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Cell Growth & Differentiation Vol. 10, 565-573, August 1999
© 1999 American Association for Cancer Research

Ecotropic Viral Integration Site-1 Is Activated during, and Is Sufficient for, Neuroectodermal P19 Cell Differentiation1

Hiroshi Kazama, Takao Kodera, Seiichi Shimizu, Hideaki Mizoguchi and Kazuhiro Morishita2

Biology Division, National Cancer Center Research Institute, Tokyo 104-0045 [H. K., T. K., S. S., K. M.]; and Department of Hematology, Tokyo Women’s Medical University, Tokyo 162-0054 [H. K., H. M.], Japan


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Expression of the ecotropic viral integration site-1 (Evi1) proto-oncogene during murine embryonal development is observed by in situ hybridization in primary head folds and neural crest-derived cells associated with the peripheral nervous system and embryonic mesoderm. To elucidate whether expression of Evi1 is involved in early neuroectodermal or mesodermal differentiation, we used murine embryonal carcinoma P19 cells as a model for the study of early embryonic differentiation. After retinoic acid (RA) treatment with aggregation, expression of Evi1 was detected during neural differentiation in P19 cells. However, Evi1 was not expressed in P19 cells during mesodermal differentiation after DMSO treatment with aggregation. Enforced expression of Evi1 in P19 cells induced neuron-specific microtubule-associated protein-2 microtubule-associated protein-2 and TrkA expression in the absence of RA under monolayer culture. After incubation with RA with aggregation, the Evi1 clones expressed microtubule-associated protein-2 continuously but did not express glial fibrillary acidic protein as an astrocyte marker protein until 12 days of culture. Thus, the overexpression of Evi1 leads to neural differentiation of P19 cells and blocks further differentiation into astrocytes by RA treatment, suggesting that Evi1 might be an important transcription factor for regulation of early neuroectodermal differentiation.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The Evi13 locus was initially identified as a common site of retroviral integration in myeloid tumors of the AKXD-23 recombinant inbred mouse strain (1) . Over 30% of murine, virally induced myeloid leukemias had viral integration in the Evi1 locus, and we isolated the Evi1 gene, which encodes a protein with 10 zinc finger motifs and an acidic domain as a potential transcription factor (2) . Transcriptional activation of the Evi1 gene was identified not only in murine leukemias but also in human acute myeloid leukemias with chromosomal translocations or inversions at the region of 3q26 (3) . In 3q21q26 syndrome, Evi1 was transcriptionally activated by t(3;3)(q21;q26), ins(3)(q21q26), and inv(3)(q21q26) (3) . Furthermore, fusion mRNAs were identified in t(3;21) as AML1/MDS1/EVI1 (5 , 6) and in t(3;12) as TEL/MDS1/EVI1 (7) . Therefore, the Evi1 gene is suspected to be an important transcription factors in the pathogenesis of murine and human myeloid leukemias.

The Mr 145,000 glycosylated Evi1 protein contains two separate DNA-binding domains, which have distinct DNA-binding specificities and gene regulation capacities (8, 9, 10) . The first DNA-binding domain binds to a GATA-like motif, and the Evi1 protein competes with the GATA1 protein for binding to suppress GATA1-induced transcriptional activation. The DNA-binding sequence of the second domain resembles that of an ETS-like motif. This domain has a weak transcriptional activation (10) and indirectly activates AP1 proteins, such as c-jun and c-fos (11 , 12) . However, genes directly regulated by Evi1 have not been identified. Although the normal function of the Evi1 gene has not yet been found, ectopic expression of the Evi1 gene in hematopoietic cells and embryonal fibroblast cells might be associated with improved survival or modulation of differentiation (11 , 13 , 14) . On the basis of these data, the Evi1 gene is thought to function as a transcription factor and a proto-oncogene for leukemia.

