FEBS Letters 588 (2014) 4071–4077
journal homepage: www.FEBSLetters.org
DISP3 promotes proliferation and delays differentiation of neural progenitor cells Martina Zíková 1, Jana Konírˇová 1, Karolína Ditrychová, Alicia Corlett, Michal Kolárˇ, Petr Bartu˚neˇk ⇑ Institute of Molecular Genetics AS CR v.v.i., Vídenˇská 1083, 142 20 Prague 4, Czech Republic
a r t i c l e
i n f o
Article history: Received 29 July 2014 Revised 4 September 2014 Accepted 25 September 2014 Available online 2 October 2014 Edited by Jesus Avila Keywords: Cancer Proliferation Neural cells Differentiation Lipids
a b s t r a c t DISP3 (PTCHD2), a sterol-sensing domain-containing protein, is highly expressed in neural tissue but its role in neural differentiation is unknown. In the present study we used a multipotent cerebellar progenitor cell line, C17.2, to investigate the impact of DISP3 on the proliferation and differentiation of neural precursors. We found that ectopically expressed DISP3 promotes cell proliferation and alters expression of genes that are involved in tumorigenesis. Finally, the differentiation proﬁle of DISP3-expressing cells was altered, as evidenced by delayed expression of neural speciﬁc markers and a reduced capacity to undergo neural differentiation. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The Dispatched 3 gene (Disp3), also known as Ptchd2 and KIAA1337, was ﬁrst reported in a human cDNA sequencing project that was focused on identifying genes encoding large proteins in the human brain . Subsequent cloning and characterization of Disp3 revealed that it encodes a 13-transmembrane domain-containing protein that is related to the Dispatched family of proteins. Further, its expression is regulated both in vivo and in vitro by thyroid hormone . In adult neural tissue, Disp3 is expressed predominantly in the hippocampus and cerebellum, with scattered expression in the cortex. In ﬁbroblasts, ectopic expression of DISP3 leads to increased cellular cholesterol levels that exhibit an altered distribution pattern. Based on these ﬁndings it was proposed that DISP3 might represent a new molecular link between thyroid hormone and cholesterol metabolism . Previous studies in mice have demonstrated that retroviral insertional mutagenesis induced by a recombinant M-MLV encoding PDGF-B results in a high incidence of glioblastomas.
Abbreviations: EGL, external germinal layer; GFAP, glial ﬁbrillary acidic protein; GNP, granule neuron precursors; M-MLV, Moloney Murine Leukemia Virus; PDGF, plate-derived growth factor; SSD, sterol-sensing domain ⇑ Corresponding author. E-mail address: [email protected]
(P. Bartu˚neˇk). 1 These authors contributed equally to this work.
One of the genes identiﬁed in this study was Disp3 . Interestingly, a subsequent study further demonstrated a link between Disp3 and oncogenesis, showing that Disp3 expression was ampliﬁed in long latency tumors . To identify the pattern and level of Disp3 expression in human brain cancer we investigated datasets from the publicly available Oncomine database (www.oncomine.org) . Analysis of the Northcott 3 , Kool et al.  and Fattet et al.  brain datasets revealed that Disp3 mRNA was signiﬁcantly elevated in human medulloblastoma. Medulloblastoma, the most common cause of oncological death in children, accounts for over 20% of all central nervous system tumors. According to the WHO classiﬁcation, medulloblastoma is deﬁned as a grade IV malignant embryonal neoplasm that arises from the cerebellum with ﬁve histological varieties . Several research groups have performed gene-expressing proﬁling of medulloblastoma and have identiﬁed four molecular subtypes: WNT, Sonic Hedgehog (SHH), Group 3, and Group 4. These molecular subtypes exhibit distinct gene expression, mutational proﬁles, and prognosis and indicate that medulloblastoma is not just one disease (reviewed in ). Given that Disp3 expression is elevated in medulloblastoma, we wished to investigate what role this gene may play in neural progenitor commitment and differentiation. To this end we used the multipotent neural progenitor cell line C17.2 originally established from cerebellar neural cells isolated from the external germinal layer (EGL) of neonatal mice that were subsequently immortalized
