1 Department of Biochemistry and Molecular Biology, University of Queensland, St Lucia Campus, QLD 4072, Australia
2 Epithelial Pathobiology Group, Centre for Immunology and Cancer Research, University of Queensland, Woolloongabba, QLD 4102, Australia
* Author for correspondence (e-mail: ross.s{at}uq.edu.au)
Accepted 20 April 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: hnRNP, Cell cycle dependence, p53, BRCA1, p21
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
More than 20 hnRNPs, many of which are characterised by possession of RNA-recognition motifs (RRMs), have been identified. The hnRNPs A/B are the major components of the 40S particles that package hnRNA. This packaging was originally envisaged to mimic that of histones in nucleosomes but this analogy appears to be inappropriate as the levels of the core proteins are not stoichiometric: they differ markedly between cell types, and their association with RNA is dependent on the nucleic acid sequence (Dreyfuss et al., 1993). However, given the role of these core particles in hnRNA packaging one might anticipate a correlation between the abundance of their proteins and the transcriptional activity in the cell. This is borne out experimentally: there is a marked difference in the concentration of hnRNP A1 between resting or slowly dividing cells and rapidly dividing cells. In the latter, hnRNPs A1 and A2 have been proposed to be present at similar levels. But hnRNP A1, which is abundant in a range of human, hamster and mouse proliferating cells, is present at markedly lower levels in confluent or resting cells, whereas hnRNP A2 is less affected (Celis et al., 1986
; LeStourgeon, 1978).
The levels of hnRNPs A/B differ not only between proliferating and resting cells: some also fluctuate during the cell cycle (Leser and Martin, 1987; Minoo et al., 1989
). In HeLa cells hnRNPs A2 and B1 are synthesized in the G1 phase, and their levels fall in G2 and M phases, with hnRNP B1 protein level falling more markedly (Kamma et al., 2001
). The relative and absolute levels of these two proteins also differ markedly between tissues, both being particularly abundant in rat brain, testis, lung, spleen and ovary (Kamma et al., 1999
; Ma et al., 2002
).
Many genes show a correlation between strong expression in proliferating cancer cells and the fluctuations in the protein level across the cell cycle (Dreyfuss et al., 1993; Whitfield et al., 2002
). The levels of hnRNP A/B proteins are of particular interest as it has been suggested that upregulation of some members of this protein family is associated, as either a cause or consequence, with cellular proliferation and cancer. The hnRNP A2 and its longer B1 isoform are expressed at an early stage in a variety of tumours and have been proposed as early markers for cancer, especially lung cancer (Fielding et al., 1999
; Mulshine et al., 2002
; Pino et al., 2003
; Sueoka et al., 1999
; Whitfield et al., 2002
; Zhou et al., 1996
) and possibly breast cancer (Zhou et al., 2001a
). The upregulation of hnRNP A2/B1 in cancer parallels its expression in lung development, supporting the view that it is an oncodevelopmental protein (Montuenga et al., 1998
). Levels of hnRNP A1 are also elevated in some cancers, including oligodendrogliomas (Xu et al., 2001
).
We report here studies of hnRNP A1, A2 and A3 expression in a range of normal and transformed cells. Until recently, the published components of hnRNP core particles excluded hnRNP A3 even though it is very closely related to hnRNPs A1 and A2 (Percipalle et al., 2002; Rappsilber et al., 2002
). The tandem RRMs of hnRNP A3 have a higher sequence identity than hnRNP A2 with hnRNP A1, and its glycine-rich region more closely matches hnRNP A2 than hnRNP A1 (Ma et al., 2002
). Moreover, hnRNP A3 binding to single-stranded oligoribonucleotides parallels that of hnRNP A2, reinforcing the close relationship between these two paralogues. The levels of these three proteins, however, varied markedly between cell types. The expression of hnRNP A2 and to a lesser extent A1, but not their mRNAs, fluctuated during the cell cycle, peaking during S phase, declining in G2 and M phases, and being restored in late G1. By contrast, hnRNP A3 levels remained constant. In general, hnRNPs A2/B1 are overexpressed in various epithelial cancer cells when compared with normal cells, but some cancers showed reduced expression of hnRNP B1 and overexpression of hnRNP A3. Suppression of hnRNP A2, but not A1 or A3, with shRNA treatment lowered the growth rate of HaCaT and Colo16 cells, whereas simultaneous downregulation of any two of the three genes had a more marked effect on cell growth. Suppression of the hnRNP A2/B1 gene did not affect the level of p53, which has been shown to be dysregulated in most cancer cells, but was accompanied by downregulation of BRCA1 and upregulation of p21, proteins that are closely linked to cell growth.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies
Rabbit polyclonal antibodies against peptides unique to hnRNPs A1, A2, B1 and A3 were raised in our laboratory. The synthetic peptides, SKSESPKEPEQLC for A1, GGNFGFGDSRGC for A2, VKPPPGRPQPDSC for A3 and KTLETVPLERKKC for B1 were conjugated to diphtheria toxin before injection into rabbits. The A1 antibody binds A1 and the minor A1B isoform (the latter not visible on Fig. 1B); the A2 antibody detects A2 and its B1 isoform and the A3 antibody recognizes all four isoforms, which are unresolved on the short gels used in these experiments. In immunofluorescence studies, a mouse monoclonal antibody against -tubulin (Sigma; used at 1:100 dilution) was used to counterstain the cell cytoskeleton, together with antibodies against hnRNPs A1, A2, B1 or A3. The secondary antibodies included FITC-conjugated goat anti-rabbit IgG (Sigma; 1:50 dilution) and Cy3-conjugated sheep anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA; 1:300 dilution). For western blotting, mouse monoclonal antibodies were used to detect BRCA1 (Oncogene, Boston, MA; 1:500 dilution), p53 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:2000 dilution), p21 (Oncogene; 1:2000 dilution), and GAPDH (Chemicon International, Boronia, Australia; 1:50,000 dilution). The secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Sigma; 1:5000 dilution) and HRP-conjugated rabbit anti-mouse IgG (Sigma; 1:5000 dilution).