Expression of the Evi1 gene was found in the renal tubules in the corticomedullary junction of the kidney and the cytoplasm of developing oocytes in adult mice (15) , and fetal expression has been reported in the urinary system, limb buds, heart, nasal pits, bronchial epithelium, and other tissues (16) . However, a recent study showed that detection of high levels of Evi1 expression was limited to the anterior section of the primary head folds at 8.5 dpc (17) . Along with the expression in other organs shown in previous studies (15) , additional expression of the Evi1 gene was observed in neural crest-derived cells associated with the peripheral nervous system and embryonic mesoderm after 9.5 dpc (17) .

Evi1 homozygous mutant embryos died at 10.5 dpc and were represented by widespread hypocellularity, hemorrhaging, and disruption in the development of the paraxial mesenchyme (17) . In addition, defects in the heart, somites, and cranial ganglia were detected, and the peripheral nervous system failed to develop. The specific loss of neurons in the superior regions of the cranial ganglia may be related to Evi1 expression in ectodermal placodes. The loss of the marginal layer of the neural ectoderm in mutant mice suggested that development or migration of neurons is inhibited within the neural ectoderm. Furthermore, it is reported that migration and differentiation of Caenorhabditis elegans HSN motor neurons are regulated by the egl-43 gene, a homologue of the murine Evi1 gene (18) . Thus, these data suggested that the Evi1 gene may play an important role in early neural development.

Here, we examined the roles of the Evi1 gene in neural development using an in vitro neural differentiation system of P19 mouse embryonal carcinoma cells (19) . We report here that expression of the Evi1 gene was transiently activated during neuroectodermal differentiation of P19 cells induced by RA treatment. However, no expression of the Evi1 gene was identified during mesodermal differentiation of P19 cells induced by DMSO treatment. Overexpression of the Evi1 gene in P19 cells showed that expression of neuron-specific MAP2 and TrkA was identified in those transformants, suggesting that the transformants clearly differentiated neural lineage cells. After RA treatment, the Evi1 clones expressed MAP2 continuously but did not express GFAP as an astrocyte cell marker protein until 12 days of culture. These data indicated that Evi1 overexpression leads the P19 cells into committed neural progenitor cells and blocks further differentiation into astrocytes. Thus, the Evi1 gene might be an important gene for regulation of the neural differentiation program.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Transcriptional Induction of Evi1 mRNA in P19 Cells during Neuroectodermal Differentiation by RA.
P19 EC cells can be induced to differentiate either into neuroectodermal cells by aggregation and addition of 1 µM RA or into mesodermal cell derivatives by addition of 1% DMSO. Therefore, we first examined the expression of the Evi1 gene in differentiating cultures of P19 cells by Northern blot analysis of RNA isolated at various times following treatment of cells with either RA or DMSO. As shown in Fig. 1Citation , Evi1 mRNA was not detectable in undifferentiated P19 cells. After aggregation and addition of 1 µM RA for 2 days and replating without RA for 2 days (day 4), a 6-kb Evi1 transcript was first detected, but levels subsequently decreased on day 6 (Fig. 1A)Citation . c-jun mRNA was detected at low levels by day 2 and then increased through day 4. After day 4, expression of the c-jun gene decreased until day 6. Expression of c-fos was also detected on day 4 and then decreased until day 6. Under the same conditions, Mash1 mRNA was not detectable in undifferentiated P19 cells but also detected from day 4 to day 6. At the same time, a 6-kb MAP2 transcript (type 2c) was detected on day 4, but a faint 9-kb band of MAP2 (type 2a + 2b) transcripts was detected at the same stage, encoding a high molecular weight full-length MAP2 protein (20) .



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Fig. 1. Expression of the Evi1, c-jun, and c-fos genes during RA or DMSO-induced differentiation of P19 EC cells. P19 cells were aggregated for 2 days and treated with 1 µM RA (A) or 1% DMSO (B) for an additional 2 days (days 1 and 2). The cells were then transferred into tissue culture dishes without additives. After incubation times of 2 and 4 days after replating (days 4 and 6), P19 cells were followed by mRNA extraction and Northern blot analysis. Five µg of mRNA were electrophoresed in each lane. The membrane was sequentially hybridized with Evi1, Mash1, MAP2 (or ActivinßA in B), c-jun, c-fos, and ß-actin probes, as described in "Materials and Methods.".

