JOURNAL OF CELLULAR PHYSIOLOGY 129:71-76 (1986)

Activated N-ras Gene Induces Neuronal Differentiation of PC12 Rat Pheochromocytoma Cells

ISABEL GUERRERO, HENRY WONG, ANGEL PELLICER, AND D A V I D E. BURSTEIN* Department of Pathology and Kaplan Cancer Center, New York University Medical

Center, New York, New York 10016

Activated mouse N-ras gene transfected into PC12 rat pheochromocytoma cells suppressed proliferation and promoted neuronal differentiation. Normal mouse N-ras in a LTR-containing vector caused differentiation with a reduced efficiency, but normal N-ras in a vector lacking LTR sequences failed to alter the PC12 phenotype. Cultures of NGF-resistant PC12 variant subline U7 also showed outgrowth of neurites and cessation of cell division following trans- fection with the mutated ras gene. The present findings suggest that ras genes can, in certain cells, play a role in promoting differentiation and sup pressing proliferation, in contrast to their established oncogenic neoplasia- promoting activity in other cells.

The PC12 line of neoplastic rat chromaf'fh cells, when exposed to nerve growth factor, ceases dividing and undergoes neuronal differentiation, marked by out- growth of long branching neuritic processes, onset of electrical excitability, and induction of several neuron- associated proteins (Greene and Tischler, 1976,1982).

We report herein transfection studies on the effects of ras genes on PC12 cell phenotype. The ras gene family has three prominent members, H-, K-, and N-ras. The first two were initially isolated from rat sarcoma viruses (Shih et al., 1978, Langebehiem et al., 1980) while the third one, N-ras, was originally described in a human neuroblastoma line (Shimizu et al., 1983).

The difference between normal and activated ras genes is the presence of a point mutation in critical bases of the coding region Warmus, 1984) that alters the struc- ture of their gene product, p21, a membrane-associated GTP binding protein, resulting in decreased GTPase activity (Sweet et al., 1984; McGrath et al., 1984). The normal and activated mouse N-ras genes that we have used in this study were isolated from an animal with a thymic lymphoma induced by the chemical carcinogen nitrosomethylurea (NMU) (Guerrero et al., 1984a). In these experiments a mutated N-ras with a C to A trans- version in the first base of codon 61 was utilized (Guer- rero et al., 1985). The activated ras genes are potent inducers of oncogenic transformation in the mesenchy- ma1 mouse NIH3T3 cell line and other rodent recipients (Shih et al., 1981; Guerrero et al., 198413).

It was anticipated that expression of activated ras genes introduced into PC12 cells might result in induc- tion of a NGF-resistant state. However, 1-2 days after the transformed mouse N-ras gene was transfected into PC12 cultures, scattered cells showed neurite outgrowth in the absence of NGF. The present studies, along with several other recent studies (Bar-Sagi and Feramisco, 1985; Noda et al., 1985; Hagag et al., 1986) lend support to a possible role of the ras gene product p21 in the

mechanism of action of NGF-induced neuronal differentiation.

MATERIALS AND METHODS Recombinant constructs

The activated mouse N-ras gene, previously isolated from a transfectant derived from an NMU-induced thymic lymphoma and its normal counterpart, isolated from the brain of the animal which developed the tumor (Guerrero et al., 1984a1, were made blunt-ended by using T4 DNA polymerase and subsequently ligated to XhoI linkers. XhoI does not have a site inside the N-ras gene and digestion with XhoI left the gene ready to be in- serted in the XhoI site of the retroviral vector tll-pVHC (kindly provided by S . Goff). Alternatively the XhoI frag- ment containing the normal or activated N-ras gene was subcloned into PIBW3, a vector containing the neo-resis- tant gene under the control of the HSV TK regulatory sequences. This plasmid vector was linearized with Bam HI at the 5' end of the neo' hybrid gene and also made blunt ended and ligated to XhoI linkers.

Gene transfer PC12 cells (passage 30-40) were plated on 60 mm.

