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[Cancer Research 64, 4171-4179, June 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

{alpha}CP-4, Encoded by a Putative Tumor Suppressor Gene at 3p21, But Not Its Alternative Splice Variant {alpha}CP-4a, Is Underexpressed in Lung Cancer

Ruben Pio1,2, Isabel Zudaire1,3, Irene Pino1,4, Zafira Castaño1, Natalia Zabalegui5, Silvestre Vicent1,4, Fermin Garcia-Amigot5, Maria D. Odero1,3, Maria D. Lozano4, Jesus Garcia-Foncillas5, Maria J. Calasanz3 and Luis M. Montuenga1,4

1 Division of Oncology and Departments of 2 Biochemistry, 3 Genetics, 4 Histology and Pathology, and 5 Biotechnology, Clinica Universitaria and School of Medicine, Center for Applied Medical Research. University of Navarra. Pamplona, Spain


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}CP-4 is an RNA-binding protein coded by PCBP4, a gene mapped to 3p21, a common deleted region in lung cancer. In this study we characterized the expression of {alpha}CP-4 and {alpha}CP-4a, an alternatively spliced variant of {alpha}CP-4, in lung cancer cell lines and non-small cell lung cancer (NSCLC) samples from early stage lung cancer patients. In NSCLC biopsies, an immunocytochemical analysis showed cytoplasmic expression of {alpha}CP-4 and {alpha}CP-4a in normal lung bronchiolar epithelium. In contrast, {alpha}CP-4 immunoreactivity was not found in 47% adenocarcinomas and 83% squamous cell carcinomas, whereas all of the tumors expressed {alpha}CP-4a. Besides, lack of {alpha}CP-4 expression was associated with high proliferation of the tumor (determined by Ki67 expression). By fluorescence in situ hybridization, >30% of NSCLC cell lines and tumors showed allelic losses at PCBP4, correlating with the absence of the protein. On the other hand, no mutations in the coding region of the gene were found in any of the 24 cell lines analyzed. By Northern blotting and real-time reverse transcription-PCR, we detected the expression of {alpha}CP-4 and {alpha}CP-4a messages in NSCLC and small cell lung cancer cell lines. Our data demonstrate an abnormal expression of {alpha}CP-4 in lung cancer, possibly associated with an altered processing of the {alpha}CP-4 mRNA leading to a predominant expression of {alpha}CP-4a. This may be considered as an example of alternative splicing involved in tumor suppressor gene inactivation. Finally, induction of {alpha}CP-4 expression reduced cell growth, in agreement with its proposed role as a tumor suppressor, and suggesting an association of this RNA-binding protein with lung carcinogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytogenetic analyses have established that genetic alterations on the short arm of chromosome 3 are among the most common abnormalities found in lung cancer. Loss of heterozygosity (LOH) at 3p, frequently at 3p21, occurs at the very earliest preneoplastic stages and has been also shown in histologically normal bronchial epithelium from patients with lung cancer and smokers without cancer (1, 2, 3) . It has been repeatedly hypothesized that persistent 3p21 LOH in lung cancer may indicate that this area harbors one or more tumor suppressor genes of which the inactivation is required for malignant transformation. This region has been extensively examined to characterize these tumor suppressor genes (4 , 5) . Although some candidates have been suggested (6, 7, 8) , the identification of tumor suppressor genes has proven elusive, and intense research efforts are currently devoted to this task. As part of this effort, in a detailed allelotyping analysis in tumors and tumor cell lines, multiple areas of discontinuous LOH throughout the 3p21 region were identified (4) .

PCBP4 is located at 3p21 and codes for {alpha}CP-4, a member of the polycytidylic acid binding protein (PCBP) family. PCBPs are RNA-binding proteins characterized by a high polycytidylic acid affinity. These are composed of two subsets in mammalian cells, hnRNPs K/J and {alpha}CP proteins (9) . The best-characterized {alpha}CP members are {alpha}CP-1 and {alpha}CP-2. These proteins are involved in several aspects of post-transcriptional gene regulation (9) . On the basis of sequence similarity, two novel {alpha}CP proteins, denoted as {alpha}CP-3 and {alpha}CP-4, have been characterized (10) . Almost simultaneously to the report identifying the new members of the {alpha}CP family, a novel cellular p53 target named MCG10 was found (11) . When MCG10 expression was induced in lung cancer cells they underwent apoptosis and cell cycle arrest in G2-M (11) . Because MCG10 mapped to 3p21, the authors suggested its potential role as a new tumor suppressor gene (11) . Sequence identity demonstrates that both {alpha}CP-4 and MCG10 are the same protein, although with some discrepancies at the NH2 terminus sequence (9) . One alternatively spliced form of {alpha}CP-4 has been described. This alternative form, termed {alpha}CP-4a, differs from {alpha}CP-4 in length and primary sequence at the COOH terminus (10) . Whereas no data on the intracellular localization of {alpha}CP-4 exist, immunofluorescence studies have revealed that {alpha}CP-4a expression is restricted to the cytoplasm in transfected HeLa cells (12) .