 
Evi1 expression was not detected in DMSO-treated P19 cells differentiating into mesodermal derivatives until day 6 (Fig. 1B)Citation . Transcripts of activinßA, which appear to be involved in mesoderm development (21) , were detected at the immature P19 cells. After DMSO treatment, expression of activinßA was not detected through day 2. After day 2, expression of the activinßA gene was detected, reached a peak on day 4, and decreased again on day 6. The expression of c-fos was at low levels throughout the culture period. However, expression of the c-jun gene was detected at low levels at day 1 and continued to increase gradually up to day 6. On the basis of these data, it appears that Evi1 expression in P19 cells was possibly associated with differentiation through the neuroectodermal lineage.

Isolation of Evi1 Stably Expressing P19 Cells.
To investigate the possible role of Evi1 protein in cell differentiation and proliferation in P19 cells, P19 cells were transfected with either the parental expression vector pEF-BOS or Evi1 cDNA cloned in pEF-BOS, together with a vector expressing the selectable neomycin resistance gene (pSV2Neo). Resistant clones were isolated after 14 days of selection with geneticin. The presence of Evi1 mRNA or protein in these clones was determined by Northern or Western blot analysis. We detected transcripts at {approx}3.5 kb in seven resistant clones (data not shown) and detected a Mr 150,000 Evi1 protein in these clones and control P19 cells with RA treatment (Fig. 2)Citation . Compared with the band of Evi1 in P19 cells 4 days after treatment with RA, the Evi1 protein band of the stable transformants were more intense. Therefore, we used these two clones (D2 and D5) for further studies.



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Fig. 2. Expression of Evi1 protein in P19 transformant cells. P19 cells were transfected as described in "Materials and Methods." Each nuclear extract was subjected to SDS-PAGE, blotted to a filter, and probed with anti-pTEV1 antibody. Control P19 EC cells (P19 EC), vector-transfected P19 clones (Mock), and Evi1-transfected clones (clones P19/D2 and P19/D5) were grown as monolayers in the absence of 1 µM RA, and control P19 cells were cultured in the presence of 1 µM RA for 4 days after RA treatment [P19 RA (+) Day 4]. A human leukemia cell line (MOLM1) expressed Evi1 highly as a positive clone.

 
Evi1 Expression Leads to Neural Differentiation of P19 EC Cells.
As shown in Fig. 3Citation , parental P19 cells and mock transformant (P19/pEF-BOS) cells kept a morphologically undifferentiated status (Fig. 3, a and c)Citation . When wild-type P19 cells were cultured with RA, aggregated for 2 days, and replated for an additional 2 days without RA (day 4), a number of neuron-like cells having long axon-like processes appeared (Fig. 3b)Citation . In contrast, the clones expressing Evi1 were longer and flat under monolayer culture without RA (Fig. 3d)Citation . To confirm the differentiation status of the Evi1 clones, we stained MAP2, which is specifically expressed in the neurally differentiated cells (22) , with a monoclonal anti-MAP2 (type 2a + 2b) antibody. In immunofluorescent staining of MAP2, no positive staining of MAP2 protein was detected in the monolayer culture of P19 cells (Fig. 4a)Citation , but positive staining of MAP2 protein was detected in a culture of P19 cells at day 4 after RA treatment (Fig. 4c)Citation . Strikingly, positive staining of MAP2 protein was detected in the Evi1 clones under the monolayer culture without RA (Fig. 4b)Citation . Also, the staining of MAP2 was detected in the clone D2 at day 4 after RA treatment (Fig. 4d)Citation . These results demonstrated that the clone expressing Evi1 could differentiate into neural cells under a monolayer culture without RA in the medium. As a result, it was suggested that overexpression of Evi1 possibly promoted neural differentiation of P19 cells.



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Fig. 3. Morphological differences among Evi1-expressing clones and control P19 cells cultured as monolayers in the absence of RA. Represented are micrographs of wild-type P19 cells (a), vector-transformant P19 clone (Mock; c), and Evi1-transformant clone (D2; d); under monolayer culture without RA. Wild-type P19 cells were cultured with RA and aggregation for 2 days and additional 2 days replating without RA (day 4; b).

