Exp Brain Res (1992) 89:408414

Experimental BrainResearch 9

Springer-Verlag1992

Characterisation of a human glioblastoma cell line (LI) expressing hypothalamic and pituitary hormones* A. Savarese 2,3, M. Annicchiarico-Petruzzelli t, G. Citro 1, G. ZupF, L.G. SpagnoiP, A. Colantoni 1, P. Vernole ~, A. Stephanou 4, R.A. Knight 5, P. GuerrierP, and G. Melino ~ Universita Rome Tor Vergata, Dip. Medicina Sperimentale, 1-00173 Rome, Italy 2 Experimental Chemotherapy Lab, Istituto Regina Elena, 1-00173 Rome, Italy 3 I.S.T., Genova, Italy 4 Neuroendocrinology, Charing Cross Hospital, London W8, UK s Brompton Hospital, London SW3, UK Received June 20, 1991 / Accepted January 20, 1992

Summary. The h u m a n glioblastoma cell line LI showed morphological features typical of its neuroectodermal origin. Cells were positive by immunofluorescence to G F A P , M H C class II, and L1 determinants. Cytogenetic analysis showed the presence o f a m o d a l c h r o m o s o m e n u m b e r of 63, ranging f r o m 58 to 69 c h r o m o s o m e s ( D N A index was 1.6). N o r t h e r n blot analysis demonstrated the presence of m R N A transcripts specific for transglutaminase C (type II or "tissue"), growthh o r m o n e releasing-hormone ( G H R H ) , insulin-like growth factor II ( I G F - I I ) , and p r o o p i o m e l a n o c o r t i n (POMC). The G H R H m R N A was present in two different sizes, one similar to the normal hypothalamic species of 0.75 kb, whilst the second species was a large transcript of approximately 10 kb size. T r e a t m e n t with 5 g M retinoic acid or 5 m M a-difluoromethylornithine for 5 days sharply reduced the growth rate and also induced modulation of the ultrastructure and antigenic profile. This cell line m a y be useful to study glial differentiation and the relationship of G H R H , I G F - I I and P O M C expression with differentiation in neuroectodermal tumours.

and/or a sex chromosome, structural abnormalities o f 9p, and double minutes (Bigner et al. 1988; Ray et al. 1989). These neoplasms exhibit little morphological evidence o f differentiation. To help gain insight into the neurobiology of malignant gliomas any additions to the approximately 20 cell lines currently available would be beneficial. Here we characterise a h u m a n glioblastoma cell line, first established and partially described by Zupi et al. (1988), derived f r o m a surgically removed central brain tumour. We report several characteristics o f this cell line including its morphology, ultrastructure, neuropeptide expression, cytogenetic abnormalities and the immunological phenotype. This cell line showed some changes in m o r p h o l o g y and antigenic profile after treatment with retinoic acid, and expressed transglutaminase C ( T G ; type II or "tissue"), growth-hormone releasingh o r m o n e ( G H R H ) , insulin-like growth factor II ( I G H - I I ) , and proopiomelanocortin (POMC).

Key words: G l i o b l a s t o m a - Retinoic acid - Proopio-

Cell cultures

melanocortin - G r o w t h hormone-releasing h o r m o n e Human

Introduction Malignant gliomas, including anaplastic astrocytomas and glioblastoma multiforme, often demonstrate nonr a n d o m karyotypic abnormalities. In adult malignant gliomas, the m o s t c o m m o n abnormalities include extra copies of c h r o m o s o m e 7, loss of c h r o m o s o m e s 10, 22 * This work was partially carried out at the Istituto Dermatologico Immacolata - IRCCS, Via Monti de Creta 104, 1-00167 Roma, Italy Offprint requests to: G. Melino

Methods

The LI cell line was established in 1988 and was partially described by Zupi et al. (1988); it was derived from a 51 year old male patient who presented with a large right fronto-temporal mass classified histologically as glioblastoma multiforme. All experiments were performed with cells at the 75th-106tu passage. Cells were grown in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated foetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin and streptomycin, 2.0 g- L- t bicarbonate and 10 mM Hepes in a humidified atmosphere with 5 % (v/v) CO2 at 37~ C. All materials for cell culture were obtained from Flow Ltd. (UK). Cell treatment for the differentiation studies was performed by plating 4x 104 cells" cm -2 in fresh growth medium, replaced daily, containing 5 gM retinoic acid (RA; Sigma Ltd. USA; stock solution 5 mM dissolved in 70 % ethanol); 0.07 % ethanol was also added to control cultures. Alternatively cell differentiation was induced with 5 mM ct-difluoromethylornithine (DFMO; gift of Merrell Dow Ltd. France) for seven days. Cell population-doubling time was assessed by cell count of replicate cultures (8 to 10 9 104 cells in 5 ml medium were seeded in 60 x 15 mm culture dishes). Cell number and viabil-

