Nucleolin As A Protooncogene That Stimulates Cell Proliferation
It has been shown that in cancer cell lines characterized by different proliferation rates, the transcriptional activity of RNA polymerase I and the expression of the major nucleolar proteins involved in the control of rRNA transcription and processing are directly related to nucleolar size and to the rapidity of cell proliferation (Derenzini et al., 1998). Several observations suggest that nucleolin is a major actor in promoting cell proliferation. First of all, its amount is correlated with the proliferative status of the cell: nucleolin level is higher in tumours and actively dividing cells (Derenzini et al., 1995; Mehes and Pajor, 1995; Roussel et al., 1994; Sirri et al., 1995, 1997) and is widely used as a marker of cell proliferation. Secondly, over expression of nucleolin cooperates with oncogenic mutant Ras in a rat embryonic fibroblast transformation assay (Takagi et al., 2005). Nucleolin can therefore be considered a bona fide proto-oncogene. Thirdly, transfection of several tumour cell lines with G-rich oligonucleotides (GROs) arrests cells in S phase (Xu et al., 2001) and the anti-proliferative activity of GROs is perfectly correlated with their ability to bind nucleolin (Bates et al., 1999; Dapic et al., 2003). The exact mechanism of action of GROs is not unravelled yet, but one likely explanation is that they sequester nucleolin, thereby preventing its binding to DNA (see below).
If these results suggest a positive role for nucleolin on proliferation, they do not indicate which of its activities are responsible for it. One hypothesis is that the stimulation of ribosome biogenesis by nucleolin is indispensable for active cell division. Indeed, a direct link between protein translation and cancer is clearly emerging (Ruggero and Pandolfi, 2003). However the studies mentioned above suggest a more direct impact of nucleolin on cell division. If part of its effects could come from its capacity to repress p53 mRNA translation (Takagi et al., 2005), a more direct role of nucleolin in DNA replication can also be considered.
1.1. Nucleolin and RNA Polymerase I Transcription
Although the higher level of nucleolin in tumours and actively dividing cells suggests that nucleolin could play a positive role in the production of ribosomal RNA, most of the studies rather show a repressive effect of nucleolin on RNA polymerase I transcription.
A strong link between the phosphorylation of nucleolin, its proteolysis and the production of ribosomal RNA has been observed (Bouche et al., 1984; Bourbon et al., 1983; Warrener and Petryshyn, 1991). The inhibition of proteolysis using leupeptin leads to a lower rRNA transcription in an in vitro transcription system (Bouche et al., 1984). In another series of experiments, the injection of nucleolin antiserum leads to 2-3.5 fold stimulation of pre-rRNA synthesis in Chironomus tentans salivary glands (Egyhazi et al., 1988), although it was not clearly demonstrated that these antibodies blocked specifically the homolog of nucleolin in this species. A model was proposed based on these observations where nucleolin was suggested to be involved in regulating elongation of rRNA transcripts. In this model, nucleolin could interact with the nascent rRNA through RNA binding domains (RBDs) and with the RNA pol I machinery through its N terminal. This interaction was envisaged as a stalling mechanism, and proteolysis of nucleolin after its phosphorylation could release the transcriptional block (Ginisty et al., 1999). However, this model was challenged by recent experiments using the xenopus oocyte system (Roger et al., 2002). The injection of a 2-4 fold excess of nucleolin in Xenopus laevis stage IV oocytes leads to a significant reduction in accumulation of 40 S pre-RNA (Roger et al., 2002). This repression was specific for RNA polymerase I, and could be obtained with a minimal pol I promoter independently of the nature of the RNA sequence that is transcribed, ruling out that the specific interaction of nucleolin with nascent pre-rRNA was required to regulate transcription elongation.
A recent study in the carp (Cyprinus carpio) shows that level of nucleolin is up regulated in cold-acclimatized carp with a concomitant nucleolar segregation and depression in rRNA transcription (Alvarez et al., 2003).
