levels were decreased twofold by 2 mM and 100-fold by
5 mM sodium butyrate treatment for 48 h (Fig. 4B).
Analysis of DNA histograms showed that cells were
enriched in the G0/G1 phase at this time point (data not
shown).
Effect of overexpression of SPRR1B in mouse and
human cells. To further investigate the role of SPRR1B
on the cell cycle, we stably transfected several cell lines
from mouse and human adenocarcinomas with
pREPSPRR1B or pREP vector as control. Flow cytometric analysis of the hygromycin-resistant, transfected cell populations showed that the 4N peak of the
pREPSPRR1B cells from CHO (Fig. 5A), CL25, and
SK-MES-1 (data not shown) cells was enlarged,
whereas the 2N peak was reduced. A sub-G1 peak was
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Fig. 3. A: cell lines from the mouse adenocarcinomas
express SPRR1B. RNA was isolated from individual
tumors, and 20 ␮g of total RNA were analyzed by
Northern blot analysis as described in MATERIALS AND
METHODS. B: mouse cell line I033 formed papillary adenocarcinomas when injected in nude mice and showed
SPRR1 expression in the same pattern as the primary
lung adenocarcinomas. In situ hybridization with the
SPRR1B AS cRNA probe showed signals in single cells,
whereas the S cRNA control showed no hybridization.
C: adenocarcinomas produced in athymic nude mice by
injection of the human adenocarcinoma cell line NCIH596 show expression of SPRR1. In situ hybridization
of these tumors with the AS cRNA probe showed a
similar pattern of expression as the primary tumor,
whereas the S cRNA control showed no hybridization.
Arrows show areas of hybridization.
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also observed in the pREPSPRR1B-transfected populations. Isolation of single clones from these transfected populations revealed a mixed population of diploid and tetraploid cells that caused an enlarged 4N
peak (Fig. 5B). In contrast, the I033 and NCI-H596 cell
lines showed no alteration in DNA ploidy, similar to
the control vector-transfected cell population for all cell
lines.
The pREPSPRR1B-transfected cells from all cell
lines expressed SPRR1B, whereas the protein was not
detected in the pREP-transfected cells (data not
shown). Five clonal cell populations were isolated from
each pREPSPRR1- and pREP-transfected CHO populations. SPRR1B was detected by Western blot analysis
using the N29 and C23 antibodies in pREPSPRR1B
cells at varying levels but not in pREP cells (Fig. 5C).
Clones 1 and 5 were tetraploid, and clones 2, 3, and 4
were diploid; however, similar SPRR1B levels were
detected in clones 1 and 2, indicating that expression
levels do not correlate with the change in ploidy. Both
light microscopy (Fig. 5, D and E) and the forward
scatter plot (not shown) reveal that the tetraploid cells
of pREPSPRR1B-transfected CHO cells were larger
than the diploid cells. However, the growth rate of both
control and SPRR1B-overexpressing tetraploid cells
did not differ.
An inducible expression system transfected into
CHO cells was used to further characterize the direct
effect of SPRR1B overexpression on the cell cycle. Six
of 51 EcRCHO clonal cell populations stably transfected with pINDSPRR1B showed SPRR1B expression
after treatment with 10 ␮M muristerone (Fig. 6A).
Clone number 17 showed the highest level of SPRR1B
AJP-Lung Cell Mol Physiol • VOL
DISCUSSION
These studies show that SPRR1B has functions not
directly associated with squamous differentiation but
that are related to the transition of cells from the cell
cycle to G0. Overexpression of this protein enhanced
the rate of entry into the G0 phase but did not noticeably affect the kinetics of the cell cycle. Our findings
also substantiate the expression of SPRR1B in adenocarcinomas from both rodents and humans.
In situ hybridization shows that the signals observed
by Northern blot analyses of adenocarcinomas
stemmed from individual or clusters of cells expressing
high levels of SPRR1B mRNA. These cells show no
squamous morphology, and expression of SPRR1B was
not restricted to a certain cell type. Therefore, its
expression may be associated with the stage of cell
cycle. This hypothesis is supported by the finding that
I033 cells arrested in G0 with low-serum medium or
with sodium butyrate expressed SPRR1B before cells
entered G0 and downregulated SPRR1B expression
along with cdc2p34 after they entered G0. Also, the
hamster homolog of SPRR1, which was previously
termed G0SPR1 and is essentially identical to other
members of SPRR1B, is expressed before entering the
G0 phase (38).