409 ity were evaluated in a Neubaucr haemocytometer chamber or in a Coulter Counter (ZBI, Italy). Tumorigenicity was evaluated as described by Zupi et al. (1988), but using CD 1 nude mice. Indirect immunofluorescence analysis was carried out using 18 mouse monoclonal and 2 rabbit polyclonal antibodies directed against neural associated antigens (see for description Carachi et al. 1987; or Melino et al. 1988)9Mechanically removed cells were incubated with antibody for 30 min at room temperature. Antibody binding was visualised by incubation with ftuorescein isothiocyanateconjugated goat anti-mouse F(ab)z immunoglobulin. Fluorescence was monitored on a Leitz Dialux 22 microscope or on a FACS-star (Becton-Dickinson, Rutherford, N J, USA) flow cytometer using Consort 30 software9 DNA content was evaluated in fixed cells (1:1 v/v solution of phosphate buffered saline, PBS, and acetone: methanol 1 : 4 v/v at - 20~ C), and the stored at 4 ~ C. Before analysis samples were incubated for 30 min at room temperature with 0.5ml RNAse (150KU'm1-1) and 0.5ml propidium iodide (1 mg" ml- 1). The red fluorescence was monitored and analysed on a FACS-star flow cytometer. The catalase activity was determined by oxygen production, measured with a Clark electrode CYSImodel 53, Italy) at 25~ C in 0.15 phosphate buffer (pH 6.0) containing 1 mM EDTA. HzO z was added at a concentration of 0.1 to 1 mM.

Transmission electron microscopy Untreated and treated (RA or DFMO for 7 days) cells in 25 cm 2 flasks were fixed wit 2 % (w/v) glutaraldehyde in 0.1 M cacodylate buffer with 4.5% (w/v) sucrose. After 2 h of fixation the flasks were washed with the same buffer and post-fixed in 1% (w/v) osmium tetraoxide for 2 h. Specimens were incubated in 1% uranyl acetate for one hour, then dehydrated through ascending alcohol concentrations. Epon 812 embedded material was cut and oriented for thin sectioning. Specimens were examined and photographed on a Philips 301 transmission electron microscope.

Cytogenetic and Northern blot analyses Non confluent cell cultures were incubated for two hours with 0.5 gg" m1-1 colchicine and then detached from the culture flasks by trypsin. Centrifuged cells were resuspended in hypotonic solution (0.075 M KC1) for 15min at 37~ C. Cells, fixed in methanol:acetic acid (3:1) for 30 rain at 4 ~ C, were washed three times and then dropped onto the slides9 These were air dried, treated mildly with trypsin in order to obtain G bands (Seabright 1971) and 50 metaphases were observed, out of which 10 karyotypes were made. Total RNA was extracted from cells by the guanidinium thiocyanate method (Chomczynski and Sacchi 1987), treated with glyoxal and electrophoresed through a 1% agarose gel in 12 mM Tris, 6 mM sodium acetate and 0.3 mM EDTA pH 7.0. Hybridisation was performed on blotted Zeta-probe membrane (Bio-Rad, UK) using a GHRH eDNA probe (git from K.A. Mayo, Salk Institute, San Diego, USA), or a genomic POMC probe (kindly provided by Dr. M. Davies, Institute of Neurology, London). IGF-II probe was from ATCC (Bethesda, USA) and TG probe was a kind gift of Dr. P. Davies (Houston, Texas, USA) and Dr. M. Piacentini (Tor Vergata University, Rome, Italy).

Results

Cell line 9rowth and morphology Cell line g r o w t h and m o r p h o l o g y by c o n t r a s t p h a s e d m i c r o s c o p y have been described previously (Zupi et al. 1988). In order to evaluate the ability o f the cells to differentiate u p o n exposure to R A or D F M O we analysed the g r o w t h and the transmission electron micro-

2e+6

2000

B t~ E

._~

~1e+6

~1000

/

j/-

\ le+5

0

9 5i 9 1i0 . days

....

