Cell, Vol. 69, 265-273,

April 17, 1992, Copyright

0 1992 by Cell Press

Abnormal Regulation of Mammalian p21 ras Contributes to Malignant Tumor Growth in von Recklinghausen (Type 1) Neurofibromatosis Jeffrey E. DeClue,’ Alex G. Papageorge,” Jonathan A. Fletcher,t Scott Ft. DiehI,* Nancy Ratner,§ William C. Vass,’ and Douglas Ft. Lowy’ Laboratory of Cellular Oncology National Cancer Institute Bethesda, Maryland 20892 tDepartment of Pathology Brigham and Women’s Hospital Boston, Massachusetts 02115 *Departments of Psychiatry and Human Genetics Medical College of Virginia Virginia Commonwealth University Richmond, Virginia 23298 §Department of Anatomy and Cell Biology University of Cincinnati College of Medicine Cincinnati, Ohio 45267-0521 l

Summary Tumor cell linesderivedfrom malignant schwannomas removed from patients with neurofibromatosis type 1 (NFl) have been examined for the level of expression of NFl protein. All three NFl linesexaminedexpressed lower levels of NFl protein than control cells, and the level in one line was barely detectable. The tumor lines expressed normal levels of p120wp and ~21’~. Although the p21 I” proteins isolated from the tumor cells had normal (nonmutant) biochemical properties in vitro, they displayed elevated levels of bound GTP in vivo. The level of total cellular GAP-like activity was reduced in extracts from the tumor line that expresses very little NFl protein. Introduction of the catalytic region of GAP into this line resulted in morphological reversion and lower in vivo GTP binding by endogenous ~21”~. These data implicate NFl protein as a tumor suppressor gene product that negatively regulates ~21” and define a ‘positive” growth role for ras activity in NFl malignancies. Introduction Neurofibromatosis type 1 (NFl) is a human genetic disease affecting 1 in 3500 people of all races (Stumpf et al., 1987). The disease affects cells of the peripheral nervous system, especially those of neural crest origin and results in a variable phenotype that may include benign neurofibromas, flat pigmented lesions (cafe au lait spots), Lisch nodules of the iris, and other, more variable abnormalities (Riccardi and Eichner, 1988). Patients with NFl are also at increased risk for development of certain malignancies, especially malignant peripheral nerve sheath tumors that can often be schwannomas. The gene for NFl , which lies at 17qll.2, was recently cloned by reverse genetics (Marchuk et al., 1991; Viskochil et al., 1990; Cawthon et al., 1990; Wallace et al., 1990), and its coding sequence

was found to contain a large region of homology with the products of the yeast IRA1 and IRA2 genes (Xu et al., 1990b) and a smaller region of homology with the GTPase activating protein (p120GAP) (Trahey and McCormick, 1987). IRA1 and IRA2 encode negative regulators of RAS in Saccharomyces cerevisiae (Tanaka et al., 1989, 1991) which may also participate in RAS effector (target) function (Mitts et al., 1991). Similarly, mammalian p120GAP functions as a negative regulator and also may be a target effector for mammalian Ras proteins (Hall, 1990; Lowy et al., 1991; Marshall, 1991). Inactivating mutations of IRA produce elevated RAS activity in yeast (Tanaka et al., 1990) while overexpression of GAP can block transformation by normal ras @hang et al., 1990) or by oncogenes that depend upon endogenous ras activity for their transforming function (DeClue et al., 1991d; Nori et al., 1991). The common domain shared by the IRA, GAP, and NFl proteins encodes an enzymatic stimulator of Ras GTPase; expression of this (catalytic) region of GAP or NFl can complement mutations in IRA (Ballester et al., 1990,1989; Martin et al., 1990; Tanaka et al., 1990; Xu et al., 1990a). These findings have raised the possibility that the NFl protein product may be a physiological regulator of mammalian Ras proteins and that the pathogenesis of NFl disease may involve abnormal regulation of Ras. The mammalian ras genes encode 21 kd protein products (~21’~~) that are attached to the plasma membrane, can bind guanine nucleotides, and catalyze the hydrolysis of bound GTP to GDP (Bourne et al., 1990, 1991; Wittinghofer and Pai, 1991). Like other guanine nucleotide-binding proteins, ~21”” is active when it is in the GTP-bound state and inactive in the GDP-bound state (Marshall, 1991). Normally only a small proportion of cellular p21raS is GTP bound (Gibbs et al., 1990; Satoh et al., 1990a, 1990b). However, mutations in ras can dramatically increase its biological activity by encoding mutant p21 rasthat accumulates much higher levels of the GTP-bound form. Most mutations appear to be activating because they reduce the intrinsic GTPase of the encoded p21 rasand render it resistant to GAP and NFl (Ballesteret al., 1990; Martin et al., 1990; Trahey and McCormick, 1987; Xu et al., 199Oa). These mutations have been found in the viral ras genes as well as in many human and animal tumors or tumor cell lines (Barbacid, 1987). Another class of activating mutation causes an increased rate of dissociation of GDP, so that the net level of GTP bound in vivo is increased. This class of mutation has been identified by in vitro mutagenesis studies of ras (Sigal et al., 1986; Walter et al., 1986). By analogy to other GTP-binding proteins, mammalian ~21”” is thought to signal to a downstream effector molecule that may itself possess or associate with an enzymatic function. At this time, GAP and NFl are candidate effector targets for mammalian p21 raS,in addition to their proposed negative regulatory role, since these molecules require an intact Ras “effector” domain for normal interaction (Adari et al., 1988; Cal& et al., 1988). Recently we described anti-NFl sera, raised against the

