Properties of two temperature-sensitive rous sarcoma virus transformed cerebellar cell lines

Brain Research, 202 (1980) 445-458

445

((,~Elsevier/North-Holland Biomedical Press

P R O P E R T I E S OF T W O T E M P E R A T U R E - S E N S I T I V E ROUS S A R C O M A VIRUS T R A N S F O R M E D C E R E B E L L A R CELL LINES

G. J. GIOTTA, J. HEITZMANN and M. COHN Developmental Biology Laboratory, The Salk Institute for Biological Studies, San Diego, Calif. 92138 (u.s.A.)

(Accepted June 19th, 1980) Key words: temperature-sensitive-Rous sarcoma - - cerebellar cells - - veratridine - - sodium-uptake

SUMMARY Cells from the cerebellum of 3-day-old BD-IX rats were obtained as permanent lines by transforming them with temperature-sensitive Rous sarcoma virus. The presence or absence of veratridine-stimulated Na+-uptake (voltage-dependent channels) was used to operationally classify them as neuronal or glial. When incubated at 34 °C, the permissive temperature for transformation, the cerebellar cells exhibit a transformed phenotype determined by anchorage independence, rounded morphology, high growth rate and absence of density-dependent inhibition of growth. In contrast, when the transformed cerebellar cell lines are kept at a temperature (38 °C) non-permissive for transformation, they exhibit a normal cellular phenotype with respect to the above properties. Moreover, changes toward neuronal morphology, increase in veratridine-stimulated Na+-uptake, decreased growth rate and the expression of the astrocyte specific protein, glial fibrillary acidic protein, suggest that a degree of differentiation is expressed at the non-permissive temperature.

INTRODUCTION Several investigators have shown that Rous sarcoma virus (RSV) can transform neural cells in vivo ~ and in vitro 25. Generally, the tumors, or cell lines established from the tumors, have been classified as glial based on morphological criteria and the presence of the putative glial specific enolase, S-10020, 25. In this report we confirm and extend these observations by showing that astrocytic glial, as well as cells with neuronal properties can be generated by transformation with RSV. RSV hitherto has not been shown to transform cells lines with neuronal properties. Our approach in deriving cell lines differs from previous work in that the two

446 lines which we describe here were obtained by transformation with RSV which ix temperature sensitive (ts) for translbrmation (ts-RSV). The rationale for using Is-RSV is based on the work of others who have shown that cells infected with ts-RSV exhibit phenotypic alterations which appear to be under control of the ts-transforming gene 5,14,1'%17,1'~.23. At the permissive temperature (PT) infected cells exhibit a transformed phenotype and similar to cells transtbrmed by other techniques, display alterations in the differentiated properties characteristic of the cell type being studied. In contrast, at the non-permissive temperature fNPT) cells transformed with ts-RSV assume a normal phenotype and express highly differentiated properties. Similarly, we show that two cerebellar cell lines transformed with ts-RSV grow at the PT displaying properties characteristic of transformed cells, whereas at the NPT they exhibit properties characteristic of the normal cell phenotype. Moreover, the parameters studied suggest that ts-RSV transformed neural cell lines express a degree of differentiation at the NPT which is not apparent, or diminished at the PT. A brief account of several cell lines transformed with ts-RSV has ah'eady appeared a~. MATERIALS AND METHODS

Chemicals Polybrene, ouabain, tetrodotoxin and scorpion venom were obtained from Sigma Chemicals. Veratridine was from K and K Chemicals, and 22Na~ from Amersham-Searle. Animals and cell lines BD-IX rats were obtained from a colony maintained at The Salk Institute: Dave Schubert kindly provided the n-ethyl-N-nitrosourea derived BD-IX rat brain cell line, BI03. This line has been characterized and shown to be neuronal 26. Previous work by other investigators has shown that the PT and NPT for transformed avian cells is 36 '~C and 41 °C, respectivelyz2,z7 while f o r transformed mammalian cells it is 33-35 '~C and 37-40 '~C10,18 respectively. The rat cerebellar cells appear morphologically transformed at 33-34 °C and morphologically normal at 38-39 °C and these were therefore considered to be the PT and NPT, respectively. Cell culture technique Rat cells were grown in Dulbecco's modification of Eagles medium (DMEM) supplemented with 10 % fetal calf serum. They were passaged using magnesium- and calcium-free phosphate-buffered saline containing 2 m M E D T A and 0.25 °//,, trypsin. Cloning in agar Cells were cloned in 0.33 o/Difco agar on top of base layer of 0.6 ?,~,agar. Agar was made up in D M E M supplemented with 10% fetal calf serum. Virus techniques Stocks of wild type Rous sarcoma virus (RSV), strain Schmidt-Ruppin subgroup

