Introduction

Annotation of the draft sequence of the human genome has opened up the possibility of applying novel genomic approaches to the characterization of molecular pathogenesis of human disorders (The genome international sequencing consortium, 2001; Venter et al., 2001). Among genomic screening methods, DNA microarray analysis has to date provided the greatest insight into leukemogenesis. This technology readily allows measurement of the expression levels of thousands of genes simultaneously (Duggan et al., 1999). Expression profiling with microarrays has thus made it possible, for example, to distinguish acute myeloid leukemia (AML) from acute lymphoid leukemia (ALL) (Golub et al., 1999), to define novel subgroups of leukemias and lymphomas (Alizadeh et al., 2000; Armstrong et al., 2002), and to identify candidate genes for leukemogenesis (Ohmine et al., 2001; Makishima et al., 2002).

An important concern in the assay of fresh specimens by microarray analysis, however, is that apparent changes in gene expression detected at different stages of carcinogenesis may actually reflect changes in cell composition rather than changes in gene expression per se. For example, whereas immature (leukemic) blasts constitute 20% of bone marrow (BM) mononuclear cells (MNCs) in individuals with leukemia, BM MNCs of normal individuals contain only a few percent immature blasts. A simple comparison by microarray analysis between normal and leukemic BM cells would, therefore, likely reveal changes in gene expression only attributable to the expansion of immature blasts in the latter. Indeed, we observed that one of the genes whose expression appeared highly specific for leukemic BM cells, compared with normal BM cells, was that for CD34, simply reflecting the expansion of CD34⁺ leukemic blasts in the leukemic BM specimen (Miyazato et al., 2001).

To eliminate such population-shift effects, we have purified and stored AC133⁺ hematopoietic stem cell (HSC)-like fractions from the BM of patients with a wide range of leukemic disorders and deposited them in our 'Blast Bank.' The suitability of such purified fractions for microarray analysis was confirmed by the observation that the CD34 gene was expressed at similar levels in AC133⁺ cells obtained from normal individuals and in those isolated from leukemic patients (Miyazato et al., 2001). Microarray analysis of Blast Bank samples has also resulted in the identification of new molecular markers for myelodysplastic syndrome (MDS) (Miyazato et al., 2001) and for chronic myeloid leukemia (CML) (Ohmine et al., 2001).

Despite its potential for identifying genes important in leukemogenesis, microarray analysis is not able to provide direct information on the abundance or post-translational modification of proteins. The transcriptional activity on a given gene is thus not always a major determinant of the expression level of the encoded protein. After exclusion of several of the most abundant proteins, a large-scale study (Gygi et al., 1999) of yeast cells determined the correlation coefficient between the amount of an mRNA and the abundance of the corresponding protein to be only 0.4. Furthermore, only four out of 28 proteins identified in a mouse cell line showed relative levels similar to those of the corresponding mRNAs (Lian et al., 2001). A thorough characterization of leukemogenesis thus requires direct determination of the accompanying changes not only in the amounts of cellular mRNAs but also in protein abundance.

In addition to discrepancies between the amounts of mRNA and protein derived from a given gene, the activities of many proteins are influenced by post-translational modifications such as phosphorylation, cleavage, glycosylation, and redox regulation. Such concerns have highlighted the importance of proteomic approaches that are able to assess changes in the abundance and post-translational modification of proteins on a large scale. One of the main current strategies in proteomics is the combination of two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). Whereas 2DE allows the screening of hundreds to thousands of proteins for changes in molecular size, isoelectric point (pI), or phosphorylation, MS then allows the identification of protein spots of interest.

However, as in the case of DNA microarray analysis, the population-shift effect is also an important consideration in proteomics. Differentiation of cells is thus accompanied by changes in the expression of a substantial number of proteins (Lian et al., 2001). A proteome comparison between two specimens with different cell compositions would therefore yield pseudopositive data that reflect only the population-shift effect. A proteomic approach to the characterization of leukemogenesis would thus ideally require the purification of background-matched cell populations from fresh specimens of leukemic patients.

