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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 80-85, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4880

Downregulation of hnRNP A2/B1 Expression in Tumor Cells under Prolonged Hypoxia

Mercedes Garayoa, Yan-Gao Man, Alfredo Martínez, Frank Cuttitta and James L. Mulshine

Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD; Department of Histology and Pathology, Carcinogenesis Unit, University of Navarra, Pamplona, Spain; and Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology, Washington DC.

Address correspondence to: Dr. James L. Mulshine, Intervention Section, Cell and Cancer Biology Branch, CCR, NCI, NIH, Bldg. 10, Room 12N226, 9000 Rockville Pike, Bethesda, MD 20892-1906. E-mail: mulshinj{at}mail.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 has been previously shown to be overexpressed in breast and lung tumors. Because hypoxia is a feature inherent in solid tumors, the regulation of hnRNP A2/B1 expression and subcellular localization under hypoxic conditions was studied on human lung and breast carcinoma cell lines. We found that sustained hypoxic treatment downregulated hnRNP A2/B1 expression in MCF7 and H157 cell lines. Northern blot analysis showed that this decay: (i) was observed as a marked diminution of transcript levels after 24–48 h of exposure to low oxygen tension; (ii) is not mediated by the transcription factor, hypoxia inducible factor-1; and (iii) is partially dependent on a higher hnRNP A2/B1 messenger RNA turnover under hypoxic than normoxic conditions. Immunocytochemical staining also showed a significant diminution of hnRNP A2/B1 staining in these cell lines after 24–48 h of hypoxia, together with a predominant loss of cytoplasmic staining. Further investigations are warranted to evaluate the relevance of modulation of hnRNP A2/B1 in hypoxic environments relative to its previously reported utility as a marker of early lung carcinogenesis.

Abbreviations: adenosine uridine, AU • deoxycytosinetriphosphate, dCTP • deformed epidermal autoregulatory factor-1, DEAF-1 • hypoxia-inducible factor 1, HIF-1 • heterogeneous nuclear ribonucleoprotein, hnRNP • messenger RNA, mRNA • 3' untranslated region, 3'UTR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins involved in various aspects of messenger RNA (mRNA) biogenesis (see reviews in Refs. 1 and 2). Among other functions, they have been reported to be involved in regulation of transcription and pre-mRNA alternative splicing in the nucleus (3, 4), in nucleocytoplasmic mRNA export (5), as well as to intervene in mRNA translation and turnover processes once in the cytoplasm (6, 7).

Several lines of evidence support a relationship between hnRNPs and both growth regulation and carcinogenesis. Some members of this family have been related to the binding and elongation of single stranded telomere repeats (8). In addition, hnRNP A2/B1 has been characterized as an early marker of lung cancer (9), since overexpression of this protein in exfoliated bronchial epithelial cells of archival sputum samples correlated with the eventual development of invasive tumor in three different clinical cohorts (10, 11). Further assessment of this attribute has emerged since: (i) hnRNP A2/B1 immunoreactivity in lung tissue was found to correlate with loss of heterozygosity for 14 lung cancer DNA markers, regardless of the normal morphologic phenotype of the cells (12); and (ii) hnRNP A2/B1 mRNA overexpression has been found to correlate with microsatellite alteration at 3p in non–small-cell lung cancer (13). In addition, hnRNP A2/B1 mRNA and protein display dynamic patterns of expression during mammalian lung development, with highest levels in primitive alveoli, and lowest levels in the adult lung (14), which is consistent with an oncofetal character for this molecule. More recently, hnRNP A2/B1 has also been evaluated as a possible marker for breast carcinogenesis (15), suggesting that the involvement of hnRNP A2/B1 overexpression may not only be of significance in lung carcinogenesis but in other epithelial systems as well.

A common feature inherent to the biologic processes of embryogenesis and carcinogenesis, both proliferative pathways where hnRNP A2/B1 is overexpressed, is the presence of focal areas of low oxygen tension (<= 2% O2) (16, 17). As a consequence of rapid cell proliferation in these situations, the distance to blood vessels exceeds the effective O2 diffusion distance and hypoxic areas arise. Several transcription factors, including hypoxia-inducible factor 1 (HIF-1), stimulatory protein 1, activator protein-1, nuclear factor {kappa}B, and p53 are known to be activated under such hypoxic conditions (see reviews in Refs. 18 and 19), and in turn, they regulate the expression of a series of genes that protect the cell and compensate for the potentially lethal hypoxic environment. Among these transcription factors, HIF-1 is known to be activated within physiologically relevant O2 concentrations (maximal response at 0.5% O2), in contrast to other transcription factors, which are induced only by very low O2 concentrations (below 0.02%), anoxic conditions, or even require reoxygenation (19).