Falcon dishes (1-2.5 million cellddish) coated with rat tail collagen and poly-D-lysine in complete medium [85% DMEM (low glucose), 10% horse serum, and 5% fetal calf serum (KC Biological)]. The following day, cells were washed once with HeBS buffer (137 mM NaC1, 5 mM KC1, 5.5 mM glucose, 0.8 mM Na2HP04, 21 mM HEPES), pH 7.05, and incubated at room temperature in 0.4 rnl of a 1:l mixture of HeBS buffer and DNN0.25 M CaC12 (Graham and Van der Eb, 1973; Wigler et al., 1979). DNA consisted of a ras-containing plasmid (0.4-2

Received April 2,1986; accepted June 5, 1986. *To whom reprint requestskorrespondence should be addressed.

0 1986 ALAN R. LISS, INC.

72

1

GUERRERO, WONG, PELLICER, AND BURSTEIN

TABLE 1. Transfection efficiencies of normal and transforming ras constructs in PC12 cells

Construct (concentration) cellddish pg/1o6 cells

1. Normal N-ras-neo (25 pg/ml)l 0 0 2. Normal N-ras-LTR (25 pg/mD2 10-20 0.9-1.8

4. Activated N-ras-LTR (0.6 pg/ml? 5. Activated N-ras-LTR (0.06 pg/m1I2

Neurite-bearing Neurite-bearing cells/

3. Normal N-ras-LTR (5 pglrnl)' 5-10 2.27-4.54 8.7 x 103 8.7 x 103

5 x 103 5 x 102

Cultures of PC12 cells (1.1-2.4 million celld60-mm dish) were transfected and scored as described in Materials and Methods. 'Construct in Figure 2B. 2Construct in Figure 2A.

- Fig. 1. Effect of NGF and activated N-ras on phenotype of PC12 cells. (A): Cells treated with 50 ng/ml NGF for 7 days. 03,C): Cells 6 and 7 days after transfection with LTRcontaining activated N-ras construct. (D): Enlarged, degenerating cell 15 days after transfection with LTR- containing activated N-ras construct. (E,F): Cells 7 days after transfection with LTR-containing normal N-ras construct.

RAS INDUCES NEURONAL DIFFERENTIATION 73

pg), and rat carrier DNA (8-10 pg), with or without a neomycin resistance plasmid (0.4 pg). After 20 min, 1.2 ml of complete medium was added and cells were incu- bated at 37°C for 4-7 hr. Cells were then osmotically shocked by removing medium and adding 2 ml of 75% DMEM/25% glycerol for 1-2 min, washed, and replated in complete medium (Schweitzer and Kelly, 1985). Transfection efficiencies were scored by counting the number of neurite-bearing cells in 40-80 high power fields, extrapolating to the total area of the dish, and dividing by the number of cells counted on the day of transfection. When fewer than 100 neurite-bearing cells per dish were present, the entire dish was scanned at low power for more accurate quantification.

RESULTS AND DISCUSSION Scattered cells (Table 1) showed outgrowth of neuritic

processes in the absence of NGF (Figs. lB, C) 1-2 days after the transforming mouse N-ras gene was trans- fected into PC12 cells by a modification of standard methods (Graham and van der Eb, 1973; Wigler et al., 1979; Schweitzer and Kelly, 1985) that grew progres- sively longer over 2 weeks in culture. We used two types of constructs containing activated N-ras. One construct contained activated N-ras flanked by Moloney leukemia virus LTR sequences (Fig. 2A). The second lacked LTR sequences (Guerrero et al., 1984a). Cells transfected with either vector appeared phenotypically indistinguisha- ble. In a typical experiment utilizing the LTRcontain- ing vector, the transfection frequency measured 2 days after gene transfer was approximately 3600 neurite- bearing cells/pg DNA/million cells (Table 1). Transfec- tion with more than 0.6 pg of DNA per culture (1-2.5 million cells), up to 10 pg, caused little further increase in the number of neurite-bearing cells per dish. The

number of neurite-bearing cells in dishes transfected with 0.06 pg/dish was about one-tenth as high as in dishes transfected with 0.6 pg/dish (Table 1). Control cultures transfected with a plasmid containing a bacte- rial gene, neo, which confers resistance to neomycin (Colbere-Garapin et al., 1981), showed no phenotypic alteration. Cells also showed a progressive increase in somatic size, prominence of nucleoli, and often, cyto- plasmic flattening. Transfectants frequently showed outgrowth of multiple neurites, and neurite branching was noted in numerous cells.

