Heparanase Is a Critical Regulator of Mitotic Spindles Required for Maintaining Chromosome Stability

Abstract

The mitotic spindle, assembled by microtubule organization center (MTOC), is a critical structure required for proper chromosome segregation during mitosis. Studies demonstrated that defects of mitotic spindle assembling would result in chromosome instability, associating with oncogenesis. In this study, we identified that heparanase (HPSE) was localized on MTOC in mitotic cells. Induced expression of HPSE in human airway epithelial cells promoted mitotic spindle formation, and silencing of HPSE expression disrupted the mitotic spindle formation, resulting in chromosome instability, including chromosome mis-segregation and increased micronuclei formation. In conclusion, HPSE is an important protein in regulation of mitotic spindle and loss of HPSE function on MTOC associates with chromosome instability, indicating that loss of HPSE on MTOC may play critical roles in oncogenesis.

Introduction

The mitotic spindle is a critical structure required for proper chromosome segregation during mitosis and is assembled by microtubule organization center (MTOC). Increased function of negative regulators of mitotic spindle assembling and the spindle checkpoint function will result in chromosome mis-segregation and micronuclei formation, a process associated with oncogenesis (Cassimeris and Skibbens, 2003; Ditchfield et al., 2003; Varmark, 2004; Viji et al., 2007; Janssen et al., 2011; Crasta et al., 2012; Granic and Potter, 2013; Roshan et al., 2014). Heparan sulfate proteoglycans (HSPGs) are macromolecules with the common characteristic of containing covalently attached heparan sulfate (HS) side chains. They function in regulation of cell proliferation, differentiation, and migration through interacting with fibronectin, laminin, collagen, and growth factors (Schuger et al., 1996; Izvolsky et al., 2003; Lin, 2004; Harisi et al., 2005). Recent studies identified that some HSPGs, including syndecan-4 (SDC4) and glypican-1, were located on MTOC or mitotic spindle (Qiao et al., 2008; Zong et al., 2009; Keller-Pinter et al., 2010), providing a functional link of HSPGs with mitotic spindle regulation. Malfunction of those HSPGs and their regulators may associate with defects of chromosomal segregation.

Heparanase (HPSE) is the only known mammalian endoglycosidase. It regulates the function of HSPGs through remodeling their HS side chain (Fernandes et al., 2014). Previous studies showed that increased expression of HPSE was found to be associated with tumor angiogenesis and metastasis through modifying the HS side chain of its substrate proteins in extracellular matrix (ECM) (Nakajima et al., 1988; Vlodavsky et al., 1994; Bergers et al., 2000; Elkin et al., 2001). Studies also identified that HPSE located in cell nuclei or cytoplasm in lung cancer tissues and the cytoplasmic location significantly correlated with poorer prognosis of the patients (Schubert et al., 2004; Cohen et al., 2008), indicating that HPSE has different functions at different cellular compartments. Whether HPSE locates on mitotic spindle and functions in maintaining chromosome stability of the cell are unknown. In this research, we found that HPSE located on MTOC and silencing of HPSE expression disrupted mitotic spindle formation, resulting in chromosomal mis-segregation and micronuclei formation. These data demonstrated that HPSE was an important regulator of the mitotic spindle, and dysregulation of HPSE may associate with induction of chromosome instability during oncogenesis.

Materials and Methods

Cell culture

Human bronchial epithelial cells (HBECs), a human airway epithelial cell line, were cultured in Keratinocyte-SFM Basal medium (GIBCO) supplemented with Bovine Pituitary Extract (GIBCO) and Recombinant Epidermal Growth Factor (GIBCO).

Calculation of mitotic cells with normal mitotic spindle (%)

pcDNA3.1 or pcDNA3.1-HPSE was transfected into cultured cells using Lipofectamine 2000 (Invitrogen, Grand Island, NY). Forty-eight hours after transfection, microtubule inhibitor nocodazole (Andreassen and Margolis, 1994) was added (final concentration: 100 nM) to the media for 18 h to disrupt the mitotic spindle, then cells were washed with PBS thrice to release nocodazole, and fixed at different time points. Immunofluorescence staining of α-tubulin and DAPI was carried out, and pictures were taken using Olympus BX51 microscope. Total numbers of mitotic cells at prophase, metaphase, and anaphase were counted (n = 200), and among those cells, the number of mitotic cells with normal mitotic spindle (Fig. 3A′) were counted. Percentage of mitotic cells with normal mitotic spindle was calculated as per following equation: (number of mitotic cells with normal mitotic spindle/total numbers of mitotic cells) × 100%. Independently repeated experiments (n = 3) were performed, and data are expressed as the mean ± standard error of the mean (SEM).