We have now prepared protein extracts from the purified AC133⁺ leukemic blasts of 13 individuals with acute leukemia or related disorders. Screening of these protein samples with 2DE and MS resulted in the identification of 10 proteins that were expressed differentially among the patients.

Results

2DE of Blast Bank samples

Our goal was to identify proteins whose abundance, relative molecular mass, or pI differs markedly among HSC-like fractions isolated from individuals with leukemia. Preliminary studies with cell lines revealed that at least 1 10⁶ MNCs were required to yield 100 spots reproducibly on 2DE gels. Given that the AC133⁺ HSC-like fraction usually constitutes a small proportion of leukemic blasts, it was not always possible to obtain such a large number of AC133⁺ cells from fresh patient specimens. Indeed, our Blast Bank contained only 13 AC133⁺ fractions that comprised 2 10⁶ cells. The clinical characteristics of the corresponding patients (five with de novo AML, four with MDS-associated AML, three with myeloproliferative disorders (MPDs) in blast crisis (BC), and one with ALL) are summarized in Table 1. All these patients died within 12 months of diagnosis.

Table 1 - Clinical characteristics of the patients subjected to proteome analysis.

Full table

Our preliminary studies also revealed that abundant proteins sometimes masked neighboring minor protein spots on the 2DE gels. To minimize such effects, we therefore first fractionated the cell samples into different subcellular components, including nuclear, mitochondrial, microsomal, and cytosolic fractions (Watarai et al., 2000). Each subcellular fraction was then independently compared by 2DE among the 13 patients. The cytosolic fractions consistently yielded 100 spots per gel and were analysed further in the present study. The comparisons of the other subcellular fractions will be described separately.

Identification of differentially expressed proteins

A representative image of a silver-stained gel, for which Melanie III software detected >200 independent spots, is shown in Figure 1a. Comparison of these spots among the gel images for the 13 patients revealed a total of 11 spots that showed a significant difference in abundance (as judged by the criteria described in Materials and methods). Peptide fingerprinting by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS of these 11 spots resulted in the identification of the corresponding 10 proteins (Table 2). They include nuclear protein that associates with the mitotic apparatus (NuMA), heat shock 70-kDa protein 5 (HSPA5), heat shock 70-kDa protein 8 (HSPA8), adenosine deaminase (ADA), aldolase A (ALDOA), triose phosphate isomerase 1 (TPI1), glutathione S-transferase pi (GST-pi), superoxide dismutase 2 (SOD2), peptidyl-prolyl isomerase A (PPIA), and heat shock 70-kDa protein 9B (HSPA9B). Two independent spots with different molecular mass and pI values were revealed to be derived from the same gene product, HSPA8 (Figure 1a).

Figure 1.

2DE analysis of Blast Bank samples. (a) The cytosolic fraction (10 g of protein) isolated from AC133⁺ leukemic blasts was subjected to 2DE on a 7.5–15% gradient gel. The scanned image of the silver-stained gel was then used to detect and compare protein spots. The positions and identities of proteins expressed differentially among leukemic patients are indicated by arrows, and the positions of molecular mass standards (in kilodaltons) are shown on the right. (b) Images of the NuMA spots for all 13 patients analysed. (c) Complementary DNAs were prepared from the AC133⁺ blasts of a healthy volunteer (normal) and those with AML, ALL, MDS-associated leukemia (MDS), CML in CP, and MPD in BC, and were subjected to real-time RT–PCR analysis with primers specific for the NuMA or GAPDH gene. The ratio of the abundance of NuMA mRNA to that of GAPDH mRNA was calculated as 2ⁿ, where n is the C_T value for GAPDH cDNA minus the C_T value for NuMA cDNA. The expression levels of NuMA mRNA for the patients with abundant expression of NuMA proteins (more than 3.0 U in Table 2) are shown as gray columns

Full figure and legend (180K)

Table 2 - Quantitation of the abundance of proteins expressed differentially among the patients.