Cell 266

ing by (nonmutant) p21 rasin the tumor cell lines. In addition to describing a novel form of activation for mammalian P21”S, the findings provide evidence that neurofibromin is a tumor suppressor gene product that negatively regulates ~21’~. In other experiments, we have shown that it is possible to complement functionally the reduction in NFl protein and induce reversion of one of the tumor lines by introducing the catalytic region of GAP. These results demonstrate that Ras activity contributes to the malignant phenotype of the tumor cells and suggest that anti-Ras therapies may be use ful in the treatment of NFl malignancies.

NFl) -

225

123456

B

Results

GAP) - 97.4 123456

C RAS ) -18.4 1

2

3

4

5

6

D

NFl)

123456 Figure 1. Expression of neurofibromin, NFl Tumor Cell Lines

GAP, and ~21‘~ in Control

and

(A-D) Lane 1, NIH 3T3; lane 2, RN-22; lane 3, HeLa; lane 4, NFI 88-3; lane 5, NFlEE-14; lane 6, NF190-8. (A-C) lmmunoprecipitations were carried out as described from lysates of cells labeled with [JSS]methionine and PS]cysteine. (A) neurofibromin was immunoprecipitated using anti-GRD serum (DeClue et al., 1991a) and analyzed on a 6% polyacrylamide gel. (6) GAP was immunoprecipitated using anti-GAP A antiserum (Ellis et al., 1990) and analyzed on an 8% gel. (C) ~21’~ was immunoprecipitated with the Y13-259 antibody (Furth et al., 1982) and analyzed on a 15% gel. Migration of molecular size standards is indicated at right in kd. (0) Western blot of neurofibromin. Protein (100 ug) from each cell line was electrophoresed on a 8% polyacrylamide geland transferred toafilter.Thefilterwasincubatedwith NFI 3’rabbit antiserum, washed, and incubated with [‘251]anti-rabbit (Amersham).

catalytic region of NFl , that were used to identify the 280 kd NFl protein product (p280NF1, now designated neurofibromin) by immunoprecipitation (DeClue et al., 1991a). Here we have used the antiserum to investigate the expression of neurofibromin in three malignant schwannoma cell lines from NFl patients. We describe a correlation between reduced neurofibromin, decreased cellular GAP-like activity in vitro, and increased in vivo GTP bind-

Expression of NFl Protein Is Reduced in NFl Tumor Cell Lines The gene responsible for NFl has been identified through the mapping of mutations in affected individuals (Viskochil et al., 1990; Wallace et al., 1990). It has been suggested that NFl malignancies arise when a second, somatic mutation occurs in the Schwann cells, as for tumor suppressor gene products such as retinoblastoma (Rb) (Weinberg, 1991). To test this possibility, we investigated whether patient-derived tumor cell lines might express reduced levels or aberrant patterns of NFl protein products. Three malignant schwannoma cell lines derived from tumors removed from patients diagnosed with NFl have been examined here; two of these schwannoma lines have recently been described (Fletcher et al., 1991; Glover et al., 1991). The presenceof neurofibromin, p120GAP, and ~21’” proteins were assayed by metabolic labeling and immunoprecipitation and analyzed on polyacrylamide gels (Figures lA, lB, and 1C). Controls included murine NIH 3T3 (lane l), a rat schwannomacell line, RN-22, which is a derivative of the RN-2 line (Pfeiffer and Wechsler, 1972) (lane 2), and the human HeLa carcinoma line (lane 3). As shown in Figure 1A, NIH 3T3 and RN-22 expressed a single strong band of NFl protein (the fainter, slower migrating band at the top is not NFl related, based on peptide mapping). HeLa cells expressed a strong comigrating NFl band and two other forms represented by weaker bands above and below it (again, the top band is not NFl related). Two alternatively spliced exons have been described for the human NFI gene (Marchuk et al., 1991; Xu et al., 1990b); the migration rates of the three different forms of NFl in human HeLa might therefore be a result of differences in splicing. Two NFl tumor lines, 88-3 and 90-8 (lanes 4 and 6, respectively) expressed less of the major form of NFI than controls, and also expressed the slower migrating form. The ST88-14 tumor line (case 7 in Fletcher et al. [1991]) (lane 5) expressed only two faint bands, corresponding to the major and faster migrating forms. In contrast, all of the cell lines expressed p120GAP (Figure 1B) and similar amounts of p21 r88(Figure 1C). Since all of the immunoprecipitations were from the same set of lysates, it is unlikely that differences in uptake or incorporation of label could account forthe reduction in neurofibromin in the NFl lines. To verify the results obtained with the immunoprecipita-