Calculation of mitotic cells with abnormal mitotic spindle (%)

HBECs were transfected with Hpse siRNA (oligo ID HSS116734 or oligo ID HSS116735; Invitrogen) or control siRNA (Cat. No. 12935-300; Invitrogen) and harvested at 48 h after transfection. Immunofluorescence staining of α-tubulin and DAPI was carried out. And pictures were taken using Olympus BX51 microscope. Total mitotic cells at prophase, metaphase, and anaphase (n = 200) were counted, and among those cells, mitotic cells with abnormal spindles (Fig. 4A–F) were counted, and the percentage of mitotic cells with abnormal spindle was calculated as per following equation: (the number of mitotic cells with abnormal spindle/total mitotic cells at prophase, metaphase, and anaphase) × 100%. Independently repeated experiments (n = 3) were performed, and data are expressed as the mean ± SEM.

Immunofluorescence staining

Cells were cultured on coverslips, harvested and immunostained as previously described (Wang et al., 2014). Primary antibodies are listed in Table 1. Fluorochrome-conjugated secondary antibodies (Alexa Fluor 488 or 594; Invitrogen) were used. Images were captured with an Olympus BX51 microscope.

Table 1.

Antibody List

Antibody name	Company	Cat. No.	Immunogen	Host species	Experiment
HPSE	Abcam	Ab85543	Human	Rabbit	WB/IF
γ-Tubulin	Sigma	T6557	Human	Mouse	IF
γ-Tubulin	Sigma	T3559	Human	Rabbit	WB
α-Tubulin	Sigma	T6199	Human	Mouse	WB/IF
Acetylated a-tubulin	Sigma	T6793	Human	Mouse	WB

HPSE, heparanase; IF, immunofluorescence; WB, Western Blot.

Western Blot

Cells lysates were harvested at 72 h after transfection, and Western Blot was performed. Antibodies are listed in Table 1. Independently repeated experiments were performed (n = 3), densitometry quantification of the Western Blot bands were carried out using Quality One Software (Bio-Rad Laboratories, Philadelphia, PA), and data are expressed as the mean ± SEM. All images are representative of corresponding experiments.

Protein immunoprecipitation assay

Immunoprecipitation assays were performed using protein G agarose beads according to the manufacturer's protocol (Roche). Briefly, total cell lysates were collected at 72 h after transfection and precleared with protein G agarose, then incubated with HPSE specific antibody for 3 h at 4°C, followed by incubation with protein G agarose for 3 h at 4°C. Binding proteins were eluted from pelleted beads in the gel-loading buffer. The γ-tubulin protein level was analyzed by Western blot. Independently repeated experiments were performed (n = 3); all images are representative of corresponding experiments.

Flow cytometry analysis

Cells were treated as previously described (Crasta et al., 2012). Briefly, cells were cultured and transfected with control siRNA or Hpse siRNA. Seventy-two hours after transfection, cells were harvested and fixed in 75% ethanol at 4°C overnight, followed by incubation in RNase A solution (50 μg/mL) at 37°C for 15 min, and stained with propidium iodide (Sigma) for 30 min in the dark at 37°C. The percentage of cells in each cell cycle phase was measured according to the S-fit program in the FACSta. Independently repeated experiments (n = 3) were performed, and data are expressed as the mean ± SEM.

Statistical analysis

All data were analyzed using SAS software (version9.0; SAS Institute, Cary, NC). Student's t-test was used to determine differences between groups. The value p < 0.05 was considered as statistically significant.