http://dx.doi.org/10.1016/j.febslet.2014.09.036 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
M. Zíková et al. / FEBS Letters 588 (2014) 4071–4077
by v-myc transduction . These progenitor cells have the potential to differentiate into neurons or glia depending on micro-environmental signals, and can thus be used as an alternative to primary brain tissue culture . 2. Materials and methods 2.1. Cell cultures and reagents C17.2 cells, a generous gift from Dietmar Spengler (Max-Planck Institute of Psychiatry, Munich, Germany), were grown in DMEM (Sigma) supplemented with 10% FCS, 5% horse serum, 2 mM L-glutamine, 100 U penicillin/ml and 100 lg streptomycin/ml (Gibco). The cells were passaged every 3rd day using 0.05% trypsin/EDTA and reseeded at a density of 1 104/cm2. For differentiation experiments cells were seeded at a density of 0.26 103/cm2 in standard growth medium. One day later the cells were washed and incubated in differentiation medium DMEM:F12 (Sigma) containing N2 supplements (Gibco), NGF and BDNF (10 ng/ml of each neurotrophic factor, R&D Systems). The human DAOY cell line was obtained from Mathias Heikenwaelder (Institute of Virology, TU Munich, Germany) and cultured in IMEM (Gibco) with 10% FBS and 100 U penicillin/ml and 100 lg streptomycin/ml. 2.2. Viral production, transduction of C17.2 cells and transfection of DAOY cells To generate Disp3-expressing C17.2 cells, the full-length human Disp3 was cloned into the retroviral vector pBABE-Hygro. Retroviruses were prepared by transfecting the expression vector into Phoenix cells using Lipofectamine reagent (Invitrogen). Viral supernatant was harvested at 48 and 72 h post transfection. C17.2 cells were incubated with virus supernatant and polybrene (4 lg/ml) for 6 h before adding the fresh medium and hygromycin was added 48 h later. To generate Disp3-expressing DAOY cells, the full-length human Disp3 was cloned into the pCDNA3 vector. DAOY cells were transfected using Lipofectamine and neomycin was added two days after transfection. The control cells were generating by transducing C17.2 and DAOY cells by empty vectors pBABE-Hygro and pCDNA3, respectively. 2.3. RT-PCR and real-time qPCR Total RNA was isolated using PureLink RNA Mini Kit (Ambion) according to the manufacturer’s protocol. For RT-PCR, 2 lg of total RNA was reverse transcribed using random hexamer primers (Invitrogen) and M-MLV Reverse Transcriptase (Promega). All qPCR reactions were performed in triplicate using the SYBR Green I Master mix and the LightCyclerÒ 480 system (Roche). 2.4. Microarray analysis The quality and concentration of RNA were measured and RNA integrity was analyzed using Agilent Bioanalyzer 2100 (Agilent). Microarray analysis was conducted using Illumina MouseRef-8 v2 Expression BeadChips according to their standard protocol. The analysis was performed in four biological replicates per group. The raw data were preprocessed using GenomeStudio software and analyzed within the limma package  of Bioconductor  as described elsewhere . A moderated t-test was used to detect transcripts differentially expressed between the treated samples and controls. As the analysis was considered exploratory, we used weak criteria to select differentially transcribed genes (P < 0.05, which corresponds to Storey’s q < 0.9 , and |log2FC| > 0.6). The microarray data were deposited in the ArrayExpress database under accession number E-MTAB-2975.
