Role of the Centrosomal MARK4 Protein in Gliomagenesis

Human gliomas are the most frequent tumours of the central nervous system (Kleihues & Cavenee, 2000). They are of neuroectodermal origin and present as different histological types and malignancy grades (Louis et al., 2007). According to the WHO (world health organization) system, astrocytoma, oligodendroglioma and mixed oligoastrocytoma are classified as differentiated gliomas, while anaplastic glioma and glioblastoma show increasing grades of malignancy (Box 1).

malignant glioma.Gliomas frequently display near-diploid (2n+/-) and/or near-tritetraploid (3n+/-)/(4n+/-) karyotypes, implicating aberrant mitotic divisions, in addition to chromosomal rearrangements.Highly polyploid subpopulations and the presence of apoptotic nuclei are also reported (Figures 1a-d).Low-grade astrocytomas and oligodendrogliomas (WHO grades I-II) show a number of chromosome aberrations quite low.When present, they involve the gain of chromosome 7, the loss of chromosomes 10, 22 and one sex chromosome (see Figures 1a, b), while structural changes affect in particular 1p (Figure 2a) and 9p (Figure 2b) chromosome arms.These chromosome abnormalities are qualitatively similar to those found in anaplastic astrocytoma (WHO grade III) and glioblastoma (WHO grade IV), but their frequency is At early mitosis, the merotelic orientation escapes the spindle mitotic checkpoint thus representing the major mechanism of chromosome mis-segregation in non-cancer cells.Usually these errors are corrected before cells enter anaphase, to preserve genome stability (Cimini et al., 2004).

Tetraploidy, centrosome amplification and spontaneous chromosomal instability in glioma
A relationship between extra centrosomes and the formation of multipolar spindles in cancer cells has been proposed by different authors (Basto et al., 2008;Cimini et al., 2004;Saunders, 2005;Sluder & Nordberg, 2004).Multipolarity in cancer cells is considered an essential transient stage prior to clustering extra centrosomes in a bipolar fashion (Brinkley, 2001).Multiple centrosomes have been detected in many types of cancer cells including glioma (Figure 4) and strongly linked to aneuploidy in a variety of studies (D'Assoro et al., 2002;Ganem et al., 2009;Ghadimi et al., 2000;Katsetos et al., 2006;Lingle et al., 2002;Magnani et al., 2009;Pihan et al., 1998).A positive linear correlation between the p e r c e n t a g e o f c e l l s w i t h s u p e r n u m e r a r y centrosomes and the extent of aneuploidy within a panel of glioblastoma cell lines is shown in Figure 5.In tumour development, aneuploidy is frequently preceded by tetraploidy, often with prolonged tetraploid precancerous status, a feature that makes it of central importance to cancer research (Margolis et al., 2003).It has been proposed that failure of cytokinesis is a key step in the formation of tetraploid karyotypes and in tumour initiation (Fujiwara et al., 2005).A tetraploid cell inherits twice the normal complement of centrosomes, a condition assessed to generate chromosomes mis-segregation in subsequent cell divisions (Ganem et al., 2007).However, tetraploid cells are observed in some normal tissues including liver and heart, indicating that cytokinesis is physiologically regulated.The possible fate of a tetraploid progeny is shown in Figure 6.To measure the occurrence of DNA damage in once-divided binucleated (BN) cells, the cytokinesis-block micronucleus cytome (CBMN Cyt) assay, an established biomarker to detect spontaneous genomic instability (Fenech, 2007), can be used.Application of CBMN Cyt to a series of glioma cell lines evidenced a high rate of micronuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss, and chromosome aberrations such as nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere endfusions determining the furrow regression, and nuclear buds (NBUDSs), a biomarker of elimination of amplified DNA and/or DNA repair complexes (Figure 8a, b).Thus, binucleated tetraploid cells may be transmitted to the progeny and enhance subsequent rounds of aberrant mitosis.