447 D and o f a ts src mutant LA90 were raised by standard procedures which consisted of infecting c/o primary or secondary chick embryo fibroblasts at a multiplicity of infection of 0.5-1 focus forming unit/cell. Virus infection was enhanced by including 2-4 big/m[ of polybrene in the medium. Titrations of virus stocks on either primary or secondary chick embryo fibroblasts were done using the agar overlay technique described by Vogt 33. Rescue of virus from infected rat cerebellar cells was performed as previously described 1°.

Virus infeetion of cerebellar cells The method used to infect rat cerebellar cells was essentially the same procedure used by Chen et al. 10 to transform rat kidney cells. The only difference in the procedure consisted of incubating the cerebellar cells in D M E M plus 15 o/,, FCS.

Cerebellar culture techniques Cerebella from 3-day-old BD-IX rats were carefully dissected out and the tissue minced into l-2 mm pieces. After washing 3 times with cold D M E M minus serum the tissue was incubated at 37 °C in D M E M with 0.25 ~ trypsin and 5 rain later disrupted by gentle up and down passage through a Pasteur pipette. The cells were incubated for 5 rain more at 37 C and the process repeated. Large pieces of tissue were allowed to settle to the bottom of the tube and the supernatant removed and added to an equal volume of D M E M with 10 ~,~fetal calf serum. Cells were spun down in a clinical table top centrifuge, washed 3 times with D M E M containing 101~ FCS and viable cells counted in a hemocytometer using trypan blue. G r o w t h cur~,,es

The growth kinetics of cells grown at either the PT or N P T were determined by seeding between 150,000-300,000 cells per 60 m m tissue culture dish in 5 ml of D M E M supplemented with 1 0 ~ fetal calf serum. Cells were kept in the same medium for the duration of the experiment in order to avoid errors in counting due to the removal of floating cells which were present in transformed cultures grown at the PT. The growth curves shown are representative examples of either 2 or 3 separate experiments which utilized duplicate tissue culture dishes for each point. The cell counts in each dish did not vary by more than +_ 15 °/o. Cell viability at either the PT or NPT, measured by trypan blue exclusion was routinely 87-95 ~,.

Protein determination Protein was determined by the Lowry method 21 using bovine serum albumin as a standard.

VeratrMine-stirnulated Na+-uptake The assay has been described previouslyS,9, 30. Basically, cells were grown at the PT in several 150 mm plates. H a l f of the plates were incubated at the PT and the remaining half at the NPT. In the case of the neuronal cell line (i.e. WCl7a) in order to assay cells kept at the N P T it was necessary to seed cells at high densities. Cells were

448 allowed to incubate at the proper temperature for 2 days, fed and then reincubated for 2 more days, removed from the culture dish with trypsin-EDTA and seeded onto 35 mm polylysine-coated tissue culture dishes at 2 : 4 10~ cells per dish. Twenty-four hours later cell lines were assayed for NaP-uptake. Veratridine and scorpion venom were used at concentrations of 2 × 10 -4 M and 100 #g/ml, respectively. The assay was conducted at 37 °C.

Glial fibrillary acidic protein ( GFA P) Antisera generated in rabbits against G F A P was the generous gilt ot" Dr. Bignami. It was used at a dilution of 1/80 as previously described 4. Briefly, cells were o/ethanol/5 5/0acetic acid for 10 min at --20 fixed prior to staining with a mixture o foe ~,J/,, "C. After fixation the cells were washed with Dulbecco's modification of Eagle's medium supplemented with 2',!o fetal calf serum and then incubated with G F A P antisera (1/80) for 30 min at 37 '~C in an atmosphere of 90 % air/10 o/~CO2. At the end of the incubation period the cells were washed thoroughly and incubated with rhodamine-labeled goat anti-rabbit lgG (Cappell Laboratories) at a dilution of 1/50 for 30 rain at 37 °C. At the end of this period the cells were washed and mounted in phosphate-buffered saline plus 10 ',?oglycerol. Controls for non-specific staining which proved negative were: (l) normal rabbit serum followed by rhodamine goat antirabbit IgG; and (2) incubation of unfixed cells with G F A P followed by rhodamine goat anti-rabbit IgG. Fluorescence was visualized using a Zeiss fluorescence microscope equipped with phase-contrast and rhodamine optics. RESULTS Three considerations led us to transform BD-IX tat cerebellar cells taken from 3-day-old animals: (1) 3 of the 5 types of neurons in the cerebellum synthesize DNA during this timO, a prerequisite for transformation; (2) minimal stable synapse formation in rats occurs prior to day 37,35 thus minimizing the amount o f cell death resulting from torn synaptic connections; and (3)by the third postnatal day, cerebellar cells are already developmentally committed to express unique functions associated with their terminally differentiated adult state ~5.