Results

The highest level of HPSE protein was identified at metaphase in epithelial cells

To explore the function of HPSE protein during mitosis, HBECs were synchronized as described previously (Whitfield et al., 2002) and harvested at different time points after releasing of cell cycle synchronization. Western blot analysis demonstrated that protein level of HPSE was dynamically changed during mitosis, associating with changes of phospho-histone H3, a mitosis marker (Demion et al., 2014) (Fig. 1A). Consistent with these findings, fluorescent immunocytochemistry staining of HPSE in HBEC demonstrated that cytoplasmic staining of HPSE was significantly increased in cells at prophase, metaphase, and anaphase (Fig. 1B′–B′″), compared with cells at interphase (Fig. 1B), and the highest level of HPSE protein was observed at metaphase. Similar findings were also identified in other cell lines, for example, HaCaT cells (Supplementary Fig. S1A–A′″; Supplementary Data are available online at www.liebertpub.com/dna). Enriched HPSE staining was observed at specific cytoplasmic regions shaped like MTOC, indicating that HPSE may play a critical role in mitotic spindle organization during mitosis. The staining pattern of a HPSE substrate, SDC4, was found to be similar to HPSE in HBEC (Fig. 1C–C′″), which is consistent with findings in other cell lines (Keller-Pinter et al., 2010). In summary, HPSE may locate on MTOC and regulate mitotic spindle during mitosis.

FIG. 1.

The dynamic change of HPSE in HBECs during mitosis. (A) Western blot analysis of HPSE protein at different time points after releasing from cell cycle synchronization. Representative figures were shown. Phospho-histone H3 (PH3) was used to indicate different cell cycle phases, and GAPDH served as loading control. (B–B’”, C–C’”) Immunofluorescence staining of HPSE (red) (B–B’”) and SDC4 (red) (C–C’”); Nuclei were counterstained with DAPI (blue). Condensed staining of HPSE and SDC4 in regions shaping like MTOC was indicated by arrows. Scale bars, 10 μm. HBECs, human bronchial epithelial cells; HPSE, heparanase; MTOC, microtubule organization center; SDC4, syndecan-4.

HPSE located on MTOC

To determine whether HPSE locates on MTOC, co-fluorescent immunohistochemistry staining of HPSE with γ-tubulin, a MTOC associating protein, was carried out in HBEC. Although HPSE staining was observed throughout the cytoplasm, enriched staining of HPSE was identified in regions with γ-tubulin staining (Fig. 2A–C), indicating that HPSE was located on MTOC. To test whether HPSE and γ-tubulin interact with each other, HBECs were transfected with pcDNA3.1-Hpse or pcDNA3.1 plasmid, respectively; cell extracts were prepared 72 h after transfection and were analyzed by immunoprecipitation with HPSE antibody. The precipitated material was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the presence of γ-tubulin was detected by western blotting with a γ-tubulin antibody (Fig. 2D, E). These data demonstrated that HPSE was located on MTOC and that it may play a critical role in regulating mitotic spindle.

FIG. 2.

HPSE interacts with γ-tubulin. (A–A’”, B–B’”, C–C’”) Co-immunofluorescence staining of HPSE (red) (A–C) and γ-tubulin (green) (A’–C’). The nuclei were counterstained with DAPI (blue) (A”–C”). Co-localization of HPSE with γ-tubulin during mitosis was indicated by arrows. (D) Co-immunoprecipitation of HPSE with γ-tubulin. GAPDH was used as loading controls. (E) A bar graph shows that γ-tubulin interacting with HPSE was significantly increased in pcDNA3.1-Hpse transfected HBECs. The mean of the control was set to 1, relative protein levels are shown as mean ± SEM. Independently repeated experiments were performed (n = 3) (*p < 0.05). Scale bars, 10 μm. SEM, standard error of the mean.

Induced expression of HPSE promoted mitotic spindle formation

To access the role of HPSE in mitotic spindle formation, pcDNA3.1-Hpse or pcDNA3.1 was transfected into HBECs, respectively, 48 h after transfection. Nocodazole, a microtubule inhibitor, was added to the media for 18 h to stop the spindle formation. Then, cells were harvested at 0- and 10-min time points after nocodazole releasing. Immunofluorescent staining of α-tubulin, a spindle marker, was carried out to show the mitotic spindle structure. At 0-min time point, metaphase spindle formation was stopped in all the mitotic cells (Fig. 3A); no difference was identified between pcDNA3.1-Hpse transfected cells and pcDNA3.1 transfected cells. At 10-min time point, some cells with normal mitotic spindle structure were visualized (Fig. 3A′); percentage of mitotic cells with normal mitotic spindle was significantly increased in cells transfected with pcDNA3.1-Hpse, compared with the controls (Fig. 3B), indicating that HPSE promoted mitotic spindle formation. Consistent with this, increased levels of proteins functioning on mitotic spindle, including α-tubulin, acetylated α-tubulin, and γ-tubulin, were identified by Western blot in HBEC transfected with pcDNA3.1-Hpse, compared with controls (Fig. 3C, D). These data demonstrated that induced expression of HPSE could promote mitotic spindle formation.