In view of all of these data, we aimed to characterize the expression of {alpha}CP-4 and its alternatively spliced form {alpha}CP-4a in normal lung and lung cancer to better evaluate its implication in lung carcinogenesis. We studied the expression of {alpha}CP-4 and {alpha}CP-4a mRNA and protein in lung cancer cell lines and NSCLC biopsies, respectively. This expression was compared with that in normal lung tissue and abnormal histological lesions such as epithelial hyperplasias. Because allelic losses are accepted as a preliminary indication of tumor suppressor gene inactivation, we determined the frequency of losses at PCBP4 locus in cancer cell lines [both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)] and NSCLC biopsies. We also searched for mutations on the coding domain sequence of the gene. Our results showed frequent lack of {alpha}CP-4 protein expression (predominantly in squamous cell carcinomas) associated with PCBP4 allele loss. Subsequently, the lack of {alpha}CP-4 was associated with poorly differentiated and highly proliferative tumors. No mutations were found in any of the lung cancer cell lines analyzed. Surprisingly, the expression of {alpha}CP-4a, the other protein derived from PCBP4 by alternative splicing, was apparently normal in lung tumors. We also confirmed the decrease in growth rate induced by {alpha}CP-4 expression. Taken together, our results indicate a possible role of {alpha}CP-4 in lung carcinogenesis and suggest the involvement of an RNA metabolism-related mechanism for inhibiting the expression of this protein.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
A range of lung cancer cell lines from the American Type Culture Collection (Manassas, VA) were used. Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin-streptomycin (Invitrogen, Carlsbad, CA). Cell lines included in the study were as follows: SCLC (H69, H82, H187, H345, H446, H510, N417, H209, and H774), adenocarcinoma (H23, H676, Calu-3, H1264, H441, and H2087), bronchioloalveolar carcinoma (A549), squamous cell carcinoma (HTB-58, H157, and H226), large cell carcinoma (H661, H1385, and H1299), and carcinoid (H720 and H727). Primary cultures of normal human bronchial epithelial cells (Clonetics, San Diego, CA) grown in supplemented epithelial cell growth media (BGEM; Clonetics) were also used.

Lung Cancer Biopsies.
Lung carcinoma samples were obtained under an institution-approved human tissue procurement protocol. The patients consisted of 5 females and 28 males ranging in age from 45 to 87 years (mean age, 62 years). All of them were current or former smokers except for 1. Most of them were patients at early stages in the moment of diagnosis (22 stage I, 7 stage II, 3 stage III, and 1 stage IV). The tumors consisted of 18 squamous cell carcinomas and 15 adenocarcinomas (including 4 bronchioloalveolar carcinomas). Tumors, adjacent areas to the tumor, and distant (usually >5 cm) normal uninvolved tissue were obtained. Samples were immersed in buffered formalin within 20 min from surgical resection. All of the samples were removed from the fixative solution after 24 h fixation. Tissues were paraffin embedded and sectioned (3 µm).

Antibody Production and Characterization.
Rabbit polyclonal antibodies against {alpha}CP-4 and {alpha}CP-4a were generated by Zymed Laboratories (San Francisco, CA). Specific COOH-terminal sequences of {alpha}CP-4 [peptide sequence (C)NGSKKAERQKFSPY] and {alpha}CP4a [peptide sequence (K)GLGGLHCQDGSS] were used for immunization. Antibodies were purified from sera by affinity chromatography after coupling of the peptides to the column gel (Sulfo Link Coupling Gel; Pierce, Rockford, IL). Pure antibodies were stored in phosphate buffer saline at 1 mg/ml. The characterization of the immunoreactivity was carried out by ELISA using the immobilized peptides. Western blotting with protein extracts from a cell line transfected with {alpha}CP4 or {alpha}CP4a cDNA showed the specificity of each antibody in the recognition of each variant.

Immunocytochemistry.
Biopsy sections (3 µm) from all of the patients were deparaffinized and incubated overnight at 4°C with a 1:1000 dilution of the rabbit polyclonal antibodies against {alpha}CP-4 or {alpha}CP-4a. After applying the ENVISION system (DAKO, Carpinteria, CA) for 30 min, the immunostaining was developed with diaminobenzidine and counterstained with Harris hematoxylin. Ki67 and p53 immunostaining were carried out using the monoclonal antibodies MM1 and DO-7, respectively (Novocastra Laboratories, Newcastle, United Kingdom). Tissue sections were microwaved at pH 6 for 15 min at highest power (1150 W) and 15 min at lowest power (700 W) to unmask the antigen. Sections were incubated overnight at 4°C with the primary antibody (MM1 diluted 1:100 or DO-7 diluted 1:80). For p53 immunostaining, the ENVISION system (DAKO) and the diaminobenzidine detection solution were applied. In the case of Ki67 immunostaining, we used a secondary antibody (1:200) containing rabbit antimouse IgG (DAKO) for 30 min, followed by an incubation with avidin-biotin complexes (DAKO). Adsorption of {alpha}CP-4 or {alpha}CP-4a antibodies in the presence of the synthetic peptides (10 µM) was used to confirm the specificity of the immunostaining.