In this study, we have investigated the modulation of hnRNP A2/B1 expression by hypoxia in human lung cancer (National Cancer Institute–lung squamous cell carcinoma [H157]) and in a breast cancer (breast adenocarcinoma) cell line (MCF7) because this molecule is reportedly overexpressed in both cancers. By analysis of the published sequence of hnRNP A2 (GenBank U09120), we identified two potential consensus sequences for HIF-1 binding sites (19) within the 5'-flanking region of the gene. Therefore, we have also investigated the putative role of this transcription factor in regulating hnRNP A2/B1 expression under conditions of different O2 tension. For these experiments, we used a previously defined model system in which immortalized embryonic fibroblasts from HIF-1{alpha} knockout and wild type mice were used. In accordance with the wide range of functions ascribed to hnRNPs in the different cellular compartments, hnRNP A2/B1 has been shown to have a nuclear and/or cytoplasmic localization in the cells (15). Therefore, special attention was paid to the distribution pattern of hnRNP A2/B1 expression within the cancer cells, as well as the nuclear and cytoplasmic staining ratio under the different experimental conditions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines, Hypoxia Treatment and Reagents
H157 and MCF7 human cell lines were purchased through the American Type Culture Collection (Manassas, VA). They were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES buffer and 100 units/ml penicillin, 100 µg/ml streptomycin (all tissue culture reagents obtained from GIBCO BRL, Gaithesburg, MD) and maintained free of mycoplasma contamination. The HIF-1{alpha} (-/-) fibroblast cell line was generated via SV-40 transformation of mouse embryonic fibroblast cells (20); HIF-1{alpha} (-/-) and HIF-1{alpha} (+/+) fibroblast cell lines were maintained with the same medium as the human tumor cell lines. Cells were cultured at 37°C in 20% O2, 5% CO2, and 75% N2 for normoxic conditions, and fresh medium was added 12 h prior to the beginning of each experiment. For hypoxic treatments, cells were cultured in a hypoxia chamber at 37°C in 1% O2, 5% CO2, and 94% N2 atmosphere for various periods of time, following previously validated methodology (21). For hnRNP mRNA stabilization studies, MCF7 cells were treated with 4 µg/ml actinomycin D (Sigma, St. Louis, MO) and then maintained under normoxic or hypoxic conditions for periods of time from 1 h to 5 h.

Immunocytochemical Study of hnRNP A2/B1 Expression
To confirm the production of hnRNP A2/B1 protein, we used the monoclonal antibody 703D4 previously reported to specifically recognize authentic hnRNP A2/B1 (9). Immunocytochemical staining was performed as previously described (22). H157 and MCF7 cells were cultured on glass slides under normoxic or hypoxic conditions for various times and then fixed in Bouin's fluid (Sigma) for 10 min at room temperature. To neutralize intrinsic peroxidase and background staining, slides were successively treated with a 3% H2O2 solution and 10% normal bovine serum for 30 min each at room temperature. Next, slides were incubated overnight at 4°C either with 703D4 at 4 µg/ml (9, 10) or with 10% normal bovine serum as a negative control. After 2 x 5 min washes in PBS, slides were sequentially incubated with the secondary antibody, streptavidin-conjugated peroxidase, and chromogen as reported (15). Finally, slides were lightly counterstained with hematoxylin, and mounted in 3% gelatin mounting medium for microscopic evaluation. For comparison of the number of hnRNP A2/B1 immunoreactive and nonreactive cells, 27 fields were randomly selected from each experimental condition to be photographed. Counting was performed on micrographs with a final magnification of 400x, and the percentages of immunoreactive and nonreactive cells for each group was calculated. The analysis of immunocytochemical images was independently assessed by two authors (Y.-G.M. and A.M.). Percentages among different groups were statistically compared with two-tailed Student's t test (P < 0.05 was considered statistically significant).