 


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Fig. 4. Immunofluorescent staining of MAP2 in wild-type P19 cells and Evi1-expressing P19 cells (D2). a, undifferentiated P19 stem cells. b, Evi1 transformant clone (D2). c, 4 days after RA treatment of wild-type P19. d, 4 days after RA treatment of Evi1 transformant clone (D2).

 
P19 Clones Expressing Evi1 Showed a Differentiated Phenotype.
To confirm that the clones expressing Evi1 showed a differentiated phenotype, we next examined their reproduction ability in soft agar colony assay. Undifferentiated P19 cells can grow in an anchorage-independent manner in a soft agar culture, and differentiated P19 cells decrease to {approx}20% of their colony numbers at day 4 after RA treatment (23) . When compared with the colony numbers of wild-type P19 cells, colony numbers of the clones expressing Evi1 were reduced by 20–30% (Fig. 5A)Citation . Also, the size of the colonies from the cells expressing Evi1 were smaller than that of the colonies from the wild-type P19 cells (Fig. 5B)Citation . Furthermore, when we determined the rate of cell growth under the monolayer culture by viable cell count, the clones expressing Evi1 grew {approx}20% slower in terms of doubling time than those of the parental and the empty vector-transfected P19 cells (Fig. 5C)Citation . These data showed that the clones expressing Evi1 were differentiated phenotypically and had decreased reproduction capacities and growth rates.



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Fig. 5. Soft agarose colony assay of parental P19 cells, the clone transfected with empty vector (Mock), and Evi1-transfected P19 cells (clones D2 and D5). A, numbers of colony formation for the parental P19 cells and transformants. Columns, numbers of colonies in each dish; bars, SD. B, micrography of wild-type P19 cells (Ba), wild-type P19 cells 4 days after RA treatment (day 4; Bb), vector-transformant P19 clone (mock; Bc), and Evi1-transformant clones D2 (Bd) grown in the soft agarose after 10 days. C, growth curve of control P19 cells, vector-transfected cells (mock), and Evi1-transfected clones (D2 and D5). Cells (1 x 104 cells/ml) were seeded in 2 ml of culture medium in individual wells of six-well plates. At each indicated time point, cell viability was determined. Data points, mean cell numbers per triplicate wells, plotted against the number of days after seeding; bars, SD. {blacksquare}, P19; •, mock; {circ}, D2; {blacktriangleup}, D5.

 
Expression of c-jun and MAP2 mRNA in the Clones Expressing Evi1.
We examined the expression pattern of the c-fos and c-jun genes in those clones expressing Evi1 because those genes were reported as being downstream genes for Evi1. Along with expression of a 3.5-kb band of the Evi1 transcripts in two transformant cells, P19 cells at day 4 expressed a 6-kb Evi1 transcript (Fig. 6)Citation . A 6-kb MAP2 transcript (type c) was detected in P19 cells and two Evi1 clones (D2 and D5) treated with RA at day 4, but a 9-kb MAP2 transcript (type 2a + 2b) was only faintly expressed in these cells, as observed by Northern hybridization. Both Evi1 clones (D2 and D5) expressed a main 2.7-kb band of the endogenous c-jun gene in a monolayer culture. The levels of expression of the c-jun gene in both clones were a little lower than that of P19 cells at day 4, although expression of the Evi1 gene in the transformants is higher than that of P19 cells under the same conditions. On the other hand, expression of the c-fos gene in the same Northern blot analysis was undetectable in the Evi1 transformants. Furthermore, we were not able to expression of c-fos in seven clones expressing Evi1. Therefore, the expression of the c-jun gene in those clones expressing Evi1 was coregulated with neural differentiation induced by Evi1, but the expression of the c-fos gene was not regulated by the expression of the Evi1 gene. It is suggested that expression of the c-jun gene might play a role in Evi1-induced neural differentiation.