Fluorescence in Situ Hybridization (FISH).
FISH analysis was performed in 21 primary tumors and the following lung cancer cell lines: H69, H82, H187, H345, H446, N417, H23, Calu-3, H1264, H441, A549, HTB-58, H157, H720, and H727. Tissue sections were obtained as indicated previously. Cultured cell lines were treated for 4 h with 0.01 g/ml Colcemid (Life Technologies, Inc., Gaithersburg, MD), treated with a hypotonic solution of 75 mM KCl, and fixed in 3:1 methanol:acetic acid. Two-color FISH analysis was performed with bacterial artificial chromosome RP11–314A5, which includes PCBP4, and with a fluorescein direct-labeled chromosome 3 centromeric probe (Qbiogene, GmbH, Heidelberg, Germany) used as control. Bacterial artificial chromosome DNA was labeled with SpectrumOrange fluorochrome (Vysis, Downers Grove, IL) by nick-translation. The localization of the bacterial artificial chromosome probe was verified by FISH on normal lymphocyte metaphases. FISH analysis was carried out as follows. Deparaffined tissue sections were pretreated with 30% sodium bisulfite (20 min at 45°C) and digested with 0.25 mg/ml proteinase K (10 min at 45°C) followed by ethanol dehydration. In the cell lines, slides were incubated in 2x SSC at 37°C for 15 min before ethanol dehydration. Samples and probes were denatured at 75°C (5 min and 12 min, respectively), and hybridization was performed at 37°C for 2 days. Posthybridization washes were performed at 73°C for 2 min in 2x SSC/0.3% NP40 and then samples were counterstained with DAPI II (Vysis). Slides were evaluated using a Zeiss Axioplan2 fluorescence microscope. To analyze tumor sections, the areas of neoplastic cells were determined by evaluating the adjacent sections with H&E staining. FISH signals were counted from 200 nuclei of the tumor regions, and the average ratio between the number of signals for the gene probe and the number of signals for the centromeric probe was calculated. Cut-offs were calculated as the mean percentage of false deletions/gains in normal cells plus three times the SD.

Mutation Analysis.
Exons 2–13 of PCBP4, the {alpha}CP-4 gene (accession no. 29793768), were sequenced using the Big Dye Terminator V1.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA). Genomic DNA from 24 lung cancer cell lines (15 NSCLC and 9 SCLC) was subjected to PCR amplification with a set of intronic primers flanking each exon. The PCR products were sequenced according to the protocol supplied by the manufacturer using an internal primer. The sequencing reactions were carried out in a Gene Amp PCR System 2400, and final products were processed with the ABI Prism377 DNA sequencer. Software used to analyze the results was ABI Prism377XL Collection, Sequence Navigator 1.0.1 and Sequencing Analysis 3.4.1.

Northern Blotting.
Total RNA was isolated from the cell lines using the guanidine isothiocyanate and cesium chloride method (13) . Fifteen µg of RNA were loaded per lane, run in 1% agarose gels containing 2.2 M formaldehyde, blotted onto nitrocellulose membranes, and baked for 2 h at 80°C. Equal loading and integrity of RNA were monitored by ethidium bromide staining of the 28 S subunit of rRNA. The human {alpha}CP4 cDNA probe (883 pb) used in this study was generated from a reverse transcription-PCR (RT-PCR) product cloned into the pDONR-201 vector (Invitrogen) and digested using SacI and XbaI. The oligonucleotide primers used for the amplification were {alpha}S-1 and {alpha}AS-1. All of the sequences of these and other primers used throughout the study are shown in Table 1Citation . The location of the primers and probes are shown in Fig. 1Citation . The RT-PCR product was sequenced to validate the integrity of the probe. Probe was labeled with [{alpha}-32P]dCTP (3000 Ci/mmol; NEN Life Science Products, Boston, MA) using the Prime-it RmT Random Primer Labeling kit (Stratagene, La Jolla, CA). Unincorporated nucleotides were removed by MicroSpin G-50 Columns (Amersham Pharmacia Biotech, Piscataway, NJ). Hybridization was carried out overnight at 42°C in a hybridization buffer containing 40% formamide (13) . After stringency washes, blots were exposed to X-ray films for various times.


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  		Table 1 Primers and probes

 


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  		Fig. 1. Scheme representing the {alpha}CP-4 cDNA structure (10) and the location of the primers and probes used in the study. The shadowed area represents the alternatively spliced sequence that originates {alpha}CP-4a.

 
Real-Time Semiquantitative RT-PCR.
Semiquantitative analysis of the {alpha}CP-4 mRNA expression was carried out by real-time RT-PCR with the ABI PRISM 7700 Sequence Detector and the software Sequence Detector version 1.6.3. (Perkin-Elmer/Applied Biosystems, Foster City, CA). RNA was isolated from all of the cell lines using the guanidine isothiocyanate and cesium chloride method. The reactions were performed with 2 µg of total RNA with minor differences from ABI 7700 manufacturer’s instructions. Relative levels of expression were determined by the Ct method. Every assay was performed in triplicate. For a separated quantification of {alpha}CP4 and {alpha}CP4a we designed specific primers for each variant (Table 1Citation ; Fig. 1Citation ). Primers for {alpha}CP-4 mRNA amplification were {alpha}S-3 and {alpha}AS-3 with the internal TaqMan probe for the amplified sequence denoted as {alpha}P-3. The primers and probe specific for {alpha}CP4a variant were {alpha}S-4, {alpha}AS-4, and {alpha}P-4. All of the experiments included a glyceraldehyde-3-phosphate dehydrogenase internal standard. The sequences of the primers and probe used for glyceraldehyde-3-phosphate dehydrogenase amplification are shown in Table 1Citation . Semiquantitative mRNA levels were expressed as a percentage relative to the glyceraldehyde-3-phosphate dehydrogenase mRNA.