On the other side, it has been demonstrated that nucleolin phosphorylation and rRNA transcription go hand in hand. Nucleolin phosphorylation could be triggered by a variety of stimuli like androgens and growth factors (Bonnet et al., 1996; Bouche et al., 1987; Issinger et al., 1988; Suzuki et al., 1985, 1991; Tawfic et al., 1994) and the phosphorylation is invariably accompanied by increased rRNA transcription and cell proliferation. All these observations suggest that indeed nucleolin could have regulatory role in RNA polymerase I transcription. However, further experiments are clearly required to clarify the function of nucleolin in rRNA transcription.
1.2. Nucleolin and RNA Polymerase II Transcription
Several reports indicate that nucleolin is also involved in some aspect of RNA polymerase II transcription. A multiprotein complex containing YY1, HMGB2 and nucleolin binds the D4Z4 repeats and could regulate the transcription of genes located on chromosome 4q35 involved in Facioscapulohumeral muscular dystrophy (FSHD) (Gabellini et al, 2002). However, these proteins do not seem to be implicated in the FSHD cases not linked to 4q35 (FSHD1B) (Bastress et al., 2005).
Cell transformation induced by the Human Papillomavirus (HPV18) could also involve nucleolin (Grinstein et al., 2002). Nucleolin binds in a sequence-specific manner to the HPV18 enhancer. Antisense inactivation of nucleolin blocks E6 and E7 oncogene transcription and selectively decreases HPV18(+) cervical cancer cell growth. Nucleolin might be involved in opening the chromatin structure of the HPV18 enhancer suggesting that through this mechanism, nucleolin functions as a regulator of HPV18 oncogene transcription and HPV18-induced cervical carcinogenesis.
Nucleolin has also been implicated in the regulation of transcription of the Krüppel-like Factor 2 (KLF2) transcription factor. KLF2 is required for developmental and cellular functions in several distinct tissue types. Its transcription involves several transacting factors, chromatin modifications, and signaling pathways. Nucleolin has been identified as a protein that binds to a palindromic response region in the KLF2 promoter. Co-immunoprecipitation experiments indicated that nucleolin interacts with additional factors involved in KLF2 gene regulation (p85 and hnRNP-D) and small interfering RNAs targeting the nucleolin sequence selectively reduced nucleolin expression and were sufficient to block the induction of KLF2.
Nucleolin together with HnRNP D has been shown to form the LR1 transcription factor. LR1 is a B cell-specific, sequence-specific DNA binding activity that regulates transcription in activated B cells (Hanakahi et al., 1997; Hanakahi and Maizels, 2000). DNA bending induced by nucleolin and hnRNP D might regulate the transcriptional activation by LR1 (Hanakahi and Maizels, 2000).
Finally, nucleolin has also been identified as a repressor of polymerase II transcription (Yang et al., 1994). Biochemical and functional studies further established that nucleolin is a transcription repressor for regulation of alpha-1 acid glycoprotein (AGP)
These different reports indicate that nucleolin could influence transcription positively or negatively. These seemingly contradictory data might be explained if one considers that nucleolin does not act as a real transcriptional activator or repressor, but rather indirectly through the regulation of chromatin structure.
Indeed, since it has been discovered, it is well known that nucleolin binds to chromatin (Olson et al., 1975; Olson and Thompson, 1983). Furthermore, it interacts with histone H1 and seems to modulate chromatin structure (Erard etal., 1988, 1990).