Virus rescue and growth in agar Cerebellar cells exposed to either wild type Schmidt-Ruppin subgroup D RSV (wt-RSV) or a ts-src mutant LA90 D at 34 °C exhibited foci of transformed cells after 2-3 weeks. In both wt-RSV and ts-RSV infected cultures foci of adherent refractile cells appeared which were capable of sustained growth. In order to show that transformation was due to ts-RSV, a rescue experiment was performed. The ts-RSV transformed uncloned culture was co-cultivated with normal chicken embryo fibroblasts and infectious virus so obtained was titered at 35 °C and 41 °C in chick embryo fibroblasts. The focus forming units/ml at 35 °C and 41 '~C were 1.0 ~, 104 and ~ 10~, respectively indicating that the transformed mammalian cells were infected with tsRSV.

449 TABLE i Agar cohmy fi~rmation by RS V-transformed uneloned cerebellar cells"

Cells were grown on 0.33 ~ Difco Bacto agar on top of a base layer of 0.6 ~ agar. The agar was in DMEM supplemented with 10~ fetal calf serum. TNTC too numerous to count; ND not determined Virus

Temperature

10~ celL~/plate

104 cells/plate

Efficiency

TS TS WT WT

34 C 38 C 34 'C 38 C 34 'C 38 'C

TNTC 0,5,6 TNTC TNTC ND ND

180, 154, 89, 138 0, 1, 0, 3 88, 97, 70, 65 101, 83, 88, 69 0,0 0,0

1.4% 0.005 % 0.8 0.99/,,, 0.0 0.0

--

As a n initial test to determine the t r a n s f o r m e d versus n o r m a l phenotype of the cells at the PT or N P T their ability to grow in soft agar was assayed. In general, transformed but not n o r m a l cells are capable of growth in soft agar 36. Table i shows that the uncloned culture transformed with ts-RSV is markedly temperature-sensitive for growth in soft agar. At the PT, the efficiency of colony f o r m a t i o n in agar is > l0 -4. In contrast, the uncloned culture transformed with wt-RSV shows the same colony forming ability at either the PT or N P T (approximately 10 2). U n t r a n s f o r m e d cerebellar cells do not grow in soft agar at either temperature, their plating efficiency being < 10 ~1. Several clones from the parent culture were established by limiting dilution and two of these, W C l 7 a and WC5, were characterized with respect to veratridinestimulated Na -uptake, expression of G F A P , growth in liquid media and morphology. Veratridine-stimulated Na+-uptake

The n e u r o n a l or glial nature of the transformed cells was defined operationally using the veratridine-stimulated Na~-uptake assay 0,3°. Previous work has shown a good correlation between the ability of a cell to generate an action potential a n d the TABLE 11 Assay ft," presence of voltage-dependent sodium channels

Na --uptake stimulated by either veratridine or veratridine plus scorpion venom. Chines

