Evidence of asymmetric cell division and inheritance in human neuroblastoma cells

Hideki Izumi1 and Yasuhiko Kaneko1

Division of Cancer Diagnosis, Research Institute for Clinical Oncology, Saitama Cancer Center, Ina, Saitama 362-0806, Japan

Edited* by Janet D. Rowley, The University of Chicago Medical Center, Chicago, IL, and approved September 19, 2012 (received for review April 2, 2012)

Asymmetric cell division (ACD) is believed to be a physiological ACD has been examined in the systems mentioned above, and these event that occurs during development and tissue homeostasis in ACD studies have provided evidence that the molecules involved a large variety of organisms. ACD produces two unequal daughter are highly conserved among invertebrates and vertebrates (6, 7). cells, one of which resembles a multipotent stem and/or pro- Although some adult stem cells divide asymmetrically in normal homeostasis, they retain the capacity to divide symmetrically to re- genitor cell, whereas the other has potential for differentiation. store stem-cell pools depleted by injury or disease, as has been ob- Although recent studies have shown that the balance between served in the nervous and hematopoietic systems (10). Moreover, it self-renewal and differentiation potentials is precisely controlled is known that the balance between stem cell self-renewal and dif- and that alterations in the balance may lead to tumorigenesis in ferentiation is precisely controlled and an imbalance leads to tu- Drosophila neuroblasts, it is largely unknown whether human cancer morigenesis in Drosophila neuroblasts (11–13). Therefore, we cells directly show ACD in an evolutionarily conserved manner. Here, investigated the behavior of ACD and symmetric cell division we show that the conserved polarity/spindle NuMA is pref- (SCD) in human neuroblastoma cells. erentially localized to one side of the cell cortex during cell division, Results generating unequal inheritance of fate-altering molecules in human neuroblastoma cell lines. We also show that the cells with a single ACD Preferentially Occurs in Human Neuroblastoma Cells with a copy of MYCN showed significantly higher percentages of ACD than Normal MYCN Copy, but Not in Cells with MYCN Amplification. We those with MYCN amplification. Moreover, suppression of MYCN first addressed whether NuMA (Nuclear Mitotic Apparatus – in MYCN-amplified cells caused ACD, whereas expression of MYCN protein) (14 19), one of the conserved polar , is local- in MYCN-nonamplified cells enhanced symmetric cell division. Fur- ized to the cell cortex as well as spindle poles during cell division by using HeLa cells as a control (16). Immunostaining experi- MEDICAL SCIENCES thermore, we demonstrate that centrosome inheritance follows a def- ments showed that NuMA is localized to the nucleus during inite rule in ACD: The daughter centrosome with younger mother interphase, as reported previously (15), and to spindle poles is inherited to the daughter cell with NuMA preferentially throughout mitosis (Fig. S1). In addition, we found that NuMA is localized to the cell cortex, whereas the mother centrosome with the also localized to both sides of the cell cortex in late metaphase older mother centriole migrates to the other daughter cell. Thus, the and this localization signal showed a high-intensity peak during mechanisms of cell division of ACD or symmetric cell division and anaphase (Fig. S1). were also stained with anti– centrosome inheritance are recapitulated in human cancer cells, and γ-tubulin antibody to avoid false results caused by uneven dyeing. these findings may facilitate studies on cancer stem cells. Thus, this result showed that HeLa cells displayed symmetrical polar cell division and that cells showing asymmetric division mitosis | CD133 were very rare when NuMA staining was used as a cell cortex marker (0.7%, n = 301). We next selected several human