The progressive increase in length of neurites, and long lengths attained by some processes were compara- ble to that seen in NGFexposed cultures (Fig. 1A) (Greene et al., 19821, and differed from the shorter pro- cesses seen in dibutyryl cyclic AMP (DBcAMPZtreated cells (Gunning et al., 1981). The percentage of neurite- bearing cells remained approximately the same over a week.

Certain significant aspects of activated N-ras-induced changes were not identical to the NGF-induced pheno- type. Between 7 and 14 days after transfection, a sub- stantial percentage of cells showed an inordinate and progressive increase in somatic size, often accompanied by the appearance of numerous cytoplasmic vacuoles and changes suggesting cellular degeneration (Fig. 1D). Between 7 and 14 days following transfection, the num- ber of neurite-bearing cells per dish typically declined, probably due to cell degeneration. However, neurite- bearing cells were seen as late as 3 weeks after transfection.

Differentiated cells in all cultures were for the most part either isolated or present in pairs, and no colonies or large clusters of neurite-bearing cells were present. These results suggest that differentiation was accom-

A

I : 'f K b 0 2 4 6 8 10 12

N-RAS LT R I H

LT R H i

Xh Ba XhCl

I I I I I I I I

NEO' , N-RAS I

Fig. 2. Mouse N-ras gene constructs used to transfect PC12 cells. (A): Mouse N-ras genes, both normal and activated (Guerrero et al., 1984) were subcloned into the retroviral vector tll-pVHC derived from the Mo-MuLV. The arrows indicate the orientation of the LTR sequences. (B): Mouse N-ras genes, both normal and activated, were subcloned into a vector containing the dominant selective marker neo". The cross hatched box represents the HSVTK gene promoter used to drive the bacterial neo' gene (stippled box). The open box is the polyadenylation

site of the HSVTK gene that is necessary to obtain correct expression of the bacterial gene in mammalian cells. The black boxes indicate the four coding exons of the N-ras gene. The wavy lines at the ends of the constructs correspond to the PBR322 plasmid derivatives used as vec- tors. The symbols are for restriction endonucleases: Xh, XhoI; Bg, BglE, C1, ClaI; Pv, PvuII; Sa, Sa l t Sm, SmaI. The underlined restric- tion site is not mapped through all the clone. The scale on top of the figure is in kilobases.

74 GUERRERO, WONG, PELLICER, AND BURSTEIN

panied by cessation of cell proliferation. Since these cells divide very slowly and cease proliferation in response to activated N-ras, we believe that the maintenance of the neuronal phenotype may be a manifestation of extended transient expression of these genes. This is consistent with the hundred-fold higher efficiency of induction of neurite formation in these experiments compared with reported transfection efficiencies for obtaining PC 12 cell clones with a dominant selectable vector (Schweitzer and Kelly, 1985).

The activity of transforming N-ras contrasts with the results of transfection with cloned DNA containing the nontransforming mouse N-ras gene. The normal gene has been subcloned in a vector containing two LTR se- quences (Fig. 2A), as well as in a vector lacking LTR sequences but containing the neo gene (Fig. 2B) (Col- bere-Garapin et al., 1981). These vectors were previ- ously tested in NIH3T3 cells. Only the construct containing LTRs caused transformed focus formation in NIH3T3 cultures, although at a low efficiency (I. Guer- rero and A. Pellicer, unpublished data). This is analo- gous to the findings of others (Chang et al., 1982; Pulciani et al., 1985) who showed that elevated levels of normal H-ras-coded p21 induced transformation of 3T3 cells. When PC12 cultures were transfected with this construct (25 ,ug/ml/dish), rare neurite-bearing cells, ap- proximately 10-20 per one million cells, were detected (Figs. lE,F), thus this construct appeared to be about 100-1000-fold less efficient at inducing PC12 differentia- tion than was activated mouse N-ras with or without LTRs. Conversely, the normal mouse N-ras gene without LTR sequences but containing the neo gene (Fig. 2B) showed no ability to induce phenotypic alternation in either NIH3T3 or PC12 cultures.

The neurodifferentiating effect on PC12 cells was not specific to N-ras nor restricted to a rodent gene. A plas- mid, ptBl (Goldfarb et al., 1982) (kindly provided by M. Perucho), containing activated Ha-ras derived from T24 human bladder carcinoma cells showed a similar ability to induce PC12 neuronal differentiation (data not shown).