Abnormal 267

Regulation

of

ras in

NFl

Tumor

Cells

NFI Tumors

Controls

Revertants

“r-----l GDP)

GTP ) 12

3

4

5

6

7

8

9

lo

Figure 2. In Vivo Guanine Nucleotide Binding Analysis of ~21”~ Isolated from Control Cell Lines, NFl Tumor Cell Lines, and Revertants of the NFl ST88-14 Line Lane 1, v-ras-transformed NIH 3T3; lane 2, NIH 3T3; lane 3, RN-22; lane 4, HeLa; lane 5, NF188-3; lane 6, NF186-14; lane 7, NF190-8; lane 8. revertant clone 4-4; lane 9. clone 4-6; lane 10, clone 5-2. Cells were labeled with [32P]phosphate, lysed, and p21 rss was immunoprecip itated with Y13-259. After washing, the bound nucleotide was released, and chromatography was carried out in 1.3 M lithium chloride. The migration of GDP and GTP is indicated at left. The figure represents a composite of samples from different experiments; however, except for lane 1, all of the samples shown were included in Tables 1 and 2.

tions, we carried out immunoblotting with a second antiserum directed against a different region of NFI, C-terminal to the catalytic region (Daston et al., 1992) (Figure 1D). The pattern obtained was strikingly similar to the immunoprecipitation analysis (Figure 1A). Again, the ST88-14 tumor line (lane 5) expressed greatly reduced amounts of NFl, while the 88-3 and 90-8 (lanes 4 and 6) expressed significantly lower levels than the control lines. We conclude that the NFl tumor lines, in particular the ST88-14 line, exhibit reduced expression of NFl protein. Ras Proteins Bind Elevated Levels of GTP in the Tumor Cell Lines Since the tumor cell lines expressed p21raS and reduced levels of a putative negative regulator of p21”s, we examined the level of GTP bound to endogenous ~21”~ in the different lines. This was done by labeling cells with [32P]phosphate, followed by immunoprecipitation of p21’“*, and thin-layer chromatography of the bound nucleotide (Figure 2; Table 1). GDP and GTP are easily resolved in this assay, as shown in a sample from v-ras-transformed NIH 3T3 (Figure 2, lane 1). In control cells (NIH 3T3, the rat schwannoma line RN-22, and HeLa), Ras proteins were

Table 1. In Vivo GTP Binding and NFl Tumor Lines’

Line

Percent

of ~21”’

GTP

Bound

in Control

Number of Determinations

NIH 3T3 RN-22 HeLa

8.8 (+/9.2 (+/8.3 (+/-

3.1) 0.8) 2.4)

5 3 4

NF188-3 NF190-8 NF188-14

16.0 (+/29.6 (+I37.8 (+I-

4.0) 4.6) 12.2)

5 3 4

a Cells were plated and labeled, and and thin-layer chromatography were Experimental Procedures. Following ground correction and correction for out before the numbers above were

cell lysis, immunoprecipitation, all carried out as described in scanning with AMBIS, backphosphate content were carried calculated.

bound predominantly to GDP (lanes 2-4) while there was a striking increase in the level of GTP bound to ~21’“” in the tumor cells (lanes 5-7). Table 1 presents quantitative data for these experiments, derived from multiple determinations for each cell line. Under these conditions, Ras in control cells bound about 8% GTP, in accord with published work (Gibbs et al., 1990). Ras proteins in the three NFl cell lines bound 16%, 30%, and 38% GTP, all substantial elevations given the normally tight regulation of ~21”” (Table 1). One possibility that could explain the elevated level of GTP bound to Ras in the tumor line8 is that one of the ras genes had undergone mutation. Since mutations that activate the oncogenic potential of ras cause impaired GTPase or increased guanine nucleotide release by the mutant proteins, we examined these properties for the endogenous Ras proteins in the NFl lines. First, the Ras proteins were immunoprecipitated with a monoclonal antibody (Y13-238) (Furth et al., 1982) that recognizes the Ras proteins encoded by rasH and rasK but does not neutralize the GTPase or nucleotide exchange activities (Rey et al., 1989; Willumsen et al., 1986). Then the bound ~21’” was loaded with [a-32P]GTP, washed, and incubated either at 37% for 15 min to assess intrinsic GTPase (Figure 3A) or at 37% for various times in the presence of 1 mM unlabeled GTP to measure guanine nucleotide release (Figure 38). After incubation, the bound guanine nucleotides were analyzed by thin-layer chromatography (Figure 3A) or by a filter-binding assay (Figure 36). As shown in Figure 3A, mutant Ras proteins from a v-ras-transformed NIH 3T3 line remained over 80% GTP bound at the end of the incubation, while the Ras protein8 from normal NIH 3T3 and the NFl lines hydrolyzed all but 30% of the GTP during the loading and incubation periods, indicating that there was no impairment of the GTPase activity of ~21”” from the NFl lines. Similarly, nucleotide exchange by ~21’“” from the NFl lines showed the same slow rate as for the p21ras from the NIH 3T3 (Figure 38). The was encoded ~21, which contains a mutation (A59T) that increases nucleotide exchange (Lacal and Aaronson, 1986) showed a faster rate of release (Figure 3B), while the N116H mutant ~21’” showed a dramatically faster rate, as described previously (Der et al., 1988) (Figure 38). The ~21’” proteins assayed from the NFl lines therefore displayed normal biochemical properties in vitro, which suggests that the elevated GTP-bound p21 raJin the NFl tumor cell lines is not due to the presence of mutant ras in the cells. Additional Characterization of the ST88-14 NFl Cell Line We wished to characterize one of the NFl lines in more detail to gain insight into the development of the malignancy and the role of Ras in this process. We chose the ST88-14 line for this purpose, since it displayed the clearest alteration in neurofibromin expression and had the highest level of GTP-bound Ras in vivo. The half-life of neurofibromin in fibroblasts is about 36 hr (DeClue et al., 1991 a); to investigate whether the greatly reduced level of NFl protein in this line might be due to instability of the encoded NFl product, we carried out a pulse-label of 30