This is only recently that a clear role for nucleolin in chromatin dynamic has been reported (Angelov et al., 2006) (Fig. 1). It was shown that nucleolin possesses a histone chaperone activity and assists chromatin remodelers like SWI/SNF and ACF in their functions. In particular, nucleosome SWI/SNF dependent sliding and remodeling are greatly enhanced in the presence of nucleolin (Fig. 1). The presence of the acidic region in the N-terminal domain of nucleolin is a characteristic of many proteins with histone chaperone activity (Philpott et al., 2000). The acidic domain is required for nucleolin chaperone activity, but is not sufficient to explain this activity since a nucleolin truncated protein containing only this acidic region does not carry the chaperone or co-remodeling activity (Angelov et al., 2006). Nucleolin is able to destabilise the histone octamer, promoting H2A-H2B dimer displacement. Interestingly, passage of RNA polymerase II through the nucleosome is greatly facilitated in presence of nucleolin (Angelov et al., 2006). H2A-H2B dimer displacement and facilitated transcription of pol II through the nucleosome are reminiscent of the activity of FACT complex (Orphanides et al., 1998). The dual properties of nucleolin to bind non-specifically to DNA sequences and to interact with histones through its highly acidic domain recapitulate the characteristics of FACT subunits (Spt16 and SSRP1) in a single polypeptide. As mentioned above, nucleolin could exert a positive or a repressive effect on transcription. Such seemingly contradictory results could be explained if one takes into account that
Nucleolin
- Nucleolin - -SWI/SNF - + + + +
Figure 1. Nucleolin acts as a histone chaperone and boosts the activity of chromatin remodeler SWI/SNF. (a) In vitro histone deposition assay shows the histone chaperone activity of nucleolin. Recombinant core histones were incubated with 5S 147 bp DNA in presence or absence of nucleolin. Note the disappearance of aggregates in the wells and the formation of nucleosomal particles in presence of increasing amount of nucleolin. (b) Sliding assay performed on centrally positioned 601 nucleosomes (250 bp). The amount of SWI/SNF used here is insufficient to slide the nucleosomes. Note that in presence of nucleolin even the suboptimal quantity of SWI/SNF is able to slide the nucleosomes
Figure 1. Nucleolin acts as a histone chaperone and boosts the activity of chromatin remodeler SWI/SNF. (a) In vitro histone deposition assay shows the histone chaperone activity of nucleolin. Recombinant core histones were incubated with 5S 147 bp DNA in presence or absence of nucleolin. Note the disappearance of aggregates in the wells and the formation of nucleosomal particles in presence of increasing amount of nucleolin. (b) Sliding assay performed on centrally positioned 601 nucleosomes (250 bp). The amount of SWI/SNF used here is insufficient to slide the nucleosomes. Note that in presence of nucleolin even the suboptimal quantity of SWI/SNF is able to slide the nucleosomes nucleolin assists remodelers like SWI/SNF in their functions, and these remodelers are involved in both activation and repression of transcription (Martens and Winston, 2003).
2. NUCLEOLIN, DNA METABOLISM AND CANCER
DNA metabolism, i.e. DNA replication, recombination and repair, is a central process in oncogenesis. Proteins that ensure an accurate replication of the genome and protect it from genotoxic agents are "caretaker" tumor suppressor (Kinzler and Vogelstein, 1997): their loss of function leads to the rapid accumulation of deleterious mutations or chromosomal abnormalities. In parallel, proteins that stop cell proliferation when the integrity of the genome is offended are "gatekeeper tumor suppressors" (Kinzler and Vogelstein, 1997). On the other side, proteins that promote entry into S phase are potential proto-oncogenes whose gain of function enhances transformation and immortalization. It should be noted that the effects of the loss of function of many DNA replication and repair enzymes are by far not restricted to carcinogenesis. For example, mutations in several RecQ helicases cause various syndromes characterized by quite different defects (Hickson, 2003): premature ageing in Werner's syndrome, dwarfism, immunodeficiency and infertility in Bloom's syndrome or skin atrophy and skeletal abnormalities in RothmundThomson syndrome.
A role for nucleolin in DNA replication, recombination and repair is suggested by its ability to bind directly both DNA and proteins involved in these processes
(see below). As we shall see now, nucleolin could function either as a "caretaker", a "gatekeeper" or a proto-oncogene, depending on the cellular context.
2.1. A Positive Role for Nucleolin in DNA Replication?
Nucleolin has been shown to bind non specifically to denatured single-stranded DNA (Sapp et al., 1986), particularly to G-rich sequences, such as those found in non-transcribed spacer region of ribosomal DNA (Olson et al., 1983), telomeric DNA (Ishikawa et al., 1993; Pollice et al., 2000) or switch regions of immunoglobulin genes (Hanakahi et al., 1997). This probably reflects a specific affinity for G quartets (Bates et al., 1999; Hanakahi et al., 1999), a conformation adopted by G-rich single strands through Hoogsteen bonding between guanines. However, this protein is also able to bind single-stranded DNA devoid of G-rich sequences such as MVMp DNA (Barrijal et al., 1992), but this target also adopts a complex secondary structure.