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Fig. 6. Expression of the various genes in the P19 clones expressing the exogenous Evi1 gene by Northern blot hybridization. RNA was isolated from those clones under monolayer culture conditions in the absence of RA, separated on formaldehyde-containing agarose gels, and analyzed by Northern blot hybridization. The expressions of Evi1, MAP2, c-jun, c-fos, and ß-actin transcripts were detected by hybridization with each respective labeled cDNA fragment.

 
Enforced Expression of Evi1 Leads to an Altered Expression of Neural and Mesodermal Markers.
The constitutive expression of Evi1 resulted in neural differentiation in monolayer culture without RA treatment, under conditions that result in wild-type P19 cells keeping an immature phenotype. Because these results suggest that the mesodermal fate might be repressed in Evi1-overexpressing cells, the expression of several markers of neural and mesodermal differentiation was analyzed by semiquantitative RT-PCR (21 , 24) in wild-type P19 cells and Evi1 clones (Fig. 7)Citation . At first, we examined expression of bHLH transcription factors [Mash1, NeuroD, and Neurogenin 1 (Ngn 1)], which are thought to regulate neural differentiation in each specific differentiation stage. Expression of Mash1 in P19 parental (Fig. 7Citation , Lane P19) and vector-infected cells (Fig. 7Citation , Lane Mock) was at a very low level and was highly induced in P19 cells at day 4. Within the clones expressing Evi1, the clones D2, D5, and C3 were expressed at the same high levels as that of P19 after RA treatment. In contrast to the expression of Mash1, transcripts of Ngn1 and NeuroD in wild-type P19 and mock cells were expressed at a low level. However, the clones D5, C1, and C3, excepting the clone D2, expressed Ngn1 and NeuroD mRNA at high levels. Because expression of these helix-loop-helix transcription factors was detected at different and limited stages during differentiation (25) , it is suggested that different expression patterns among those clones might reflect alternative differentiation stages.



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Fig. 7. Constitutive expression of human Evi1 in P19 cells results in an altered expression of neural and mesodermal markers. Expression of transcription factors (Mash1, Ngn1, and NeuroD), TrkA, and ActivinßA were detected by the semiquantitative RT-PCR method as described (24) . Wild-type P19 EC cells (P19), vector-transfected P19 clones (mock), and Evi1-transfected clones (clones D2, D5, C1, and C3) were grown as monolayers in the absence of RA, and control P19 cells were cultured 4 days after RA treatment [day 4; P19 RA (+)]. The amount of RNA in each reaction was normalized with transcripts of the 36B4 gene that is unresponsive to retinoid treatment.

 
To confirm the neural differentiation phenotype of Evi1 clones under the monolayer culture without RA treatment, we further examined expression of two other genes in the same samples; the ActivinßA gene, as a mesodermal marker protein (21) , and TrkA (the nerve growth factor receptor gene), as a neural differentiation marker protein (26) . The undifferentiated P19 and mock cells highly expressed ActivinßA transcripts; however, the Evi1 clones and P19 cells at day 4 hardly expressed ActivinßA. Particularly, expression levels of two bHLH proteins (NgnI and NeuroD) showed an inverse relationship to that of ActivinßA. On the other hand, the expression of TrkA was detected at higher levels in all Evi1 clones than those of control P19 and mock cells. Thus, all Evi1 clones were clearly differentiated into neuron-like cells associated with up-regulation of TrkA mRNA and with downregulation of ActivinßA mRNA. On the basis of these results, expression of bHLH genes among those clones might be not directly regulated by expression of the Evi1 gene but might be influenced by differences of the stage of neural differentiation specific to each Evi1 transformant clone.