YS-RSV-derived WC 17a WC5 EN U-derived B103

Veratridine

Veratridine } scorpion venom

PT

NPT

PT

NPT

3.0 < 10 ~~ ' 0

9.4 × 10 ~ 0

2.0 × 10 8

3.0 y 10 s

9.0 ,< l0 o

7.2 >.~ l0 ~

5.0 .. l0 8

5.0 x 10 8

450 rate of Na ~-uptake in the presence of veratridine a°. Thus, to a first approximation this assay offers a means of screening cells as to their excitable nature. Table 11 is a summary of the data. Clone W C I 7 a grown at the PT exhibits an initial rate of veratridine-stimulated N a - - u p t a k e of 3.0 i*: 10 :~ tool Na~/mg total cellular protein/min. Moreover, if WC17a is kept at the N P T and then assayed there is at least a 3-fold increase in rate. It is important to point out that the rates observed for WCITa are characteristic of cells capable of generating an action potential. Thus, we classify. W C I 7 a as a neuronal clone. In contrast, clone WC5 does not show ~eratridinestimulated Na ~-uptake at either the PT or NPT. Although this finding is consistent with WC5 being glial the possibility exists that this clone may have been derived from fibroblasts or other non-neural cells associated with the cerebellum. Experiments described below show, however, that this is not the case since WC5 exhibits the astrocyte-specific protein, glial fibrillary acidic protein (GFAP). F r o m the present studies it is important to point out that ts-RSV can translbrm cells with neuronal properties, that is to say, which exhibit veratridine-stimulated N a uptake. Moreover, the N a ~ flux property of W C I 7 a is increased at the N P T compared to the PT. As a control to show that this is due to temperature abrogation of the src gene product and not simply to temperature alone, the N-ethyl-N-nitrosourea (ENU) derived neuronal line B1032~ was analyzed at the two temperatures, PT and NPT. This line was used since to date we have not succeeded in isolating neuronal cell lines with wt-RSV. Table II shows that B103 does not exhibit a significant increase in

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j

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Fig. 1. Tetrodotoxin (TTX) inhibition of veratridine-stimulated Na~-uptake of clone WC17a. Cells were incubated at the PT ( C ~ - - - - ~ , ) or N P T ( V A - - ~ I ) , and then assayed for Na+-uptake in the presence of T T X as described in Materials and Methods. Note that the concentration of T T X which gives half-maximal inhibition of Na+-uptake is 2 and 4 10 -8 M for cells incubated at the PT or NPT, respectively.

451

Fig. 2. Expression of G F A P in WC5 cells. Cells were kept for 4 days at either the PT or N PT then fixed and processed for staining as described in the Materials and Methods. Frames A and B show phasecontrast or fluorescence pictures respectively of WC5 kept at the NPT. Note that not all the cells stain uniformly for G F A P and that despite their monolayer appearance in phase-contrast, they appear multilayered by G F A P staining. Frames C and D show phase-contrast or fluorescence pictures respectively of WC5 kept at the PT. Note the absence of fluorescence in frame D. :~,~ 1650.

452 the rate of veratridine-stimulated Na~-uptake at the NPT. Although this finding suggests that the increase in rate observed for clone WC17a is not simply the result of keeping cells at the higher temperature, before a definite statement can be made, cells transformed with wt-RSV which exhibit veratridine-stimutated Na~-uptake must be identified and assayed at the PT and NPT. In addition to veratridine, we also examined the effects of scorpion venom plus veratridine on Na+-uptake. Scorpion venom acts synergistically with veratridine, and a combination of the two usually gives larger Na + fluxes than veratridine alone 8. Clone WC17a was tested at the PT and NPT. Table I1 shows that WC17a cultured at the PT exhibits substantial increases in Na+-uptake in the presence of scorpion venom plus veratridine when compared to that observed for veratridine alone. Lastly, since veratridine and scorpion venom are thought to act by holding voltage-dependent Na + gates open, the influx of Na + in the presence of veratridine, 50

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WCI7a

38 =C

2 DAYS

Fig. 3. G r o w t h rates of W C l 7 a a n d W C 5 . 3 . 10~ cells were seeded in a 60-mm tissue culture dish and at the a p p r o p r i a t e times cell n u m b e r determi ne d using a h e m o c y t o m e t e r . W C I T a : • . . . . • , P T: ~ - - - - , N PT. W C 5 : IZ . . . . - i , PT; " - - - U , N P T. After 3 days at the N P T W C 1 7 a cultures were shifted to the PT a n d cells c o u n t e d 24, 48 a n d 72 h l a t e r; (.::?~....... i ).

453 or veratridine plus scorpion venom should be inhibited by the highly specific Na + channel blocker, tetrodotoxin (TTX)2L Fig. 1 shows that for clone WC17a the fluxes induced by veratridine plus scorpion venom are half maximally inhibited by 2 and 4 × 10 s M T T X when cells are kept at either the PT or NPT, respectively. These values are in the range of those found to half-maximally inhibit action potential generation in nerve preparations from different species 13 and of other cultured neuronal cell lines 3°. Na ~uptake induced by veratridine alone or with veratridine plus scorpion toxin is a stable property of clone WC17a and maintained invariant over 6 months of continuous culture.

Glial fibrillary acidic protein (GFAP) WC17a and WC5 were screened for the presence of the astrocyte-specific protein, G F A P 3. Both lines were incubated at either the PT or N P T for 4 days before testing. W C I 7 a was negative regardless of the temperature it was kept at. In contrast WC5 was positive if kept at the N P T (Fig. 2B) but negative at the PT (Fig. 2D). This finding shows that WC5 is probably a cerebellar astrocyte. Moreover, preliminary experiments suggest that the expression of G F A P is linked to the expression of the src gene product since we have recently isolated phenotypic revertants of WC5 which behave as transformcd cells at both the PT or NPT. These cells do not exhibit G F A P at either temperature (Giotta and Cohn, in preparation).