Cell Growth Assay.
H1299 cells that inducibly express {alpha}CP-4 or {alpha}CP4a were generated as follows. Full-length {alpha}CP-4 or {alpha}CP-4a cDNA was cloned into a retroviral vector (pLTR) containing the two components of the reverse tetracycline-regulated system (14) . H1299 cells were infected with the retrovirus as described (14) . The effect of {alpha}CP-4 or {alpha}CP-4a overexpression on cell growth was studied using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based colorimetric assay cell growth proliferation kit (Roche, Mannheim, Germany). Briefly, cells were seeded at a density of 1 x 103 cells/well in 100 µl of culture medium into 96-well plates and allowed to adhere. The next day, expression of the protein was induced with doxycycline (1 mg/ml). Growth was measured daily up to 6 days, replacing the medium after 3 days of incubation. At each day, 10 ml of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-labeling reagent were added per well. After incubation for 4 h, the solubilization solution (100 µl) was added to each well, and the plate was additionally incubated overnight. Absorbance was measured using a microtiter plate reader.

Statistical Analysis.
Qualitative data obtained from the biopsies were analyze by Kendall’s correlation test. Data obtained from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide growth assay were analyzed by Student’s t test. A P < 0.05 was considered to be significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
{alpha}CP-4 Expression in Lung Cancer Biopsies.
To characterize the {alpha}CP-4 expression in biopsies we developed a polyclonal antibody specific for the COOH-terminal sequence of the protein. By immunocytochemistry, we analyzed biopsies from 33 NSCLCs, 15 adenocarcinomas, and 18 squamous cell carcinomas. We determined the presence of {alpha}CP-4 in both cancer cells and uninvolved lung tissue (Fig. 2, A–F)Citation . In normal lung bronchial epithelium a noticeable granular cytoplasmic expression, mostly in the apical region, was observed (Fig. 2, A and B)Citation . In contrast, normal alveoli showed no immunoreactivity. In the case of malignant tissue, 67% of cases lacked {alpha}CP-4 expression (47% adenocarcinomas and 83% squamous cell carcinomas). As observed in normal bronchioli, the location of the protein was cytoplasmic in the positive tumors (Fig. 2F)Citation , except for 1 adenocarcinoma in which a moderate nuclear staining was also detected. Interestingly, in hyperplastic bronchiolar epithelial tissue the protein was absent (Fig. 2C)Citation , except for 1 case in which we found a positive hyperplastic bronchiole next to an adenocarcinoma also expressing the protein. In areas of dysplasia found in our series, expression of {alpha}CP-4 was not found. On the other hand, there was an inverse correlation (Kendall’s {tau}-b coefficient = 0.507; P = 0.001) between the presence of {alpha}CP-4 in the tumors and their proliferative status determined by Ki67 expression (Fig. 3)Citation . A highly significant correlation (Kendall’s {tau}-b coefficient = 0.509; P < 0.001) was also found between the expression of {alpha}CP-4 and the histological grade of the tumor, where poorly differentiated tumors were associated to loss of {alpha}CP-4. Although {alpha}CP-4 has been identified as a p53 target gene, no correlation was found between {alpha}CP-4 expression and abnormal nuclear accumulation of p53, assessed by immunocytochemistry techniques (Kendall’s {tau}-b coefficient = 0.069; P = 0.720).



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  		Fig. 2. Immunocytochemical analysis shows that in lung cancer {alpha}CP-4 expression is different from {alpha}CP-4a expression. A, {alpha}CP-4 is present in the cytoplasm of bronchiolar epithelium cells. B, detail from previous image showing the accumulation of {alpha}CP-4 immunoreactivity close to the apical surface of the bronchial cells. C, {alpha}CP-4 expression is lost in hyperplastic bronchial epithelium. D, in the transition from normal bronchiolar epithelium (arrows) to tumor (arrowheads) a loss of {alpha}CP-4 expression can be observed. E, an adenocarcinoma with absence of {alpha}CP-4 expression. F, {alpha}CP-4 protein is located in the cytoplasm in a bronchioloalveolar carcinoma. G, example of a squamous cell carcinoma with a strong nuclear immunoreactivity for {alpha}CP-4a. H, expression of {alpha}CP-4a in the cytoplasm of cells from a bronchioloalveolar carcinoma. Original magnification of A, E, F, G, and H: x500; B: x1250; C and D, x125.

 


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  		Fig. 3. A, immunocytochemical staining for {alpha}CP-4 and Ki67 in serial reverse-face sections from two representative non-small cell lung cancers (bronchioloalveolar carcinoma and squamous cell carcinoma) shows an opposite expression of both proteins. Original magnification: x125. B, graph showing the correlation (n = 25; Kendall’s {tau}-b coefficient = 0.507; P = 0.001) between the expression of {alpha}CP-4 and the extension of Ki67 expression in non-small cell lung cancer biopsies.