Evi1 Blocks Astrocyte Differentiation.
To determine the differentiation capability of the Evi1 transformants, we treated the Evi1 clones and control P19 cells with RA with aggregation. The expressions of GFAP, as an astrocyte marker, and MAP2, as a neuron marker protein, were both determined by immunofluorescent staining. As shown in Fig. 4Citation , control P19 cells and the Evi1 clone D2 expressed MAP2 at day 4 (Fig. 4, c and d)Citation . At day 12, most of the differentiated neuron-like cells disappeared, and >80% of nonneurally differentiated P19 cells were immunoreactive to an anti-GFAP antibody (Fig. 8, Cc and Ac)Citation . However, the clone D2 was not at all immunoreactive to an anti-GFAP antibody at day 12 (Fig. 8Ad)Citation . On the other hand, control P19 cells did not express MAP2 at day 12 (Fig. 8Aa)Citation ; however, the clone D2 still kept its immunoreactivity to MAP2 (Fig. 8Ab)Citation . To confirm the expression of GFAP in both cells, we performed Northern blot hybridization to total RNA from both cells at each differentiation stage. At days 8, 10, and 12, control P19 cells abundantly expressed a 2.7-kb band of GFAP mRNA, but the clone D2 did not express GFAP at all (Fig. 8B)Citation . Morphologically, the clone D2 at day 12 seemed to keep almost the same shape as that of day 4 (Fig. 8, Cb and Cd)Citation , although control P19 cells converted from neuron-like cells at day 4 to astrocytes at day 12 (Fig. 8, Ca and Cc)Citation . Taken together, the results showed that forced expression of the Evi1 gene in P19 cells promoted differentiation into immature neural progenitor cells, but constitutive expression of the Evi1 gene inhibited further differentiation pathways by RA treatment. Thus, the Evi1 gene is suspected to be an important transcription factor for neural differentiation.



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Fig. 8. No expression of GFAP in the Evi1 clone after RA treatment for 12 days detected by immunofluorescence staining and Northern blot hybridization A, immunofluorescent staining of MAP2 was not detected in wild-type P19 cells 12 days after RA treatment (day 12; Aa) but was detected in the Evi1 clone (D2) under the same conditions (Ab). On the other hand, immunofluorescent staining of GFAP was detected in wild-type P19 cells 12 days after RA treatment (day 12; Ac), but was not detected in the Evi1 clone (D2) under the same conditions (day 12; Ad). B, Northern blot hybridization of GFAP against total RNA from wild-type P19 cells and the Evi1 clone (D2), which were cultured for indicated dates after RA treatment. C, micrographs of wild-type P19 cells at days 4 (Ca) and 12 (Cc) and those of the Evi1 clone (D2) at days 4 (Cb) and 12 (Cd).

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In this study, we first demonstrated that Evi1 gene expression was induced by RA treatment in P19 cells and that Evi1 transformants displayed changes in their morphology by expression of MAP2 with a differentiated phenotype. Along with the cells undergoing a certain degree of neural differentiation in the absence of RA, the bHLH transcription factors (Mash1, NeuroD, and Ngn1; Refs. 25 and 27, 28, 29 ) were expressed at different levels in clones expressing Evi1, which also expressed TrkA and little ActivinßA. Furthermore, the Evi1 clones did not express GFAP 12 days after RA treatment with aggregation and did not convert to astrocytes. These results suggest that the expression of Evi1 in P19 cells may induce several genes essential for neural differentiation pathways. Taken together, these results suggest that the Evi1 gene is an important transcription factor that regulates neural differentiation.

It was reported that expression of the Evi1 gene enhanced AP1 activity in P19 cells and particularly activated expression of c-fos and c-jun transcripts indirectly (11 , 12) . When RA was added to the medium of P19 cells, transcripts of both c-fos and c-jun genes were activated from day 2 to day 4. However, when we determined expression of both genes in the clones expressing Evi1 by Northern hybridization, we detected expression of the c-jun gene but not of the c-fos gene. Interestingly, it was reported that overexpression of the c-jun gene in P19 cells leads to mixed populations of endoderm- and mesoderm-like cells but not to neuroectodermal cells (30) . Therefore, expression of the c-jun gene is thought to be important for regulating the endodermal or mesodermal differentiation pathway. In particular, embryos lacking c-jun die at midgestation to late gestation and exhibit impaired hepatogenesis and altered fetal liver erythropoiesis, suggesting that an essential function of c-jun is hepatogenesis (31 , 32) . We could not, however, conclude whether transcriptional activation of the c-jun gene by enforced Evi1 expression is related to regulation of the neural differentiation event in P19 cells.