2.5. Primers The following primer sequences were used: gapdh 50 -CCATGAC AACTTTGGCATTG-30 and 50 -TCCCCACAGCCTTAGCAG-30 , ubb 50 -AT GTGAAGGCCAAGATCCAG-30 and 50 -TAATAGCCACCCCTCAGACG-30 , disp3 50 -CAGCAGCTTTGACCTCTTCA-30 and 50 -GCAACATCTGCAGGAAGGA-30 , bIII-tubulin 50 -TGGACAGTGTTCGGTCTGG-30 and 50 -C CCTCCGTATAGTGCCCTTTG-30 , gfap 50 -TGAGGCAGAAGCTCCAAGA30 and 50 -CCAGGGTGGCTTCATCTGC-30 , nestin 50 -AGGCTGAGAACTC TCGCTTGC-30 and 50 -GGTGCTGGTCCTCTGGTATCC-30 , epb4.1l3 50 GCCCAGCTATCAGAAGATATCAC-30 and 50 -AGCTCTGACTGCACCGTG TAGGA-30 , prelp 50 -GAACTTCTAGCTGGCTCTCT-30 and 50 -GGATGCC CTCATGATCCAGG-30 . 2.6. Proliferation assay Cell proliferation was measured using a thymidine incorporation assay. Cells were plated at a density of 3 103 cells/well in 96-well plates. After 12 h, cells were pulsed with [methyl-3H] thymidine (UJV Rez) and cultured for an additional 6 h. Cells were harvested onto Filtermat using a FilterMate Harvester. Incorporated radioactivity was quantiﬁed using MicroBeta2 Microplate scintillation counter (PerkinElmer). 2.7. Expression of recombinant DISP3 protein and production of antibody The expression vector encoding amino acids 692–1115 of DISP3 (NP_001186105.1) was transformed into BL21(DE3) Escherichia coli cells. The recombinant His-DISP3 protein was expressed, puriﬁed and used to immunize New Zealand rabbits as described earlier . Immune sera were collected three weeks after the ﬁnal immunization and puriﬁed using a Melon gel puriﬁcation kit (Thermo Scientiﬁc). 2.8. Immunoblotting and immunoﬂuorescence Protein samples were separated by SDS–PAGE using 4–15% precast gradient polyacrylamide gels (Biorad). Proteins were transferred onto nitrocellulose membranes, which were subsequently blocked in a solution containing 5% non-fat milk dissolved in TBS and 0.05% Tween-20 (TBST). Filters were incubated in primary antibody diluted in 1% milk/TBST overnight before being washed and incubated with secondary antibody (ECL kit, GE Life Sciences). For the detection of proteins via immunoﬂuorescence, cells were grown on glass coverslips, ﬁxed in 3% paraformaldehyde and permeabilized with 0.2% Triton X-100 before incubation with primary antibodies: polyclonal anti-DISP3 antibody (see above) or mouse anti-nestin (1:400, Millipore), mouse anti-bIII tubulin (1:1000, R&D systems), mouse anti-GFAP (1:400, Sigma), mouse anti-GAPDH (1:2000, GeneTex). Staining was visualized by the biotin-streptavidin method using streptavidin conjugated to Alexa Fluor 555 (Invitrogen). 2.9. Neutral lipid staining Neutral lipid staining was carried out in ﬁxed C17.2 cells using Nile Red (ﬁnal concentration 1 lg/ml) and analyzed using the Operetta High-Content Imaging System (PerkinElmer). Results were evaluated using Columbus software (PerkinElmer). 3. Results 3.1. Ectopic expression of DISP3 in C17.2 neural stem cells C17.2 cells overexpressing DISP3 were generated by retroviral transduction. To conﬁrm increased Disp3 expression, RT-PCR was
M. Zíková et al. / FEBS Letters 588 (2014) 4071–4077
used to analyze the mRNA isolated from infected C17.2 cultures (Fig. 1A). DISP3 protein levels were examined using a rabbit polyclonal antibody generated against a highly conserved C-terminal region of DISP3. As a control, a previously utilized DISP3 antibody generated against the N-terminal region was also used . Immunoblotting of protein extracts from both control and DISP3expressing cells showed a signiﬁcant increase in the level of a protein of approximately 150 kDa in DISP3-expressing cells (Fig. 1B). The same staining pattern was obtained using the antibody against the DISP3 N-terminal region (data not shown). Immunoﬂuorescence staining of ﬁxed cells similarly demonstrated an increase in the expression of DISP3 in overexpressing cells and its localization predominantly restricted to the cytoplasm (Fig. 1C). 3.2. DISP3 overexpression promotes proliferation of neural cells During routine cultivation of C17.2 cells we observed that cells ectopically expressing DISP3 seemed to proliferate faster than control cells. To quantitate cell proliferation, a thymidine incorporation assay was performed. The proliferation rate of C17.2 cells ectopically expressing DISP3 was increased approximately 1.7-fold compared to control cells (Fig. 2A). Furthermore, when a human medulloblastoma cell line (DAOY) that normally expresses very low levels of endogenous DISP3 was transfected with a Disp3 expression vector, its proliferation rate increased even further (1.9-fold, Fig. 2B). Taken together, these results suggest that DISP3 overexpression may provide a growth advantage by increasing cell proliferation. 3.3. DISP3 overexpression leads to altered gene expression in C17.2 cells Microarray analysis was used to identify genes whose expression was altered by the ectopic expression of DISP3. Total RNAs from control and DISP3 C17.2 cell lines were isolated and hybridized to the Illumina Mouse Expression Chips. Comparison of the resultant expression proﬁles established a list of genes whose expression was signiﬁcantly modiﬁed by increased DISP3 expression (Fig. 3A). QPCR was used to conﬁrm the reduction in mRNA
Fig. 2. Ectopically expressed DISP3 promotes cell proliferation in neural cells. Cell proliferation of control and DISP3 cells was measured by a thymidine incorporation assay in C17.2 (A) and DAOY (B) cell lines. Bars represent the average of at least three independent experiments, with error bars representing standard deviation and the level of statistical signiﬁcance (⁄⁄⁄P < 0.001). Control (empty vector).