Full table

We then examined whether clinical parameters of the patients were related to the intensity of any of the 11 protein spots. The expression level of NuMA was significantly related to whether the number of abnormal chromosomes was 3 or <3 (P=0.017; Student's t-test). The images of the NuMA spots for all 13 patients are shown in Figure 1b. NuMA is a nuclear protein that accumulates in the pericentrosomal region of the mitotic spindle and plays an important role in the assembly of mitotic asters (Compton and Cleveland, 1993; Gaglio et al., 1997; Du et al., 2001).

Since we could obtain only several thousands of AC133⁺ cells from the BM aspirates of healthy volunteers, it was impossible to assess the protein level of p240^NuMA directly in the AC133⁺ fractions of normal individuals. Instead, here we have quantified the abundance of NuMA mRNA by the 'real-time' reverse transcription–polymerase chain reaction (RT–PCR) method. The abundance of NuMA mRNA relative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was examined among the AC133⁺ HSC-like fractions obtained from a healthy volunteer and those with CML in chronic phase (CP), in addition to the specimens subjected to 2DE (Figure 1c). Although the mRNA level of NuMA did not precisely correlate with the corresponding protein level, there was a tendency that its mRNA was abundant in the samples with a high expression of NuMA protein (shown in gray columns).

The proportion of malignant blasts is below 5% in BM MNCs of patients with CML in CP, and, therefore, such BM aspirates could yield only 1 10³ 1 10⁴ of AC133⁺ cells, which were not enough for the protein analysis. However, since these patients had only one chromosomal anomaly, t(9;22), they were chosen to be included in this RT–PCR analysis. The expression level of the NuMA gene was negligibly low in the HSC-like fractions of a normal individual and most patients with CML in CP, which indirectly supports the low expression of NuMA protein in these specimens.

Aneuploidy associated with NuMA expression

The role of NuMA in mitosis suggested that aberrant induction of NuMA expression in leukemic blasts might contribute to the chromosomal instability apparent in such cells. To examine directly the effects of NuMA overexpression, we transfected mouse myeloid 32D cells (Greenberger et al., 1983) with a vector that confers Zn²⁺-dependent expression of human NuMA. Cell clones transfected with this vector (32D-NuMA#1 to 32D-NuMA#7) or the corresponding empty vector (32D-MT#1 to 32D-MT#4) were selected by culture in the presence of G418, after which 0.1 mM Zn²⁺ was added to the culture medium. Immunoblot analysis with antibodies to NuMA revealed marked expression of p240^NuMA in the 32D-NuMA clones, but not in the 32D-MT clones (Figure 2a). The electrophoretic mobility of NuMA expressed in the 32D-NuMA clones was identical to that of p240^NuMA detected in human kidney 293 cells transiently transfected with the pMT-NuMA vector.

Figure 2.

Conditional expression of NuMA in 32D cells. (a) Stable transfectants of 32D cells were isolated for pMT-CB6 (MT) or pMT-NuMA (NuMA) vectors. Total cell lysates (10 g of protein per lane) prepared from cells incubated overnight in the presence of 0.1 mM ZnSO₄ were subjected to immunoblot analysis with antibodies to NuMA. Total cell lysates prepared from human kidney 293 cells transiently transfected with pMT-CB6 or pMT-NuMA were similarly analysed. The position of p240^NuMA is indicated on the right. (b) The transfected 32D clones were cultured overnight with (+) or without (-) ZnSO₄ and then analysed for the induction of NuMA expression as in (a)

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The Zn²⁺ dependence of NuMA expression in each 32D-NuMA clone was verified by incubating cells in the absence or presence of ZnSO₄. Immunoblot analysis revealed a marked dependence of NuMA expression on the presence of Zn²⁺ for all 32D-NuMA clones (Figure 2b; data not shown). The amount of NuMA in 32D-NuMA#1 cells, for example, incubated overnight in the absence or presence of Zn²⁺ was 12.8 and 59.8 times, respectively, that of endogenous p240^NuMA in 32D-MT cells.