neuroblastoma cell lines with or without MYCN amplifi- euroblastoma is one of the most common childhood solid cation. The MYCN gene status was confirmed in each cell line by Ntumors and has a broad spectrum of clinical behavior: Some fluorescence in situ hybridization (FISH) (Fig. S2). We contin- cases have a good prognosis with minimal therapy, whereas others uously examined the expression levels of MYCN protein in the have a very poor prognosis despite aggressive therapy (1, 2). Cases neuroblastoma cell lines by immunoblotting and immunostaining are also classified into three groups: low-, intermediate-, and high- (Fig. 1A and Fig. S3). Both results showed that, whereas MYCN risk groups, based on the clinical and biological characteristics expression levels were very low in the cell lines with a normal (1, 2). This incredible heterogeneity defied explanation until MYCN copy [NB69, SK-N-SH, SH-SY5Y (a subclone of SK-N- molecular genetic and biochemical analyses of tumors began to SH), (20) and SK-N-FI], MYCN expression levels were high but shed light on these disparate clinical behaviors. Of many genetic slightly different among MYCN-amplified cell lines [TGW, SK- and biochemical features of neuroblastomas, MYCN oncogene N-BE (2), CHP-212, and SK-N-DZ (Fig. 1A and Fig. S3)]. amplification has been correlated with an aggressive phenotype β-Catenin staining showed that, whereas the tight junctions were and a poor outcome (1, 2). Recent studies have shown that MYCN completely formed in cells with a single copy of MYCN (SH- shows not only oncogenic activity but also plays a central role in SY5Y), those were partly [TGW and SK-N-BE (2)] or com- self-renewal of normal neural stem and precursor cells (3, 4). pletely (SK-N-DZ) disrupted in cells with MYCN amplification Neuroblastoma arises from the cells that normally make up an (Fig. S3). In addition, expression of CD133, a putative neural embryonic structure called the neural crest (5). The neural crest stem cell marker (21), was positive in all MYCN-amplified cell cells consist of multipotent and migratory cell populations that give lines except for CHP-212 (Fig. 1A). However, CD133 expression rise to diverse cell lineages including Schwann cells, melanocytes, was negative in cell lines with a normal MYCN copy (Fig. 1A). craniofacial cartilage and bone, smooth muscle, peripheral and en- For the CHP-212 cell line, we may consider that, whereas CHP212 teric neurons, and glia (5). Thus, neural crest cells serve as multi- potent stem cells that differentiate into mature neural tissues. It is now suspected that the multipotent neural crest cells might contribute to Author contributions: H.I. and Y.K. designed research; H.I. performed research; H.I. and neuroblastoma tumorigenesis due to aberrant MYCN expression (5). Y.K. analyzed data; and H.I. and Y.K. wrote the paper. Asymmetric cell division (ACD) is a physiological process fl during development and tissue homeostasis in a large variety of The authors declare no con ict of interest. model organisms such as Drosophila, Caenorhabditis elegans, and *This Direct Submission article had a prearranged editor. mouse brain and skin epidermis (6, 7). In addition, ACD has been Freely available online through the PNAS open access option. reported in human hematopoietic stem and progenitor cells and 1To whom correspondence may be addressed. E-mail: [email protected] or lung cancer cells (8, 9). A stem cell will divide asymmetrically, with [email protected]. one of its daughters retaining self-renewal potential, whereas the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. other proceeds to differentiate into transit-amplifying cells. To date, 1073/pnas.1205525109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1205525109 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 Fig. 1. Asymmetric cell division (ACD) occurs in human neuroblastoma cells with a normal MYCN