expression of erythrocyte protein band 4.1-like 3 (Epb4.1l3, 4.1B) and proline arginine-rich and leucine-rich repeat protein (Prelp). Compared to control cells, the relative expression of Epb4.1l3 and Prelp in C17.2 DISP3 cells was decreased by 27% and 45%, respectively (Fig. 3B). In situ hybridization data obtained from the Allen Brain Atlas demonstrate that both genes are expressed within the mouse brain (Fig. 3C, http://www.brain-map.org). Speciﬁcally, Epb4.1l3 exhibits prominent expression in the hippocampus, olfactory bulb and Purkinje cells of the cerebellum. Prelp is expressed similarly, albeit at much lower levels. Interestingly, Epb4.1l3 is frequently lost in a variety of human tumors including meningioma and is thought to act as a negative regulator of tumor progression . Prelp is a member of the small leucine-rich proteoglycan family that is involved in the biology of meningioma and was found to be differentially expressed in glioblastoma multiforme and meningioma brain tumors . 3.4. Disp3 overexpression affects the differentiation of neural cells
Fig. 1. C17.2 cells ectopically express DISP3. C17.2 cells were infected with a retrovirus encoding human DISP3 and empty retroviral vector (control). Expression was conﬁrmed by RT-PCR (A), western blot (B) and immunoﬂuorescence (C). Gapdh served as a loading control (A, B). Cells were stained with a polyclonal DISP3 antibody (red), and DAPI (blue) was used to stain nuclei (C).
Neural progenitor C17.2 cells have the capacity to differentiate into neurons or glial cells and when cultured in the standard growth medium show typical morphology of undifferentiated cells. Cells cultured under differentiation conditions change their morphology with a fraction of cells generating dense cell bodies – prospective neurons and astrocytes (Fig. 4A) growing on top of the progenitor cells. In undifferentiated C17.2 cells the type-IV intermediate ﬁlament nestin, which is used to characterize progenitor cells , was detected by qPCR and immunoﬂuorescence staining in both control and DISP3-expressing cells. Upon neural differentiation into both cell types, nestin mRNA levels signiﬁcantly decreased over time. Compared to control cells, nestin expression levels were only higher in DISP3-expressing cells during the initial phases of differentiation with no difference seen after day 3 (Fig. 4B line graph, day 0 of differentiation is highlighted in the bar graph). Whilst nestin mRNA differences between control and DISP3 cells were not statistically signiﬁcant, there was an obvious difference detected at the protein level. Compared to day 0, control cells showed a signiﬁcant decrease in nestin staining by day 2, which was not observed in DISP3-expressing cells (Fig. 4B). To identify neurons during neuronal differentiation, we analyzed bIII-tubulin (part of the microtubular complex, )
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however, the levels were already reduced by day 3 (Fig. 4D). These results were corroborated by morphological and immunoﬂuorescence studies, which revealed signiﬁcantly reduced GFAP staining in DISP3-expressing cells at differentiation day 7 (Fig. 4D). 3.5. Disp3 overexpression leads to accumulation of lipids in neural cells It has previously been shown in ﬁbroblasts that ectopic expression of DISP3 leads to an increase in the number and size of lipid droplets and an accumulation of neutral lipids . To evaluate the lipid content in control and DISP3 C17.2 cells we stained them with Nile Red, a dye that speciﬁcally associates with neutral lipids. Fluorescent images show that relative to control cells, DISP3 cells stain more intensively (Fig. 5A). To quantify these results we analyzed cells using the high-content analysis. The results shown in Fig. 5B demonstrate that in comparison to control cells there was a signiﬁcant increase in Nile Red staining intensity in DISP3 cells, suggesting these cells contain a higher neutral lipid content (Fig. 5B). 4. Discussion
Fig. 3. DISP3 expression alters gene expression in C17.2 cells. (A) List of genes with altered gene expression in DISP3-overexpressing cells over control cells obtained by microarray expression proﬁling. (B). Relative expression of Epb4.l3 and Prelp in control and DISP3 cells was conﬁrmed by quantitative RT-PCR. Results are an average of at least three independent experiments with error bars representing standard deviation (⁄⁄P < 0.01, ⁄⁄⁄P < 0.001). Control (empty vector). (C) Representative sagittal section of an adult mouse brain hybridized with antisense Epb4.l3 and Prelp probes. Images were obtained from the Allen Brain Atlas (http:// www.brain-map.org).