Cell

268

NFl) 1234 Figure Lines

4. Expression

of neurofibromin

in Tumor

and

Lymphocyte

Lane 1, HeLa; lane 2, NFl88-14 tumor line; lane 3, control lymphocyte line from non-NFI patient; lane 4, lymphocyte line from NFI patient whose tumor gave rise to the 813-14 line. lmmunoprecipitation was carried out as in Figure 1A.

B

Figure 3. GTP Hydrolysis Isolated from NFl Tumor

and Nucleotide Release by ~21’” Cell Lines and Control Lines

Proteins

lmmunoprecipitations of ~21” from unlabeled cell extracts were carried out with the Yi 3-238 monoclonal antibody. This antibody precipitates the 021 encoded bvra.s”and ra.f but not ra.9 (Furth et al.. 1987). The imm;noprecipitatedRas proteins were boundiith [@P]GiP anb incubated at 37% for 15 min (A) or various times in the presence of 1 mM unlabeled GTP (B). For (A), the amounts of GDP and GTP were determined by chromatography as for Figure 2, and the percent GTP bound at the end of the incubation period is shown. For (B), the [32P]nucleotide bound to Ras was quantified in a filter-binding assay, and is shown as %b. v-ras, (G12R, A59T)rasn; H116, (N116H)rasn.

min, followed by immunoprecipitation. If neurofibromin in the cell line were unstable, we would have expected to see more normal levels of neurofibromin after this pulse, but as had been true of the steady-state condition, the amount of neurofibromin following the pulse was also greatly reduced compared with control cells under these conditions (data not shown). The defect in this line, therefore, appears to involve reduced production of neurofibromin, rather than production of an unstable protein product. To determine whether all cells in the NFl patient expressed low levels of the NFl protein or whether these low levels are present only in the tumor cells, we examined the expression of neurofibromin in Epstein-Barr virusimmortalized lymphocytes from the NFl patient whose tumor gave rise to the ST88-14 line (Figure 4). Control EBV-immortalized lymphocytes were compared to EBVimmortalized lymphocytes from the NFl patient by labeling cells for 24 hr and immunoprecipitating with the NFl antiserum. We observed the same pattern of neurofibromin in HeLa and ST88-14 tumor cells as in Figure 1 (Figure 4, lanes 1 and 2). Control (non-NFl) lymphocytes (lane 3) expressed significant levels of neurofibromin, and

the level of neurofibromin in the NFl patient lymphocytes was approximately one-half that in the control lymphocytes (lane 4). These data support a model in which one mutant NF7 allele is present in all cells of the patient, and a “second-hit” in the normal allele occurred at some point during the development of the malignancy. Since the reduced expression of neurofibromin in the NFl lines correlated with increased proportion of GTP bound to the Ras proteins, cell extracts from the ST88-14 line should contain less total GTPase-stimulating activity than control extracts. Therefore, we prepared cellular extracts from the ST88-14 and RN-22 schwannoma lines and incubated different amounts of the extracts with [aJ’P]GTP-bound, bacterially synthesized p21ras (Figure 5). In the absence of any added extract, the p21ras remained 70% GTP bound following the incubation (Figure 5, left columns). Addition of small amounts of RN-22 extract accelerated the GTP-to-GDP hydrolysis in a concentrationdependent manner, while extract from the ST88-14 line had little or no activity in this assay (Figure 5, right panels). These results suggest that although GAP is expressed in the ST88-14 schwannoma line (and RN-22), the major

z -5

1

I

1

RN-22 Figure 5. GAP-Like Activity Schwannoma Lines

in Extracts

STSS-14 from the RN-22

and STEE14

Cell extracts were prepared as described in Experimental Procedures, and their protein content was determined. Bacterially expressed ~21” was purified, bound with [a-“P]GTP, and incubated with the indicated amount of lysate protein for 12 min at 30%. The Ras protein was then immunoprecipitated, and the bound guanine nucleotide was analyzed by thin-layer chromatography. Bar graph indicates the percent GTP bound at the end of the incubation.