copy, but not in the cells with MYCN amplification. (A) Immunoblot of MYCN and CD133 in human neuroblastoma cells. Both MYCN and CD133 expressions were detected in the MYCN-amplified cells, whereas the cells with a normal MYCN copy showed no expression of MYCN and CD133. Immunoblot of γ-tubulin served as a loading control. (B) Representative images of symmetric distribution of NuMA during the late stage of mitosis in TGW cells. γ-Tubulin is red, NuMA is green, and DNA is blue. Arrowheads show spindle poles. Arrows show the distribution of NuMA on the cell cortex. Double arrow shows a spindle pole distance. (C) Representative images of asymmetric distribution of NuMA during the late stage of mitosis in the SH-SY5Y cells. Centrosome is red, NuMA is green, and DNA is blue. Arrowheads show a spindle pole. Arrows show the distribution of NuMA on the cell cortex. Double arrow shows spindle pole distance. (D) Representative images of ACD at anaphase in the SH-SY5Y cells. Pan-cadherin (Pan-Cad) is red, NuMA is green, and DNA is blue. Arrows show the distribution of NuMA on the cell cortex. (E) Representative images of ACD and symmetric cell division (SCD) at anaphase in TGW cells. Pan-cadherin (Pan-Cad) is red, NuMA is green, and DNA is blue. Arrows show the cell cortex distribution of NuMA. (F) Quantification of cells with ACD in human neuroblastoma cells during the late metaphase and anaphase. Error bars represent SEM from three experiments, P < 0.0001. (Scale bars, 10 μm.)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1205525109 Izumi and Kaneko Downloaded by guest on September 27, 2021 with the double minutes form of MYCN amplification is derived immunostaining studies of the cells with (TGW) or without from the primary site (22), the other MYCN-amplified cell lines (NB69, SK-N-SH, SH-SY5Y, and SK-N-FI) MYCN amplification with the homogeneously staining region form of MYCN amplifi- using an antibody for the mother centrosome marker, ODF2/ cation are derived from bone marrow metastasis (www.atcc.org). cenexin (26). ODF2/cenexin is acquired by young mother cen- By using these cell lines, we tested whether these neuroblastoma trioles in G2/M transition and during mitosis, and the level of cell lines showed asymmetric distribution of NuMA to the cell ODF2/cenexin remains higher in the centrosome with old mother fi cortex. In all cell lines with MYCN ampli cation, the NuMA centriole than in the one with young mother centriole (Fig. S6) crescent was localized to both cell cortexes during mitosis (Fig. 1B (26). Interestingly, our results showed that, whereas the mother and Fig. S4). However, in the cell lines with a normal MYCN copy centrosome migrated to the daughter cell without a NuMA cres- number, asymmetric distribution of NuMA to one side of the cell cent, the daughter centrosome stayed at the other daughter cell C cortex was observed during the late mitotic stages (Fig. 1 and with a NuMA crescent in a larger population of ACD cells (Fig. 2 Fig. S4). In addition, we also examined immunostaining analysis – using anti-pan cadherin antibody as a cell membrane marker to A F). In addition, it is known that NuMA crescent cells stayed as avoid false images caused by uneven dyeing and found asym- self-renewal neuroblasts, whereas the others had more differen- tiation potential in Drosophila neuroblast system (17, 18). metric distribution of NuMA, as expected (Fig. 1 D and E and Fig. fi S4). We additionally carried out similar experiments by using Therefore, by analogy, our ndings may also suggest that NuMA several single clones isolated from parental cell lines with (TGW) crescent cells have more self-renewal potential than the other or without (SH-SY5Y) MYCN amplification because a specific cells, as reported previously (14). population of cells within the cultured cells could retain the ca- pacity to undergo ACD. As a result, we found that each clone Knockdown