|
Immunocytochemistry
Cells cultured on coverslips were collected at S, G2, M and G1 phases, washed twice in chilled phosphate-buffered saline (PBS, pH 7.0), fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature and rinsed twice with cold PBS prior to immediate staining or storage at 4°C. Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes, rinsed twice with PBS, then incubated in 50 mM ammonium chloride in PBS for 10 minutes before washing again with PBS. Following a 30-minute block in 0.2% bovine serum albumin (BSA) in PBS, cells were incubated in the primary antibody diluted in PBS containing 0.2% BSA for 1 hour at 37°C and then washed four times with PBS. The cells were incubated with secondary antibodies diluted in PBS containing 0.2% BSA for 30 minutes in the dark. Finally, coverslips were mounted with Vectashield containing 4',6-diamidino-2-phenylindole hydrochloride (DAPI) (Vector Laboratories, Burlingame, CA) before being visualized with BioRad Radiance 2000MP confocal and multiphoton microscope (BioRad, Hercules, CA).
Western blotting
Cells were lysed in a total protein extraction buffer (Hoek et al., 1998). Equal amounts of protein (20 µg/lane for detection of hnRNPs A1, A2 and A3, and 30 µg/lane for B1) were separated on SDS/12% polyacrylamide gels (Gradipore, Sydney, Australia) and transferred onto polyvinylidene difluoride membranes (Millipore, Sydney, Australia). The membrane was then incubated overnight at 4°C in a blocking buffer (58 mM Na2HPO4, 17 mM NaH2PO4, 68 mM NaCl, 0.1% Tween-20 and 1% skimmed milk powder). The membrane was probed with antibodies against hnRNP A/B proteins in the blocking buffer for 1 hour at room temperature, washed four times with the blocking buffer mixed with 0.5% BSA, incubated in blocking buffer containing an HRP-conjugated goat anti-mouse antibody for 45 minutes and washed again. The target proteins were detected with the ECL-PLUS western blotting detection system (Amersham Biosciences, Buckinghamshire, UK) and recorded on X-ray film. The membrane was then stripped in 62.5 mM Tris-HCl, pH 6.8, 100 mM ß-mercaptoethanol and 2% SDS in a 60°C oven, followed by a thorough wash with 0.1% Tween-20 in PBS. GAPDH on the stripped membrane was detected using a mouse anti-GAPDH monoclonal antibody. Band intensities were assessed using an imaging densitometer to determine the relative abundance of the proteins, using GAPDH as a loading control. To detect BRCA1, 100 µg/lane of total protein was separated on an SDS/5% polyacrylamide gel. Western blotting for BRCA1 was performed using a mouse anti-BRCA1 monoclonal antibody buffered with 5% skimmed milk powder in PBS containing 0.15% Tween-20. The mouse monoclonal antibody against p53 and p21 was diluted in 5% skimmed milk powder/PBS and 5% BSA, respectively.
Northern blotting
Total RNA was isolated using TRIzol reagent (Invitrogen), separated on 1.2% agarose/formaldehyde gels and transferred onto nylon membranes (Amersham Biosciences). The probes for BRCA1 and p21 were labelled by random-primer synthesis with Klenow DNA polymerase in the presence of [32P]dCTP. A p21 cDNA fragment from 1653 to 1856 bases downstream of the starting codon, and the first 1 kb of the coding region of BRCA1 mRNA were used as templates. Hybridization was performed in Rapid-hyb buffer (Amersham Biosciences) and the signals were visualized by autoradiography. Signals of the mRNAs of interest were normalized to the levels of ß-actin mRNA.
Real-time PCR
Total RNA isolated by TRIzol reagent was reverse-transcribed to produce cDNA using Superscript III (Invitrogen). mRNAs of interest were quantified by SYBR green real-time PCR on an ABI 7700 Sequence Detector (Applied Biosystems, Foster City, CA) using ß-actin mRNA as a normalization control. The specific primers (Proligo, Lismore, Australia or GeneWorks, Adelaide, Australia) were: hnRNP A1 forward, 5'-GGAGAAGCCATTGTCTTCGGA-3' and reverse, 5'-GCATAGGATGTGCCAACAATCA-3'; hnRNP A2 forward, 5'-GAGTCCGCGATGGAGAGAGA-3' and reverse, 5'-GATCCCTCATTACCACACAGTCTGT-3'; hnRNP A3 forward, 5'-CATAGAAGTTATGGAAGACAGGCAGAG-3' and reverse, 5'-GGCCTTTTTCACTTCACAATTATGC-3'; hnRNP B1 forward, 5'-GGAGAAAACTTTAGAAACTGTTCCTTTG-3' and reverse, 5'-GCTTTCCCCATTGTTCGTAGTAGT-3'; ß-actin forward, 5'-CGTTACACCCTTTCTTGACAAAACC-3' and reverse, 5'-GCTGTCACCTTCACCGTTCCA-3'; p21 forward, 5'-TAGCAGCGGAACAAGGAGTCA-3' and reverse, 5'-GCCAGTATGTTACAGGAGCTGGAA-3'.