Cytogenomics of gliomas
Chromosomal instability can be detected by different techniques, including conventional karyotyping, fluorescence in situ hybridization (FISH), spectral karyotyping (SKY) and array-based comparative genomic hybridization (aCGH) analyses.The classic assay to monitor and quantify chromosome aberrations is karyotyping (see Figure 2).The in situ hybridization technique with fluorescently labelled probes targeting specific chromosomes is commonly applied on fixed glioma cells, allowing the analysis of chromosomes of interest cell by cell.Examples of FISH analysis in glioma cell lines are shown in Figure 9. Aneuploidies are rapidly detectable by interphase FISH as well as by quantification of micronuclei formed by chromosomes that lagged behind during a previous mitosis (Figure 10).The technique of array-CGH is considered the most powerful tool for identifying copy number changes of genetic material, since it combines high resolution and large scale genomic analysis, characteristics that are not combined by conventional approaches.Since it allows a quantification of amplifications and deletions, pointing through human genome databases directly to the affected genes, aCGH technology is more and more used in the study of tumours for the identification of potentially causative cancer genes.aCGH studies have been applied to gliomas and have successfully complemented previously published metaphase-CGH, SKY and LOH (loss of heterozigosity) analyses (Bredel et al, 2005;Cowell et al., 2004aCowell et al., , 2004b;;Kitange et al., 2005;Nigro et al., 2005).Integration of the results has demonstrated an excellent correlation between the findings obtained through this genomic approach and those obtained by alternative techniques, stressing the usefulness and overall accuracy of aCGH as compared to classic previously widely employed analyses (Cowell et al., 2004a(Cowell et al., , 2004b)).Comparative analysis of elaborated aCGH data led to identify copy number changes shared by various glioma grades as well as aberrations apparently related to progression to glioblastoma (GBM) (Roversi et al., 2006).

Non-random chromosomal aberrations in gliomas: The 19q13 abnormalities
Over the last decade, molecular approaches including mutation screening, LOH and aCGH analyses have led to identify the most frequently recurring genomic imbalances associated with each WHO glioma subtype (Kitange et al., 2005;Koschny et al., 2002;Shapiro, 2002) and hence the driver genes acting in pathways involved in glioma development, either in the initiation stages (Tp53 and Ras by PDGF-NF1) or in malignant progression (Rb-CDKN2-CDK4) (Collins, 2004;Zhu & Parada, 2002).Comprehensive genomic characterization by integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in >200 glioblastomas has then refined the definition of human glioblastoma genes and core pathways (The Cancer Genome Atlas [TGCA] Research Network, 2008).Deletion of chromosome 19q is nevertheless of particular interest, as it is shared by all three glioma subtypes, occurring in approximately 75% of oligodendrogliomas, 45% of mixed oligoastrocytomas and 40% of astrocytomas (von Deimling et al., 1992(von Deimling et al., , 1994)), where it is associated with the transition from low-grade to anaplastic tumours (Ohgaki et al., 1995;Ritland et al., 1995;Smith et al., 1999) Box 2. TSGs: tumour suppressor genes.
At the cytogenetic level, chromosome 19q abnormalities are more frequently detectable in GBM than in low grade glioma, with 19q13 as the most affected region, as shown in Table1.
Furthermore, similarly to oligodendroglioma, combined LOH of 1p and 19q was found to define a small subset of GBM patients with a significantly better survival, even if their tumours were not morphologically distinguishable from the bulk of GBMs (Schmidt et al., 2002).This finding has been translated into significant advance in the prognosis and treatment of oligodendrogliomas (van den Bent, 2004).A candidate tumour suppressor region has been assigned by LOH to 19q13.3 (Hartmann et al., 2002), but no positional or functional candidate gene in this band has yet been appointed.
Only recently an integrated analysis of human glioblastoma multiforme with the application of next generation sequencing technology disclosed a new marker associated with an increase in overall survival, represented by recurrent mutations in the active site of isocitrate dehydrogenase 1 (IDH1) in a large fraction of young patients with secondary GBM (Parsons et al., 2008).