of MYCN Expression Causes ACD in Human Neuroblastoma showed no significant difference in the incidence of asymmetric Cells with MYCN Amplification. As mentioned above, it is suggested distribution of NuMA in mitosis compared with parental cell lines that the absence of MYCN protein expression decreases the self- (Fig. S5). Thus, MYCN-nonamplified cell lines showed asym- renewal population and maintains a balance between stem cells metric cell distribution of NuMA far more frequently than and differentiated progeny via ACD. Conversely, in the MYCN- fi < MYCN-ampli ed cell lines (P 0.0001) (Fig. 1F). amplified cells, ACD was hardly detectable because of the self- renewal ability given by MYCN. These findings lead to the idea Centrosome Inheritance in the ACD of Human Neuroblastoma Cells. that MYCN may be a key regulator that controls the balance of There is some controversy about the patterns of centrosome whether cells display ACD or SCD. To test this idea, MYCN inheritance in the process of ACD because different results have expression was knocked down with short-hairpin RNA (shRNA) been reported among studies using different model systems. In in MYCN-amplified cells [SK-N-BE (2) and SK-N-DZ (Fig. S7)]. the G1 phase during the cell cycle, a centrosome contains one After 72 h of shRNA transfection, the down-regulation of MYCN MEDICAL SCIENCES mother centriole and one daughter centriole. After centriole fi A A duplication, three generations of are present: an older was con rmed by immunoblotting (Fig. 3 and Fig. S7 ), and the mother, a younger mother, and two new daughters. The cen- status of cell division was investigated by immunostaining (Fig. 3 B and Fig. S7B). Our findings showed that the numbers of cells with trosome with the older mother centriole is termed the mother fi – centrosome. In the case of Drosophila melanogaster male germ the ACD phenotype signi cantly increased in both the MYCN line stem cells, the mother centrosome stays at a stem cell that is knocked-down cell lines compared with that in the control cells anchored to the niche, whereas the daughter centrosome migrates (Fig. 3 C and Fig. S7C). Moreover, the pattern of centrosome in- to the cells at the opposite side with differentiation potential (23). heritance was also conserved in these cells: The mother centro- However, in the case of Drosophila melanogaster neuroblasts, the some migrated to the daughter cells without NuMA crescent (Fig. mother centrosome does not stay at the daughter cell with self- 3 B and D and Fig. S7 B and D). Importantly, CD133 expression renewal capacity, but migrates to the daughter cell with differ- was also down-regulated in both MYCN-amplified cells when entiation potential (24, 25). To address this issue, we conducted MYCN expression was knocked down (Fig. 3A and Fig. S7A).

Fig. 2. Centrosome inheritance in ACD of human neuroblastoma cells. (A) Representative images of symmetric (Upper) and asymmetric (Lower) cell di- vision in the SH-SY5Y cells. ODF2/cenexin is red, NuMA is green, and DNA is blue. Arrowheads show the mother centrosome because, during mitosis, one old mother centriole (at the right in the upper panel and the left in the lower panel) stained more brightly for ODF2/cenexin than the other (young mother centri- ole). In all images, NuMA and ODF2 were in the same focal plane. (B) Representative images of ACD in the NB69 cells. Arrowheads show a centrosome. (C) Rep- resentative images of the ACD in SK-N-SH cells. Arrowheads show a mother centrosome. (D) Repre- sentative images of ACD in the SK-N-FI cells. Arrow- heads show a mother centrosome. (E) Representative images of ACD in the TGW cells. Arrowheads show a mother centrosome. (F) Quantification of centro- some inheritance during ACD in “the opposite side type” and “the same side type” in human neuro- blastoma cells. Data represent average ratios from three experiments. (Scale bars, 10 μm.)