Even though nucleolin has no or very little affinity for double-stranded DNA, it binds efficiently Matrix-Attachment Regions (MARs) (Dickinson and Kohwi-Shigematsu, 1995). Nevertheless, MARs, which mediate attachment of chromosomes to the nuclear matrix contain a sequence with an unusually high base unpairing potential (Galande, 2002), and nucleolin binds far more efficiently the T-rich single strand of MARs (Dickinson and Kohwi-Shigematsu, 1995).
Nucleolin lacks a characteristic DNA binding domain (Ginisty et al., 1999). Its non-specific affinity for DNA is conferred by two different domains: its four RNA binding domains, particularly the 3rd and the 4th ones, and its C-terminal GAR domain (Hanakahi et al., 1999; Sapp et al., 1989). Of importance, these properties were determined in vitro with the native protein purified from cell extracts or recombinant truncated proteins; they are likely to be altered in vivo by interaction with other DNA binding factors (Dempsey et al., 1998) and/or by post-translational modifications.
What could be the function of nucleolin in DNA replication? An intriguing possibility is that nucleolin may be a component of the DNA replication machinery as it was found associated with a DNA synthesome (Applegren et al., 1995). Some authors attributed a helicase activity to human nucleolin and identified it as human helicase IV (Tuteja et al., 1995 ). A possible ortholog of nucleolin in Pea displayed a similar activity which mapped to its GAR domain, even though it has no sequence homology with any other helicase (Nasirudin et al., 2005). These results should, however, be interpreted cautiously as the activity described was far less efficient than that of typical helicases, while other groups simply did not detect it (Ginisty et al., 1999). Moreover, two other studies demonstrated an antagonistic activity of nucleolin that favors complementary strand annealing (Hanakahi et al., 2000; Sapp et al., 1986). The reason for the discrepancy is not clear: either nucleolin does have antagonistic activities depending on its post-translational modifications, or some activities reported are conferred by contaminants.
However, one recent paper reports a genuine activity of nucleolin in replication (Seinsoth et al., 2003). In this study, the authors demonstrate that nucleolin forms a ternary complex with the SV40 helicase T-antigene and endogenous topoi-somerase I. Nucleolin could mediate the cohesion of this bipartite holoenzyme helicase complex, thus enhancing plasmid unwinding. It is not known at present whether nucleolin could play a more general role in endogenous replication initiation complexes or replication forks, through its helicase activity, its histone chaperone properties (Angelov et al., 2006) or some yet unknown functions. Nevertheless, this notion is supported by the fact that nucleolin is a MAR binding protein (Dickinson and Kohwi-Shigematsu, 1995; Olson et al., 1983) and a bona fide component of nuclear and nucleolar matrix (Dickinson and Kohwi-Shigematsu, 1995; Gotzmann et al., 1997) where DNA replication is believed to occur (Cook, 1999; Falaschi, 2000). It is noteworthy that MAR-binding proteins are frequently involved in cancer (Galande, 2002), but one has to keep in mind that MARs and nuclear matrix likely play equally important roles in other cellular processes, namely regulation of transcription (Falaschi, 2000).
At last, nucleolin might play a specific role in telomeric replication and maintenance, as suggested by two types of data. First, it binds telomeric repeat (TTAGGG)n in vitro (Ishikawa et al., 1993; Pollice et al., 2000), with a marked preference for the single-stranded form. Secondly, it interacts in vitro and in vivo with hTERT (Khurts et al., 2004), the protein catalytic component of human telom-erase. This interaction takes place both in the cytoplasm and in the nucleolus, where it could promote the assembly of hTERT with the RNA subunit hTERC. As a conclusion, many data regarding the involvement of nucleolin in DNA replication are indirect and an experimental demonstration through knockdown or knockout studies is still awaited.