shRNA and transfection
The selected shRNA sequences targeting hnRNP A1, A2 and A3 mRNAs were 5'-AGCAAGAGATGGCTAGTGC-3', 5'-CGTGCTGTAGCAAGAGAGG-3' and 5'-AGAGAGCTGTTTCTAGAGA-3', respectively. BLAST alignment showed no sequence identity with other transcripts of human genes. An oligonucleotide (5'-CGTACGCGGAATACTTCGA-3') targeting firefly luciferase mRNA was used as a negative control. The synthesized oligonucleotides (GeneWorks) were annealed and inserted into pSUPER plasmids following established protocols (Brummelkamp et al., 2002). For transfection, exponentially growing cells were seeded into a six-well plate at a density of 360,000 cells/well and cultured for 20 hours before the medium was replaced with OptiMEM with 5% FBS. shRNA plasmids (3.0 µg) were mixed with 7.5 µl Lipofectamine 2000 (Invitrogen) and incubated at room temperature for 20 minutes before being applied to cells. The medium was changed back to RPMI 1640 with 10% FBS 6 hours after transfection, and cells were cultured for another 66 hours before harvesting, unless otherwise specified.
MTT proliferation assay
Cells were trypsinized 48 hours after transfection, counted and replated into 96-well plates at 1500 cells/well (Time 0 started at this point). After being re-plated for 3 hours, cells were sampled at 6-hour intervals to perform an MTT proliferation assay. For each time point, six wells were used for each treatment; 10 µl methylthiazoletetrazolium (MTT; Sigma; 5 mg/ml in PBS) was added to three and an equal volume of PBS to the other three as a control. The cells were cultured in the presence or absence of MTT for another 3 hours and lysed in an SDS-HCl solubilization buffer (10% SDS in 0.01 M HCl) for 18 hours at 37°C before measuring the absorbance at 570 nm. The experiment was repeated three times for each cell line.
TUNEL assay
Cell apoptosis was determined with an in situ cell death detection kit (Roche Diagnostics Australia, Castle Hill, Australia) following the manufacturer's protocol.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lysates were prepared from synchronously growing Colo16 and HaCaT cells harvested at intervals after being released from a double thymidine block. The progression through the cell cycle was determined by flow cytometry. The S, G2, M and G1 phases of Colo16 cells started at 0, 7, 8 and 10 hours post-synchronization, respectively, whereas HaCaT cells entered G2, M, and G1 phases 1 hour later than Colo16 cells (Fig. 1A). Proteins were isolated from cell lysates, separated by SDS/polyacrylamide gel electrophoresis and immunoblotted using antibodies raised against peptides from hnRNPs A1, A2, B1 and A3. The specificities of these antibodies were confirmed by western blotting (Fig. 1B). hnRNPs A1, A2 and B1 all manifested cell cycle-specific changes though their profiles differed (Fig. 1C). The amount of hnRNP A1 was relatively stable during most of the cell cycle except for a clear decrease in early G1 phases for both cell lines, whereas hnRNP B1 expression dropped markedly during the short period from G2 to M phases, remained low in early G1 and slowly increased from late G1. In contrast to a previous observation in HeLa cells (Kamma et al., 2001), a dramatic change of hnRNP A2 expression was detected in both cell lines, though Colo16 cells expressed the lowest level of hnRNP A2 in G2 phase, and HaCaT cells in G1. No change was detected in the hnRNP A3 protein level at different cell division stages (Fig. 1C).
In view of the significant fluctuation of hnRNP A2 and B1 proteins in different cell cycle stages, we also investigated the localization of these proteins in the nucleus and cytoplasm. Colo16 cells collected at S, G2, M and G1 phases were immunostained with the antibody that recognizes hnRNPs A2 and B1. The predominantly nuclear localization for both proteins was not significantly different in S, G2 and G1 phases (Fig. 2). During M phase, the proteins were almost uniformly distributed in the cell body as a result of collapse of the nuclear envelope. Interestingly, hnRNPs A2 and B1 were found at lower levels around the chromosomes than other parts of M phase cells (Fig. 2).
|
|
Expression of hnRNPs A1, A2, B1 and A3 in cancer and normal cells
The levels of hnRNPs A2 and B1, or B1 alone in some cases, are elevated in lung, breast cancers and lymphomas (Sueoka et al., 1999; Sueoka et al., 2001
; Tani et al., 2002
; Zhou et al., 2001a
). Nevertheless, controversy still exists on whether hnRNP B1 or both A2 and B1 are overexpressed in these tissues. We therefore assessed the amounts of hnRNP A/B proteins by western blotting in normal keratinocytes, and in cancer or immortalized cell lines. The expression patterns of hnRNPs A1, A2, B1 and A3 differed considerably between various cell lines, though all were epithelial in origin (Fig. 4). The breast cancer cell line, T-47D, expressed higher levels of all of the detected proteins compared with the immortalized cells, MCF10a, whereas another breast cell line, MCF7, showed only a higher hnRNP B1 level. Upregulation of hnRNP A2 or B1, or both, was detected in all cancer cell lines compared to normal keratinocytes or immortalized cells, whereas A1 was less abundant in MCF10a, MCF7 and two prostate cell lines. Higher levels of hnRNP A3 were found in Colo16, A549 and T-47D cells. Significantly higher levels of hnRNP B1, which is overexpressed in a variety of cancers (Sueoka et al., 1999
; Sueoka et al., 2001
; Tani et al., 2002
), were detected in Colo16, A549, T-47D and RPMEI cells.