 
Status of PCBP4.
Deletion at PCBP4 was determined by FISH using bacterial artificial chromosome RP11314-A5 labeled with SpectrumOrange. A fluorescein-labeled chromosome 3 centromeric probe was used as control. To established the cutoff of false deletion/gain results in the biopsies we carried out FISH analysis of 5 normal-appearing bronchiolar epithelial areas coming from the histologically normal regions of the samples from which tumors were obtained. For cell lines, blood samples from 5 healthy donors were also used as controls. Cutoff was calculated as mean percentage of false deletions/gains in controls plus three times the SD. In tissue sections, the percentage of false deletions (percentage of normal nuclei with a ratio below 1) was 12.4 ± 6.4 (mean ± SD), and thus a cutoff of 31.6% was used. This implies that tumors showing <31.6% of nuclei with a ratio below 1 were considered normal, whereas tumors with >31.6% of their nuclei with a ratio below 1 were considered to have PCBP4 deletion. On the other hand, percentage of normal nuclei with a ratio >1 (false gain) was 11.1 ± 5.1 (cutoff for gains 26.4%). In cell lines, the cutoff level for deletion was 4.1 (1.7 ± 0.8) and for gain was 5.4% (2.6 ± 0.95). Fifteen cell lines (both SCLC and NSCLC cell lines) were analyzed. The G banding analysis revealed complex karyotypes with structural rearrangements and frequent polyploidy. FISH analysis in SCLC cell lines showed PCBP4 allele deletion in H446, whereas H69, H82, H187, N417, and H345 were normal. In NSCLC cell lines, 4 of them showed deletion at PCBP4 (H23, A549, H720, and H441), 4 were normal (H1264, H727, HTB-58, and Calu-3), and in 1 case (H157) a gain of the gene was detected. In the NSCLC biopsies, 21 cases could be analyzed. One of 10 adenocarcinomas and 6 of 11 squamous cell carcinomas showed lost of one PCPB4 allele. We did not detect normal epithelial cells (alveolar or bronchiolar) with deletion of PCBP4, even in proximity to tumor cells.

When we compared the status of PCBP4 to the presence of {alpha}CP-4 in each tumor, a significant correlation (Kendall’s {tau}-b coefficient = 0.612; P < 0.001) existed between both parameters, showing a clear association between heterozygosity retention and expression of {alpha}CP-4 (Table 2)Citation .


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  		Table 2 Contingency table showing {alpha}CP-4 immunoreactivity versus PCBP4 deletiona

 
We then searched for mutations in the exonic sequence of PCBP4 (exons 2–13). We analyzed 15 NSCLC (H23, A549, H720, H358, H1264, H460, H157, H226, H2087, H727, H661, Calu-3, H441, HTB-58, and H676) and 9 SCLC (H209, N417, H446, H69, H345, H510, H82, H187, and H774) cell lines and found no mutations.

To evaluate the involvement of other mechanisms in the absence of {alpha}CP-4 expression we determined the presence of the {alpha}CP-4 mRNA by Northern blot analysis in normal human bronchial epithelial cells and in SCLC and NSCLC cell lines. {alpha}CP-4 mRNA was detected in all of them, with some cancer cell lines expressing high message levels. This fact was particularly evident in SCLC cell lines (Fig. 4)Citation .



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  		Fig. 4. Northern blot analysis of {alpha}CP-4 mRNA expression in normal human bronchial epithelium (NHBE) cells, small cell lung cancer (SCLC) cell lines and non-small cell lung cancer (NSCLC) cell lines. Fifteen µg of total RNA were added per lane, and ethidium bromide staining of 28 S was used to control equal loading and RNA integrity.

 
{alpha}CP-4a Expression in Lung Cancer.
Due to the lack of {alpha}CP-4 protein expression in most NSCLC tumors we investigated whether that was also the case for the alternative splice variant {alpha}CP-4a. By RT-PCR and using primers flanking the alternatively spliced region at exon 13 ({alpha}S-2 and {alpha}AS-2; Table 1Citation ; Fig. 1Citation ), we separately analyzed the expression of the {alpha}CP-4 splicing variants, {alpha}CP-4 and {alpha}CP-4a. Both {alpha}CP-4 and {alpha}CP-4a mRNA variants were detected in all of the cell lines as 550- and 350-bp bands, respectively (Fig. 5)Citation . Their identities were confirmed by cloning and sequencing of the band from H82 cell line. With this technical approach, the expression level of the {alpha}CP-4a transcript seemed to be lower than the expression of the {alpha}CP-4 transcript. This was confirmed with a semiquantitative real-time RT-PCR analysis carried out in some of the cell lines (Fig. 6)Citation . The primer design allowed us to distinguish between the two alternative splice forms (Fig. 1Citation ; Table 1Citation ). Interestingly, the ratio between both splice forms varied widely between the different cell lines (from 53.6 for H510 to 5.9 for H69 and H1385) suggesting a post-transcriptional regulation. We developed a polyclonal antibody for the specific COOH-terminal sequence of {alpha}CP-4a that does not recognize {alpha}CP-4. Immunocytochemical analysis in normal lung epithelium demonstrated that {alpha}CP-4a expression pattern was similar to what was observed for {alpha}CP-4, in which alveoli did not show immunoreactivity, and bronchioli presented cytoplasmic expression. However, in contrast to what was found for {alpha}CP-4 in tumors, {alpha}CP-4a was observed in all of the NSCLCs. The localization of the protein widely varied between samples, with tumors showing the protein in the nucleus (Fig. 2G)Citation , tumors showing cytoplasmic localization (Fig. 2H)Citation , and tumors expressing the protein in both cell compartments.