2.2. Nucleolin as a Stress-Responsive Tumour Suppressor?
An unexpected role of nucleolin in an S-phase checkpoint triggered by various stresses was recently uncovered. The story began with the discovery that heat shock causes a dramatic redistribution of nucleolin from the nucleolus to the nucleoplasm in HeLa cells (Daniely and Borowiec, 2000). This relocalization is very quick, since it starts as early as 5 minutes after a 15 minute heat shock at 44°C and is only transient, lasting around 1 hour. These results were subsequently confirmed in other cell lines and extended to other cell stresses such as exposition to gamma-irradiation or camptothecine, a topoisomerase I inhibitor (Daniely and Borowiec, 2000; Wang et al., 2001). This phenomenon is nevertheless stress-selective, since it is not triggered by UV irradiation or hydroxurea exposure.
How specific stresses provoke nucleolin redistribution remains an open question. A block of ribosomal transcription cannot account for the whole phenomenon as shutdown of RNA polymerase I activity by actinomycin D induces rather a nucleolar-to-cytoplasmic change in nucleolin localization (Daniely and Borowiec, 2000). Interestingly, the relocalization is dependent on p53 since it does not occur in cell lines that lack p53 (Daniely et al., 2002). Activated p53 interacts transiently with nucleolin after heat shock or gamma irradiation, through its last 30 C-terminal amino acids (Daniely et al., 2002) known to bind several DNA repair proteins. The precise cell compartment where this interaction takes place is not known for the moment as nucleolin shuttles between the cytoplasm, the nucleoplasm and the nucleolus (Borer et al., 1989). However, stress-activated nucleoplasmic p53 most likely binds nucleolin and prevents its import into the nucleolus, resulting in its accumulation onto the nuclear matrix (Daniely et al., 2002). What induces the transient interaction in either compartment is also not known at present, but it is highly probable that nucleolin itself undergoes specific post-translational modification after stress, such as serine phosphorylation by casein kinase II (Kim et al., 2005).
What is the biological meaning of this transient relocalization? Heat shock causes a transient arrest of DNA replication and kills cells preferentially in S-phase (Wang et al., 2001). Elevated temperature has pleitotropic effects, including inhibition of origin firing, elongation step, histone deposition into chromatin and ligation of replication intermediates. These effects cannot be explained by mere alterations of the chromatine substrate, as lysates of heated cells fail to replicate exogenous plasmids carrying the SV40 origin of replication (Wang et al., 2001): this in vitro assay accurately reproduces natural DNA replication, requiring only the addition of SV40 T-antigen. One of the main components affected by heat shock is RPA, the primary single-stranded DNA binding protein of eukaryotic cells which is necessary for both initiation and elongation steps of chromosomal DNA replication (Iftode et al., 1999). Indeed, addition of recombinant hRPA to the cell lysates reverses the inhibition (Wang et al., 1998). Interestingly, nucleolin was shown to interact in vitro with hRPA via its GAR domain and this interaction is induced in vivo upon heat shock (Daniely and Borowiec, 2000; Kim et al., 2005). Since there is a striking correlation between the kinetics of replication inhibition, nucleolin mobilization and formation of nucleolin-RPA complex, it is tempting to build the following model: heat shock would provoke p53-dependent nucleolin redistribution to the nucleoplasm where it could sequester RPA and thereby inhibit origin unwinding and replication elongation (Daniely et al., 2002). Importantly, nucleolin inhibits hRPA without affecting its single-stranded DNA binding activity (Daniely and Borowiec, 2000), which suggests that it sterically prevents interaction of hRPA with another factor. This model is strengthened by the observation that nucleolin-RPA complexes are preferentially located outside replicating regions (Daniely and Borowiec, 2000).
However, although this model is very attractive, it is still a matter of debate (Kim et al., 2005). First of all, co-immunoprecipation experiments proved that the formation of a nucleolin-RPA complex occurs in the nucleolus as well as in the nucleoplasm (Kim et al., 2005). Secondly, the interaction can be detected in a cell line which lacks p53 expression. Thirdly, hydroxyurea also induces RPA-nucleolin interaction without mobilizing nucleolin. On the other hand, mutant forms of Nucleolin that are constitutively mislocalized outside the nucleolus also constitu-tively interact with RPA, provided they retain the GAR domain (Kim et al., 2005).
