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

1Lovelace 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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
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.

squamous; quiescence; differentiation; adenocarcinomas; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone


THE SPRR GENE FAMILY maps to human chromosome 1q21 [PDB] , 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). Numerous 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 growth-arresting 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 the very poorly understood mechanisms involved in the transition of cells from the cell cycle to the G0 phase.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
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-µm 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; DuPont-NEN, 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 µg/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 · 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 x 105 cpm/µl of either the sense or antisense cRNA probes. Tissue sections were hybridized for 16 h at 55°C, then washed as follows: 1) 5x SSC, 10 mM DTT, 30 min, 55°C; 2) 50% formamide, 2x SSC, 10 mM DTT, 30 min, 65°C; 3) RNase A (20 µg/ml in 0.5 M sodium chloride, 10 mM Tris · HCl, pH 8.0, 5 mM EDTA), 30 min, 37°C; 4) repeat step 3 wash (minus RNase); 5) repeat step 2 wash; 6) 2x SSC, 15 min, 22°C; and 7) 0.1x 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 µg/ml insulin (Collaborative Research Products), 5 µg/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 µg/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 µg/ml propidium iodide (Sigma) and 100 µg/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 autoradio-graphic 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 x 105 cells were 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 pREP-transfected cells was started by adding 50 µg/ml hygromycin (Boehringer Mannheim, Indianapolis, IN) to the culture medium. Clonal cell populations from pINDSPRR1B-transfected EcRCHO cells were selected using zeocin (100 µg/ml) and neomycin (300 µg/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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
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 2 of 15 (13%) Be-induced lung adenocarcinomas showed SPRR1B expression. (Fig. 1C).



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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 µg 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.

 

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.



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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).

 

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 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).

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 morphology 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.



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Fig. 3. A: cell lines from the mouse adenocarcinomas express SPRR1B. RNA was isolated from individual tumors, and 20 µg 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.

 

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 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).



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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 µg 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 µg 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.

 

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 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.



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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).

 

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 µM muristerone (Fig. 6A). Clone number 17 showed the highest level of SPRR1B expression; therefore, further experiments were carried out using this clone.



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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 µg 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 µM muristerone were needed to induce SPRR1B protein expression in clone 17. Muristerone at 1, 3, and 6 µM (lanes 1, 2, and 3, respectively) did not sufficiently induce SPRR1B proteins, and 10 µM (lane 5) did not further increase its expression compared with 8 µM (lane 4). C: at least 24 h of incubation with 8 µM muristerone was necessary to induce SPRR1B expression. Treatment with 8 µM 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 µM 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.

 

A minimum of 8 µM 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 µM 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).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
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 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 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 NNK-induced 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.

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 microtubule formation. It is also possible that SPRR1B interacts with cyclin-dependent kinase-cyclin complexes that ensure for cells in G2 not to undergo an extra round of S phase before cells undergo mitosis (reviewed in Ref. 30). One reason for not isolating cells with higher ploidy than 4C could stem from the fact that the 4C cells are stable and survive to form a population, whereas cells with higher ploidy are not viable.

Although ploidy change was observed in stably transfected CHO cells, which were grown over many cell doublings to establish cell populations, no change in ploidy was detected when SPRR1B was expressed in an inducible fashion for only 6 days. Although the change of ploidy may result from prolonged overexpression of SPRR1B, expression levels in stably transfected cells were not correlated with the change in ploidy. Why SPRR1B overexpression causes ploidy change in stably transfected cells but enhances entry into G0 when overexpressed for short time periods is unclear.

Collectively, these studies show that SPRR1B plays a role in the entry of cells into G0 and may be coupled 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 mechanisms that drive cells into G0 are largely unknown, although it is an important state as cells undergo differentiation (17). Identifying SPRR1B-interacting proteins may contribute significantly to our understanding of cellular factors that promote entry into G0.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study was supported by National Institute of Environmental Health Sciences Grant ES-09237.


    ACKNOWLEDGMENTS
 
The authors thank Yoneko Knighton (Lovelace Respiratory Research Institute) for preparing the tissue samples.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Tesfaigzi, Lovelace Respiratory Research Inst., 2425 Ridgecrest Dr., SE, Albuquerque, NM 87108 (E-mail: ytesfaig{at}lrri.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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