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  		Fig. 5. Reverse transcription-PCR amplification demonstrates the expression of the predicted 550- and 330-pb products corresponding to {alpha}CP-4 and {alpha}CP-4a, respectively, in normal human bronchial epithelium (NHBE) cells, small cell lung cancer cell lines and non-small cell lung cancer cell lines. A subsequence sequencing of both products from H82 mRNA confirmed the identity of the amplified sequences.

 


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  		Fig. 6. A, real-time PCR for the expression of the alternatively spliced variants {alpha}CP-4 and {alpha}CP-4a mRNAs in lung cancer cell lines and normal human bronchial epithelium (NHBE) cells. The Y axis represents the relation between {alpha}CP-4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (in percentage). B, ratio between the expression of the two alternatively spliced products from the {alpha}CP-4 pre-mRNA. SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer.

 
Effect of {alpha}CP-4 Overexpression on Cell Growth Rate.
It has been described previously that MCG10 induces growth suppression in the lung cancer cell line H1299 (11) . Although {alpha}CP-4 and MCG10 are the same protein, some discrepancies at the NH2 terminus amino acid sequence has been published (9) . Therefore, we wanted to evaluate whether {alpha}CP-4 has a similar effect on cell growth regulation. We examined the growth rate of {alpha}CP-4- and {alpha}CP-4a-inducible transfectants using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Overexpression of {alpha}CP-4 or {alpha}CP-4a was induced with 1 µg/ml of doxycycline in cells stably transfected with the retroviral vector pLTR containing the complete coding domain sequence for {alpha}CP-4 or {alpha}CP-4a. The cell rate was measured daily up to 6 days from the beginning of the induction. As observed in Fig. 7Citation A, {alpha}CP-4 overexpression was capable of reducing cell proliferation after 4 days from induction. This activity was not apparently shared with its alternative splice variant {alpha}CP-4a (Fig. 7B)Citation .



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  		Fig. 7. Effect of {alpha}CP-4 and {alpha}CP-4a overexpression on H1299 cell growth. Proliferation of {alpha}CP-4 (A) or {alpha}CP4a (B) inducible clones was evaluated using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based method. Cells were plated (103 cells/well) into 96-well plates and cultivated in the presence or absence of doxycycline (1 µg/ml). Data are presented as a function of absorbance (reflecting cell numbers) versus time (days after induction). Results are representative of four independent experiments and values are expressed as mean; bars, ±SD. **, P < 0.01; ***, P < 0.001.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Consistent allelic losses at 3p21 strongly support the hypothesis that one or more tumor suppressor genes essential for the development of lung cancer reside within this chromosomal region. Identification of these genes would be crucial to elucidate the molecular mechanisms of lung carcinogenesis. This information will be also useful for the development of molecular-based techniques for early detection of premalignant and malignant cells. Several candidate genes in 3p21 have already been identified as potential tumor suppressors. Most of the research efforts have focused on a 630-kb region defined by homozygous deletion (15, 16, 17) . In a previous study aimed to identify new potential tumor suppressor genes, 19 genes found in this region and located within a deleted overlap region of 370 kb were tested for mutations and loss of expression in lung cancer (5) . Several of them showed reduced or null expression in lung cancer cell lines, although none of them presented a frequent mutation rate. In additional studies, promoter hypermethylation was proven as a common mechanism for the silencing of these genes. Additional functional data have reinforced the role of some of these candidates as tumor suppressor genes (6 , 7 , 18, 19, 20, 21, 22) . By using 28 3p markers, Wistuba et al. (4) identified other discrete regions of allele loss where other tumor suppressor genes may be located.

In our study we have evaluated the expression of {alpha}CP-4, a candidate tumor suppressor gene located at 3p21 and identified recently (10 , 11) . In accordance with the possible role of {alpha}CP-4 as a tumor suppressor for lung tumors, we have detected a frequent lack of {alpha}CP-4 expression in NSCLCs, preferentially in squamous cell carcinomas. The loss of {alpha}CP-4 expression was associated with poorly differentiated and highly proliferative tumors. Due to the predominant presence of early stage tumors in our series, our data indicate that the alteration in {alpha}CP-4 occurs at the earliest clinically detectable phases of lung carcinogenesis. This is supported by the absence of {alpha}CP-4 expression in abnormal bronchial lesions such as hyperplasias. As a component of the p53 tumor suppressor pathway it could be argued that the lack of {alpha}CP-4 is a consequence of the functional alteration of p53 in the tumor. However, we have not found a correlation between p53 abnormalities (determined as p53 accumulation by immunocytochemistry) and lack of {alpha}CP4. Moreover, in some of the studied cases p53 alteration with simultaneous {alpha}CP4 expression was found. On the other hand, some tumors with apparently normal p53 (determined by the absence of p53 staining) were negative for {alpha}CP4. This indicates that p53 is not a necessary factor for {alpha}CP-4 transcription. In addition, the expression of {alpha}CP-4a, the alternative splice form, seems to be unaffected by p53 status, as normal bronchial epithelium, premalignant lesions, and tumors showed a strong expression of the protein.