Moreover, overexpression of the same mutants inhibit DNA replication and block the cells at the G1/S-phase transition (Kim et al., 2005), emphasizing the potential role of nucleolin mobilization. It is therefore highly probable that two different processes help the formation of RPA-nucleolin complexes after a genotoxic stress: a post-transcriptional modification of nucleolin that renders the GAR domain of nucleolin accessible to RPA, and its p53-dependent relocalization to the nucleoplasm where a higher amount of RPA is available. Of importance, nucleolin relocalization is transient and lasts far less than replication inhibition (Daniely and Borowiec, 2000). This means that nucleolin-RPA interaction is only an initial event and that other mechanisms account for prolonged replication inhibition.
As a conclusion, vertebrate nucleolin has acquired new functions in cell cycle control, compared to its yeast homologs GAR2 and NSR1. Nucleolin is a genuine stress-responsive protein which functions in a new S-phase checkpoint. This could relate to a more general function of the nucleolus as a stress sensor that enables coupling of cell metabolism (through ribosome biogenesis and therefore protein translation) and proper cell division (through control of DNA replication). Though paradoxical, this function is not incompatible with other results describing nucleolin as a proto-oncogene that promotes cell replication in normal conditions: depending on its post-translational modifications triggered by the phase of the cell cycle or the environment, nucleolin could play antagonistic roles. Of interest, other nucleolar proteins involved in ribosomal biogenesis display an additional role in cell cycle control. For example, nucleophosmin/B23 is rearranged or mutated in a number of hematological malignancies. Its inactivation in mouse leads to embryonic lethality and carcinogenesis, caused by unrestricted centrosome duplication and genome instability (Grisendi et al., 2005).
2.3. A Function for Nucleolin in DNA Repair and Recombination?
Several enzymatic properties of nucleolin support a role for this protein in DNA repair and recombination: it is able to bend DNA (Hanakahi and Maizels, 2000) and can either unwind double-stranded DNA or enhance annealing of complementary DNA strands (Hanakahi and Maizels, 2000; Nasirudin et al., 2005; Sapp et al., 1986; Tuteja etal., 1995).
Consistent with this idea, nucleolin was found in a B-cell specific recombination complex (Borggrefe et al., 1998). It also forms a complex with hRNP-D that binds switch regions of immunoglobulin genes in B lymphocytes which undergo class-switch recombination (Hanakahi et al., 1997). If nucleolin is clearly not the endonuclease of this recombination process, it still could have an accessory function such as structuring the switch regions or recruiting other factors.
Interestingly, nucleolin interacts with several key DNA repair and recombination proteins. First of all, it interacts with topoisomerase I through its N terminus (Bharti et al., 1996; Edwards et al., 2000). This interaction does not modify the enzymatic activity of topoisomerase I per se, but it could play important role in its predominantly nucleolar localization as it was demonstrated in yeast (Edwards et al., 2000). Nucleolin could thus modulate rDNA recombination, an important (but not unique) function of topoisomerase I. Nucleolin also interacts with p53 (Daniely et al., 2002), YB-1 (Gaudreault etal., 2004) and RPA (Daniely and Borowiec, 2000), three proteins notably involved in DNA repair. At last, a recent paper describes a surprising interaction between nucleolin and Rad51 (De et al., 2006), a RecA homolog required for homologous recombination. Most strikingly, the authors of this study show that inhibition of nucleolin by electroporation of a blocking antibody impairs intra-plasmid homologous recombination activity and sensitizes the cells to a topoisomerase II inhibitor, a phenotype quite similar to that observed after Rad51 inactivation by the same approach (De et al., 2006).
Nucleolin could therefore contribute to genome stability in two different ways: by modulating the activities of several DNA repair enzymes through physical interaction and by enhancing the translation of some of these proteins after a genotoxic stress (Yang et al., 2002).