Northern Blot Analysis
Immediately after treatment for the indicated times, cells were washed once in PBS and total RNA was extracted using the guanidine isothiocyanate and cesium chloride method (23). A total of 15 µg of RNA were loaded per lane, run in 1% agarose gels containing 2.2 M formaldehyde, blotted by capillarity onto nitrocellulose membranes, and baked for 2 h at 80°C. The human hnRNP A2/B1 probe was a full-length complementary DNA for hnRNP A2 (15). The generation of a complementary DNA probe for human adrenomedullin, used here as an internal control for a hypoxia-inducible gene, has been described elsewhere (21). Probes were labeled with [{alpha}-32P]-deoxycytosinetriphosphate (dCTP) by random priming and hybridization was performed overnight at 42°C in a hybridization buffer containing 40% formamide (23). After stringency washes, blots were exposed to Kodak XAR film at –80°C for varying times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunocytochemical Staining
Immunocytochemical staining showed that the expression of hnRNP A2/B1 in H157 cells started to decline at 24 h of hypoxic treatment, and this decay became more prominent after 48 h of hypoxic condition as manifested by the weaker cytoplasmic intensity (Figure 1A and 1B). The statistical comparison of the number of cells with nuclear or cytoplasmic immunoreactivity after 48 h of normoxic or hypoxic conditions is listed in Table 1. It is shown that the percentage of H157 cells with only nuclear staining for hnRNP A2/B1 in normoxic conditions is not significantly different from that of cells exposed to 48 h of hypoxia. In contrast, the percentage of cells with exclusive cytoplasmic staining drops significantly in cells maintained in hypoxia for 48 h as compared with that of cells maintained for the same time under normoxic conditions (P < 0.001). Similarly, cells with both nuclear and cytoplasmic immunoreactivities were more widely represented in cells under normoxic than hypoxic conditions (P < 0.001). Furthermore, the frequency of cells displaying no hnRNP A2/B1 immunoreactivity under hypoxic conditions was significantly greater than that of those under normoxic conditions (P < 0.001; Table 1). A similar pattern of response for hnRNP A2/B1 immunoreactivity was seen in MCF7 cells under normoxic and hypoxic conditions (data not shown). In conclusion, the diminution of hnRNP A2/B1 immunoreactivity under hypoxia seems to be mainly due to a reduction of cytoplasmic staining, whereas nuclear staining remains unchanged.



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  		Figure 1. Demonstration of hnRNP A2/B1 immunoreactivity in H157 cell line after 48 h of culture under normoxic (A) or hypoxic (B) conditions. Note a clear reduction in immunocytochemical staining in cells subjected to hypoxia (B). x400 magnification.

 

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  		TABLE 1 Comparison of heterogeneous nuclear ribonucleoprotein A2/B1 immunopositive compartments in H157 cells after 48 h in normoxia or hypoxia

 
Downregulation of hnRNP A2/B1 mRNA in Tumor Cell Lines after Prolonged Hypoxia
Analysis of mRNA expression by Northern blotting in H157 cells showed that hnRNP A2/B1 transcript levels present in normoxic conditions were maintained after 24 h exposure to hypoxia; however, these levels were markedly decreased after 48 h of hypoxic treatment (Figure 2A). MCF7 cells showed a similar pattern of regulation, although hnRNP A2/B1 mRNA levels already exhibited a decay at 24 h exposure to hypoxia (Figure 2B). In contrast, transcript levels of adrenomedullin, a hypoxia-inducible gene, were elevated in both cell lines after exposure to 1% O2, and thus are shown here for comparative purposes (Figures 2A and 2B).



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  		Figure 2. Downregulation of hnRNP A2/B1 mRNA under hypoxia in H157 (A) and MCF7 cells (B). Northern blots were first probed for hnRNP A2/B1 expression and subsequently for human AM, a previously reported hypoxia-inducible gene (21), to compare both responses. Ethidium bromide staining of 28 S rRNA was used to assess for equal loading and RNA integrity.

 
hnRNP A2/B1 Transcripts Are Destabilized under Hypoxia
To investigate the mechanistic basis for the reduction of hnRNP A2/B1 mRNA expression by hypoxia, MCF7 cells were cultured in the presence of actinomycin D, a compound known to inhibit RNA synthesis, and then maintained under normoxic or hypoxic conditions from 1 to 5 h. hnRNP A2/B1 mRNA decayed more rapidly under hypoxic than under normoxic conditions (Figure 3). In fact, transcript levels in normoxia after actinomycin D addition remained very stable and exhibited little difference in expression over time. Densitometry analysis of the degradation of hnRNP A2/B1 mRNA in this experiment was performed, and the pooled data resulted in a calculated half-life of hnRNP A2/B1 mRNA of 16.5 h under normoxia and 4.0 h under hypoxia.



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  		Figure 3. hnRNP A2/B1 mRNA is less stable in hypoxic than in normoxic conditions. MCF7 cells were treated with actinomycin D and then maintained in either normoxic or hypoxic conditions for the indicated times. Ethidium bromide staining of 28 S rRNA was used to assess for equal loading and RNA integrity.