The effect of transforming N-ras was further tested on a previously analyzed NGF-resistant PC 12 subline, U7, which arose spontaneously in PC12 cultures (Burstein and Greene, 1982a). U7 cultures exposed to NGF in complete medium (15% serum) variably show short cy- toplasmic projections and only occasional neurite-bear- ing cells, and fail to stop dividing in NGF. However, they show extensive differentiation in response to NGF under growth-arresting conditions, such as cultivation in serum-free medium or complete medium containing the growth-arresting compounds cytosine arabinoside (Burstein and Greene, 1982a) or hydroxyurea (Fig. 3A). Furthermore U7 cells manifest a mitogenic response to NGF under growth-restrictive conditions (medium con- taining 0.5-2% serum) (Burstein and Greene, 1982b). U7 cells transfected by activated N-ras with or without LTR sequences showed a pattern of neurite outgrowth, cessation of division, and somatic enlargement analo- gous to that seen in PC12 cultures. The transfection efficiencies were similar to efficiencies of PC12 cells (data not shown). After several days of culture, a high proportion of U7 cells transfected with the activated N- ras acquired markedly enlarged, often bizarrely shaped, cell bodies (Figs. 3B,C). Such bizarre-appearing cells are

Fig. 3. Effect of NGF and activated N-ras on phenotype of U7 cells. (A): U7 cells were cultured for 12 days on collagen without poly-D- lysine in the presence of NGF, 50 ng/ml, and hydroxyurea, 0.3 mM. cB,C): U7 cells (passage 50-60) were plated and transfected as explained in Materials and Methods and photographed 7 days after transfection.

not common in U7 cultures induced to differentiate when exposed to NGF under growth-arresting conditions (Fig. 3A). Cultures transfected with the control neo-gene-con- taining plasmid or with normal mouse N-ras covalently linked to this plasmid (Fig. 2B) showed no neuronally differentiated cells.

These findings suggest new concepts about the actions of ras oncogenes. First, the ability of ras to either pro- mote or suppress proliferation appears to be dependent

RAS INDUCES NEURONAL DIFFERENTIATION 75

upon the target cell, and specifically the nature of cellu- lar pathways activated by the ras gene product p21. This may be analogous to the ability of NGF to act either as a mitogen or as a differentiation-promoting antimito- genic signal (Greene and Tischler, 1976; Burstein and Greene, 1982b; Boonstra et al., 1983; Lillien and Claude, 1985).

Recently, reports have appeared linking the other ras oncogenes (Bar-Sagi and Feramisco, 1985; Noda et al., 1985) as well as v-src (Alema et al., 1985) to PC12 differ- entiation. One of the reports indicates that microinjec- tion of p21 coded by an activated human Harvey ras gene induced PC12 neurite growth, whereas injection of normal human Harvey ras-coded p21 (at a concentration that was shown by immunofluorescence to be consider- ably greater than the normal level of PC12 p21) failed to alter the PC12 phenotype (Bar-Sagi and Feramisco, 1985).

In another set of experiments Noda et al. (1985) have obtained similar results by infecting PC12 cells with either Harvey or Kirsten murine sarcoma viruses (Ha- and Ki-MSV) and showing that they can trigger neurite outgrowth, cessation of division, and induction of acetyl- colinesterase and resting membrane potential. From this study no information about the effect of the normal ras gene could be obtained. More recently, the role of the ras gene product in the pathway triggered by NGF has been strengthened by a report indicating that anti-pal antibody can block NGF-induced differentiation (Hagag et al., 1986).

In the report using microinjection of bacterial made p21, the product from the normal human Harvey ras gene failed to induce differentiation although present at apparently higher abundance than native PC12 p21 (Bar-Sagi and Feramisco, 1985). In contrast, in the pres- ent study, the normal mouse N-ras gene possessed neu- rite-promoting activity when situated in a LTR- containing construct, although at a significantly re- duced rate. One possible explanation for this discrep- ancy might be the limited number of cells that were analyzed in the microinjection study (Bar-Sagi and Fer- amisco, 1985).

PC12 cells are relatively specific in their responsive- ness to NGF, a physiologic developmental signal of sym- pathetic neuronal differentiation (Levi-Montalcini and Angeletti, 1968). Fibroblast growth factor is the only other characterized polypeptide growth factor that can cause a limited degree of neurite outgrowth in PC12 cells, although not of the magnitude induced by NGF Vogari et al., 1985).