Identification of MARK4 gene through refined FISH mapping of 19q13 breakpoints
FISH studies of structural 19q chromosomal rearrangements in glioma (Magnani et al., 1999) and a detailed analysis of the breakpoints underlying the 19q13 alterations in the MI-4 glioblastoma cell line, led to identify a 19q13.2intrachromosomal duplication of the MAP/microtubule affinity-regulating kinase 4 (MARK4) gene (Beghini et al., 2003) (Figure 11).Genomic profiling by means of array-CGH interrogation of 25 primary glioma cell lines including the MI-4 GBM cell line (Roversi et al., 2006) revealed that the BAC clone encompassing MARK4 at 19q13.2 (Figure 12) is included in a "gain" region in a few of the tested cell lines and confirmed MARK4 duplication in the MI-4 glioblastoma cell line (Figure 13).The combined FISH and array-CGH results provided the rationale for investigating a possible role of the serine-threonine kinase MARK4 in glioma.It's worth of note that this gene, belonging to the so called "kinome", maps at the centromeric boundary of the 19q13.3LOH region in glioma.

The family of MARK kinases
MARK4 (MAP/microtubule affinity-regulating kinase 4) is a member of the MARKs family, constituted in mammals by four serine-threonine kinases (MARK1-4) which are able to phosphorylate the microtubule-associated proteins (MAPs, including Tau, MAP2, MAP4 and doublecortin) (Drewes et al., 1997).Microtubules (MTs) are cytoskeleton cylindrical structures formed by and tubulin dimers; dimers can quickly assemble or disassemble, causing the microtubules to grow or shorten and making them very dynamic.MAPs association stabilizes the MTs; when MARK kinases link a phosphate group to MAPs (phosphorylation), MAPs cannot associate to MTs any longer, thus microtubules become more instable and disassemble (Figure 14).charged residues, which may interact with negatively charged regions of cytoskeletal proteins, MARK catalytic domain or MARK CD domain (Tochio et al., 2006) with an inhibitory effect.It has been proposed it could be involved in protein localization to the membrane, being identified as a domain that binds membrane anionic phospholipids, in particular phosphatidylserine (Moravcevic et al., 2010).(Timm et al., 2008); in addition, phosphorylation by CaMKI (calcium/calmodulin-dependent protein kinase I) activates MARK2 (Matenia & Mandelkow, 2009).On the contrary, phosphorylation by the glycogen synthase kinase 3 (GSK3 ) on the serine residue in the activation loop, by aPKC (atypical protein kinase C) in the spacer region or by Pim1 kinase, down-regulates MARK activity (Matenia & Mandelkow, 2009;Timm et al., 2008).Finally, interaction between MARK catalytic domain and other proteins/MARK domains (such as 14-3-3 proteins, PAK5, MARK UBA and KA1 domains) inhibits MARK activity (Marx et al., 2010).

MARKs functions
Since MARK kinases regulate the affinity between MAPs and MTs, they are implicated in several cellular processes involving the microtubules, such as cytoskeleton dynamics, neuron motility (Schaar et al., 2004), and microtubule-dependent transport of proteins, vesicles and organelles (Mandelkow et al., 2004).Microtubules also play an important role in centrosome formation (Box 3) and in the correct distribution of the chromosomes in the two daughter cells during cell division (mitosis and cytokinesis; Box 4).
Tau is a microtubule-associated protein particularly expressed in the central nervous system.The aggregation of hyperphosphorylated Tau has been demonstrated to form insoluble neurofibrillary tangles (Chin et al., 2000;Gamblin et al., 2003) which are characteristic of Alzheimer's disease.MARKs role in this pathology has been evaluated in many studies, demonstrating, as an example, MARK co-localization with neurofibrillary tangles (Chin et al., 2000).MARK2 is involved in establishing cell polarity, cooperating in the organization of the epithelial structure of liver, kidney and stomach (Cohen et al., 2004;Matenia & Mandelkow, 2009), and regulating axon formation in neuronal cells (Chen et al., 2006).Experiments in mice demonstrated that MARK2 is also implicated in many physiological functions, such as fertility, homeostasis of the immune system, memory, growth and metabolism (Bessone et al., 1999;Hurov et al., 2001;Hurov & Piwnica-Worms, 2007;Segu et al., 2008).MARK3 plays an important role in cell signaling and cell cycle control: phosphorylation of some proteins by MARK3 induces their binding to 14-3-3 proteins thus regulating many cellular pathways (Bachmann et al., 2004;Müller et al., 2001).