|
|
|
The effect of suppression of hnRNP A1, A2 and A3 was first assessed by cell counting. Colo16 cells were transfected with pS-A1, pS-A2, pS-A3 or a combination of two of them, and the cell number was compared with cells transfected with pS and pS-Luc after 72 hours post-transfection. Reducing hnRNPs A1 or A3 did not affect cell growth whereas suppression of A2 isoforms caused a small but statistically significant reduction in cell number (Fig. 7). Simultaneous suppression of any two of the hnRNPs slowed cell proliferation, with the combination of pS-A1 and pS-A2 affecting cell growth most significantly (Fig. 7). Secondly, an MTT proliferation assay, in which expression of hnRNP A2 and its alternative splicing isoforms was suppressed, was performed to support the above data. Colo16 and HaCaT cells were transfected as above, and after 48 hours, the cells were re-plated into 96-well plates. Between 3 and 45 hours after re-plating, cells were sampled at 6-hour intervals for MTT proliferation assays. A decrease in the proliferation rate of cells treated with pS-A2 was observed, whereas pS and pS-Luc had no impact (Fig. 8 and Table 1).
|
|
|
The slower proliferation of the pS-A2-treated Colo16 cells was unlikely to have resulted from apoptosis because the TUNEL assay did not show significant increases in apoptotic cells (data not shown). Flow cytometry also revealed similar percentages of apoptotic cells in the treatments with pS, pS-Luc and pS-A2 (data not shown).
hnRNP A2 suppression inhibits expression of BRCA1 but induces p21
The cell growth retardation caused by suppression of hnRNP A2 isoform expression led us to explore the effect of these proteins on several known cell cycle regulation factors. We chose p21 because it not only controls the cell cycle progression through its inhibitory binding to cyclin/cyclin-dependent kinase complexes, the driving force for cell cycle progression, but also inhibits DNA synthesis and proliferating cell nuclear antigen (PCNA) activity (Pan et al., 1995; Waga et al., 1997
), which is pivotal for nucleic acid metabolism (Kelman, 1997
). Cell cycle checkpoints are regulated through p21 by p53, which activates cell apoptosis in the presence of irreparable DNA damage, to inhibit tumour development (el-Deiry et al., 1993
). BRCA1 can also regulate p21 expression by binding to the p21 promoter region (Somasundaram et al., 1997
), and loss of BRCA1 function is associated with cell cycle defects and p21 induction in the mouse (Hakem et al., 1996
) and p21 overexpression in human tumours (Sourvinos and Spandidos, 1998
). We compared the expression levels of p53, BRCA1 and p21, which are known to be involved in cell growth regulation, in Colo16 cells before and after pS-A2 transfection. Western blotting showed that the p53 level was maintained (Fig. 9A) but BRCA1 decreased markedly (Fig. 9B) in Colo16 cells when hnRNP A2 isoforms were suppressed. Analysis of BRCA1 gene expression by northern blotting confirmed that this suppression resulted from the deficiency of hnRNP A2 family proteins (Fig. 9C). On the other hand, expression of the cyclin-dependent kinase inhibitor p21, increased significantly at both protein and mRNA levels (Fig. 10) following suppression of hnRNP A2. These results suggest a linkage between the functions of hnRNP A2 isoforms in cell proliferation and the expression of cell growth regulators such as BRCA1 and p21, which are known to control the growth of various types of cells (el-Deiry et al., 1993
; Zhang et al., 1995
).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cancer cell lines showed widely varying levels of the hnRNP A/B proteins. Although these cell lines were all epithelial in origin, they displayed different expression patterns for hnRNPs A2 and B1 (Fig. 4). Two cell lines, Colo16 and T-47D, expressed high levels of both hnRNPs A2 and B1. In accord with previous immunohistochemistry studies (Sueoka et al., 1999; Sueoka et al., 2001
), lung cancer cells overexpressed hnRNP B1 rather than A2 (Fig. 4). On the other hand, hnRNP B1 was reduced in the prostate cancer cell line, LNCAP, but not in an immortalized prostate epithelial cell-line RPMEI (Fig. 4). This result, which parallels that observed in adult T-cell lymphoma/leukaemia (Tani et al., 2002
), suggests that increased hnRNP B1 expression is not a universal phenomenon in different kinds of cancer. Overexpression of hnRNP B1 may be useful for the diagnosis of lung cancer as previously suggested (Hamasaki et al., 2001
; Sueoka et al., 1999
; Sueoka et al., 2001
), but is clearly inappropriate for other cancer types. In addition we observed overexpression of hnRNP A3 in three out of seven cancer cell lines.
Proliferation-dependent expression of hnRNP A1 in different cell types at both the protein (Celis et al., 1986; LeStourgeon, 1978; Loeb et al., 1976
; Martin et al., 1979
) and mRNA levels (Biamonti et al., 1993
; Buvoli et al., 1988
) has been reported. Levels of hnRNP A2 mRNA also increase in proliferating fibroblasts compared to the quiescent state (Biamonti et al., 1993
; Martin et al., 1979
). The low levels of hnRNPs A1 and A2 in slowly dividing cells led to the proposition that they may play a role during cell division and growth. Our observation of significant fluctuations in hnRNP A1, A2 and B1 expression during the cell cycle (Fig. 1C) presents further evidence that these proteins are specifically required at certain stages of the cell cycle. However, the levels of hnRNP A1, A2, B1 and A3 gene transcripts did not change significantly during cell division, except for that of hnRNP A3 in the G1-phase of HaCaT cells, suggesting that the modulation of their protein expression occurred at the post-transcriptional level, through changes in the rate of translation or protein degradation. This is the first demonstration of post-transcriptional regulation of these genes during the cell cycle.