One alternative mechanism to be considered involves possible activation of adenylate cyclase. DBcAMP can cause outgrowth of processes that are less stable than NGF-promoted neurites (Gunning et al., 1981; Togari et al., 1985). Several significant NGF-induced changes in PC12 cells, including onset of priming (Greene et al., 1982; Gunning et al., 1981; Burstein and Greene, 1978, 1982a) and pronounced induction of MAP1 (Greene et al., 1983; Drubin et al., 19851, are not caused by DBcAMP. In yeast, ras appears to activate adenylate cyclase Cl'oda et al., 19851, although in certain mamma- lian cells, a role of p21 as a regulatory component of adenylate cyclase has been ruled out (Beckner et al., 1985). The length of ras-induced neurites more closely resembled that of NGF-induced neurites than DBcAMP-

promoted processes. Furthermore Hagag et al. (1986) have recently demonstrated that anti-p21 antibody blocks PC12 differentiation by NGF but not the out- growth triggered by DBcAMP.

The present studies on U7 cells indicate that a cell line that is resistant to NGF under normal growth con- ditions can differentiate in response to activated N-ras. An interpretation of this finding, which is consistent with the hypothesis of ras p21 being part of the NGF pathway, is that the defect in the NGF mechanism in U7 cells could precede or involve a pal-mediated step. The presence or absence of responsiveness to N-ras ap- pears to be a useful means of analyzing other NGF- responsive and -nonresponsive lines with neuronal prop- erties or potential, including other PC12 mutants and neuroblastoma lines. However, not all lines of neuronal or neuroblastic origin are induced to differentiate by activated N-ras since this gene was initially identified in a human neuroblastoma cell line (Shimizu et al., 1983).

Our findings indicate that N-ras-induced phenotypic changes in PC12 and in U7 cells do not precisely dupli- cate the changes induced by NGF, since differences in cell size and shape and degenerative changes are seen in activated N-ras transfectants that are not character- istic of NGF effects. NGF-stimulated cells undergo in- crease in somatic size (Greene and Tischler, 1976; Greene et al., 1982), but somatic appearance stabilizes as cells acquire a neuronal phenotype, and differentiated PC12 cells can persist for many weeks in NGF-treated cul- tures. The persistent increase in cell size and relatively rapid degeneration of activated N-ras-transfected PC 12 cells both suggest a lack of regulation compared to cells induced to differentiate by NGF.

The ability of normal N-ras in a LTR-containing over- expression vector to induce PC12 differentiation appears to be analogous to the ability of this gene in the same vector, to induce transformation of 3T3 cells. Normal mouse N-ras in a vector lacking LTR sequences fails to cause either 3T3 transformation or PC 12 differentia- tion. These analogous findings suggest the possibility that the p21-mediated step may be identical in both PC12 and 3T3 cells despite the Merent phenotypic changes ultimately triggered by this protein in these cells.

The PC12 cell system is a well-studied model of neu- ronal differentiation. A variety of chemical probes that can modulate the NGF response (Burstein and Greene, 1978; Seeley et al., 1984; Burstein et al., 1985), as well as straightforward means for selecting phenotypic vari- ants with altered NGF responses (Burstein and Greene, 1982a, b, Bothwell et al., 1980; Green et al., 1986) make PC12 cells useful for mechanistic studies. Further use of such probes and genetic approaches to the ras-induced effects on PC12 cells may help elucidate the mechanisms of action of this important set of genes as well as reveal heretofore unexplained molecular aspects of neuronal differentiation.

ACKNOWLEDGMENTS We thank George Rosa and Robert Lake for excellent

technical assistance. Isabel Guerrero is a Fulbright fel- low. Angel Pellicer is an Irma T. HirschL'Monique Weill- Caulier awardee. Sponsored by NIH grant CA36327 (A.P.), NIH grant NS21648 (D.E.B.), NCI Clinical Inves-

76 GUERRERO, WONG, PELLICER, AND BURSTEIN

tigator award CA01025 (D.E.B.), American Cancer So- ciety Local Institutional grant-in-aid m-14-z (D.E.B.), and Research support grant RR05399 (D.E.B.).

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