Abnormal 269

Regulation

of ras in NFl

Tumor

Table 2. In Vivo GTP Binding of ~21”” from NFl Tumor Line ST68-14a,b

Line

Percent

GTP

NF188-14 NF186-14(v-ras)

37.8 (+I44.1 (+I-

GAP-C revertants NF18E14 clone 4-4 NF188-14 clone 4-6 NF18814 clone 5-2

11.9 (+I-- 3.1) 9.6 (+I- 1.6) 11.6 (+I- 5.3)

in Cell Clones

Bound

12.2) 16.2)

a Values were determined as for Table DThe v-ras-expressing line was derived helper virus, while the GAP-C revertants

Cells

Derived

Number of Determinations 4 3 3 3 4

1. by infection with amphotropic were derived by transfection.

contributor to the GTPase acceleration in these extracts is NFl protein. The detergent dodecyl maltoside has been shown to inhibit the catalytic region of NFl but not GAP, and can therefore be used to discriminate between these GAPS (Bollag and McCormick, 1991). We found that the majority of GTPase-stimulating activity in the RN-22 schwannoma extract was inhibited by 5 mM dodecyl maltoside (data not shown), defining it as “NFl-like.” We conclude that NFl is an important regulator of ~21”” in Schwann cells, and the reduction of neurofibromin in the ST88-14 cell line can be detected as a loss of enzymatic activity in in vitro assays using extracts from these cells. Expression of the Catalytic Region of GAP Can Induce Reversion of the ST88-14 Tumor Line Having demonstrated an increased proportion of Ras protein in the GTP-bound form in the NFl tumor lines, we sought to determine its biological significance by increasing or decreasing the Ras activity in the cells. To increase Ras, we introduced the v-ras gene into the ST88-14 tumor line. The resulting cell line had only asmall increase in the percentage of GTP-bound Ras, to 44%, versus 38% in the parental ST88-14 line (Table 2). The v-fas line resembled the ST88-14 line in its morphology, although it was somewhat more refractile and grew at a slightly higher frequency in agar suspension (data not shown). These marginal differences suggested either that Ras activity might not be making a biologically significant contribution to the growth properties of the cells (if, for example, neurofibromin were required for Ras target function) or that the already high level of Ras activity had not been increased significantly by was. To distinguish between these possibilities, we sought to reduce the level of GTP-bound Ras in the ST88-14 line. If the ST88-14 line lacked Ras target, reducing Ras activity should have little effect on the growth properties of the cells. If, on the other hand, the already high level of GTPbound Ras contributes to the growth of the tumor cells, reducing the Ras activity should inhibit their growth. We have described asubcloneof GAP, encoding the (C-terminal) catalytic region, linked to a G418 resistance marker, that can efficiently down-regulate normal Ras in vivo (DeClue et al., 1991d; Zhang et al., 1990). We introduced this DNA clone into the ST88-14 line by transfection and

Figure 6. Characterization of NF18814 Expressing the GAP C-Terminus

Line and Revertant

Clones

Identity of clones is designated above each pair of panels. (A) Cellular Morphology of NF188-14 line (top) and revertant clones expressing the GAP C-terminus. Cells were plated at 5 x 105 per 60 mm dish 2 days prior to microscopy. Original magnification was 85 x (B) Anchorageindependent growth of NF188-14 line and revertant clones. Cells (7 x IO4 per ml) were seeded in 0.4% agar in a 60 mm dish and grown for 15 days prior to microscopy.

selected colonies of cells that were resistant to G418. Many of the drug-resistant colonies displayed a flat, nonrefractile morphology that was clearly different from that of the parental ST88-14 line. Three of these revertant cell clones (designated 4-4, 4-8, and 5-2) were expanded into cell lines and subjected to additional analysis. Figure 8A shows the morphology of the parental ST8814 line and the three revertant clones. While the ST88-14 cells were rounded, refractile, and piled on top of each other, the cells in the revertant clones were flatter, less refractile, and grew as a monolayer. When the ST88-14 cells were seeded in agar suspension (Figure 86, top), they formed colonies l-2 mm in diameter at a frequency of approximately 1 x 1 O-‘. The revertant cells grew more poorly in agar (Figure 8B, bottom three panels) and formed colonies at a frequency that was reduced 5-to lo-fold. The revertant cells contained three distinct karyotypic changes found in the ST88-14 line (data not shown), but no additional changes. To confirm the expression of the 38 kd GAP C-terminus protein in the revertants, cell lysates were

43NGAP-c 29-

12 Figure vertant

34

5

7. Expression of the 38 kd GAP C-Terminal Clones Derived from the NF188-14 Line

Protein

in Re-

Lane 1, NIH 3T3; lane 2, NF188-14; lane 3, revertant clone 4-4; lane 4, clone 4-6; lane 5, clone 5-2. Protein (100 ug) from each cell line was electrophoresed on a 12% polyacrylamide gel and transferred to a filter. The filter was incubated with the RH6-2A anti-GAP serum, washed, and incubated with [‘Z51]anti-rabbit (Amersham). Migration of molecular size standards is indicated at left in kd.