FIG. 3.

Induced expression of HPSE promoted mitotic spindle formation. (A) Representative picture of abnormal spindles in nocodazole treated cells. (A′) Representative picture of normally formed mitotic spindle in cells released from nocodazole treatment. (B) Percentage of mitotic cells with normal mitotic spindle was calculated as described in Materials and Methods section. A bar graph shows that percentage of mitotic cells with normal mitotic spindle was significantly higher in pcDNA3.1-Hpse transfected HBECs, compared with pcDNA3.1 transfected HBECs 10 min after nocodazole releasing (*p < 0.05). (C) Western blot analysis of spindle components: α-tubulin, acetylated α-tubulin, and γ-tubulin. GAPDH served as loading control. (D) Densitometric quantification of the protein levels revealed that protein levels of α-tubulin, acetylated α-tubulin, and γ-tubulin were significantly increased, compared with controls. The mean of the control was set to 1; relative protein levels are shown as mean ± SEM (*p < 0.05). Scale bars, 20 μm.

Silencing of HPSE expression resulted in mitotic spindle defects

To further identify the function of HPSE in mitotic spindle, Hpse siRNA or control siRNA was, respectively, transfected into HBECs. Cells were harvested 72 h after transfection, and immunofluorescent staining of α-tubulin was carried out. Mitotic spindle structure was outlined by α-tubulin staining. Compared with controls, percentage of mitotic cells with abnormal mitotic spindles, including cells with decreasing α-tubulin staining and cells with collapsed spindle, was significantly increased in Hpse silenced cells (Fig. 4A–G). Consistent with these findings, Western blot demonstrated that proteins functioning in spindle assembling, including α-tubulin, acetylated α-tubulin, and γ-tubulin, were significantly decreased in Hpse silenced cells, compared with controls (Fig. 4H, I). These results demonstrated that HPSE was critical for mitotic spindle.

FIG. 4.

Silencing of HPSE expression induced abnormal mitotic spindle. (A–A”, B–B”, C–C”, D–D”, E–E”, F–F”) Immunofluorescence staining of α-tubulin (green) (A’–F’) in Hpse siRNA or control siRNA transfected HBECs. Nuclei were stained with DAPI (blue) (A–F). Mitotic cells with abnormal mitotic spindle, including collapsed spindle structure and weakening of α-tubulin staining, were observed in Hpse silenced cells (arrows). (G) A bar graph shows that percentage of mitotic cells with the abnormal spindle was significantly increased in Hpse silenced cells (N = 200, *p < 0.05). (H) Western blot analysis of spindle proteins: α-tubulin, acetylated α-tubulin, and γ-tubulin. GAPDH served as loading control. (I) Densitometric quantification of the protein levels revealed that α-tubulin, acetylated α-tubulin, and γ-tubulin were significantly decreased, compared with controls. The mean of the control was set to 1; relative protein levels are shown as mean ± SEM (*p < 0.05). Scale bars, 10 μm.

Silencing of HPSE expression induced chromosome mis-segregation

Mitotic spindle abnormalities would lead to chromosome mis-segregation, resulting in micronuclei formation (Crasta et al., 2012). To know whether HPSE silencing causes chromosome mis-segregation, immunofluorescence staining of α-tubulin and DAPI was performed in Hpse siRNA or control siRNA transfected HBECs. Lagging chromosomes were observed in Hpse silenced cells at anaphase (Fig. 5B, arrowheads), indicating that Hpse silencing affected the microtubule-chromosome attachment. Significantly increased incidence of micronuclei was observed in Hpse siRNA transfected HBECs compared with control (N = 200, *p < 0.05) (Fig. 5G). Chromosome mis-segregation would activate spindle checkpoint and induce G2/M cell cycle arrest. In agreement with this, G2/M arrest was observed in Hpse silenced HBECs by flow cytometry analysis (Supplementary Fig. S2). Taken together, these observations indicated that HPSE played a significant role in maintaining chromosome stability.