Izumi and Kaneko PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 We additionally carried out similar experiments by using a MYCN-ΔLZ in the cells (Fig. 4F), and the status of the cell MYC inhibitor, 10058-F4, which was originally identified as a division was analyzed by immunostaining (Fig. 4G). When MYCN small molecular compound and interferes with the dimerization was overexpressed in the SH-SY5Y cells, CD133 expression was of MYC-Max (27). Subsequently, it was reported that 10058-F4 up-regulated (Fig. 4F). This result is not surprising because the decreased both levels of both MYC mRNA (45%) and MYC SH-SY5Y cells are known to show the neuroblastic phenotype protein (50%) expression to the endogenous levels (28). Therefore, (5). In contrast, CD133 expression was not up-regulated in the we exposed 10058-F4 to TGW cells and conducted immunoblotting cells transfected with the MYCN-ΔLZ expression vector (Fig. 4F) and immunostaining experiments. As expected, 10058-F4 down- or MYCN-induced Tet21N cells (Fig. 4A). The different ex- regulated MYCN expression in TGW cells (Fig. S8A). Moreover, pression status of CD133 may be due to the different phenotypes the ratio of cells with the ACD phenotype significantly increased represented by the two cell lines (neuroblastic or substrate-ad- in 10058-F4-treated cells and became similar to that of cells herent), although they were derived from the same parental cell with the ACD phenotype in the MYCN knocked-down cells (Fig. line, SK-N-SH. In addition, when MYCN was overexpressed, the S8 B and C). cells showing the ACD phenotype were significantly decreased (Fig. 4 G and H). However, even when MYCN-ΔLZ was over- Overexpression of MYCN Suppresses ACD and Enhances SCD in Human expressed, the ratios of the cells showing the ACD phenotype Neuroblastoma Cells with a Normal MYCN Copy. We next tried to were similar between the control and MYCN-ΔLZ–transfected induce MYCN expression by using the tetracycline-repressible cells (Fig. 4 G and H). These results strongly suggested that MYCN expression cell line, which is termed Tet21N (29). The MYCN controls the balance of cell fate in terms of whether cells Tet21N cell line is derived from a neuroblastoma cell line, SH- display ACD or SCD via its transcriptional function. EP (the substrate-adherent type), which is a subclone of SK-N- SH (the neuroblastic type) with a normal MYCN copy (5, 20). Discussion The Tet21N cells showed the ACD phenotype when MYCN In the present study, we found that human neuroblastoma cell expression was not induced (Fig. 4 A and B). At 72 h after in- lines without MYCN expression showed the ACD phenotype. duction of MYCN expression, which was confirmed by immu- Recent studies showed that human neuroblastoma cells contain noblotting (Fig. 4A), the cell division status was analyzed by tumor-initiating cells whose phenotype resembles that of cancer immunostaining (Fig. 4B). As expected, the induction of MYCN stem cells, including features such as self-renewal, induction of expression resulted in the suppression of the ACD phenotype multilineage cell differentiation, and high drug efflux capacity and enhanced the self-renewal potential (Fig. 4 B and C). In (32, 33). Therefore, the present finding may add the ACD phe- addition, the manner of centrosome inheritance was also con- notype to the features of cancer-initiating/stem cells that are pres- served in the cells when MYCN expression was not induced (Fig. ent in the neuroblastoma cell lines with a normal copy of MYCN. 4 D and E). Our findings showed that whereas MYCN overexpression sup- The major function of MYCN is known to be transcription, pressed ACD and enhanced SCD, the down-regulation of MYCN which regulates the expression and repression of a large variety caused ACD and suppressed SCD in human neuroblastoma cells. of downstream target (30). Finally, we examined whether These results are reasonable because the overexpression of MYCN the transcription of MYCN is responsible for the ACD or SCD may overproduce cancer-initiating/stem cells with high frequency phenotype. We tried to induce overexpression of MYCN (wild- through SCD, leading to the emergence of accumulating cancer type) or MYCN-ΔLZ vector, which is a mutant with a deletion stem cells (10). The MYC gene family is a key transcriptional of the leucine zipper domain (31) and cannot interact with Max, factor for producing the induced-pluripotent stem cells (34), and resulting in transcription deficiency in the cells with a normal the present study using the wild-type or deletion-mutant type of MYCN copy (SH-SY5Y: the neuroblastic type). After 72 h of MYCN vector showed that the transcriptional activity of MYCN transfection with the MYCN or MYCN-ΔLZ expression vector, is involved in the enhanced accumulation of the self-renewal immunoblotting confirmed the overexpression of MYCN or population through SCD. Neuroblastoma with MYCN amplification

Fig. 3. The knockdown of MYCN expression causes ACD in human neuroblastoma cells with MYCN am- plification. (A) Immunoblot of MYCN and CD133 in SK-N-BE (2) cells transfected with control or MYCN shRNA. Immunoblot of γ-tubulin served as a loading control. (B) Representative images of SCD in SK-N-BE (2) transfected with control shRNA (Top) and ACD in SK-N-BE (2) transfected with MYCN shRNA (Middle). γ-Tubulin is red, NuMA is green, and DNA is blue. Arrows show the distribution of NuMA on the cell cortex. Representative images of centrosome in- heritance during SCD in SK-N-BE (2) transfected with MYCN shRNA (Bottom). ODF2/cenexin is red, NuMA is green, and DNA is blue. An arrowhead shows a mother centrosome. Arrows show cell cortex dis- tribution of NuMA. (C) Quantification of cells with ACD during the late metaphase and anaphase in SK- N-BE (2) transfected with control or MYCN shRNA. Error bars represent SEM from three experiments, P = 0.002. (D) Quantification of centrosome inheritance during the ACD in “the opposite side type” vs. “the same side type” in SK-N-BE (2) transfected with MYCN shRNA. Error bars represent SEM from two experiments, P = 0.05. (Scale bars, 10 μm.)