The sequence similarity of hnRNP A3 to A1 and A2 (Good et al., 1993; Ma et al., 2002
) raised the question of whether A3 is similarly modulated to the latter two during the cell cycle. In Xenopus, hnRNP A3 mRNA was most abundant in the ovary and late embryonic stages in parallel with A1 and A2, suggesting A3 may contribute to rapid cell proliferation in embryogenesis (Good et al., 1993
). In addition, an in vitro overlay assay showed that hnRNP A3 bound to protein kinase C (Rosenberger et al., 2002
), which is involved in the control of cell division and differentiation (Clemens et al., 1992
; Fishman et al., 1998
; Schwantke et al., 1985
; Watters and Parsons, 1999
), suggesting a role for A3 during certain stages of cell division. Our experiments, however, showed no apparent change in either mRNA or protein level of hnRNP A3, and its function during the cell cycle remains to be elucidated.
The effects of downregulation of hnRNPs A1, A2, B1 and A3 provided further evidence that these proteins play important roles in cell growth. Specific shRNA suppression of hnRNP A1 or A3 had no effect, suggesting that neither is essential for cell growth. Our results for hnRNP A1 are consistent with previous findings in the mouse erythroleukaemic CB3 cell line (Ben-David et al., 1992) and in HeLa and HCT116 cells (Patry et al., 2003
). However, suppressing expression of hnRNPs A1 and A3 at the same time did affect cell growth, suggesting that these two proteins may functionally compensate for each other. This result is in accord with the high sequence identity of these two proteins (Ma et al., 2002
).
Colo16 and HaCaT cells with lowered levels of hnRNP A2 isoforms were found to grow at a slower rate, though they can still proliferate (Figs 7 and 8), in contrast to the results of Chabot and co-workers who found that suppressing hnRNP A2 by siRNA duplexes had no effect on the proliferative capacity of HeLa and HCT116 cells (Patry et al., 2003). This difference may be due to the variable performance of siRNAs in different cell lines. In this study, Colo16 cells showed a suppression efficiency of 70-95% (based on western blotting results) for hnRNP A2 shRNA, depending on the degree of cell confluency before transfection. Cells with a simultaneous reduction in A1 and A2 exhibited more significant growth retardation compared to those lacking A2 alone. Similar synergistic effects with hnRNPs A1 and A2 have been observed in HeLa and HCT116 cells (Patry et al., 2003
). However, there was no such effect when shRNAs against hnRNP A2 and A3 were used together: the growth rate was comparable to that of cells treated with A2 shRNA alone.
Simultaneous suppression of hnRNPs A1 and A2 in HeLa cells results in apoptosis through their effect on the telomere capping process (Patry et al., 2003). Earlier proteomic studies also suggested that hnRNPs A1 and A2/B1 are involved in the
CD95-induced apoptosis in Jurkat T cells because of the translocation, cleavage and dephosphorylation of these proteins during apoptosis (Brockstedt et al., 1998
; Hermann et al., 2001
; Thiede et al., 2002
). In our study, apoptosis was not detected by either the TUNEL assay or flow cytometry analyses. Colo16 cells deficient in A1/A2, A1/A3, A2/A3 or A2 alone were able to proliferate, but at a lowered rate, and no truncated forms of these proteins were detected in our western blots, suggesting a mechanism differing from that observed in Jurkat T cells (Brockstedt et al., 1998
; Thiede et al., 2001
; Thiede et al., 2002
). The mechanism behind the growth retardation observed in A2-suppressed Colo16 cells is unknown. As hnRNPs A/B proteins play a major role in splicing, trafficking and export of mRNA out of nucleus, their suppression may impede the mRNA processing and trafficking, causing a reduction in cell growth rates.
In our study we have examined the effects of the hnRNP A2 proteins on a group of cell growth regulation factors, including p53, p21 and BRCA1, which have been implicated in the uncontrolled proliferation of tumour cells (Hakem et al., 1996; Perez-Roger et al., 1999
; Ullrich et al., 1992
). The changes in expression of p21 and BRCA1 in cells with reduced levels of hnRNP A2 are consistent with the observed reduction in proliferation rate of Colo16 cells, and also correlated with the well-established negative roles that two proteins play in cell growth (Hakem et al., 1996
; Vermeulen et al., 2003
). Although the hnRNP proteins may affect cell proliferation through BRCA1 and p21, there are multiple pathways regulating cell growth, and it will be of interest to see how widespread the effects of hnRNPs are.
In summary, our results demonstrate that hnRNP A/B proteins, especially A2 and B1, are dysregulated in a wide range of epithelial cancer cells. Their expression and that of hnRNP A1, are cell cycle-dependent, whereas the hnRNP A3 level is constant during cell division. We have also presented evidence that hnRNPs A2 and B1 play an important role in cellular proliferation, in accord with their effects on expression of p21 and BRCA1. Defining the mechanisms behind these interactions will aid understanding of the functions of hnRNP A2/B1 during cell growth as well as their dysregulation in some cancers.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ben-David, Y., Bani, M. R., Chabot, B., De Koven, A. and Bernstein, A. (1992). Retroviral insertions downstream of the heterogeneous nuclear ribonucleoprotein A1 gene in erythroleukemia cells: evidence that A1 is not essential for cell growth. Mol. Cell Biol. 12, 4449-4455.[Abstract]
Biamonti, G., Bassi, M. T., Cartegni, L., Mechta, F., Buvoli, M., Cobianchi, F. and Riva, S. (1993). Human hnRNP protein A1 gene expression. Structural and functional characterization of the promoter. J. Mol. Biol. 230, 77-89.[CrossRef][Medline]
Brockstedt, E., Rickers, A., Kostka, S., Laubersheimer, A., Dorken, B., Wittmann-Liebold, B., Bommert, K. and Otto, A. (1998). Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by caspase 3. J. Biol. Chem. 273, 28057-28064.