1'5

0

0

2'0 days

3'0

Fig. 1A, B. Growth rate of LI cells. A Shows the growth pattern in vitro in the absence (--) or presence (....) of 5 gM RA. The error bar shows the s.e.m, of two flasks, each counted five distinct times. B Shows the growth in nude mice of untreated cells (--) and cells pretreated for 5 days with 5 gM RA (....). Tumour growth was evaluated daily using a caliper to assess the diameter; on day 33 the mice were sacrificed in order to analyse tumour weight and histology. The error bar shows the s.e.m, of five mice in each group scopy o f u n t r e a t e d and treated cells. D F M O p r o d u c e d a complete g r w o t h arrest (data n o t shown); this effect has been described also in other neurocrest-derived t u m o u r s (Melino et al. 1988). Figure l a shows the in vitro g r o w t h pattern o f L I cells. R A (5 g M ) increased the d o u b l i n g time f r o m 24 to 41 h a n d decreased the flask saturation density f r o m 2 - 106 to 1 " 106 cells 9 plate -1. Similar results were also o b t a i n e d in vivo in nude mice, as shown in Fig. lb. All mice developed t u m o u r s , a l t h o u g h those injected with cells pretreated for 5 days with R A showed 4 days delay in t u m o u r a p p e a r a n c e a n d a slightly reduced t u m o u r g r o w t h rate. H o w e v e r the g r o w t h in vivo o f untreated and R A - p r e t r e a t e d cells resulted in a very similar h o m o g e n e o u s histological a n d cytological pattern (data n o t shown). The m o r p h o l o g y o f the cell line derived f r o m the t u m o u r is s h o w n in Fig. 2, left panels. U n t r e a t e d cells s h o w e d a low degree o f differentiation, with small scattered g r o w t h cones. Indeed, by transmission electron m i c r o s c o p y the cell culture was f o r m e d o f a u n i f o r m p o p u l a t i o n , each cell being characterised by a large oval nucleus with one to f o u r huge nucleoli. The nuclear: cytoplasmic ratio was greater t h a n 1 : 1. The c y t o p l a s m was irregularly elongated a n d s h o w e d n u m e r o u s m i t o c h o n dria and a smaller a m o u n t o f r o u g h endoplasmic retic u l u m ( R E R ) . The c y t o p l a s m was dense with polyrib o s o m e s a n d showed a b u n d a n t catalase crystals. Some cells showed a variable n u m b e r o f a u t o p h a g i c vacuoles and tertiary lysosomes. The cell surface was at times decorated by short microvilli. P u n c t i f o r m cell to cell c o n t a c t could also be seen. In order to evaluate whether the cells could differentiate in vitro, as described for other n e u r o e c t o d e r m a l cells (Melino et al. 1988), we grew the cells for seven days in the presence o f 5 ~tM retinoic acid or 5 m M D F M O . There was a clear m o r p h o l o g i c a l response to retinoic acid, as s h o w n in Fig. 2, right panels. The nuclear: cytoplasmic ratio b e c a m e less t h a n 1 : 1. The nuclei showed smaller nucleoli and the c y t o p l a s m was larger t h a n in untreated cells and irregularly fusiform. The c y t o p l a s m often tended to f o r m elongated processes in c o n t a c t with

410

Fig. 2A-C. Left panels. Electron micrograph of untreated LI cells. A,c Low field magnification. Most of the area is occupied by cells with a low degree of differentiation with abundant microvilli (bar 3 gm). B,e Cell to cell junction, showing a desmosome. Ferritin granules are present in the cytoplasm (bar 0.5 gm). C,c Catalase crystals at high magnification (bar 0.2 gm). Right panels. After 5

days RA treatment of 5 ~tM concentration, LI cells show a more differentiated phenotype characterised by an incrased number of cytoplasmic processes (panel A,ra, bar 4 gm). B,ra Elongated cytoplasmic processes (bar 2 gin). C,ra Neurosecretory granules (bar 1 gm)

411 Table 1. Modulation of the antibody staining pattern of LI cells

Catalase activity in LI cells 9

20

Antibody

r

Target

E o D.