MARK4
MARK4 is the less characterized member among MARK proteins.It has been discovered by Kato and colleagues in 2001 among a few genes whose expression resulted significantly increased in hepatocarcinoma cells with elevated -catenin levels in their nucleus (Kato et al., 2001).MARK4 gene is located on chromosome 19q13.2,consists of 18 exons and encodes at least two isoforms, namely MARK4S and MARK4L, originated by alternative splicing (Kato et al., 2001) (Figure 16).mRNA splicing is a complex process consisting in the removal of introns, which are non-coding sequences, and in the joining of exons, the coding sequences, to generate the "edited" mRNA ready to be translated into a protein. MARK4S ("short") protein is the native isoform, consisting of all the 18 exons, and is 688 aminoacid-long with predicted molecular weight of 75.3 kilo Daltons (kDa);  MARK4L protein derives from skipping of exon 16, which causes a shift of the reading frame1 with a downstream stop codon, originating a longer protein (752 aminoacids; predicted molecular weight: 82.5 kDa).Both MARK4L and S share the same protein structure of MARKs, with 90% sequence homology in the kinase domain.The two isoforms differ in the C-terminal tail, since MARK4L includes the kinase-associated 1 domain as the other MARK proteins, whereas MARK4S contains a domain with no homology to any known structure (Kato et al., 2001;Moroni et al., 2006) (Figure 16).Actually, MARK4 has less sequence homology in the Cterminus compared to the other MARKs; nevertheless MARK4L tail seems to fold in a similar shape, suggesting that the role of the C-terminal region may apply also to MARK4L (Marx et al., 2010).

MARK4 regulation
Phosphorylation by LKB1, in the activation loop, activates MARK4, while polyubiquitination of MARK4 inhibits the kinase activation (Al-Hakim et al., 2008).Furthermore, as MARK4 interacts with aPKC (Brajenovic et al., 2008), it could be phosphorylated and inactivated by this kinase as reported for MARK2 and MARK3.

MARK4 interactors and hypothetical functions
By tandem affinity purification and immunoprecipitation experiments, near twenty proteins have been identified as putative MARK4 interactors (Brajenovic et al., 2008).Among them, PKCλ and Cdc42 are implicated in cell polarity control and TGF IAF (transforming growth factor -inducing anti-apoptotic factor) is thought to be a hortologue of Miranda, a protein involved in the asymmetric division of neuroblasts in Drosophila.MARK4 interacts with the 14-3-3η isoform (Angrand et al., 2006;Brajenovic et al., 2008) of 14-3-3 proteins, which control multiple cellular processes by binding phosphorylated proteins and could directly regulate MARK4 or act as bridges among different pathways.Other MARK4 interactors are ARHGEF2, a cytoskeleton binding protein, and Phosphatase 2A, which is associated to microtubules and regulates Tau (Brajenovic et al., 2008).MARK4 protein has been also found to co-localize and co-precipitate in complex with , , and tubulin, myosin and actin (Brajenovic et al., 2008;Trinczek et al., 2004).
As the other MARK members, MARK4 phosphorylates MAPs, increasing microtubule dynamics; therefore, as also suggested by the interactions above reported, MARK4 may be involved in many processes involving microtubules, such as cytoskeleton dynamics.