 
The Hypoxic Downregulation of hnRNP A2/B1 mRNA Is not Directly Dependent on HIF-1
One of the factors implicated in the transcriptional response to lowered oxygen levels is HIF-1. This transcription factor is a heterodimer consisting of the constitutively expressed aryl hydrocarbon receptor nuclear translocator/HIF-1ß and the hypoxically responsive component of the complex (HIF-1{alpha}) (19, 24). Comparison of HIF-1 DNA binding sites from 13 different genes reported to be under HIF-1 control revealed a decabase consensus motif (BR CGTG VBBB) as proposed by Wenger and Gassmann (19). Sequence analysis of the 5' flanking region of the hnRNP A2 gene (GenBank accession no. U09120) led us to identify two putative HIF-1 binding sites with no mismatch from the proposed consensus sequence (positions: -944 [CGCCCACGCC], -809 [GGCGTGGGGC]), and three putative sites with only one base mismatch outside the CGTG core (positions: -1,136 [CGCGTGtGGC], -932 [GCGCCACGaA] and -446 [GGGGCACGgA]); sequence analysis was performed on both sense and antisense orientation, and nucleotide positioning of HIF-1 binding sites was based on the hnRNP A2 transcriptional start site as +1. We therefore tested the possibility that HIF-1 could drive the hypoxic downregulation of hnRNP A2/B1 mRNA under hypoxia, using an HIF-1{alpha} knockout mouse fibroblast cell line and its wild-type counterpart (20). However, as it is shown in Figure 4, wild-type HIF-1{alpha} (+/+) and null HIF-1{alpha} (-/-) fibroblasts reveal a parallel level of downregulation for hnRNP A2/B1 mRNA under hypoxia. These results suggest that modulation of hnRNP A2/B1 expression by low O2 tension is not dependent on the transcription factor HIF-1.



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  		Figure 4. Northern blot analysis on HIF-1{alpha} wild-type and knockout mouse cell lines. Both HIF-1{alpha} (+/+) and HIF-1{alpha} (-/-) cells showed a similar hnRNP A2/B1 mRNA expression over the hypoxia time course, with a marked decrease of hnRNP A2/B1 transcripts at 48 h of oxygen deprivation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The common mechanistic feature that allows hnRNPs to account for their wide diversity of functions is their ability to recognize and interact with discrete DNA and RNA sequence elements to form RNP complexes, which subsequently modulate the accessibility or interactions of other trans-acting factors (2). In this sense, hnRNPs expression and localization could significantly influence gene expression by affecting RNA biogenesis and processing. Our interest in this study was to explore for potential differential regulation of hnRNP A2/B1 expression under hypoxic compared with normoxic conditions, because solid tumors frequently grow in low O2 environments (17). hnRNP has been found to be overexpressed in lung and breast cancer cell lines and tissues with interesting changes in cellular localization (9, 12, 14). A significant decay was observed for both hnRNP A2/B1 mRNA and protein levels in both H157 and MCF7 cell lines after 24 and 48 h of hypoxic treatment. To characterize the mechanism by which this downregulation occurs, we studied the half-life of the hnRNP A2/B1 message and found a rapid degradation under hypoxia, whereas in normoxic conditions this message was very stable. It is known that post-transcriptional regulatory mechanisms, specifically modulation of mRNA stability, can play a major role in gene expression (25). In many cases, it has been proven that mRNA turnover depends on mRNA binding proteins that specifically recognize adenosine uridine (AU)-rich elements or other sequence motifs in the 3' untranslated region (3'UTR), and either suppress or favor RNase degradation. Interestingly enough, hnRNP proteins are members of such RNA-binding transacting factors. Some of these proteins (hnRNP A1, C, and D) promote rapid mRNA turnover (6, 26), whereas other members of the family (such as hnRNP L) have a predominantly stabilizing effect (7). Of particular interest is that hnRNP A2 has also been shown to bind to the 3'UTR AU-rich elements of glucose transporter-1 mRNA and to mediate its rapid decay under ischemic or hypoglycemic conditions (27). In this sense, we can speculate that the observed downregulation of hnRNP A2/B1 expression after hypoxic treatment would diminish its destabilizing mRNA function, and may even allow the binding of other RNA-stabilizing proteins to the 3'UTR of the transcripts involved. Furthermore, if we consider that a similar scenario takes place in lung and breast solid tumors, the decrease of the destabilizing role of hnRNP A2/B1 mRNA due to the tumor-hypoxic environment could be a key element contributing to the upregulated expression of hypoxia-inducible genes with hnRNP A2/B1-binding elements in their transcripts. It is known that hypoxia-inducible genes (e.g., genes involved in glucose and energy metabolism, angiogenesis, and cellular proliferation and survival) are upregulated in solid tumors, and are essential for tumor cells to overcome the hypoxic stress and to achieve tumor growth (17, 28).