A previous study found that SPRR1 expression is not
quantifiable or inducible in 12 human bronchogenic
carcinoma cell lines (9). Another study found that expression of SPRR1 is markedly reduced with progression of tumorigenicity (24). However, these studies
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Fig. 4. Expression of SPRR1B is downregulated as cells enter quiescence. A: RNA was isolated from I033 cells 0, 24, and 48 h after
they were switched to low-serum medium, and 20 ␮g of RNA were
analyzed by Northern blotting with the SPRR1B and cdc2p34 probes.
GAPDH was used to show the loading of RNA in each lane. B: RNA
was isolated from I033 cells 48 h after treatment with 2 (lane 1) or 5
mM (lane 2) sodium butyrate or with nothing (lane 3) as control, and
20 ␮g of RNA were analyzed by Northern blotting with the SPRR1B
and cdc2p34 probes. GAPDH was used to show the loading of RNA in
each lane.
expression; therefore, further experiments were carried out using this clone.
A minimum of 8 ␮M muristerone was necessary to
induce SPRR1B after 24 h of treatment and a further
increase in muristerone concentration did not change
SPRR1B levels in the cells (Fig. 6B). No SPRR1B was
detected at 16 h postexposure; however, SPRR1B was
induced 24 h after exposure to 8 ␮M muristerone.
Treatment for longer periods of time did not affect
levels of SPRR1B expression (Fig. 6C).
Because SPRR1B mRNA levels are downregulated
when cells reach 100% confluence (38), the effect of
maintaining increased SPRR1B expression during
growth and after confluence was investigated. The rate
of cell growth was identical in SPRR1B-overexpressing
muristerone-treated and ethanol-treated control cells
(data not shown). At various stages of confluence, protein was isolated from a portion of the cells treated
with muristerone or ethanol. SPRR1B was not detected
in control, ethanol-treated EcRCHO cells but was
found in cells exposed to muristerone. SPRR1B levels
were expressed at similar levels during exponential
growth and after confluence (Fig. 6D). Entry of cells
into G1/G0 at 100% confluence was reproducibly (four
independent experiments) enhanced in cells overexpressing SPRR1B compared with controls treated with
ethanol only (Fig. 6E). Enhanced entry into G1/G0 of
muristerone-treated CHO cells transfected with vector
alone was not observed (data not shown).
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were conducted in cell culture and did not investigate
expression at different stages of the cell cycle. The
importance of this strategy is substantiated by the fact
that although expression of SPRR1 was not observed in
NCI-H596 in culture, SPRR1B was detected in the
adenocarcinomas that formed in nude mice using this
cell line. In contrast, although some established mouse
cell lines from neoplastic cells, I033 and CL25, expressed high levels of SPRR1 mRNA in culture, expression was restricted to clusters or individual cells in
tumors formed in nude mice. Together, these findings
suggest that expression of SPRR1 in solid adenocarcinomas of the lung may be regulated by contact with
neighboring cells and is dependent on the stage of the
cell cycle.
The fact that overexpression of SPRR1B caused a
higher percentage of CHO cells to enter G0/G1 faster
AJP-Lung Cell Mol Physiol • VOL
than control cells directly links SPRR1B as a factor for
entry into quiescence. The downregulation of SPRR1B
after cells reach quiescence and its role in enhancing
entry into G0 suggest that SPRR1 may be coupled
primarily to signals responsible for withdrawal from
the proliferative state rather than the final stages of
cellular quiescence and is likely not a part of the cell
cycle control in normally proliferating cells. The single
cells that express SPRR1B in adenocarcinomas and
early neoplasias may therefore represent cells that are
in the process of entering G0. Interestingly, there appears to be a trend that a higher percentage of NNKinduced tumors showed SPRR1B expression compared
with Be-induced adenocarcinomas, suggesting that fewer
cells in Be-induced tumors are entering G0. Whether this
difference results from the presence of different growth
factors or differentiation signals is unknown.
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Fig. 5. Overexpression of SPRR1B changes
ploidy in Chinese hamster ovary (CHO) cells.
DNA content analysis of CHO cells transfected
with pREP and pREPSPRR1B. Flow cytometric
analysis of stably transfected populations shows
a large peak with 4N DNA content and a sub-G1
peak (arrow, A), and flow cytometry after isolation of clonal populations (numbers 1 and 2)
shows that the pREPSPRR1B-transfected cell
population consists of a mixture of diploid and
tetraploid cells (B). C: stably transfected CHO
cells show expression of SPRR1B protein at varying levels. No expression of SPRR1B was detected on the Western blot using the N29 and
C23 antibodies in pREP cells (lane C), whereas
the 5 pREPSPRR1B clones (lanes 1–5) expressed
the protein at different levels. D: tetraploid cell
populations were larger than the diploid cells.