Up-regulation of MARK4L in glioma
MARK4 gene is expressed ubiquitously in human tissues, with particularly elevated levels in brain and testis (Kato et al., 2001).Few MARK4 expression studies are reported in literature; they were performed with nonquantitative methods, such as northern blot (Kato et al., 2001;Schneider et al., 2004;Trinczek et al., 2004) and semi-quantitative competitive PCR (polymerase chain reaction) (Moroni et al., 2006), on different organisms (human, rat and mouse tissues) not always allowing to discern between the two MARK4 isoforms.MARK4 transcriptional variants are differentially regulated in human tissues, especially in the central nervous system: MARK4S is the predominant isoform in mouse and human brain, while MARK4L has been found highly expressed in neural progenitors and in gliomas (Beghini et al., 2003;Moroni et al., 2006).By a semi-quantitative approach MARK4L has been found up-regulated in glioma tissue samples (fragments of glial tumours excised from patients) and glioma cell lines, of different malignancy grades, including the MI-4 GBM cell line carrying the MARK4 duplicated gene as detected by FISH and aCGH analysis.MARK4L has been also found highly expressed in neural progenitors and down-regulated during their glial differentiation into astrocytes, suggesting that it might be necessary for proliferation, being thus highly enriched in proliferating or undifferentiated cells (Beghini et al., 2003) (Figure 17).Protein kinase activation, often caused by gene amplification and/or mutation, is frequently associated to cancer initiation and progression, as most kinases are involved in cell proliferation.Although array-CGH analyses on glioma cell lines showed that the BAC clone encompassing MARK4 at 19q13.2 is included in a "gain" region in a few of the tested cell lines, it did not evidence MARK4 copy number variations, except for the MI-4 GBM cell line (Roversi et al., 2006).Only a few MARK4 alterations are reported in the literature, namely two missense mutations (aminoacidic substitution) in exon 12 (R377Q and R418C in the spacer region), two silent mutations (no aminoacidic substitution) in exons 5 (Y137Y) and 9 (I286I) (kinase domain), while one intronic mutation (exon 8 +5 C>T; kinase domain) has been found in a few tumour samples (Greenman et al., 2007).In addition, only a splice-site mutation (exon 13 +1 G>A; spacer region) has been identified in one among 91 glioblastoma samples (TGCA Research Network, 2008).However, CpG methylation and/or promoter amplification have not yet been investigated.Based on this evidence, neither amplification nor mutations of MARK4 gene seem to be the cause of its reported sustained expression in glioma samples.

MARK4 sub-cellular localization in glioma cell lines
Recently, immunofluorescence analyses with a specific anti-MARK4L antibody highlighted multiple sub-cellular localizations for the endogenous MARK4L protein in glioma cell lines (Magnani et al., 2009).

Centrosome localization
It has been assessed that, under microtubule-stabilizing conditions, MARK4L localizes in the perinuclear region of glioma cell lines.By co-localization experiments with both anti-MARK4L and anti-tubulin (the main centrosomal protein) antibodies, this perinuclear localization has been demonstrated to correspond to the centrosome (Magnani et al., 2009), as shown in Figure 18 (Box 3).This result confirms previous data referring to exogenous MARK4 protein conjugated to GFP (green fluorescent protein), which has been shown to co-localize with microtubules and centrosomes of CHO (Chinese hamster ovary) and neuroblastoma cell lines (Trinczek et al., 2004), in contrast to MARK1, MARK2 and MARK3 that exhibit uniform cytoplasmic localization.Furthermore, it has been demonstrated that the association with the centrosome is independent from microtubules, since it is not abolished when microtubules are depolimerized by nocodazole treatment (Magnani et al., 2009).The endogenous MARK4L localizes both at normal interphase centrosomes (Figure 18) as well as at the aberrant centrosomes frequently observed in glioma cell lines (see Figure 4), suggesting a possible link between the alternatively spliced kinase and the mitotic instability frequently observed in human glioma.Two abnormal centrosome configurations are reported: a random one (multiple centrosomes randomly distributed) and a clustered one (multiple centrosomes collected in a single large aggregate) (Magnani et al., 2009), as depicted in Figure 19.