In our study, we have also detected PCBP4 allele losses in 33% of lung cancer cell lines and primary NSCLCs. Using FISH analysis we compared the number of PCBP4 signals to the number of chromosome 3 centromeric signals to establish the existence of PCBP4 deletions. Because no allele-specific typing of the tumors has been performed, the PCPB4 deletions cannot undoubtedly be associated with LOH. This is particularly true in the case of cell lines showing chromosome 3 polyploidies. However, most primary tumors retain a normal number of chromosome 3 centromeres, indicating that there has not been chromosome 3 duplication (only 1 of 7 specimens with deletion of PCBP4 had a clear chromosome 3 polysomy). In this case, when we detect the loss of one PCBP4 signal, LOH is the most likely situation. Therefore, we can conclude that our results demonstrate frequent PCBP4 deletions most likely associated with LOH at this particular genomic region.

Although 3p allelic losses occur at very early preneoplastic stages of lung cancer, it is accepted that these losses are progressive, and advanced lesions and tumors often have deletions in most of the arm or the entire arm, whereas early lesions have more focal deletions. The NSCLC biopsies studied in our work corresponded preferentially to early stage cases (~90% of the specimens were obtained from patients diagnosed at stages I and II). Therefore, LOH at PCBP4 seems to be an early change in lung carcinogenesis and not a mere consequence of gross 3p alterations associated with advanced lung cancers. A clear relation between PCBP4 allele loss and the expression of {alpha}CP-4 has been found in the present study where the deletion of PCBP4 is associated with a loss of {alpha}CP-4 expression. In the classical two-hit model for inactivation of a tumor suppressor gene, the loss of one allele is frequently accompanied by an inactivating mutation of the retained allele (23) . We have analyzed the presence of mutations in PCBP4 (exons 2–13) in 24 lung cancer cell lines without detecting any alteration. This is common when searching for a tumor suppressor gene at 3p in lung cancer, where none or very rare mutations have been found in the potential tumor suppressors (5) . In fact, there is a general agreement that failing in detecting mutations in a limited number of samples does not necessarily exclude the role of the gene as a tumor suppressor. In sporadic human tumors, events such as transcriptional silencing by promoter hypermethylation or haploinsufficiency are alternative molecular mechanisms for gene inactivation. These processes result in absence or low mRNA levels due to a reduced transcription of the gene. All of the lung cancer cell lines analyzed in our study expressed {alpha}CP4 mRNA, even in the absence of one PCBP4 allele. In fact, in some lung cancer cell lines the mRNA levels were abnormally high when compared with those in primary cultures from normal bronchial cells. Therefore, our data do not suggest a role of promoter hypermethylation or haploinsufficiency in the {alpha}CP-4 inactivation. Another mechanism that can affect the expression of a potential tumor suppression gene is the alteration of the RNA processing machinery. Alternative splicing of pre-mRNA is an important regulatory mechanism to control gene expression and to generate proteins with distinct functions. Tumorigenesis and tumor progression have been related to the expression of alternatively spliced mRNAs encoding altered forms of proteins such as cyclin D1, mdm2, Fhit, TSG101, vascular endothelial growth factor, and CD44 (24, 25, 26, 27, 28, 29) . For that reason, we evaluated the expression of {alpha}CP-4a, the other protein derived from PCBP4 by alternative splicing, in lung cancer. We have been able to demonstrate that {alpha}CP-4a mRNA is expressed by lung cancer cell lines and, surprisingly, the protein is present in all lung tumors (although its intracellular location may change from normal to tumors). Considering together both {alpha}CP-4 and {alpha}CP-4a results, we can speculate that the lack of {alpha}CP-4 expression in lung cancer may be due to changes in the processing of the {alpha}CP-4 mRNA that lead to a predominant expression of {alpha}CP-4a.

Only speculation can be made about the mechanisms responsible for the altered processing of the {alpha}CP-4 mRNA. Many intracellular proteins are involved in the processing of the mRNA, and any alteration in their expression or function in cancer may change the processing of mRNAs such as that for {alpha}CP-4. Heterogeneous nuclear ribonucleoproteins are RNA binding proteins involved in mRNA splicing, localization, stabilization, nuclear-cytoplasm transport, and translation. In NSCLCs we have found a progressive increase in heterogeneous nuclear ribonucleoproteins A1, A2/B1, C1/C2, and K expression from normal lung epithelium to hyperplastic lesions and tumors (30) . Other heterogeneous nuclear ribonucleoproteins proteins and RNA binding proteins have been related to tumors (29 , 31, 32, 33, 34, 35) . Altered expression of alternative splicing regulatory factors has also been reported in in vitro models of cellular transformation (36) . The modifications in the expression of these RNA processing proteins in lung cancer may alter the fate of some mRNAs important for normal cell physiology giving rise to proteins associated with tumor formation and/or maintenance. For example, Bcl-xL and Bcl-xS are splice variants produced by alternative splicing of Bcl-x pre-mRNA. Whereas Bcl-xL is antiapoptotic, Bcl-xS has been shown to produce cell death (37) . In many malignancies, Bcl-xL is the predominant form, with low or undetectable levels of Bcl-xS (38, 39, 40, 41) .