expression. As expected, the level of bIII-tubulin mRNA varied in the course of the 7-day differentiation, with a sharp increase in bIII-tubulin expression between days 2–5 (Fig. 4C). Interestingly, whilst both cell types followed the same bIII-tubulin expression trend, DISP3-expressing cells consistently exhibited a reduced bIII-tubulin level compared to the control cells (Fig. 4C line graph, differentiation day 0 is highlighted in bar graph). These data obtained at the mRNA level were also conﬁrmed morphologically, when DISP3 cells were unable to form proper neurites and showed reduced bIII-tubulin immunoﬂuorescence staining after seven days of culture (note differences in appearance of neurons in control and DISP3 cells on differentiation day 7 in Fig. 4C). Glial ﬁbrillary acidic protein (GFAP) is a type-III intermediate ﬁlament that has previously been used to distinguish astrocytes . During astrocyte differentiation, GFAP mRNA levels signiﬁcantly increased from undetectable levels at day 0 to relatively high levels in control cells by day 7 (Fig. 4D). GFAP expression in DISP3-expressing cells followed a similar pattern to control cells;
In this study we examined the role of DISP3 in the proliferation and differentiation of the multipotent neural progenitor cell line, C17.2. These cells were originally established from cerebellar neural cells isolated from the EGL of neonatal mice  and are often used as an alternative to primary brain tissue cultures, which are a heterogeneous mixture of cell types . Within the EGL of the cerebellum there is a transient zone containing granule neuron precursors (GNPs), which differentiate into granule neurons. It is widely thought that GNPs whose growth/proliferation is altered during cerebellar development are at the cellular origin of medulloblastoma tumors [23,24]. Ectopic expression of DISP3 in C17.2 cells resulted in proliferation rates that were 1.7-fold higher than those of control cells. These results were further conﬁrmed using the human medulloblastoma cell line DAOY, suggesting that increased DISP3 expression promotes cellular proliferation. Gene expression proﬁling of DISP3 C17.2 cells revealed that the expression of Epb4.1l3 and Prelp mRNA was signiﬁcantly reduced. Epb4.1l3 is a member of the 4.1 superfamily of proteins that link transmembrane proteins to the actin cytoskeleton and are frequently lost in brain, lung, breast and prostate cancers . In a screen for genes involved in prostate cancer metastasis, it was shown that Epb4.1l3 expression is reduced in highly metastatic tumors . Moreover, other members of this family have been shown to enhance the metastatic potential of bone and soft tissue sarcomas , . Prelp was found as a gene that is differentially expressed in glioblastoma multiforme and meningioma. These two primary brain tumors are histologically and pathologically distinct with different prognoses , . Prelp is overexpressed in meningioma together with other members of the small leucinerich proteoglycan family . This family of proteins are known to function in tumor progression; however, their precise role varies according to the tumor type. Prelp was also identiﬁed in a functional genetic screen initiated to identify genes that allow cells to bypass cellular senescence  and later was additionally proposed to be involved in the development of accelerated aging syndrome, Hutchinson-Gilford progeria, . Thus, the downregulation of Epb4.1l3 and Prelp, two genes directly involved in tumorigenesis, by increased DISP3 expression in neural cells correlates well with the ability of DISP3 to promote cell proliferation. A genome-wide siRNA analysis of p16 modulators in normal ﬁnite human mammary epithelial cells revealed that Disp3 siRNA
M. Zíková et al. / FEBS Letters 588 (2014) 4071–4077
Fig. 4. Ectopic expression of DISP3 in C17.2 cells alters the expression of cell type speciﬁc markers. (A) Multipotent C17.2 neural stem cells give rise to neurons and astrocytes (oligodendrocytes were not detectable). Cell type speciﬁc markers analyzed were nestin, bIII-tubulin and GFAP. (B) Quantitative RT-PCR analysis of nestin mRNA in control and DISP3 cells during differentiation days 0–7; Ubb was used as a reference gene. Nestin mRNA levels at day 0 is shown. Immunoﬂuorescent images of cells stained with nestin (red) and DAPI (blue; to stain nuclei) during differentiation day 0 and 2. (C) Quantitative RT-PCR analysis of bIII-tubulin mRNA in control and DISP3-expressing cells during differentiation days 0–7; Ubb was used as a reference gene. bIII-tubulin mRNA levels at differentiation day 0 are shown. Bars represent the average of at least three independent experiments, with error bars representing standard deviation (⁄P < 0.05). Immunoﬂuorescent images of cells stained with bIII-tubulin (red) at differentiation days 0 and 7. (D) Quantitative RT-PCR analysis of GFAP mRNA in control and DISP3 cells during differentiation days 0–7; Ubb was used as a reference gene. Immunoﬂuorescent images of cells stained with GFAP (red) at differentiation days 0 and 7. Control (empty vector).