Midbody localization
The centrosome association is maintained during the entire course of mitosis, as MARK4L co-localizes with tubulin in all the cell cycle phases.The anti-MARK4L antibody is also detected in the midbody, a microtubule structure forming at the contact point between the two daughter cells at the end of the cell division.These data demonstrate that the kinase is endogenously associated with the centrosomes during the whole cell cycle and concentrates thereafter into the midbody during cytokinesis (Magnani et al., 2009) (Figure 20) (Box 4).Box 5.
The overall immunofluorescence data on endogenous MARK4L protein confirm the previous evidence on its centrosome association and highlight two novel localization sites of MARK4L: the nucleolus and the midbody (Magnani et al., 2009).
Immunoblotting with anti-MARK4L antibody on centrosomes, midbody and nucleoli isolated by biochemical fractionation from glioblastoma cell lines confirmed the presence of MARK4L protein in each fraction, validated by antibodies specific for each cell structure: anti-tubulin antibody for centrosomes, anti-tubulin for the midbody ( tubulin, together with tubulin, accounts for 30% of midbody proteins) and anti-nucleolin for the nucleolus (Magnani et al., 2009).
The localization pattern of MARK4L delineated by the above studies suggests that the kinase may take part in cell cycle progression and influence the microtubules, particularly those affecting the centrosome and midbody.MARK4L association with the nucleolus in glial tumours is very interesting, since MARK4L could have a functional impact on this organelle, being requested for its building and maintenance like other protein kinases, as well as it could be spatially regulated by alternate translocation in and out the nucleolus.Many proteins are indeed sequestered in the nucleolus and then released according to a temporally regulated activity, since they must exert their function in certain phases of the cell cycle (Visintin & Amon, 2000).Last, the nucleolar localization of a protein may also influence its stability, protecting the protein from proteasomal degradation, since proteasomes are present in the nucleoplasm but not in nucleoli (Wojcik & DeMartino, 2003).

Conclusion
A few remarks can be drawn from the above synthesis on cytogenomics of human gliomas and the MARK4 cell cycle gene as a likely "player" in gliomagenesis.
Gliomas are one of the most intractable tumours due to their "complex identity": as it has been beautifully underlined, the generation -since the earliest glioma stages -of multiple cell populations with different genotypic and phenotypic features makes unlikely to succeed therapeutic strategies targeting only clones with "dominant" or "average" characteristics of the cell population (Noble & Dietrich, 2004).The intrinsic genomic heterogeneity of human glioma has first been disclosed cytogenetically, as documented by a huge number of studies which across two decades have used the cytogenetic tools suitable to monitor the intratumour cell heterogeneity and to discern "recurrent" and potentially causative chromosomal rearrangements.A few of these rearrangements entered the diagnostic and prognostic flow chart of gliomas, others allowed to identify crucial genes which mutations or imbalance are the signature of a specific glioma type or glioma malignancy stage.In line with a research pathway that has been reiterated for several genes of relevance in cancer, focus on MARK4 has been pinpointed by cytogenetics and deepened by multiple tools ranging from gene-targeted molecular to genomic and cytogenomic analyses.Despite its nature of serine-threonine kinase gene, MARK4 has not be found mutated or affected by copy number alterations in glioma, while its encoded proteins represented by two different isoforms, MARK4S and MARK4L, could be featured as a potential target of dysregulation in tumours due to its dual nature.The latter isoform, produced by alternative splicing, has been found up-regulated in glioma and shown to display sub-cellular localizations, namely the centrosome, the midbody and the nucleolus, which strictly associate it with the process of cell division.Interestingly, alternative mRNA splicing has been considered a mechanism not only increasing proteomic complexity but also involved in cancer, through mechanisms of oncogenes/tumour suppressors activation/inactivation or through the generation of CIN (López-Saavedra & Herrera, 2010).CIN is a general property of aneuploid cancer cells and is generated by defects in different processes, among which the regulation of the number of centrosomes, the dynamics of microtubules attachment to the kinetochores and the overall control of cell cycle.Defects in centrosomal number and structure have been well documented in gliomas (D'Assoro et al., 2002;Katsetos et al., 2006;Magnani et al., 2009) raising the issue whether the increased MARK4L isoform, a gene involved in microtubule dynamics, may concur to errors in chromosomal segregation driving gliomagenesis.Recent application of multidimensional technological approaches has comprehensively highlighted the scenario of glioma genes and core pathways.However, despite the impressive advances, the links between genes alteration and cellular behavior are yet hampered by the multiplicity of the genetic lesions and the interconnections among the different affected pathways.Hopefully ongoing and next years research will compose the puzzle promising to translate into the clinical set the unraveled glioma pathomechanisms.

Acknowledgement
We thank the Associazione italiana per la ricerca sul cancro (AIRC) for supporting this work (grant n 4217 to LL for 2008).