Photomicrographs of CHO cells that overexpress
SPRR1B and show a DNA trace of diploid (D)
and tetraploid cells (E).
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Fig. 6. Inducible expression of SPRR1B causes enhanced entry into
quiescence. A: 6 clonal populations of CHO cells with the heterodimer of ecdysone receptor (EcRCHO) transfected with
pINDSPRR1B show inducible expression of SPRR1B when treated
with muristerone. Proteins were extracted from clonal cell populations that were treated with muristerone (M) or ethanol (E, the
solvent of muristerone); 100 ␮g of protein were analyzed by Western
blotting using the C23 antibody. A cross-reacting protein shows that
the same amount of protein was loaded on each lane. B: 8 ␮M
muristerone were needed to induce SPRR1B protein expression in
clone 17. Muristerone at 1, 3, and 6 ␮M (lanes 1, 2, and 3, respectively) did not sufficiently induce SPRR1B proteins, and 10 ␮M (lane
5) did not further increase its expression compared with 8 ␮M (lane
4). C: at least 24 h of incubation with 8 ␮M muristerone was
necessary to induce SPRR1B expression. Treatment with 8 ␮M
muristerone for 0, 3, 6, or 16 h (lanes 1–4) did not induce SPRR1B
expression, but continued treatment of cells for 24, 28, 32, and 48 h
(lanes 5–8) induced SPRR1B production; however, expression was
not augmented by treatment for longer than 24 h. D: cells were
treated either with ethanol only (lanes 1–4) or with 8 ␮M muristerone in ethanol (lanes 5–12) and harvested at 50% (lanes 1, 5, and
6), 70% (lanes 2, 7, and 8), 100% (lanes 3, 9, and 10) and 2 days after
they had reached confluence (lanes 4, 11, and 12). Two populations
from each of the muristerone-treated cells were analyzed for inducible SPRR1B expression, and the rest of the cells were fixed in
ice-cold methanol for analysis by flow cytometry. SPRR1B was not
detected in cells treated with ethanol only but was expressed in
muristerone-treated cells at 70 and 100% confluence and 2 days
postconfluence. E: flow cytometric analysis of cells at 50% (1 day),
70% (2 days), 100% (4 days), and 2 days after they had reached
confluence (6 days) shows that a higher portion of cells were in G0/G1
at 100% confluence following treatment with muristerone compared
with cells treated with ethanol as control. Results are expressed as
averages ⫾ SE from 4 different experiments. No difference in the
percentage of cells at G0/G1, S, or G2/M at any other stages of
confluence. * Significantly different from control, P ⬍ 0.05.
AJP-Lung Cell Mol Physiol • VOL
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Support for the role of SPRR1B during withdrawal
from the cell cycle is also derived from studies showing
that expression of this protein correlates with the cessation of proliferation and induction of terminal differentiation in the periderm (20). In addition, Gadd 33
mRNA, a transcript homologous to SPRR1 (11), is
induced by exposure to DNA base-damaging agents
such as the alkylating agent methylmethane sulfonate
(12, 13) and in response to growth arrest treatments by
depletion of medium of growth factors and many nutrients or treatment with prostaglandin A2 (19). The
increase in mRNA levels appears to stem from increased RNA stability (19). Similarly, we have previously determined that downregulation of SPRR1B by
retinoids in tracheobronchial epithelial cells is a result
of decreased mRNA half-life (2). Thus posttranscriptional regulation has a significant role in regulating
mRNA levels of this gene.
Although SPRR1B overexpression in some stably
transfected cell lines caused a change in ploidy, e.g.,
CL25, no such effect was observed in others, e.g., I033.
Treatment of CHO cells with sodium arsenite during
the G2 phase (16) or transfection of Chinese hamster
cells with SV40 (18) also induces changes in ploidy.
This change in ploidy results from disrupted spindle
integrity, causing cells to enter interphase prematurely without segregation of chromatids. SPRR1B is
rich in cysteine (11%) and may, when overexpressed,
interact with sulfhydryl groups of tubulin-disrupting
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The authors thank Yoneko Knighton (Lovelace Respiratory Research Institute) for preparing the tissue samples.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
DISCLOSURES
This study was supported by National Institute of Environmental
Health Sciences Grant ES-09237.
18.
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when overexpressed for short time periods is unclear.
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with proteins responsible for withdrawal from the proliferative state. This protein is likely not a part of the
cell cycle, because no difference was observed in the
growth rate of any SPRR1B-overexpressing cell. The
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understanding of cellular factors that promote entry
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