Here, we demonstrated that expression of the Evi1 gene was activated in P19 cells during neural differentiation by RA responsiveness. Therefore, it is possible that the Evi1 gene is one of the downstream genes for the RA signal transduction pathway. RA receptor and retinoid X receptor heterodimers activate transcription of target genes by binding to RAREs and are believed to transduce the retinoid signal to the transcription machinery and the chromatin template through transcriptional intermediary factors (33) . When we determined nucleotide sequences of {approx}1 kb of the promoter region of the murine and human Evi1 gene, typical RAREs were not found in either promoter region (data not shown). Interestingly, a RARE was found at the promoter region of the c-jun gene and two factors (DRF1 and DRF2) were found to bind to the element that is necessary for RA-mediated up-regulation of the c-jun gene during the differentiation of F9 cells (34) . A part of the second Evi1 responsive element (CTCATC) was contained in the RARE (TTACCTCATCCCGTGAGC), and we found, by gel mobility shift assay, that purified GST-Evi1 protein could bind to the RARE of the c-jun gene promoter region, and also that the shifted band was supershifted by adding Evi1 antibody (data not shown). Therefore, we are now investigating whether expression of the Evi1 gene is related to transcriptional activation of the c-jun gene during RA-mediated differentiation in P19 cells.

Evi1, which is expressed in multiple tissues of different origins at various stages of development, could be involved as a regulator in the control of differentiation of multiple cell lines. Ectopic expression of the Evi1 gene in the hematopoietic cells is important in the pathogenesis of myeloid leukemias. Because the Evi1 gene is involved in regulating neural differentiation, expression of the Evi1 gene in hematopoietic cells might modulate the differentiation program in myeloid cells or erythroid cells, which were demonstrated by the results using 32Dcl3 cells (13 , 14) , normal bone marrow cells (17) , or preleukemic cells (data not shown). Further molecular analysis is clearly required to elucidate the actual roles played by Evi1 in neural differentiation, and this might be helpful for determining how the Evi1 gene is involved in leukemogenesis.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Lines and Cell Culture Conditions.
The EC cell line P19 (18) was kindly provided by Drs. A. Sugiyama (Science University of Tokyo, Tokyo, Japan) and T. Uetsuki (Osaka University, Osaka, Japan). Routine culturing and differentiation induction of P19 cells were performed as described previously (28 , 35) . Upon reaching confluence, P19 cells were replated at 5 x 105 cells/ml in 100-mm nontissue culture-type, bacteriological-grade dishes. After 48 h, the aggregates were collected, resuspended in fresh medium (DMEM-10% FCS) containing 1 µM RA or 1% DMSO, and transferred into new 100-mm bacteriological-grade dishes (day 0). After an additional 48 h (day 2), the aggregates were collected again and were distributed into tissue culture-grade dishes in fresh medium without RA or DMSO. The medium was changed every second day. Soft agar growth assays were performed, as described previously (23) .

mRNA Preparation and Northern Blot Analysis.
mRNA was extracted from cells growing in culture using the FastTrack 2.0 mRNA isolation kit according to the manufacturer’s instructions (Invitrogen, San Diego, CA). Approximately 5 µg of mRNA were denatured with 37% formamide-15% formaldehyde, electrophoresed on 1% agarose gel containing 17% formaldehyde, and then transferred to nylon filters. The filters were hybridized with a [{alpha}-32P]dCTP-labeled human Evi1 probe (36) and exposed to Kodak Biomax film at -70°C. Pre-hybridization, hybridization and post-hybridization washes were performed as described (37) .

Probes.
The cDNAs for Evi1 were isolated from murine leukemia cells (2) or from human endometrial carcinoma cells (36) ; c-jun and c-fos were provided by Dr. S. Hirai (Yokohama City University, School of Medicine, Yokohama City, Japan); MAP2 was provided by Dr. M. Nakahuku (The Graduate School of Biological Sciences, Nara Institute of Science and Technology); and Mash1, Ngn1, and NeuroD were kindly provided by Drs. R. Kageyama (Institute for Virus Research, Kyoto University, Kyoto, Japan) and F. Guillemot (IGRMC, France).