immunoblotted with an antiserum that recognizes an epitope in the catalytic region of GAP (Halenbeck et al., 1990) (Figure 7). While lysates of NIH 3T3 or ST891 4 cells made no GAP C-terminus protein (lanes 1 and 2) the three revertants all expressed a strong immunoreactive band at 38 kd (Figure 7, lanes 3-5). We conclude that the reversion of the tumor line was induced by expression of the catalytic region of GAP. To test whether the mechanism of reversion did indeed involve the down-regulation of Ras, we examined the in vivo GTP binding of p21 rae in the revertant cell lines. We found that the level of GTP bound to Ras was reduced from 38% in the ST891 4 line to 9%-i 1% in the revertants (Table 2) a level that was similar to control cells (Figure 2). We conclude that the reversion was most likely a direct result of this reduction in Ras-GTP, and that Ras contributes a positive signal toward the growth of the tumor cells. Consistent with the analysis of the intrinsic GTPase activity and guanine nucleotide release assays on the cell lines (Figure 3) the results also demonstrate that the GTP bound to Ras protein in the cells is sensitive to negative regulation by the catalytic domain of GAP, which further strengthens the conclusion that the Ras protein in the cell line is nonmutant.

Our analysis of three cell lines derived from malignant human schwannomas in NFl patients shows that each line possessed high concentrations of GTP-bound ~21’“” and low levels of neurofibromin. The control cell lines, which included a rat schwannoma cell line, contained significantly higher levels of neurofibromin and lowerconcentrations of ~21” in the active, GTP-bound form. We have also detected normal levels of neurofibromin and GTPbound ~21” in several cell lines derived from non-NFl human melanomas (unpublished data), which, like schwannomas, are neural crest-derived tumors. Therefore, the low levels of neurofibromin and high concentrations of GTP-bound ~21’~ observed in the human NFl lines are not obligatory features of neural crest tumors in general or schwannomas in particular. Many reports have identified tumors in which a fes allele

has been mutated, leading to high concentrations of GTPbound Ras. With the vast majority of these Ras mutants, the intrinsic GTPase activity of Ras is reduced, and the protein is resistant to negative regulation by the catalytic activity (GTPase acceleration) of GAP and NFI The p21 ‘as in the NFl lines described here also contains abnormally high concentrationsof the active, GTP-bound form. In contrast with previously described situations, however, p21 ‘as in the NFl lines appears to be nonmutated, since the protein analyzed possessed normal intrinsic GTPase activity and guanine nucleotide dissociation rate, and the GTPbound protein in the ST88-14 line was shown to be sensitive in vivo to negative regulation by the catalytic domain of GAP. This conclusion raises the possibility that p21raS may be activated via inappropriate regulation in other types of tumors. Since p21 ‘=does not appear to be mutationally activated in the NFl lines, it is likely that the increased levels of GTP-bound Ras are a consequence of diminished negative regulation by NF-1. The level of GTP-bound Ras correlated inversely with the amount of NFl protein in the four schwannoma lines (the three NFl lines plus the rat schwannoma line). The ST8914 line had barely detectable amounts of NFl and significantly higher levels of GTP-bound Ras than the two NFI lines with more modest reductions in neurofibromin. It is therefore most likely that the reduced levels of neurofibromin in the NFl tumor lines underlie the activation of p21 ‘as, analogous to the effects in S. cerevisiae of IRA disruptions on RAS (Tanaka et al., 1990). It remains formally possible that mutational inactivation of GAP catalytic activity might account for some of the impaired negative regulation of p21 rasin the NFl lines, but there was no correlation between the levels of p120GAP in the cell lines and that of GTP-bound ~21”~. We noted a striking reduction in total GTPase accelerating (GAP-like) activity in extracts of the ST88-14 NFl line, compared with the rat schwannoma line. As was true of GTP-bound Ras, the GAP-like activity correlated with the relative amount of neurofibromin, rather than with p120GAP, since both lines contain similar levels of the latter protein. The results suggest that NFl may be a particularly important regulator of ~21”” in schwannomas (and presumably also in Schwann cells) and that the catalytic activity of pl20GAP may be much lower in Schwann cells than in nonneural crest cells. In fibroblasts, the catalytic activity of p120GAP appears to be sufficient for negative regulation of Ras @hang et al., 1991). These findings are consistent with the cell-type restriction of NFl disease. We are currently investigating possible mechanisms by which GAP may be less active as a negative regulator in Schwann cells, such as by posttranslational modification or proteinprotein interactions (Moran et al., 1991). The high concentration of GTP-bound Ras contributes to the transformed properties of the ST88-14 line, since introduction of the GAP catalytic domain induced morphological reversion of the cells and reduced their GTP-bound Ras to near normal levels. The ability of the GAP catalytic domain to suppress the transformed phenotype and the high levels of GTP-bound Ras suggest that strategies