2.4. Nucleolin as a Cell Surface Receptor
Although nucleolin is found almost exclusively within the nucleolus, increasing evidence indicates its presence at the surface of various cell types. While it can be argued that the detected protein could be a closely related antigen in some reports, several laboratories unequivocally identified nucleolin, using a combination of western blot, immunofluorescence, immunogold labeling, FACS analysis and micro-sequencing of the purified protein (Callebaut et al., 1998b; Dumler et al., 1999; Hirano et al., 2005).
Although the presence of nucleolin at the cell surface could appear at first surprising, there are now a growing number of nuclear proteins involved in DNA metabolism and chromatin structure that have been shown to be present on the cell surface. Examples include the Ku protein which is involved in multiple nuclear processes (Dalziel et al., 1992), nucleophosmin (Brandt et al., 2004), Nopp140 (Kubler, 2001) and the chromosomal associated protein HMGB1 which was recently discovered to be a crucial cytokine that mediates the response to infection, injury and inflammation (Lotze and Tracey, 2005). Interestingly, auto-antibodies to human nucleolin (Valdez et al., 1995), nucleophosmin (Chung and Utz, 2004), Ku proteins (Kelavkar et al., 2002; Reeves et al., 1991) and even to nucleosomal particles (Amoura et al., 2000; Ghirardello et al., 2004) were found associated with auto immune diseases like the Systemic lupus erythematosus.
Difficulties in detecting nucleolin on the cell surface could be explained by its very low concentration in this compartment (Hovanessian et al., 2000). Moreover, this cell surface expressed nucleolin protein has a different isoelectric point and it is recognized by only one monoclonal antibody (mAb D3) in its native conformation (Hovanessian et al., 2000). This probably reflects specific post-translational modifications undergone by nucleolin on the cell surface. Consistent with this hypothesis, extracellular nucleolin is a substrate of ecto-protein kinases including casein kinase II (Dumler et al., 1999; Jordan et al., 1994). Interestingly, indirect evidence suggests that nucleolin shutlles back and forth between the nucleus, the cytoplasm and the cell surface. Indeed, mAb D3 anti-nucleolin antibodies are quickly internalized by living cells (Hovanessian et al., 2000) and eventually gain access to the nucleolus (Deng et al., 1996).
These observations are quite amazing since nucleolin lacks a hydrophobic signal sequence (Ginisty et al., 1999) They raise two questions: how is nucleolin attached to the cell surface and how is it translocated across the membranes? First of all, nucleolin can be dissociated from the membrane at high salt concentrations (Harms et al., 2001). Thus, it is likely to bind integral membrane or GPI-anchored proteins through electrostatic interactions. The mechanisms of its externalization and subsequent re-internalization remain completely obscure for the moment. Externalization of nucleolin occurs through a mysterious alternative secretory pathway which is insensitive to inhibitors of the transport within the ER-Golgi network but requires physiological temperatures (Hovanessian et al., 2000). Of note, such alternative pathways have already been described for several proteins, namely interleukin-1ß and FGF-2 (Nickel, 2005). After internalization, nucleolin is detected within cytoplasmic smooth vesicles (Dumler et al., 1999; Hovanessian et al., 2000) and colocalizes with EEA1, a marker specifically associated with clathrin in early endosomes (Legrand et al., 2004). Nucleolin may therefore use the classical endocytic pathway in an initial step owing to its association with the actin cytoskeleton (Hovanessian et al., 2000) and unidentified membrane-anchored proteins. It would subsequently penetrate the cytosol through an unknown mechanism.