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1205525109 Izumi and Kaneko Downloaded by guest on September 27, 2021 Fig. 4. Overexpression of MYCN suppresses ACD and enhances SCD in human neuroblastoma cells with a normal MYCN copy. (A) Immuno- blot of MYCN and CD133 in Tet21N cells with or without tetracycline (1 μg/mL) treatment (Tet) for 72 h. Immunoblot of γ-tubulin served as a loading control. (B) Representative images of ACD in Tet21N cells trea- ted with tetracycline (Tet+), and SCD in Tet21N cells without tetracycline treatment (Tet−). γ-Tubulin or pan- cadherin is red, NuMA is green, and DNA is blue. Arrows show the dis- tribution of NuMA on the cell cortex. (C)Quantification of cells with ACD during the late metaphase and anaphase in Tet21N cells with (Tet+) or without tetracycline (Tet−). Error bars represent SEM from three experiments, P = 0.0005. (D)Repre- sentative images of SCD (Upper) and ACD (Lower) in Tet21N cells treated with tetracycline (Tet+). NuMA is green, ODF2/cenexin is red, and DNA is blue. Arrows show the distribution of NuMA on the cell cortex. An arrowhead shows a mother centrosome. (E)Quantifica- tion of centrosome inheritance dur- ing ACD in “the opposite side type” MEDICAL SCIENCES vs. “the same side type” in Tet21N cells treated with tetracycline. Error bars represent SEM from two ex- periments. (F) Immunoblot of MYCN and CD133 in SH-SY5Y cells trans- fected with a control vector (vector), pCMV-MYCN, or pCMV-MYCN-ΔLZ vector. Immunoblot of γ-tubulin served as a loading control. (G)Rep- resentative images of the ACD in SH- SY5Y transfected with the control vector (vector), those of SCD in SH-SY5Y transfected with the MYCN vector (MYCN), and those of ACD in SH-SY5Y transfected with the MYCN-ΔLZ vector (MYCN-ΔLZ). NuMA is green, MYCN is red, and DNA is blue. Arrows show the dis- tribution of NuMA on the cell cortex. (H)Quantification of cells with ACD during late metaphase and ana- phase in SH-SY5Y transfected with a control vector (vector), MYCN vector (MYCN), or MYCN-ΔLZ vector (MYCN-ΔLZ). Error bars represent SEM from three experiments, P = 0.0218 and P = 0.0031, respectively. (Scale bars, 10 μm.)