Brummelkamp, T. R., Bernards, R. and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553.
Buvoli, M., Biamonti, G., Tsoulfas, P., Bassi, M. T., Ghetti, A., Riva, S. and Morandi, C. (1988). cDNA cloning of human hnRNP protein A1 reveals the existence of multiple mRNA isoforms. Nucleic Acids Res. 16, 3751-3770.[Abstract]
Celis, J. E., Bravo, R., Arenstorf, H. P. and LeStourgeon, W. M. (1986). Identification of proliferation-sensitive human proteins amongst components of the 40 S hnRNP particles. Identity of hnRNP core proteins in the HeLa protein catalogue. FEBS Lett. 194, 101-109.[CrossRef][Medline]
Clemens, M. J., Trayner, I. and Menaya, J. (1992). The role of protein kinase C isoenzymes in the regulation of cell proliferation and differentiation. J. Cell Sci. 103, 881-887.
Dallaire, F., Dupuis, S., Fiset, S. and Chabot, B. (2000). Heterogeneous nuclear ribonucleoprotein A1 and UP1 protect mammalian telomeric repeats and modulate telomere replication in vitro. J. Biol. Chem. 275, 14509-14516.
Dreyfuss, G., Matunis, M. J., Pinol-Roma, S. and Burd, C. G. (1993). hnRNP proteins and the biogenesis of mRNA. Annu. Rev. Biochem. 62, 289-321.[CrossRef][Medline]
Dreyfuss, G., Kim, V. N. and Kataoka, N. (2002). Messenger-RNA-binding proteins and the messages they carry. Nat. Rev. Mol. Cell Biol. 3, 195-205.[CrossRef][Medline]
el-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W. and Vogelstein, B. (1993). WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817-825.[CrossRef][Medline]
Erlitzki, R. and Fry, M. (1997). Sequence-specific binding protein of single-stranded and unimolecular quadruplex telomeric DNA from rat hepatocytes. J. Biol. Chem. 272, 15881-15890.
Fielding, P., Turnbull, L., Prime, W., Walshaw, M. and Field, J. K. (1999). Heterogeneous nuclear ribonucleoprotein A2/B1 up-regulation in bronchial lavage specimens: a clinical marker of early lung cancer detection. Clin. Cancer Res. 5, 4048-4052.
Fishman, D. D., Segal, S. and Livneh, E. (1998). The role of protein kinase C in G1 and G2/M phases of the cell cycle (review). Int. J. Oncol. 12, 181-186.[Medline]
Good, P. J., Rebbert, M. L. and Dawid, I. B. (1993). Three new members of the RNP protein family in Xenopus. Nucleic Acids Res. 21, 999-1006.[Abstract]
Goto, Y., Sueoka, E., Chiba, H. and Fujiki, H. (1999). Significance of heterogeneous nuclear ribonucleoprotein B1 as a new early detection marker for oral squamous cell carcinoma. Jpn. J. Cancer Res. 90, 1358-1363.[Medline]
Hakem, R., de la Pompa, J. L., Sirard, C., Mo, R., Woo, M., Hakem, A., Wakeham, A., Potter, J., Reitmair, A., Billia, F. et al. (1996). The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse. Cell 85, 1009-1023.[CrossRef][Medline]
Hamasaki, M., Kamma, H., Wu, W., Kaneko, S., Fujiwara, M., Satoh, H., Haraoka, S., Kikuchi, M. and Shirakusa, T. (2001). Expression of hnRNP B1 in four major histological types of lung cancers. Anticancer Res. 21, 979-984.[Medline]
Hermann, R., Hensel, F., Muller, E. C., Keppler, M., Souto-Carneiro, M., Brandlein, S., Muller-Hermelink, H. K. and Vollmers, H. P. (2001). Deactivation of regulatory proteins hnRNP A1 and A2 during SC-1 induced apoptosis. Hum. Antibodies 10, 83-90.[Medline]
Hoek, K. S., Kidd, G. J., Carson, J. H. and Smith, R. (1998). hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA. Biochemistry 37, 7021-7029.[CrossRef][Medline]
Kamma, H., Horiguchi, H., Wan, L., Matsui, M., Fujiwara, M., Fujimoto, M., Yazawa, T. and Dreyfuss, G. (1999). Molecular characterization of the hnRNP A2/B1 proteins: tissue-specific expression and novel isoforms. Exp. Cell Res. 246, 399-411.[CrossRef][Medline]
Kamma, H., Satoh, H., Matusi, M., Wu, W. W., Fujiwara, M. and Horiguchi, H. (2001). Characterization of hnRNP A2 and B1 using monoclonal antibodies: intracellular distribution and metabolism through cell cycle. Immunol. Lett. 76, 49-54.[CrossRef][Medline]
Kelman, Z. (1997). PCNA: structure, functions and interactions. Oncogene 14, 629-640.[CrossRef][Medline]
Krecic, A. M. and Swanson, M. S. (1999). hnRNP complexes: composition, structure, and function. Curr. Opin. Cell Biol. 11, 363-371.[CrossRef][Medline]
LaBranche, H., Dupuis, S., Ben-David, Y., Bani, M. R., Wellinger, R. J. and Chabot, B. (1998). Telomere elongation by hnRNP A1 and a derivative that interacts with telomeric repeats and telomerase. Nat. Genet. 19, 199-202.[CrossRef][Medline]
Leser, G. P. and Martin, T. E. (1987). Changes in heterogeneous nuclear RNP core polypeptide complements during the cell cycle. J. Cell Biol. 105, 2083-2094.[Abstract]
LeStourgeon, W. M. B. A., Christensen, M. E., Walker, B. W., Poupore, S. M. and Daniels, L. P. (1978). The packaging proteins of core hnRNP particles and the maintenance of proliferative cell states. Cold Spring Harb. Symp. Quant. Biol. 42, 885-898.[Medline]
Loeb, J. E., Ritz, E., Creuzet, C. and Jami, J. (1976). Comparison of chromosomal proteins of mouse primitive teratocarcinoma, liver and L cells. Exp. Cell Res. 103, 450-453.[CrossRef][Medline]
Ma, A. S., Moran-Jones, K., Shan, J., Munro, T. P., Snee, M. J., Hoek, K. S. and Smith, R. (2002). Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein. J. Biol. Chem. 277, 18010-18020.