Our knowledge on the cellular activity of {alpha}CP-4 is limited. In the search for novel cellular p53 target genes, MCG10 was identified as a gene specifically induced by wild-type p53 and DNA damage. In a lung cancer cell line (H1299) that inducibly overexpressed MCG10 it was found that this protein was able to increase apoptosis and cell cycle arrest in G2-M (11) . All of these data suggested that MCG10 may be a potential mediator of p53 tumor suppression. MCG10 and {alpha}CP-4 are the same protein, although MCG10 is truncated at the NH2 terminus. We have now confirmed the growth inhibition effect mediated by {alpha}CP4 overexpression. It can be suggested that the loss of {alpha}CP-4 expression found in the NSCLC biopsies would result in stimulation of cell proliferation with diminution of apoptosis. This would participate in the development of the malignant phenotype. This is in agreement with the correlation that exists between the lack of {alpha}CP-4 expression and the proliferative status of the tumor. The relevance of the loss of this protein in the prognosis of lung cancer merits examination.

Although nothing is known about the molecular mechanisms that mediate {alpha}CP-4 activity, it is known that disruption of the polycytidylic acid binding domains is necessary for inducing apoptosis and cell cycle arrest (11) . The best-known members of the {alpha}CP family are {alpha}CP-1 and {alpha}CP-2, also known as heterogeneous nuclear ribonucleoproteins E1/E2 or PCBP1/2. These proteins are involved in several biological processes mediated by their binding to C-rich single-strand motif in the RNA (9) . These proteins are linked to mRNA stabilization, translational activation, and translational silencing. Through the binding to specific mRNAs, {alpha}CP-4 may control the fate of specific mRNAs, regulating the cellular processes in which their proteins are involved (i.e. apoptosis and cell cycle arrest). {alpha}CP-4a, the alternatively spliced variant of {alpha}CP-4, differs in the primary COOH-terminal sequence from {alpha}CP-4. {alpha}CP-4a is shorter and does not contain potentially important {alpha}CP-4 motifs such as proline-rich domains, a nuclear export signal and a nuclear localization signal (11) . Interestingly, our data suggest a distinct effect of {alpha}CP-4 and {alpha}CP-4a on cell growth. Whereas {alpha}CP-4 expression is associated with reduced cell proliferation, in our study, {alpha}CP-4a overexpression did not apparently affect growth rate. Considering also its expression and location in lung tumors, this result strongly suggests that in vivo {alpha}CP-4a activity is different from that of {alpha}CP-4.

Although our data support that {alpha}CP-4 acts as a tumor suppressor protein, more detailed functional analyses such as gene transfer and gene disruption should be performed to clarify the role of {alpha}CP-4 in carcinogenesis. Additional studies must also be conducted to demonstrate that the replacement of its expression suppresses tumorigenicity in vivo. These functional evidences are required for the establishment of {alpha}CP-4 as a tumor suppressor gene of which the inhibition is important for malignant transformation. It should also be interesting to determine the involvement of the splice variant {alpha}CP-4a in lung carcinogenesis. Besides, an understanding of the regulatory mechanisms of alternative splicing occurring along with neoplastic transformation may help to elucidate some of the causes of this and other abnormal gene expressions in lung cancer. Understanding these mechanisms might also pave the way to restoring the function of tumor suppressor genes affected by splicing alterations.

In conclusion, our results, together with previous data implicating MCG10 in apoptosis and cell cycle arrest (11) , suggest a role of {alpha}CP-4 as a new tumor suppressor associated with lung carcinogenesis. Among the abnormalities described in our work, we have found frequent PCBP4 deletions in lung cancer. In the case of a NSCLC series, these deletions were associated with lack of {alpha}CP-4 expression, whereas {alpha}CP-4a was expressed in all of the tumors. Our data suggest that dysregulation of {alpha}CP4 mRNA metabolism may account for this modification, creating an abnormal expression pattern, which helps cancer growth. Additional studies on the {alpha}CP-4 functions and gene alterations would contribute to the elucidation of the molecular mechanisms involved in lung malignant transformation and would help in the improvement of prevention, detection, and treatment strategies for lung cancer.


   ACKNOWLEDGMENTS
 
We thank Amaya Lavin for technical assistance and Dr. Iñaki Martin-Subero for helping us with the FISH technique. We also thank Dr. Wenceslao Torre, Dr. Javier Zulueta, Dr. Maria Jose Pajares, Natalia Rey, and Usua Montes for their work in the collection of the NSCLC biopsies.


   FOOTNOTES
 
Grant support: "UTE project CIMA," and Fondo de Investigaciones Sanitarias (# 00/0835 and PI021116), Red Temática de Investigación Cooperativa de Centros de Cáncer del Ministerio de Sanidad y Consumo (# C03/10), and Departamento de Salud del Gobierno de Navarra (# 38/2003).

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

Requests for reprints: Ruben Pio, Department of Biochemistry, School of Medicine, University of Navarra, Irunlarrea 1, Pamplona 31080, Spain. Phone: 34-948-425600. Fax: 34-948-425649. E-mail: rpio{at}unav.es

Received 9/22/03. Revised 2/26/04. Accepted 3/24/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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