FIG. 5.

HPSE silencing induced chromosome mis-separation. (A–A”, B–B”) Immunofluorescence staining of α-tubulin (green) in Hpse siRNA or control siRNA transfected HBECs. Nuclei were stained with DAPI (blue) (A–B). Lagging chromosome was shown with arrowhead. (C–C”, D–D”, E–E”, F–F”) Representative figures of Hpse silenced cells with micronuclei at different cell cycles were shown, and micronuclei were indicated by arrow. (G) Micronuclei counting were carried out as described in Materials and Methods section. A bar graph shows that the micronuclei were significantly increased in Hpse silenced cells (N = 200, *p < 0.05). Scale bars, 10 μm.

Discussion

Defect of mitotic spindle is one of the critical causes of chromosome instability associating with oncogenesis (Venkatesan et al., 2015). In this study, we identified that HPSE locates on MTOC and is indispensable for proper segregation of chromosomes during mitosis; loss of MTOC localization of HPSE associates with chromosome instability.

HPSE is an important protein involved in regulation of mitotic spindle

Previous studies demonstrated that HPSE was a glycosidase and functions in cell proliferation, differentiation, and migration through modifying HSPGs located in different cellular compartments (Gingis-Velitski et al., 2004; Kobayashi et al., 2006; Nobuhisa et al., 2007; Zheng et al., 2015). In late endosome and lysosome, HPSE involved in the processing and recycling of heparin sulfate from internalized HSPGs (Goldshmidt et al., 2002; Cohen et al., 2005). In ECM, HPSE played a key role in both normal and disease processes such as cell migration, wound healing, angiogenesis, and inflammation (McKenzie, 2007). In the nucleus, it was thought to play a role in nuclear HS degradation and stimulation of gene expression (Schubert et al., 2004). In this research, HPSE was found to be enriched at M-phase during mitosis and co-localized with γ-tubulin on MTOC. HPSE silencing caused mitotic spindle defects, including decreasing of spindle protein levels (α-tubulin, acetylated α-tubulin, and γ-tubulin) and disruption of spindle structure, which in turn resulted in chromosome mis-segregation and micronuclei formation. This study identified a new cellular localization of HPSE and demonstrated that HPSE was an indispensable protein required for maintaining normal mitotic spindle structure. Loss of HPSE function on MTOC may play critical roles in chromosome instability during oncogenesis. The underline mechanism needs to be further explored.

Loss of HPSE function on MTOC associated with chromosome instability

Recent studies demonstrated that micronuclei, formed from chromosome mis-segregation and spindle checkpoint defect, can persist overall for several generations and generate more chromosome breaks and DNA mutations (Crasta et al., 2012). These provided strong mechanistic evidence that increased rates of chromosome mis-segregation and spindle checkpoint defect can accelerate oncogenesis. Our study demonstrated that loss of HPSE function on MTOC caused the defects in mitotic spindle, which consequently affected the chromosome segregation and resulted in increased micronuclei formation. These data indicated that loss of HPSE function on MTOC could be one cause of chromosome instability associating with oncogenesis. A significant G2/M arrest has been observed in Hpse silenced cell, indicating that chromosome mis-segregation caused by Hpse silencing activated spindle checkpoint machinery, and the defects could be partially corrected by intact function of spindle checkpoint. Compared with molecules functioning in negative regulation of both chromosome segregation and spindle checkpoint, including TAO1 and ZM447439 (Ditchfield et al., 2003; Viji et al., 2007; Roshan et al., 2014), silencing of Hpse resulted in much less changes of chromosome instability.

A large number of researches clearly link increased expression of HPSE to the process of tumorigenesis in a lot of cancers, including breast, colon, gastric, pancreatic, thyroid and prostate, and so on (Nadir and Brenner, 2014). In the present study, we identified that HPSE was localized on MTOC and that silencing of HPSE disrupted the mitotic spindle formation, resulting in chromosome instability, including chromosome mis-segregation and increased micronuclei formation. Whether the loss of MTOC localization of HPSE existed in cancer tissues and whether loss of MTOC localization of HPSE correlated with poor prognosis of cancer need to be further explored.