Martin, T., Jones, R. and Billings, P. (1979). HnRNP core proteins: synthesis, turnover and intracellular distribution. Mol. Biol. Rep. 5, 37-42.[CrossRef][Medline]
Minoo, P., Sullivan, W., Solomon, L. R., Martin, T. E., Toft, D. O. and Scott, R. E. (1989). Loss of proliferative potential during terminal differentiation coincides with the decreased abundance of a subset of heterogeneous ribonuclear proteins. J. Cell Biol. 109, 1937-1946.[Abstract]
Montuenga, L. M., Zhou, J., Avis, I., Vos, M., Martinez, A., Cuttitta, F., Treston, A. M., Sunday, M. and Mulshine, J. L. (1998). Expression of heterogeneous nuclear ribonucleoprotein A2/B1 changes with critical stages of mammalian lung development. Am. J. Respir. Cell Mol. Biol. 19, 554-562.
Mulshine, J. L., Cuttitta, F., Tockman, M. S. and De Luca, L. M. (2002). Lung cancer evolution to preinvasive management. Clin. Chest Med. 23, 37-48.[CrossRef][Medline]
Pan, Z. Q., Reardon, J. T., Li, L., Flores-Rozas, H., Legerski, R., Sancar, A. and Hurwitz, J. (1995). Inhibition of nucleotide excision repair by the cyclin-dependent kinase inhibitor p21. J. Biol. Chem. 270, 22008-22016.
Patry, C., Bouchard, L., Labrecque, P., Gendron, D., Lemieux, B., Toutant, J., Lapointe, E., Wellinger, R. and Chabot, B. (2003). Small interfering RNA-mediated reduction in heterogeneous nuclear ribonucleoparticule A1/A2 proteins induces apoptosis in human cancer cells but not in normal mortal cell lines. Cancer Res. 63, 7679-7688.
Percipalle, P., Jonsson, A., Nashchekin, D., Karlsson, C., Bergman, T., Guialis, A. and Daneholt, B. (2002). Nuclear actin is associated with a specific subset of hnRNP A/B-type proteins. Nucleic Acids Res. 30, 1725-1734.
Perez-Roger, I., Kim, S. H., Griffiths, B., Sewing, A. and Land, H. (1999). Cyclins D1 and D2 mediate myc-induced proliferation via sequestration of p27(Kip1) and p21(Cip1). EMBO J. 18, 5310-5320.
Pino, I., Pio, R., Toledo, G., Zabalegui, N., Vicent, S., Rey, N., Lozano, M. D., Torre, W., Garcia-Foncillas, J. and Montuenga, L. M. (2003). Altered patterns of expression of members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family in lung cancer. Lung Cancer 41, 131-143.[CrossRef][Medline]
Rappsilber, J., Ryder, U., Lamond, A. I. and Mann, M. (2002). Large-scale proteomic analysis of the human spliceosome. Genome Res. 12, 1231-1245.
Rosenberger, U., Lehmann, I., Weise, C., Franke, P., Hucho, F. and Buchner, K. (2002). Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus. J. Cell Biochem. 86, 394-402.[CrossRef][Medline]
Schwantke, N., Le Bouffant, F., Doree, M. and Le Peuch, C. J. (1985). Protein kinase C: properties and possible role in cellular division and differentiation. Biochimie 67, 1103-1110.[Medline]
Shyu, A. B. and Wilkinson, M. F. (2000). The double lives of shuttling mRNA binding proteins. Cell 102, 135-138.[CrossRef][Medline]
Somasundaram, K., Zhang, H., Zeng, Y. X., Houvras, Y., Peng, Y., Wu, G. S., Licht, J. D., Weber, B. L. and El-Deiry, W. S. (1997). Arrest of the cell cycle by the tumour-suppressor BRCA1 requires the CDK-inhibitor p21WAF1/CiP1. Nature 389, 187-190.[CrossRef][Medline]
Sourvinos, G. and Spandidos, D. A. (1998). Decreased BRCA1 expression levels may arrest the cell cycle through activation of p53 checkpoint in human sporadic breast tumors. Biochem. Biophys. Res. Commun. 245, 75-80.[CrossRef][Medline]
Sueoka, E., Goto, Y., Sueoka, N., Kai, Y., Kozu, T. and Fujiki, H. (1999). Heterogeneous nuclear ribonucleoprotein B1 as a new marker of early detection for human lung cancers. Cancer Res. 59, 1404-1407.