Cell cultures and preparation of human metaphase chromosomes
Glioma cell lines were derived from primary tumour post-surgery specimens and subsequently maintained by serial passages in RPMI 1640 medium containing 5% Fetal Calf Serum at 37°C in a 5% CO 2 atmosphere.Most of the cell lines were used within the first 30 passages.
Metaphase spreads were obtained on both fresh tumours and cultured cell lines, harvested when "peak" mitotic activity was observed; usually, a 16-hour treatment with Colcemid at a final concentration of 0.01-0.02mg/ml is employed (Magnani et al., 1994).

Fluorescence in situ hybridization (FISH) analysis
Fluorescence hybridization with genomic DNA has proven to be a powerful tool for identification of chromosome rearrangements in cancer cells.Potential applications include detection of chromosome-specific aneuploidy in metaphase and interphase cells, quantification of the frequency of chromosome translocations and/or aneuploidy as a measure of genetic damage, and detection of diagnostically and prognostically relevant chromosomal lesions.Detection of translocations between human metaphase chromosomes is possible by using cocktails of chromosome-specific sequences that hybridize more or less uniformly along the chromosome.The coverslips are then removed and fluorescein-avidin DCS is applied.The coverslips are put back in their original places and the slides incubated 20 min at 37°C.They are then washed in BN buffer at 45°C.The intensity of biotin fluorescence is amplified by adding a layer of biotinylated goat anti-avidin antibody followed, after washing as above, by another layer of fluorescein-avidin DCS.After washing in BN buffer a fluorescence anti-fade solution is added.The DNA counterstain [4,6-diamidino-2-phenylindole (DAPI) or propidium iodide] is included in the anti-fade solution (Magnani et al., 1999;Pinkel et al., 1986).

Immunofluorescence
Immunofluorescence analyses enable to visualize, by fluorescence microscopy, the subcellular localization of a specific protein in cultured cells.The target protein is recognized by an antibody, which in turn is conjugated to a fluorochrome emitting fluorescent light.Briefly, cells are grown on glass chamber slides, then permeabilized (with solvents that extract lipids from the membranes allowing antibodies to reach a sub-cellular structure) and fixed (in order to protect the cell structure from eventual damages and to "freeze" cells in their current state).Afterwards, cells are incubated with bovine serum albumin (BSA) to block non-specific binding of antibodies.Glass slides are then incubated with a primary antibody specific to the target protein, then with a secondary antibody conjugated to the fluorochrome and finally observed under the microscope (Magnani et al., 2009).

Biochemical fractionation and immunoblotting
By biochemical fractionation we mean the whole techniques that allow to separate and isolate intact cellular components.It usually consists in carefully breaking the cell membrane with homogenizers and isotonic/hypotonic solutions, so that intact organelles can come out, and in separating cellular components by centrifugation, on the basis of differences in their mass and specific weight.Centrosome, midbody and nucleoli isolation protocols are described in Magnani et al., 2009 and based on methods respectively by Moudjou & Bornens, 1994;Chu & Sisken, 1977;Muramatsu et al., 1963.In particular, for midbody isolation cells are synchronized in mitosis by nocodazole treatment and then released from mitotic arrest in nocodazole-free medium, so that after 30 minutes near 90% of cells had formed the midbody.
After membrane breaking, all the passages are done at 4°C and with protease inhibitors, in order to prevent protein degradation, possibly exerted by released proteases.Proteins extracted from centrosome, midbody and nucleolus fractions are then analyzed by immunoblotting.Proteins are first separated, according to their molecular weight, by SDS-PAGE (Sodium Dodecyl Sulphate -PolyAcrilamide Gel Electrophoresis): this technique allows proteins to migrate, driven by electric current, in a porous gel, with speed depending exclusively on their size.Afterwards, separated proteins are transferred onto a membrane, incubated with a blocking solution (BSA or milk) to prevent non-specific binding of antibodies and then incubated with appropriate antibodies (immunoblotting).The primary antibody is specific to the target protein and is recognized by the secondary antibody conjugated to HRP (horse radish peroxidase).Antibodies are detected by covering the membrane with a peroxide/enhancer solution, which is oxidized by HRP and emits light signals.