Examination of the morphology of 32D transfectants revealed that the induction of NuMA expression in some cells resulted in a marked increase in cell size and in the formation of multiple nuclei (Figure 3a); effects suggestive of the development of aneuploidy. Flow cytometric analysis of DNA content revealed that incubation of 32D-NuMA transfectants with Zn²⁺ for 2 days led to mitotic block (a decrease in the proportion of cells in S phase of the cell cycle, and an increase in the proportion of those in G₂–M); such an effect was not observed in 32D-MT cells (Table 3). Furthermore, induction of NuMA expression in transfected cells was associated with an increase in the proportion of cells with an abnormal (4(n)) DNA content (Table 4). These data thus suggested that overexpression of NuMA perturbs cell cycle progression by inhibiting mitosis and thereby results in the development of aneuploidy.

Figure 3.

Induction of aneuploidy and apoptosis by expression of p240^NuMA. (a) Mouse 32D cell clones transfected with pMT-CB6 (MT#1) or pMT-NuMA (NuMA#3) were cultured for 4 days in RPMI 1640 medium supplemented with 10% FBS and IL-3 (25 U/ml) either in the absence (-) or presence (+) of 0.1 mM ZnSO₄. The cells were then stained with Wright–Giemsa solution and examined by light microscopy. Scale bar; 30 m. (b) Mouse 32D cell clones transfected with pMT-CB6 (MT#4) or pMT-NuMA (NuMA#1) were cultured as in (a) in the absence (-) or presence (+) of ZnSO₄, and total cell number was determined at the indicated times. (c) Mouse 32D cell clones transfected with pMT-CB6 (MT#1 and #4) or pMT-NuMA (NuMA#1 and #3) were cultured as in (a) in the presence of IL-3 and ZnSO₄ for 0, 2, or 4 days, as indicated. Genomic DNA was then isolated, subjected to agarose gel electrophoresis through a 2% gel, and stained with ethidium bromide. Genomic DNA was also examined for 32D cells in which apoptosis was induced by an overnight deprivation of IL-3 (right-most lane). Lane M, DNA size markers (50-bp ladder, Invitrogen). (d) Human kidney 293 cells transfected with pEGFP (EGFP) or pEGFP/NuMA (NuMA-EGFP#1–3) were subjected to the analysis with a fluorescent microscope. Scale bar; 20 m

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Table 3 - Cell cycle analysis of NuMA-expressing cells.

Full table

Table 4 - Effect of NuMA induction on the proportion of cells with aneuploidy.

Full table

We also noticed that induction of p240^NuMA expression was accompanied by the appearance of cells with apoptotic features (Figure 3a) as well as by marked decreases in both the rate of cell growth (Figure 3b) and cell viability (data not shown). The increase in the prevalence of apoptosis in NuMA-overexpressing cells was confirmed by the detection of an increased extent of internucleosomal DNA fragmentation by agarose gel electrophoresis of genomic DNA (Figure 3c).

In our 2DE analysis, NuMA was identified within the cytoplasmic fractions, while the protein was originally reported to be accumulated in the nucleus. To determine its subcellular localization, p240^NuMA fused with enhanced green fluorescent protein (EGFP) (NuMA-EGFP) was expressed in human kidney 293 cells. As shown in Figure 3d, EGFP was expressed diffusely within cells. With regard to NuMA-EGFP, it was localized in the nucleus (but not in the nucleolus) in the majority of transfected cells (NuMA-EGFP#1). There was, however, a fraction of cells that had NuMA exclusively within their cytoplasm (NuMA-EGFP#2–3). Therefore, NuMA can be localized within the cytoplasm in living cells. Since such cells with cytoplasmic NuMA had a round shape, they may be at specific stages of the cell cycle. It would be an intriguing issue to address whether subcellular localization of p240^NuMA is regulated in a cell cycle-dependent manner.