Growth properties As the nervous system matures, the neuronal population loses the capacity to divide whereas it is generally believed that the glial population does not. Thus, an analysis of the growth properties of WC17a and WC5 may give further insight to their neuronal or glial natures. Therefore, we decided to determine what effcct incubation at the N P T has on the growth rate of clones WCI7a and WC5. Their growth rates at the PT and N P T are shown in Fig. 3. At the PT the clones grow with doubling times of 24 h for WC5 and 40 h for clone WC17a. Upon reaching confluence neither clone shows density-dependent inhibition of growth, a characteristic of the transformed state. At the NPT, clone WC5 has a doubling time of 20 h but at confluence shows good density-dependent inhibition of growth, a characteristic of the untransformed state. In striking contrast, clone W C I 7 a does not grow at the NPT. The cells remain perfectly viable at the N P T as shown by their exclusion of trypan blue. Most surprising is that the growth-inhibited state established at the N P T is irreversible for WCI7a. This finding suggests that abrogation of transformation may permit the differentiation of WCI 7a to an end neuronal cell incapable of growth and therefore unable to re-establish cell division. The glial clone WC5, however, grows readily if switched from the N P T to the PT (data not shown).

Morphological alterations Morphological criteria are often used to characterize the differentiation of cells in vitro. For instance, neuroblastoma cells which grow as round cells in suspension can be induced to flatten down onto the tissue culture substratum, extend neurites and

454

Fig. 4. Effect of temperature on the morphology of ts-RSV-transformed clones WCI7a and WC5~ Frames A and B show W C I 7 a at the PT and D and E at the NPT. Frame C shows WC5 at the PT and F at the NPT. Frames A, D and E were taken from cells seeded at the same density on the same day a nd then kept at the PT (A) or N PT (D and E) for 4 days at which time pictures were taken. Note the bipola r processes (arrows). Frame B shows the mo~ phology of WC17a at low density. Some cells also e×tend small processes (arrows). All pictures were taken with phase-contrast optics. 800

s i m u l t a n e o u s l y e x h i b i t i n c r e a s e d levels of' a c e t y l c h o l i n e s t e r a s e e S , " ; L S i m i l a r m o r p h o logical changes are also seen with pheochromocytoma

cells a f t e r t r e a t m e n t w i t h n e r v e

g r o w t h f a c t o r , a n d s i m u l t a n e o u s l y t h e s e cells a c q u i r e e l e c t r i c a l e x c i t a b i l i t y a n d a n increased

sensitivity to

acetylcholinelL

reasoned that if ts-RSV-transformed

With

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cells e x p r e s s a n o r m a l d e v e l o p m e n t a l p r o g r a m a t

455 the NPT, this should be seen as a change in morphology towards a more differentiated state. Indeed, Fig. 4 shows that the morphology of clones W C I 7 a and WC5 is markedly temperature-sensitive. At the PT, independent of cell density, WC17a displays cells with a round and refractile morphology (Fig. 4A and B). These cells are strongly auto-adherent but poorly adherent to the tissue culture dish. Occasionally, those cells which adhere to the tissue culture substratum extend processes. In comparison, glial WC5 at the PT exhibit a spindle-shaped morphology (Fig. 4C). At the NPT, both clones exhibit marked changes in morphology. Neuronal WC17a after 24 h at the N P T starts to flatten down onto the tissue culture substratum, small protuberances appear from the cell body, and by the end of 48 h nearly all cells have a flat morphology. If the cells are left for another 48 h at the N P T some cells in the culture exhibit no further alteration in morphology while others extend processes (Fig. 4D and E). Approximately 25-501!0 of the cells extend such processes. The processes are usually 3-4 times the length of the cell body and in many instances are bipolar as well as bifurcated and impinge on neighboring cells. Moreover, near the end of the 4-day period, many cultures which contain numerous cells with processes also show bright cell bodies (Fig. 4E) seen in phase-contrast. By comparison with clone WCI7a, glial WC5 does not undergo a protracted period of morphological change at the NPT. Indeed at the NPT, at the end of 24 h nearly all cells have flattened out and if kept at the N P T for longer times do not exhibit further alterations in morphology (Fig. 4F). The changes in morphology observed for either WC 17a or WC5 at the N P T are not caused simply by an increase in temperature since cells infected with wt-RSV do not display drastic changes in morphology over the temperature range studied nor do a variety of cell types such as neuroblastoma (N-18) or the N-ethyI-N-nitrosoureaderived BD-IX neuron cell line, B-103 (data not shown). Further, no temperaturedependent changes in morphology occur in untransformed cerebellar cultures. DISCUSSION We have shown that rat cerebellar ceils can be transformed in vitro by Rous sarcoma virus (RSV) which is temperature-sensitive for transformation, and that the two cell lines described, W C I 7 a and WC5, exhibit neuronal or glial properties respectively based on whether they display veratridine-stimulated tetrodotoxin-inhibitable Na~-uptake, a measure of voltage-dependent sodium gates. Hitherto RSV has not been shown to transform neurons. Thus our work suggests that the range of neural cells transformable by RSV may be wider than previously suspected. It is important to keep in mind, however, that other investigators have shown that RSV produces mostly g[iomas, specifically astrocytomas, and a spectrum of other neuroglial tumors6, '~5. In this light it is possible that W C I 7 a represents a precursor cell exhibiting both neuronal and glial properties. As such incubating W C I 7 a at the N PT may drive it partly along a neuronal differentiation pathway in the absence of in vivo signals which normally would result in a bona fide gila cell. In order to determine if this is correct it will be necessary to develop markers to ascertain the lineage of cells in the cerebellum.