What could be the function of this protein at the cell surface? Independent affinity chromatography experiments suggested that nucleolin is a receptor for plenty of ligands, including several growth factors and adhesion molecules. It is doubtful that a single nucleolar protein is endowed with so many activities and the physiological relevance of these results awaits further confirmation. However, several experimental data support the idea that nucleolin is a genuine surface receptor in at least a few cases. First of all, a few putative ligands induce clustering of nucleolin at the cell surface: midkine (Said et al., 2002), HIV particles (Nisole et al., 2002a,b), lactoferrin (Legrand et al., 2004) and pleiotrophin (Said et al., 2005). Secondly, the same molecules and two others (urokinase (Dumler et al., 1999) and F3 peptide (Christian et al., 2003)) are co-internalized with nucleolin and this process can be blocked by anti-nucleolin antibodies. Nucleolin could thus act as a bridge between the cell surface and the nucleus, guiding growth factors such as midkine, lactoferrin and FGF-2 directly to the effector compartment where they would modulate the expression of ribosomal genes for example. This would explain why many tumour cells and angiogenic endothelial cells display an apparent accumulation of nucleolin at the cell surface (Christian et al., 2003): abnormal relocalization of nucleolin would confer a competitive advantage by increasing the access to growth factors. At the same time, this observation makes nucleolin an attractive target for cancer therapy. Indeed, F3, a tumor homing peptide that binds specifically to nucleolin, and mAb D3 antibodies injected intravenously selectively accumulate in tumour and angiogenic vessels (Christian et al., 2003; Joo et al., 2005).
Cell surface expression of nucleolin could also have a pathophysiological significance as it has been shown to interact with several viruses and enteropathogenic bacteria. Depending on the micro organism, nucleolin may promote either their initial attachment to the cell surface (Callebaut et al., 1998a,b; de Verdugo et al., 1995; Sinclair et al., 2006; Sinclair and O'Brien, 2002, 2004) or their entry in the host cell (Bose et al., 2004; Nisole et al., 2002a). A well documented example is the relationship between nucleolin and HIV. Nucleolin binds to V3 loop of gp120 and to a pseudo-peptide, HB-19, which mimics its structure (Callebaut et al., 1998a). As a consequence, HB-19 (Nisole et al., 2002a) as well as other putative ligands of nucleolin such as midkine (Callebaut et al., 2001), lactoferrin (Legrand et al., 2004) and pleitrophin (Said et al., 2005) inhibits HIV attachment to the cell surface of mammalian cells and subsequent viral entry. Therefore, nucleolin could be a target of interest to design new antiviral molecules, but this would require first a deeper understanding of the physiological role of nucleolin at the cell surface.
3. CONCLUDING REMARKS: NUCLEOLIN AS A NEW PHARMACOLOGICAL TARGET
The recent findings of the localisation of nucleolin at the cell surface of cancer cells and its involvement in DNA replication have promoted the development of anti-cancer drugs targeting nucleolin and the use of nucleolin as a marker for the diagnosis of cancer.
For many years, the detection of AgNOR proteins by immuno cytochemistry has been used by pathologists to predict the clinical outcome of some cancer diseases. Nucleolin, together with nucleophosmin is mostly responsible for this specific silver staining and is, therefore, an interesting marker for diagnosis of cancer. More recently, the apparent preferential expression of nucleolin on the cell surface of cancer cells, is leading to the development of new tools for diagnosis of cancer cells. In particular detection of nucleolin at the cell surface using a monoclonal antibody was used in the Nucleolin OncoMarker kit (Assay Designs).
Several molecules with potential pharmaceutical activities targeting nucleolin function have been also developed. One can mention for example the development of the aptamer AS1411 (also known as AGRO100) by the company Antisoma (England). These aptamers are G-rich oligonucleotides, which can form G-quartet structures. They affect cell proliferation by inhibiting DNA replication (Xu et al., 2001). It is claimed that these aptamers bind nucleolin on the cell surface of cancer cells, and then inhibit nucleolin function in cell proliferation by inhibiting DNA replication or another unknown process. Interestingly, these aptamers AS1411 reduced tumour growth in xenograft models of both renal and lung cancers and are currently being tested in phase I study with patients with kidney and lung cancers.
By targeting nucleolin on the cell surface, HB-19 pseudo-peptides (Nisole et al., 2002a) inhibit HIV attachment to the cell surface of mammalian cells and subsequent viral entry, demonstrating that nucleolin could also be an interesting pharmaceutical target to prevent viral infection.
These examples demonstrate that nucleolin could be a therapeutic target for the development of innovative molecules against cancer and virus infection. The newly described function of nucleolin in chromatin dynamics and gene expression is potentially another function of nucleolin that could be targeted for the search of specific inhibitors of cell proliferation.
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