Discussion

We have compared the protein profiles of HSC-like fractions derived from individuals with various leukemia-related disorders. Given that the proteome is strongly influenced by cell differentiation (Lian et al., 2002), a simple comparison of BM MNCs from different patients is likely to result in the identification of proteins whose apparent change in expression actually reflects a difference in the cellular composition of the specimens. To prevent such a complication, we isolated highly immature BM cells on the basis of their surface expression of AC133. Comparison of such background-matched fractions should eliminate pseudopositive data that might result from different proportions of leukemic blasts in BM or from differences in cell lineage to which the leukemic blasts are committed (Miyazato et al., 2001).

We identified 10 proteins that were expressed differentially among the leukemic blasts from the 13 patients examined. Three of these proteins (HSPA5, HSPA8 and HSPA9B) belong to the heat shock protein family of 70 kDa (HSP70), and two of them (GST-pi and SOD2) function in redox regulation. HSPA5 (also known as glucose-regulated protein 78 (GRP78) or immunoglobulin heavy chain-binding protein (BiP)), HSPA8 (heat shock cognate protein 70 (HSC70), HSP73, or lipopolysaccharide-associated protein 1 (LAP1)), and HSPA9 (HSP75, mortalin, or GRP75) all function as molecular chaperones. Their expression is induced by a variety of cellular stressors and they are thought to facilitate protein folding and oligomerization. In addition, however, whereas forced expression of one of the two isoforms of HSPA9, HSPA9A (MOT1), induces cell senescence (Kaul et al., 1995), that of HSPA9B (MOT2), which differs from HSPA9A by only two amino acids, promotes cell cycle progression and malignant transformation (Kaul et al., 1998). Although it is not known how MOT proteins induce malignant transformation, the stress-induced tyrosine phosphorylation of these proteins suggests that they function downstream of protein tyrosine kinases. Many molecular chaperones have also been shown to possess antiapoptotic activity (Jaattela, 1999). An increased expression of HSP70 family proteins in leukemic blasts might thus be directly linked to leukemogenesis or to the development of resistance to chemotherapeutic drugs.

The redox state of cells reflects a precise balance between the production of reactive oxygen species and the activity of reducing agents, the latter of which include thiol-based buffers and SOD (Davis et al., 2001). GST functions in cellular detoxification by catalysing the conjugation of reduced glutathione (GSH) to target molecules. In addition, GST-pi has been linked to chemoresistance to doxorubicin (Volm et al., 1992) and to cisplatin (Okuyama et al., 1994), and GST isoforms have been shown to be directly regulated by c-Jun NH₂-terminal kinase (JNK), also known as stress-activated protein kinase (SAPK) (Adler et al., 1999). SOD2, also known as manganese-dependent SOD (MnSOD), is a mitochondrial enzyme that catalyses dismutation of the superoxide anion into O₂ and H₂O₂. Overexpression of this enzyme has been detected in cancer cells (Pang et al., 1997) as well as in neurons of individuals with autosomal recessive parkinsonism (Matsumine et al., 1997; Shimoda-Matsubayashi et al., 1997). It remains to be determined whether an increased expression of GST-pi or SOD2 contributes to leukemogenesis.

NuMA was one of the most abundant soluble proteins in the leukemic blasts from some of the patients analysed in the present study. Given that NuMA is essential for the formation of the mitotic spindle, our observation that the abundance of this protein in leukemic blasts was related to the number of chromosomal abnormalities suggested that aberrant NuMA activity might result in a failure of cells to complete mitosis and lead to the development of chromosomal instability. We indeed demonstrated such effects of NuMA by inducing its expression in mouse 32D cells. However, the forced expression of NuMA also resulted in G₂–M arrest and apoptosis. The mere overexpression of NuMA does not therefore appear to be sufficient for malignant transformation to leukemic cells. Other genetic events that promote cell cycle progression or protect cells from apoptosis are likely also required to overcome the proapoptotic activity of NuMA. Chromosomal instability associated with NuMA expression may increase the chance of such genetic events, the occurrence of which may be reflected in changes in the proteome detected in our study.