Footnotes

Acknowledgments

This research was supported by internal funds from the West China Second University Hospital and grants from the National Natural Science Foundation of China (NSFC81572754 and NSFC81322001, H.W.) and Fundamental Research Funds for the Central Universities (Excellent young scholars program, 2014SCU04B04, H.W.).

Disclosure Statement

No competing financial interests exist.

References

Andreassen

P.R.

, and Margolis

R.L.

(1994). Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells. J Cell Biol, 127, 789–802.

Bergers

, Brekken

, McMahon

, Vu

T.H.

, Itoh

, Tamaki

, et al. (2000). Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol, 2, 737–744.

Cassimeris

, and Skibbens

R.V.

(2003). Regulated assembly of the mitotic spindle: a perspective from two ends. Curr Issues Mol Biol, 5, 99–112.

Cohen

, Atzmon

, Vlodavsky

, and Ilan

(2005). Heparanase processing by lysosomal/endosomal protein preparation. FEBS Lett, 579, 2334–2338.

Cohen

, Doweck

, Naroditsky

, Ben-Izhak

, Kremer

, Best

L.A.

, et al. (2008). Heparanase is overexpressed in lung cancer and correlates inversely with patient survival. Cancer, 113, 1004–1011.

Crasta

, Ganem

N.J.

, Dagher

, Lantermann

A.B.

, Ivanova

E.V.

, Pan

, et al. (2012). DNA breaks and chromosome pulverization from errors in mitosis. Nature, 482, 53–58.

Demion

, Thireau

, Gueffier

, Finan

, Khoueiry

, Cassan

, et al. (2014). Trpm4 gene invalidation leads to cardiac hypertrophy and electrophysiological alterations. PLoS One, 9, e115256.

Ditchfield

, Johnson

V.L.

, Tighe

, Ellston

, Haworth

, Johnson

, et al. (2003). Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J Cell Biol, 161, 267–280.

Elkin

, Ilan

, Ishai-Michaeli

, Friedmann

, Papo

, Pecker

, et al. (2001). Heparanase as mediator of angiogenesis: mode of action. FASEB J, 15, 1661–1663.

10.

Fernandes

D.S.T.

, Gomes

A.M.

, Paschoal

M.E.

, Stelling

M.P.

, Rumjanek

V.M.

, Junior

A.R.

, et al. (2014). Heparanase expression and localization in different types of human lung cancer. Biochim Biophys Acta, 1840, 2599–2608.

11.

Gingis-Velitski

, Zetser

, Flugelman

M.Y.

, Vlodavsky

, and Ilan

(2004). Heparanase induces endothelial cell migration via protein kinase B/Akt activation. J Biol Chem, 279, 23536–23541.

12.

Goldshmidt

, Nadav

, Aingorn

, Irit

, Feinstein

, Ilan

, et al. (2002). Human heparanase is localized within lysosomes in a stable form. Exp Cell Res, 281, 50–62.

13.

Granic

, and Potter

(2013). Mitotic spindle defects and chromosome mis-segregation induced by LDL/cholesterol-implications for Niemann-Pick C1, Alzheimer's disease, and atherosclerosis. PLoS One, 8, e60718.

14.

Harisi

, Dudas

, Timar

, Pogany

, Timar

, Kovalszky

, et al. (2005). Invasive growth and topoisomerase-switch induced by tumorous extracellular matrix in osteosarcoma cell culture. Cell Biol Int, 29, 959–967.

15.

Izvolsky

K.I.

, Shoykhet

, Yang

, Yu

, Nugent

M.A.

, and Cardoso

W.V.

(2003). Heparan sulfate-FGF10 interactions during lung morphogenesis. Dev Biol, 258, 185–200.

16.

Janssen

, van der Burg

, Szuhai

, Kops

G.J.

, and Medema

R.H.

(2011). Chromosome segregation errors as a cause of DNA damage and structural chromosome aberrations. Science, 333, 1895–1898.

17.

Keller-Pinter

, Bottka

, Timar

, Kulka

, Katona

, Dux

, et al. (2010). Syndecan-4 promotes cytokinesis in a phosphorylation-dependent manner. Cell Mol Life Sci, 67, 1881–1894.