Sueoka, E., Sueoka, N., Goto, Y., Matsuyama, S., Nishimura, H., Sato, M., Fujimura, S., Chiba, H. and Fujiki, H. (2001). Heterogeneous nuclear ribonucleoprotein B1 as early cancer biomarker for occult cancer of human lungs and bronchial dysplasia. Cancer Res. 61, 1896-1902.
Tani, H., Ohshima, K., Haraoka, S., Hamasaki, M., Kamma, H., Ikeda, S. and Kikuchi, M. (2002). Overexpression of heterogeneous nuclear ribonucleoprotein B1 in lymphoproliferative disorders: high expression in cells of follicular center origin. Int. J. Oncol. 21, 957-963.[Medline]
Thiede, B., Dimmler, C., Siejak, F. and Rudel, T. (2001). Predominant identification of RNA-binding proteins in Fas-induced apoptosis by proteome analysis. J. Biol. Chem. 276, 26044-26050.
Thiede, B., Siejak, F., Dimmler, C. and Rudel, T. (2002). Prediction of translocation and cleavage of heterogeneous ribonuclear proteins and Rho guanine nucleotide dissociation inhibitor 2 during apoptosis by subcellular proteome analysis. Proteomics 2, 996-1006.[CrossRef][Medline]
Tockman, M. S., Mulshine, J. L., Piantadosi, S., Erozan, Y. S., Gupta, P. K., Ruckdeschel, J. C., Taylor, P. R., Zhukov, T., Zhou, W. H., Qiao, Y. L. et al. (1997). Prospective detection of preclinical lung cancer: results from two studies of heterogeneous nuclear ribonucleoprotein A2/B1 overexpression. Clin. Cancer Res. 3, 2237-2246.[Abstract]
Ullrich, S. J., Anderson, C. W., Mercer, W. E. and Appella, E. (1992). The p53 tumor suppressor protein, a modulator of cell proliferation. J. Biol. Chem. 267, 15259-15262.
Vermeulen, K., Van Bockstaele, D. R. and Berneman, Z. N. (2003). The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif. 36, 131-149.[CrossRef][Medline]
Waga, S., Li, R. and Stillman, B. (1997). p53-induced p21 controls DNA replication. Leukemia 11 Suppl. 3, 321-323.[Medline]
Watters, D. J. and Parsons, P. G. (1999). Critical targets of protein kinase C in differentiation of tumour cells. Biochem. Pharmacol. 58, 383-388.[CrossRef][Medline]
Weighardt, F., Biamonti, G. and Riva, S. (1996). The roles of heterogeneous nuclear ribonucleoproteins (hnRNP) in RNA metabolism. BioEssays 18, 747-756.[CrossRef][Medline]
Whitfield, M. L., Sherlock, G., Saldanha, A. J., Murray, J. I., Ball, C. A., Alexander, K. E., Matese, J. C., Perou, C. M., Hurt, M. M., Brown, P. O. et al. (2002). Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol. Biol. Cell 13, 1977-2000.
Wu, S., Sato, M., Endo, C., Sakurada, A., Dong, B., Aikawa, H., Chen, Y., Okada, Y., Matsumura, Y., Sueoka, E. et al. (2003). hnRNP B1 protein may be a possible prognostic factor in squamous cell carcinoma of the lung. Lung Cancer 41, 179-186.[CrossRef][Medline]
Xu, X., Joh, H. D., Pin, S., Schiller, N. I., Prange, C., Burger, P. C. and Schiller, M. R. (2001). Expression of multiple larger-sized transcripts for several genes in oligodendrogliomas: potential markers for glioma subtype. Cancer Lett. 171, 67-77.[CrossRef][Medline]
Yan-Sanders, Y., Hammons, G. J. and Lyn-Cook, B. D. (2002). Increased expression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP) in pancreatic tissue from smokers and pancreatic tumor cells. Cancer Lett. 183, 215-220.[CrossRef][Medline]
Zhang, W., Grasso, L., McClain, C. D., Gambel, A. M., Cha, Y., Travali, S., Deisseroth, A. B. and Mercer, W. E. (1995). p53-independent induction of WAF1/CIP1 in human leukemia cells is correlated with growth arrest accompanying monocyte/macrophage differentiation. Cancer Res. 55, 668-674.[Abstract]
Zhou, J., Mulshine, J. L., Unsworth, E. J., Scott, F. M., Avis, I. M., Vos, M. D. and Treston, A. M. (1996). Purification and characterization of a protein that permits early detection of lung cancer. Identification of heterogeneous nuclear ribonucleoprotein-A2/B1 as the antigen for monoclonal antibody 703D4. J. Biol. Chem. 271, 10760-10766.
Zhou, J., Allred, D. C., Avis, I., Martinez, A., Vos, M. D., Smith, L., Treston, A. M. and Mulshine, J. L. (2001a). Differential expression of the early lung cancer detection marker, heterogeneous nuclear ribonucleoprotein-A2/B1 (hnRNP-A2/B1) in normal breast and neoplastic breast cancer. Breast Cancer Res. Treat. 66, 217-224.[CrossRef][Medline]
Zhou, J., Nong, L., Wloch, M., Cantor, A., Mulshine, J. L. and Tockman, M. S. (2001b). Expression of early lung cancer detection marker: hnRNP-A2/B1 and its relation to microsatellite alteration in non-small cell lung cancer. Lung Cancer 34, 341-350.[CrossRef][Medline]