Current proteomics techniques are limited in their sensitivity for protein detection. In our study, no single 2DE image yielded >300 independent protein spots, which is far fewer than the total number of human proteins predicted from sequencing of the human genome (30 000–40 000 proteins without taking into account the products of alternative RNA splicing). Further improvements in proteomics tools and their application to the direct comparison of protein profiles among background-matched cell fractions prepared from fresh specimens should provide an insight into the intracellular events that underlie malignant transformation in human leukemias.

Materials and methods

Cell culture

Mouse myeloid 32D cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and mouse interleukin-3 (IL-3; 25 U/ml; Invitrogen). Cells transfected with pMT-CB6-based vectors were subjected to selection with the same medium containing G418 (1 mg/ml; Invitrogen). Human kidney 293 cells (American Type Culture Collection) were cultured in DMEM/F12 medium (Invitrogen) supplemented with 10% FBS and 1 mM L-glutamine.

Isolation of Blast Bank samples

With written informed consent, BM MNCs had been isolated by Ficoll-Hypaque density gradient centrifugation from the individuals with various hematopoietic disorders. AC133⁺ cells were purified from such MNCs and stored in our Blast Bank with the use of an immunoaffinity chromatography for AC133 as described previously (Miyazato et al., 2001). Briefly, MNCs were labeled with magnetic bead-conjugated antibody toward AC133 (AC133 MicroBeads; Miltenyi Biotec, Auburn, CA, USA) in phosphate-buffered saline supplemented with 3% FBS and 2 mM EDTA. The cells were then loaded onto miniMACS magnetic cell separation columns (Miltenyi Biotec), and AC133⁺ cells were purified according to the manufacturer's instruction. To examine the enrichment of AC133⁺ cells, portions of the MNC and AC133⁺ cell preparations were stained with Wright-Giemsa solution or analysed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) for the expression of CD34, CD38, and AC133. At the end of March 2003, Blast Bank contained 411 independent AC133⁺ fractions, including 141, 95, and 60 specimens derived from individuals with de novo AML, MDS, or CML, respectively.

2DE of subcellular fractions

A total of 13 AC133⁺ cell preparations (2 10⁶–1 10⁷ cells) were subjected to subcellular fractionation by sequential centrifugation as described previously (Watarai et al., 2000). In brief, cells were suspended in 300 l of a solution containing 20 mM HEPES-NaOH (pH 7.4) and 0.25 M sucrose, and homogenized by 30 strokes in a Dounce homogenizer. The resulting homogenate was centrifuged at 1000 g for 10 min, the resulting pellet was saved as the nuclear fraction, and the resulting supernatant was further centrifuged at 10 000 g for 10 min. The pellet from this second centrifugation was saved as the mitochondrial fraction, and the supernatant was centrifuged at 100 000 g for 60 min to yield the microsomal fraction (pellet) and cytosolic fraction (supernatant). All subcellular fractions were independently subjected to isoelectric focusing on IPG DryStrips (pH 3–10 NL; Amersham Biosciences, Uppsala, Sweden) with a Multiphor II electrophoresis system (Amersham Biosciences). The separated proteins were then subjected to SDS–PAGE through 7.5–15% gradient gels (Bio-Craft, Tokyo, Japan) and visualized by silver staining (Shevchenko et al., 1996).