Abnormal 271

Regulation

of ras in NFl

Tumor

Cells

based on catalytic or noncatalytic antagonism of ras function (Gibbs, 1991) might have therapeutic potential in NFl malignancies. The response of Schwann ceils to high fas activity appears to depend upon the physiological status of the cells. Introduction of v-fas by itself in primary rodent Schwann cells was reported to result in cell cycle arrest, but when was was introduced together with a nuclear oncogene, v-ras transformed the cells (Ridley et al., 1988). In the RN-22 rat schwannoma line, introduction of mutationally activated v-ras induced dramatic morphological transformation and increased anchorage-independent growth (unpublished data). Based on these results, both RN-22 and ST88-14 (and presumably the other NFl tumor lines) may have undergone events involving nuclear oncogenes that allow for increased GTP-bound Ras to stimulate cell growth. Consistent with this hypothesis, published studies have identified mutations in cellular ~53 genes that altered the ~53 coding sequence in 2 of 8 malignant NFl tumors or tumor lines (Menon et al., 1990; Nigro et al., 1989), including the 88-3 line described here. We verified the mutation in the 88-3 line but did not detect any mutations in p53genesfrom the ST88-140r the90-8 lines (unpublished data). It seems likely that another cellular gene(s) that can cooperate with ras may be altered in the NFl malignancies that do not contain mutated ~53. Our results support the idea that neurofibromin is a tumor suppressor gene product that regulates p21’“, in which case NFl patients would be analogous to those who have single mutant alleles of ~53 (Li-Fraumeni syndrome) or Rb (juvenile retinoblastoma). Lymphocytes from the patient whose tumor gave rise to the ST88-14 cell line expressed more neurofibromin than did the tumor line, but less than control lymphocytes, which suggests that the mutant allele in the lymphocytes synthesizes little or no stable neurofibromin (the unavailability of any family history for this case makes it impossible to determine whether this represents a familial or sporadic case). The barely detectable levels of neurofibromin in the ST88-14 line imply that during development of the malignancy, a second event occurred involving the normal NFl allele: an independent mutation of the normal allele, a gene conversion event, or a reduction to hemizygosity. The latter possibility is supported by the observation that there are no intact copies of chromosome 17 present in the ST88-14 tumor cells, but they do contain a single, grossly intact NFl gene on a satellite chromosome (J. E. Reynolds, J. A. Fletcher, C. H. Lytle, L. Nie, C. C. Morton, and S. R. Diehl, unpublished data). Consistent with the protein analysis presented here, Reynolds et al. have detected NFl mRNA from ST88-14 by PCR techniques, but little if any intact mRNA is seen in Northern blots. The appearance of malignant schwannomas in NFl patients is a low frequency event, especially at the level of the individual cell, which makes it likely that multiple independent genetic events contribute to their development. However, the development of benign neurofibromas and the otherstigmataof NFl disease are much morecommon in NFl patients. Although cytogenetic evidence implies

that the normal allele is not mutated in most benign neurofibromas (Menon et al., 1990), the Schwann cells from the benign tumors are abnormal, since they possess angiogenie and invasive properties (Sheela et al., 1990). The roles of p21’@, neurofibromin, and other gene products need to be explored in the context of these benign lesions. Specifically, the proposed role of neurofibromin as adownstream target molecule of p21raS, in addition to its role as a negative regulator of p21’“, must be analyzed (Bollag and McCormick, 1991). The demonstration that Ras is biologically active in the ST88-14 line, which has a barely detectable level of neurofibromin, strongly suggests that p21 rasin this line is signaling via a downstream target other than neurofibromin. However, the current studies do not exclude the possibility that NFl might represent a second downstream Ras target that is not absolutely required in the ST88-14 line. Experimental Cell Culture

Procedures and Introduction

of Exogenous

Genes

NIH 3T3, HeLa, and RN-22 lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (GIBCO). The 88-3 and 908 neurofibrosarcoma lines (provided by Thomas Glover) were grown in DMEM with 15% fetal calf serum. The ST6E14 neurofibrosarcoma line was grown in an RPM XXI-based medium as described (Fletcher et al., 1991). Lymphocyte lines were grown in RPM1 1640 with 10% fetal calf serum. For photomicroscopy, cells were plated at a density of 5 x IO5 per 60 mm dish and grown for 2 days, then photographed. To assay agar colony formation, cells were trypsinized and brought to a concentration of 7 x 105 per ml. Five milliliter base layers of 0.6% (w/v) agar in DMEM plus 10% fetal calf serum were poured and allowed to harden. The cell suspensions were diluted I:10 in 0.4% (w/v) agar (again in DMEM plus 10% fetal calf serum), and 4 ml of the diluted cell suspension was plated on top of the base layers. The cultures were fed twice weekly with 4 ml of 0.4% agar in DMEM plus 10% fetal calf serum, and photographed and counted after 15 days. The v-fasH gene was introduced into the ST86-14 line by infection with a virus stock encoding v-ra.9 linked lo a gene conferring G416 resistance. This stock was derived by transfection of NIH 3T3 with the plasmid pBW1423 (which has the same structure as pBW1631 [Stone et al., 19661 except for the G12R and A59T mutations in ras”) and infection with an amphotropic murine leukemia virus. The revertant clones that express the GAP C-terminal protein were generated by transfection with plasmid pJDC103 (DeClue et al., 1991d) using the Lipofectin reagent (GIBCO) according to manufacturer’s specifications. G416 selection was carried out for two weeks in the presence of 100 @ml G416 (GIBCO). Cell Labeling, Immunopreclpltatlon, and In Vlvo Guanlne Nucleotide Binding Analysis Labeling of adherent cells with [35S]amino acids, cell lysis, and immunoprecipitation with anti-GAP or anti-NFl sera were all carried out as described (DeClue et al., 1991a). RPMI-based labeling medium (ICN) was used instead of DMEM for the ST66-14 and the lymphocyte lines. Lymphocytes were labeled the same way except that 8 x lo8 cells were labeled in 3 ml and harvested by centrifugation. Immunoprecipitation of ~21’~ with the monoclonal Y13-259 (Furth et al., 1962) was carried out as described (DeClue et al., 1991 c). For all immunoprecipitations, portions of IySateS containing equal numbers of acidprecipitable cpm were compared. In vivo guanine nucleotide binding was assayed as described (DeClue et al., 1991 b), except that cells were plated at a density of 1.2 x 10’ per cm* and labeled for 10 hr with 2% dialyzed fetal calf serum (rather than 10%). Chromatographs were analyzed on an AMBIS radie analytic imaging system, and the levels of GTP bound were calculated after subtracting background levels of radioactivity and correcting for the different phosphorus content of GDP and GTP.