is known to show aggressive behavior, and the present findings cells with MYCN amplification (Fig. S3). The complete tight that MYCN overproduces CD133-positive cells through SCD may junctions and the high frequency of ACD were found in the cells explain one of the mechanisms behind the aggressiveness and with a normal copy of MYCN, indicating that such cells might poor outcome of MYCN-amplified tumors. receive an extrinsic clue for ACD through cell–cell contact (39). We speculate that MYCN is one of the components that We showed that the ACD ratios ranged from 1.7 to 30% in the enhance the SCD phenotype, and that there may be additional neuroblastoma cell lines with or without MYCN amplification. positive regulators; one such candidate is Bmi-1, which is over- ACD is a very smart strategy because it maintains appropriate expressed in MYCN-amplified cells (35–37) and required for the numbers of self-renewal and differentiated cells with a single self-renewal of stem cells in the peripheral and central nervous division; however, a disadvantage of this strategy is that it leaves system (38). In addition, β-catenin staining in the present study stem cells unable to expand in number (10). The ACD ratio of shows that whereas the tight junctions were completely formed in 30% may be the upper limit for the neuroblastoma cell lines cells with a single copy of MYCN (SH-SY5Y), those were partly without MYCN expression because the cell lines will lose their [TGW and SK-N-BE (2)] or completely (SK-N-DZ) disrupted in self-renewal population if more than 30% of the cells undergo

Izumi and Kaneko PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 ACD. Interestingly, the human CD34+ CD133+ hematopoietic In summary, we showed here that it is possible to analyze stem and progenitor cells showed ∼20% ACD (8). The per- asymmetric cell division in human cancer by using cultured neu- centages of ACD are similar to those of ACD identified in the roblastoma cells. The manners of cell division of ACD or SCD and present study of neuroblastoma cell lines. centrosome inheritance conserved in Drosophila neuroblasts are We showed that a MYC inhibitor, 10058-F4, suppressed MYCN recapitulated in human cancer cells. Our findings may accelerate expression, CD133 expression, and SCD and caused ACD in a studies on the molecular mechanism of asymmetric cell division in neuroblastoma cell line. These findings suggest that certain ther- human stem cells. apeutic agents could affect the balance between ACD and SCD in tumor-initiating cell populations. Materials and Methods In the case of ACD, there is controversy over the pattern of Detailed materials and methods are provided in SI Materials and Methods. centrosome inheritance because different results were reported among various model systems: the mother centrosome remained Cell Lines and Transfections. All cell lines were maintained in complete me- in the daughter cells with self-renewal potential in Drosophila male dium DMEM, supplemented with 10% (vol/vol) FBS, penicillin [100 units/mL germ line stem cells (23) and in developing mouse brain neocortex (vol/vol)] and streptomycin [100 μg/mL (vol/vol)] in an atmosphere containing cells (40), whereas the mother centrosome migrated into the 5% CO2 at 37 °C. daughter cells with differentiation potential in Drosophila neu- The shRNAs and vector DNAs were transfected using lipofectamine 2000 roblasts (24, 25). Because NuMA (Mud: NuMA ortholog in (Invitrogen) according to the manufacturer’s instructions. Drosophila) is known to bind to apical polar proteins to ensure – the apical basal orientation of the mitotic spindle in Drosophila Indirect Immunofluorescence. Indirect Immunofluorescences were performed neuroblasts and mouse dermal epidermis cells (14, 18, 19), our as previously described (42). findings suggest that the pattern of centrosome migration in neuroblastoma was consistent with that of centrosome migration Immunoblot Analyses. reported in Drosophila neuroblasts. We would explain these con- Immunoblot analyses were performed as previously flicting findings by the following possibility. ODF2/cenexin, which described (42). was used as an old mother centrosome marker in our experiment, is known to be a component of appendages in the mother cen- ACKNOWLEDGMENTS. We thank Dr. M. Schwab for providing Tet21N cells, Dr. T. Kamijo for providing anti–N-Myc antibody, and K. Ono and H. Odagawa triole. Because ODF2/cenexin has differentiation activity such as for technical assistance. This work was supported in part by Grants-in-Aid for formation of primary cilia (41), multipotent stem cells might not Scientific Research from the Ministry of Education, Science, Sports and Culture tolerate the mother centrosome because of its differentiation of Japan and The Kawano Masanori Memorial Foundation for the Promotion potential. These issues should be addressed in the near future. of Pediatrics, Japan.

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