Fig. 1 .
Fig. 1.(a) The G-banded, near diploid karyotype of MI-4 GBM (glioblastoma multiforme) cell line (Magnani et al., 1994), showing trisomy of chromosome 7, monosomy of chromosome 10 and a complex rearrangement involving chromosomes 1, 9 and 19.(b) The G-banded, near tetraploid karyotype of MI-4 cell line, displaying several chromosome losses and structural rearrangements including marker chromosomes.(c) Representative polyploid metaphase from MI-60 GBM cell line, characterized by a high frequency of hyperdiploid cells.(d) Apoptotic and large nuclei of MI-60 cell line.

Fig. 2 .
Fig. 2. (a) Chromosome 1 rearrangements of both p and q arms observed in different glioma cell lines by G-banding.(b) Rearrangements of chromosome 9p, sharing the loss of p21 band, observed in different glioblastoma cell lines by G-banding.

Fig. 3 .
Fig. 3. Proposed events of lagging chromosomes in cancer cells with extra centrosomes through merotelic kinetochore orientation.(top) In the presence of extra centrosomes (three instead of two, as example), merotelic kinetochore orientation may occur: one kinetochore is bound by spindle microtubules from two centrosomes (right) instead of just one (left).(bottom) As cells move to mitosis and cluster extra centrosomes in a bipolar spindle, many attachment errors persist into anaphase, leading to lagging chromosomes.

Fig. 4 .
Fig. 4. Immunofluorescence with anti-tubulin antibody (red) of representative glioblastoma cell lines, showing (a) multiple centrosomes; (b) multipolar spindles; (c) a mitotic bipolar spindle in which centrosomes are larger than the normal one (likely extra centrosomes clustered into two spindle poles), a condition that favours mitotic stability and neoplastic growth; (d) normal centrosomes and a mitotic bipolar spindle configuration.The nuclei are counterstained with DAPI (4',6-di amidino-2-phenyl indole) (blue).

Fig. 8 .
Fig. 8. Photomicrographs of glioma cell lines showing (a) typical binucleated cells with nucleoplasmic bridges and (b) binucleated cells with micronuclei and nuclear buds.

Fig. 10 .
Fig. 10.(a) Interphase FISH with centromeric probes of chromosomes 7 and 10 showing trisomy of chromosome 7 and monosomy of chromosome 10 in MI-4 GBM cell line.(b) Interphase FISH with whole chromosome 19 painting probe showing a micronucleus labelled by chromosome 19 material.DNA is counterstained with DAPI (blue).

Fig. 11 .
Fig. 11.19q13.2intrachromosomal duplication of MARK4 in the MI-4 GBM cell line detected by G-banding and FISH analysis using a whole chromosome painting 19 probe and a MARK4-specific cosmid clone.

Fig. 13
Fig. 13.(left) Chromosome 19q array-CGH of MI-4 GBM cell line, showing duplicated MARK4 gene (red star) and the common LOH region in glioma.(right) Schematic representation of MARK4 position on chromosome 19, at the boundary of 19q13.3LOH region.

Fig. 14 .
Fig. 14.Schematic representation of microtubules.Assembled and tubulin dimers form the microtubules, stabilized by MAP association.When MAPs are phosphorylated, they are no more able to bind microtubules, which disassemble.

Fig. 15 .
Fig. 15.Schematic representation of MARK protein structure.Boxes are not drawn to scale.

Fig. 16 .
Fig. 16.Alternative splicing of exon 16 gives origin to MARK4 isoforms.When exon 16 is included in the mRNA, the stop codon is inside exon 18 and the encoded protein, MARK4S, lacks the KA1 domain at the C-terminal tail (left); when exon 16 is skipped, a shift of the reading frame occurs, changing the stop codon and generating a longer MARK4L protein, which has the classical KA1 domain (right).

Fig. 19 .
Fig. 19.Anti-MARK4L (green; left) and anti-tubulin (red; middle) antibodies showing colocalization signals in abnormal centrosomes of glioma cell lines.Both the abnormal centrosome configurations are reported: the random one (top) and the clustered one (bottom).The nuclei are counterstained with DAPI (blue, right).

Fig. 20 .
Fig. 20.Co-localization of MARK4L (green) and tubulin (red) proteins at the midbody (arrow) during the cytokinesis of a glioma cell.The nuclei are counterstained with DAPI (blue).