Cell 272

Western Blot Analyses Preparation of unlabeled cell lysates, determination of their protein content, and electroblotting were carried out as described (Zhang et al., 1990). For NFI immunoblotting, the filter was blocked, and antibody incubations were carried out as described (DeClue et al., 1991 d), except that 1 ug/ml antibody was used for the primary incubation, and [‘“ljanti-rabbit (Amersham) was used in place of [‘-llprotein A. lmmunoblotting with the anti-GAP RH6-ZA(supplied by F. McCormick) was carried out the same way except that the antibody was used at 1:lOOO.

Wigler, M. (1989). Genetic yeast. Cell 59, 681-666.

preparation of Cell Extracts and GTPasa Activation Assays cells were plated at a density of 1 x IO’ per cm2 in 175 cm* flasks and grown for two days, and 5 flasks were used for preparation of extracts. Cells were rinsed with phosphate-buffered saline, scraped in phosphate-buffered saline, and centrifuged. The cell pellets were resuspended in 1 ml TNMN buffer (20 mM Tris-HCI [pH 7.4],100 mM NaCI, 5 mM Mg%, 0.5% NP-40) and after a portion was removed for protein content determination, the extracts were frozen at -70%. GTPase activation assays were performed as previously described (Zhang et al., 1991) with minor modifications. In a 50 pl reaction consisting of TNMN plus 0.5 mM dithiothreitol and 0.5 mg/ml bovine serum albumin, bacterially expressed and purified c-rasH p21 prebound with [@P]GTP (1500 Cilmmol; ICN) was incubated with increasing concentrations of cell extract at 37% for 12 min, then chilled at 0%. Following immunoprecipitation with Yl3-259 (as above), immunoprecipitates were washed, solubilized in 1% SDS, 20 mM EDTAand chromatographed. Chromatographs were analyzed on an AMBIS radioanalytic imaging system.

Bourne, H. R., Sanders, superfamily: a conserved 125-132.

D.A., and McCormick, F. (1990). switch for diverse cell functions.

Bourne, H. R., Sanders, superfamily: conserved 349, 117-I 27.

D.A., and McCormick, F. (1991). TheGTPase structure and molecular mechanism. Nature

Determlnatlon of GTP Hydrolysis and Nucleotlde Release by Endogenous Ras Proteins in the NFl Tumor Lines One confluent T-75 flask of each cell type was lysed, and the ~21’~’ proteins were immunoprecipitated with the monoclonal Y13-236 as described (DeClue et al., 199lb). lmmunoprecipitates were washed three times with TNMN, resuspended in 50 l~l TNMN containing 1 mM dithiothreitol, 16 pglml aprotinin, and 0.5 mglml bovine serum albumin, and incubated with 0.5 pl(5 uCi) [a-PP]GTP for 30 min at 30%. These conditions result in a significant proportion of GTP bound to normal p21 being converted to GDP during the binding period. The immunoprecipitates were then washed 4 times with TNMN and placed at 37% for 15 min. Then the immunoprecipitates were washed twice with TNMN, once with 10 mM Tris-HCI (pH7.5) 20 mM MgCI,, boiled in 10 pl of 10 mM EDTA, 1% SDS, and chromatographed. The guanine nucleotide release assays were carried out in the same manner except that binding of [a-32P]GTP was carried out for 30 min at 30%. The binding reactions were split into several equal portions, and unlabeled GTP was added to 1 mM. After incubation at 37% for the appropriate time, the reactions were stopped by the addition of 500 ul ice-cold 25 mM Tris-HCI (pH 7.5) 0.25 mM MgC&, and the [“PJGTP bound to ~21” was captured on filters (BA85; Schleicher and Schuell) and counted in liquid scintillant. Acknowledgments We thank Frank McCormick for the RH6-2A anti-GAP serum and Thomas Glover for the 68-3 and 90-8 tumor cell lines. S. Ft. D. is supported by the Thomas F. Jeffress and Kate Miller Jeffress Memorial Trust, grant IN-105N from the American Cancer Society, and the Massey Cancer Center core support grant NCI CA 16059. 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 USC Section 1734 solely to indicate this fact. Received

December

II,

1991; revised

January

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