Plasmid Constructions.
The full-length cDNA of the human Evi1 gene was isolated from the cDNA library of acute myeloid leukemia cells with inv(3)(3q21q26) (38) . For construction of Evi1 expression plasmids, the cDNA of Evi1 was inserted into the XbaI site of pEF-BOS, which was driven by polypeptide chain elongation factor 1a, a powerful mammalian expression vector (22) .

Transfection Strategy.
P19 cells were transfected with plasmid pEF-BOS-Evi1 or pEF-BOS (no insert) bearing a neomycin-resistant gene using the lipofection method (Life Technologies, Inc.). Parent cells (4 x 106) were seeded into a 100-mm culture dish and incubated in 2 ml of Opti-MEM (Life Technologies, Inc.) with 15 µl (1 µg/µl) of Lipofectin and 2 µg of the expression vectors. Eighteen h after transfection, Opti-MEM was changed with 10 ml of growth medium. After 48 h, the cells were replated and exposed to 0.6 mg/ml G418 (Geneticin; Life Technologies, Inc.). Each of the G418-resistant colonies was transferred separately to individual 60-mm culture dishes. The cells were subsequently maintained in G418-containing medium and expanded for further analyses.

Western Blot Analysis.
A nuclear extract of the cells was prepared as described (4) . Fifty µg of protein were separated by 5–15% gradient SDS-PAGE and electroblotted to nylon membranes (Immobilon; Millipore). After being blocked with 5% skim milk and 0.1% Tween 20 in Tris-buffered saline, the membranes were incubated with 1:250 dilution of anti-pTEV1 rabbit polyclonal antibody (39) . The blot was subsequently probed by the ECL Western blotting detection system (Amersham).

Indirect Immunofluorescence Microscopy.
For indirect immunofluorescent staining, cells were cultured on coverglass and fixed in ice-cold methanol for 15 min. The fixed cells were washed with PBS (-) and incubated with monoclonal anti-MAP2 (2a + 2b) IgG (Sigma Chemical Co.) for 1 h at room temperature. After extensive washing with PBS (-), the cells were incubated with the second antibody (FITC-coupled rabbit antimouse) for 40 min at room temperature, and prepared for immunofluorescence assay by mounting in Immunon, after repeating the washing with PBS. The immunofluorescence assay was performed with a LSM-40 (Carl Zeiss) microscope.

RT-PCR.
Semiquantitative RT-PCR and Southern blotting were performed as described (21 , 24) . The sequences of the PCR primers and PCR conditions for Mash1, NeuroD, Ngn1, TrkA, and 36B4 used in Fig. 7Citation were kindly provided by Dr. P. Chambon (21) .


    Acknowledgments
 
We are grateful to Drs. Ryuichiro Kageyama, Masato Nakafuku, Akinori Sugiyama, Francois Guillemot, Taichi Uetsuki, Shuichi Hirai, and Pierre Chambon for providing cell lines, plasmids, and primer sequences. We thank Drs. Issay Kitabayashi, Jun Yokota, Masato Nakafuku, and Taichi Uetsuki for suggestions and critical reading of this manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a Grant-in-Aid for the 2nd-term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare and Grants-in-Aid from the Ministry of Health and Welfare and the Ministry of Education, Science, Sports and Culture of Japan. Back

2 To whom requests for reprints should be addressed, at Biology Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. Phone: 81-3-3542-2511 ext. 4655; Fax: 81-3-3542-2511; E-mail: kmorishi{at}gan2.ncc.go.jp Back

3 The abbreviations used are: Evi1, ecotropic viral integration site-1; dpc, days postcoitum; RA, retinoic acid; MAP2, microtubule-associated protein-2; GFAP, glial fibrillary acidic protein; EC, embryonal carcinoma; RT-PCR, reverse transcriptase-PCR; bHLH, basic helix-loop-helix; RARE, RA response element. Back

Received for publication 3/16/99. Revision received 6/ 3/99. Accepted for publication 6/ 7/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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