The immunocytochemical staining pattern of H157 cells after sustained hypoxic treatment reveals a diminution in the overall number of immunoreative cells consistent with our mRNA observations. These experiments were performed using the same antibody and assay methodology as our previous reports (12–15). In particular, after hypoxic treatment, percentages of cells with both nuclear and cytoplasmic staining or those with cytoplasmic staining alone decrease, whereas the percentage of cells showing only nuclear staining remains unaffected. These observations are suggestive of a predominant decrease of hnRNP A2/B1 levels in the cytoplasm, which could be attributable to reimport of hnRNP A2/B1 molecules to the nucleus and/or proteolytic degradation of cytoplasmic hnRNP A2/B1. Relocalization of immunoreactive hnRNPA2/B1 from the nucleus to the cytoplasm has been observed after treatment of breast cancer cell lines with retinoic acid (14), suggesting that translocation between the nuclear and cytoplasmic compartments may constitute a common mechanism of hnRNP A2/B1 regulation.

Based on the finding of at least two putative HIF-1 binding motifs in the hnRNP A2/B1 promoter region, we evaluated whether this transcription factor could be involved in the transcriptional regulation of hnRNP A2/B1 under hypoxia. However, under the hypoxic conditions tested, this seems not to be the case, because an HIF-1{alpha} knockout cell line showed a similar hnRNP A2/B1 mRNA expression to its wild-type counterpart. This finding was not surprising because HIF-1 has been reported to induce the transcriptional activation of many target genes (19), but to the best of our knowledge, no evidence has yet been found that it may directly function as a transcriptional repressor. Repression of HIF-1 transcriptional activation requires the interaction of HIF-1 with other transcription factors, such as p53 (29) or the murine single-minded–2 gene product (30). Other genes, such as aldehyde dehydrogenase-3, are also downregulated in certain cell types under hypoxic conditions; but interaction of HIF-1 with cis-acting elements within the aldehyde dehydrogenase-3 gene could not be proven (31). Therefore, although HIF-1 is the primary regulator of the adaptive responses to hypoxia, it seems likely that hnRNP A2/B1 mRNA decay is mediated by another transcription factor. In fact, HIF-1–independent mechanisms have been identified in the transcriptional regulation of several genes under hypoxic conditions (32–34), or after treatment with hypoxia mimetics (35). Recently, a nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)–related (NUDR) protein has been found to act as a transcriptional repressor of the hnRNP A2/B1 promoter (36); however, no current information suggests this kind of activity for nuclear DEAF-1–related factor under hypoxic conditions. The results of the actinomycin D experiments (Figure 3) suggest that destabilization of hnRNP A2/B1 transcripts is at least partially responsible for the decrease of hnRNP A2/B1 mRNA levels under hypoxia. It has also been reported that hnRNP A2 is regulated by direct interaction with the product of the von Hippel Lindau tumor suppressor gene through a mechanism involving ubiquitination and targeting to proteasome degradation (37).

In conclusion, we show that sustained hypoxic treatment (24–48 h) downregulates the expression of hnRNP A2/B1 in the lung carcinoma cell line H157. This same dynamic was also observed in a control breast cancer epithelial cell line (MCF7), suggesting that this hnRNP A2/B1–dependent biology is not exclusive to the respiratory compartment. This downregulation was observed as a decrease of mRNA levels as assessed by Northern blot analysis, which is not directly mediated by the HIF-1 transcription factor, and is partially dependent on hnRNP A2/B1 transcript destabilization. Immunocytochemical staining of both cell lines under prolonged hypoxic treatment revealed a predominant reduction of cytoplasmic levels of hnRNP A2/B1. These experiments suggest the possible existence of particular hypoxia-response genes subject to regulation by the compartmental expression of hnRNP A2/B1. The implications of the regulation of hnRNP A2/B1 expression at the level of mRNA stability make this an interesting finding that merits further investigation, especially in regard to the use of this molecule as a marker of early lung cancer.


   Acknowledgments
 
The authors are thankful to Drs. H. Ryan and R. Johnson for the HIF-1{alpha} knockout mouse fibroblast cell line, and to Dr. L. M. Montuenga for critical reading of the manuscript.

Received in original form April 17, 2002

Received in final form August 14, 2002


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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