456 It is important to note that while veratridine-stimulated Na+-uptake is an excellent correlate of excitability and is widely used to classify cell lines 24,3°,3I,:~~ before the excitable nature of a cell line can be unambiguously established it is necessary to do electrophysiologic testing. Nonetheless, the validity ot" using this assay for classifying cell lines is supported by studies in which veratridine-stimulated Na~-uptake has been shown to correlate better than 90~,; with electrophysiologic testing 30. A question of considerable interest is to what extent do the ts-RSV transformed neural cells express true differentiated properties at the NPT. Clearly, WCI7a and WC5 exhibit transformed and normal phenotypes at the PT and NPT respectively. However, the parameters (growth in agar, morphology, etc.) one uses to assess the normalcy of a phenotype may or may not be coupled to the expression of differentiated properties specific to that cell type. Nonetheless, the increase in veratridinestimulates Na + flux and changes in morphology exhibited by WCI7a at the NPT a priori might be expected of a neuron induced to differentiate. Although the extent of the changes are not great it is difficult to know how large a change should be expected since the state of neuronal differentiation prior to transformat!on is not easily ascertained. In this regard, it is noteworthy that erythroblasts transformed with a temperature-sensitive mutant o f avian erythroblastosis virus exhibit significant increases in hemoglobin levels at the NPT but which are still only 2 8'~i of that expressed in mature erythrocytes Iv. Perhaps the expression of G F A P in WC5 at the NPT is more exemplary of an expected change in a cell induced to differentiate. Detailed data concerning the expression of G F A P in WC5 wilt be presented elsewhere. Lastly, it is important to mention the advantages of using ts-RSV over other presently available techniques to establish neural cell lines. Most neural cell lines now in use have been derived by adapting spontaneous tumors to tissue culture, the classic example being the C1300 mouse neuroblastoma e,27 or transforming neural cells with carcinogenic agents 12.2t~. Adapting spontaneous tumors or carcinogen-induced tumors to tissue culture suffers from two major disadvantages. First, the specific anatomical site of the origin of the cells derived is poorly known. Second. inherent in the transformed nature of the lines may be alterations in normal neural cell physiology. In contrast, the procedure of establishing neural cell lines with ts-RSV does not suffer from either drawback. Cell lines can be established from discrete regions of the central nervous system at the PT and when shifted to the NPT these cells express normal cellular properties. ACKNOWLEDGEMENTS We wish to thank several investigators who helped make this work possible: Amico Bignami for the G F A P antisera, David Schubert for the BD-IX ENU derived cell line, Bart Sefton for advice concerning viral techniques and Bill Stallcup for advice with the Na + flux assay. Special thanks are due to Peter K. Vogt for the gifts of virus, for carrying out the virus rescue experiments and for critical comments concerning the manuscript.

457 This work was supported bya grant from the NCI to GJG (no. IR23 CA2439301 CBY) and Grant 1 R01 CA19754, awarded by the National Cancer Institute, DHEW, and Grant 1 T32 CA09254, awarded by the National Cancer Institute, DHEW to M.C.

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