18.

Kobayashi

, Naomoto

, Nobuhisa

, Okawa

, Takaoka

, Shirakawa

, et al. (2006). Heparanase regulates esophageal keratinocyte differentiation through nuclear translocation and heparan sulfate cleavage. Differentiation, 74, 235–243.

19.

Lin

(2004). Functions of heparan sulfate proteoglycans in cell signaling during development. Development, 131, 6009–6021.

20.

McKenzie

E.A.

(2007). Heparanase: a target for drug discovery in cancer and inflammation. Br J Pharmacol, 151, 1–14.

21.

Nadir

, and Brenner

(2014). Heparanase multiple effects in cancer. Thromb Res, 133 Suppl 2, S90–S94.

22.

Nakajima

, Irimura

, and Nicolson

G.L.

(1988). Heparanases and tumor metastasis. J Cell Biochem, 36, 157–167.

23.

Nobuhisa

, Naomoto

, Okawa

, Takaoka

, Gunduz

, Motoki

, et al. (2007). Translocation of heparanase into nucleus results in cell differentiation. Cancer Sci, 98, 535–540.

24.

Qiao

, Yang

, Meyer

, and Friedl

(2008). Glypican-1 regulates anaphase promoting complex/cyclosome substrates and cell cycle progression in endothelial cells. Mol Biol Cell, 19, 2789–2801.

25.

Roshan

L.S.

, Naoka

, Anna

, Nicolas

, Joanna

, and Viji

M.D.

(2014). TAO1 kinase maintains chromosomal stability by facilitating proper congression of chromosomes. Open Biol, 4, 130108.

26.

Schubert

S.Y.

, Ilan

, Shushy

, Ben-Izhak

, Vlodavsky

, and Goldshmidt

(2004). Human heparanase nuclear localization and enzymatic activity. Lab Invest, 84, 535–544.

27.

Schuger

, Skubitz

A.P.

, Gilbride

, Mandel

, and He

(1996). Laminin and heparan sulfate proteoglycan mediate epithelial cell polarization in organotypic cultures of embryonic lung cells: evidence implicating involvement of the inner globular region of laminin beta 1 chain and the heparan sulfate groups of heparan sulfate proteoglycan. Dev Biol, 179, 264–273.

28.

Varmark

(2004). Functional role of centrosomes in spindle assembly and organization. J Cell Biochem, 91, 904–914.

29.

Venkatesan

, Natarajan

A.T.

, and Hande

M.P.

(2015). Chromosomal instability—mechanisms and consequences. Mutat Res Genet Toxicol Environ Mutagen, 793, 176–184.

30.

Viji

M.D.

, Frank

, Grzegorz

, Mathew

E.S.

, Chen

, Anthony

, et al. (2007). A functional genomic screen identifies a role for TAO1 kinase in spindle-checkpoint signaling. Nat Cell Biol, 9, 556–564.

31.

Vlodavsky

, Mohsen

, Lider

, Svahn

C.M.

, Ekre

H.P.

, Vigoda

, et al. (1994). Inhibition of tumor metastasis by heparanase inhibiting species of heparin. Invasion Metastasis, 14, 290–302.

32.

Wang

, Liu

, Yang

, Guo

, Liu

, Li

, et al. (2014). Loss of TACSTD2 contributed to squamous cell carcinoma progression through attenuating TAp63-dependent apoptosis. Cell Death Dis, 5, e1133.

33.

Whitfield

M.L.

, Sherlock

, Saldanha

A.J.

, Murray

J.I.

, Ball

C.A.

, Alexander

K.E.

, et al. (2002). Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol Biol Cell, 13, 1977–2000.

34.

Zheng

, Ruan

, Zhao

, Chen

, Pan

, and Liu

(2015). Heparanase is involved in proliferation and invasion of ovarian cancer cells. Cancer Biomark, 15, 525–534.

35.

Zong

, Fthenou

, Wolmer

, Hollosi

, Kovalszky

, Szilak

, et al. (2009). Syndecan-1 and FGF-2, but not FGF receptor-1, share a common transport route and co-localize with heparanase in the nuclei of mesenchymal tumor cells. PLoS One, 4, e7346.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.06 MB