M. Zíková et al. / FEBS Letters 588 (2014) 4071–4077
proteins. Recently it was shown that during cell division the lipidome changes . Nile Red analysis of the lipid content of C17.2 cells revealed that ectopic expression of DISP3-expressing cells results in increased cell staining, suggesting that these cells have a higher neutral lipid content relative to control cells. DISP3 belongs to a class of sterol-sensing domain (SSD) containing proteins. It is known that SSDs bind sterols and that a mutation within this domain results in abnormal cholesterol and other lipid accumulation . Although there is no evidence demonstrating that DISP3 binds lipids directly, it is known that its overexpression does increase the cellular lipid content . Our ﬁndings clearly demonstrate that DISP3 expression alters cell proliferation; however, we are unable to determine whether this is due to the altered lipid content or whether the altered lipid content is a consequence of the increased cell proliferation. Nevertheless, our study demonstrates that ectopic expression of DISP3 elicits multiple effects on neural cells, promoting proliferation and preventing differentiation. Acknowledgement
Fig. 5. Neutral lipid content is enhanced in DISP3-expressing cells. (A) Nile Red staining (orange) in control and DISP3-expressing cells at differentiation day 0. DAPI (blue) was used to stain nuclei. (B) The Operetta High-Content Imaging System was used to quantify the neutral lipid content of control and DISP3expressing cells. Bars represent the average of at least three independent experiments with error bars representing standard deviation (⁄P < 0.05). Control (empty vector).
expression is capable of inducing cell-cycle arrest . Whilst p16 expression is low during development, it progressively increases in a wide variety of tissues during animal aging . p16 acts as a tumor suppressor by reducing the proliferative capacity of cells, allowing them to enter senescence. Evidence of the p16 tumor suppressor activity is observed when it is inactivated, leading to the genesis of a wide spectrum of human cancers . We have demonstrated that ectopic expression of DISP3 alters the fate of C17.2 neural progenitor cells by promoting cell growth and inhibiting neural differentiation. Undifferentiated C17.2 cells express the stem cell marker nestin and the neuronal marker bIII-tubulin, but little to no expression of the glial marker GFAP has been detected. In contrast, undifferentiated DISP3-expressing cells exhibited reduced bIII-tubulin expression, which remained lower than control cells even after incubation in differentiation medium. These results suggest that DISP3 overexpression reduces the ability of C17.2 cells to efﬁciently differentiate. Interestingly, bIII-tubulin mRNA was signiﬁcantly attenuated not only during the initial stages of differentiation, but remained consistently lower than in control cells throughout the 7-day time course. Whilst GFAP mRNA levels were negligible in both cell types initially, incubation in differentiation medium resulted in a large increase in expression. However, GFAP expression was signiﬁcantly attenuated in DISP3-expressing C17.2 cells. Immunoﬂuorescence and morphological analysis of these cells during differentiation conﬁrmed the mRNA expression ﬁndings showing that DISP3-expressing cells were unable to form neurite outgrowths and exhibited a ﬂat cell shape consistent with undifferentiated cells. There is a growing body of evidence suggesting the existence of a link between an altered lipid metabolism and oncogenesis . The increased cellular lipid content may inﬂuence tumor growth by increasing the pool of building blocks available for membrane biosynthesis, altering the number of lipid soluble signaling molecules and enabling the post-translational modiﬁcation of
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