Protein identification

Stained gels were scanned with an LAS1000 image analyser (Fujifilm, Tokyo, Japan) to generate a digitized image file. Protein spots in the image were detected with the use of Melanie III software (GeneBio, Geneva, Switzerland) with manual adjustment, and were quantified by the same software. The intensity of each spot on a gel was normalized on the basis of the total signal intensity for that gel. The total number of spots detected in one gel image was 16859 (means.d.). A protein spot was classified as differentially expressed if (1) the intensity of the spot was 0.5 arbitrary units in at least one of the 13 image files and (2) the spot intensity ratio was 5 for comparison between any pair of gel images. The 11 spots that fulfilled these criteria were excised from the gels and subjected to protease digestion and peptide fingerprinting by MALDI-TOF MS (Voyager ET STR; Applied Biosystems, Foster City, CA, USA). The determined molecular masses of peptides were compared with an in-house nonredundant protein database maintained by Kirin Brewery Co. Ltd.

Quantitative real-time RT–PCR analysis

Total RNA was extracted from the purified AC133⁺ cells with the use of RNAzol B (Tel-Test, Inc., Friendswood, TX, USA), and a portion of the RNA was converted to double-stranded cDNA by the SuperScript Choice System (Life Technologies, Gaithersburg, MD, USA). The cDNAs were then subjected to PCR with a QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA, USA). The amplification protocol comprised incubations at 94°C for 15 s, 60°C for 30 s (54°C in the case of NuMA cDNA), and 72°C for 60 s. Incorporation of the SYBR Green dye into PCR products was monitored in real time with an ABI PRISM 7700 sequence detection system (PE Applied Biosystems, Foster City, CA, USA), thereby allowing determination of the threshold cycle (C_T) at which exponential amplification of PCR products begins. The C_T values for cDNAs corresponding to GAPDH and NuMA genes were used to calculate the abundance of NuMA mRNA relative to that of GAPDH mRNA. The oligonucleotide primers for PCR were 5'-GTCAGTGGTGGACCTGACCT-3' and 5'-TGAGCTTGACAAAGTGGTCG-3' for GAPDH cDNA, and 5'-AAAATAGCCTCATCAGCAGCTTGG-3' and 5'-CCAGCTTCT-GGCTCTTCTCTGACT-3' for NuMA cDNA.

Conditional expression of NuMA in 32D cells

The full-length cDNA for human NuMA was inserted into the pMT-CB6 vector (kindly provided by T Inaba, Hiroshima University, Hiroshima, Japan), which allows Zn²⁺-dependent expression of exogenous genes in eucaryotic cells. The resulting pMT-NuMA vector or the empty vector was then introduced into 32D cells by electroporation as described previously (Yamashita et al., 1996). After culture for 2 weeks in the presence of G418, seven and four independent cell clones were isolated from cells transfected with pMT-NuMA or the empty vector, respectively. For the induction of NuMA expression, transfectants were cultured overnight in the presence of 0.1 mM ZnSO₄. Proteins were extracted from the transfectants and subjected to immunoblot analysis as described previously (Yamashita et al., 1996); the latter was performed with rabbit polyclonal antibodies to NuMA (Gaglio et al., 1997). The DNA profiles of cells were examined with a FACScan processor (BD Biosciences, San Jose, CA, USA) and cell cycle distribution was determined with the ModFIT program (BD Biosciences).

Subcellular localization of p240^NuMA

The NuMA cDNA spanning its total coding sequence was amplified by PCR with Pfu DNA polymerase (Stratagene, La Jolla, CA, USA), and inserted into the pEGFP-N1 vector (BD Biosciences), giving rise to pEGFP/NuMA that encodes a NuMA protein tagged C-terminally with EGFP. The human kidney 293 cells were transfected with pEGFP-N1 or pEGFP/NuMA by the calcium phosphate method, and cultured for 48 h. EGFP or NuMA-EGFP was then detected by the IX71 fluorescent microscope (Olympus, Tokyo, Japan) equipped with a digital CCD camera DP50 (Olympus).

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Acknowledgements

This work was supported in part by a grant for Research on the Human Genome, Tissue Engineering, and Food Biotechnology and a grant for the Second-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor, and Welfare of Japan; by the Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan; and by a grant from the Research Foundation for Community Medicine, Japan. JO is a research resident of the Japan Health Sciences Foundation.