.c~176 //']~N

RA

Y4 1

O

YAN

"6 E r

[]

0 100

500

1000

H 2 0 2 (microMotar)

Fig. 3. Catalase activity of LI lysed cells is increased by RA and DFMO. In vitro treatments with 5 gM RA or 5 mM DFMO were performed for 5 days. The experiment was performed at three different concentrations of hydrogen peroxide, as reported, and the error bar indicates the s.e.m, of triplicate measurements. The figure shows one of the two experiments performed

other cells. It was lighter t h a n in controls, containing fewer p o l y r i b o s o m e s a n d a larger a m o u n t o f R E R . M i t o c h o n d r i a were also well represented. Sometimes neurosecretory granules associated with the Golgi apparatus could be seen. A l t h o u g h the catalase crystals were reduced in quantity, the catalase activity increased u p o n R A treatment, as s h o w n in Fig. 3. This suggests the release o f active e n z y m e f r o m its deposits. T r e a t m e n t with D F M O induced structural changes very similar to treatment with R A , but induced higher n u m b e r s o f p r i m a r y lysosomes.

ct-GFAP (*) A2B5 Da2 Leu 10 UJ127.11 UJ181.4 PI153.3 M1N1 UJ223.8 UJ13A 5.1.H.11 Leu 3 Leu 2a a-SlO0 (*) UJ308 UJ167.11 a Yhy 1 p170 aEGFr 4.36

GFAP au HLA-DR HLA-DQ L1 L1 gp20 au au NCAM NCAM CD4 CD8 SIO0 au au Thy-I gpl70 EGF-r au

Positivity

Modulation by

(a)

(13)

RA (Y)

DFMO (7)

3 (2) 3 (3) 3 (2) 1 (2) 3 (4) 2 (4) 1 (4) 1 (3) 1 (4) 0 (3) 0 (3) 1 (2) 0 (2) 0 (2) 0 (3) 0 (3) 0 (3) 0 (2) 0 (2) 0 (2)

90 63 44 14 30 30 21 9 14 -

4 (2) 4 (3) 4 (2) 1 (2) 3 (4) 2 (4) 1 (4) 1 (3) 1 (4) 2 (3) 2 (3) -

3 (2) 1 (3) 3 (2) 1 (2) 3 (4) 2 (4) 1 (4) 1 (3) 1 (4) 0 (3) 0 (3) -

The results shown in the table are expressed on an arbitrary scale from 0 to 5: the number of experiments performed is shown in brackets. Controls were performed by omitting the primary antibody. All experiments were performed using non-trypsinised living cells; only for GFAP and S-100 the cells were pre-fixed in acetone: methanol (1:4) mixture before staining, au=antigen unknown; (e) = intensity of fluorescence by UV-light microscopy; (,8)= % of positive cells by FACS analysis; (7) = 5 gM RA or 5 mM DFMO were supplemented daily to the cultures for 5 days (see methods section for details) ; (*) = both antibodies showed a negative staining at establishment (Zupi et al. 1988)

Immunological phenotype In order to evaluate the expression o f typical neural-associated antigens indirect i m m u n o f l u o r e s c e n c e was perf o r m e d using various m o n o c l o n a l antibodies. Table 1 shows the positivity f o u n d on the cell line using a panel o f 20 different antibodies. The cell line s h o w e d a positive reaction with antibodies G F A P , UJ127.11, UJ181.4, UJ223.8, PI153.3, M 1 N 1 , A2B5, D A 2 , L e u l 0 a n d Leu3. In the original p a p e r (Zupi et al. 1988) Glial Fibrillary Acidic Protein and S-100 protein were b o t h negative on LI cells, as were the fresh biopsies f r o m which the cell line was derived. It is o f interest t h a t the L I cells were G F A P negative at passage 15 and, following the in vitro a d a p t a tion, they b e c a m e positive at passage 106. W e also investigated whether retinoic acid or a-difluoromethylornithine could m o d u l a t e the expression o f these antigens, as already observed in vitro for n e u r o b l a s t o m a cells (Melino et al. 1991). To this end we included also antibodies U J 13A a n d 5.1.H. 11, specific for the neural cell adhesion molecule N C A M . As indicated in Table 1, R A e n h a n c e d the expression o f the antigens recognised by A2B5 and D A 2 . It is n o t e w o r t h y that N C A M was negative on untreated cells, b u t b e c a m e present after R A treatment. W h e t h e r this p h e n o m e n o n was related to the u n m a s k i n g o f cryptic determinants, or to de novo synthesis regulated by R A - i n d u c e d gene expression t h r o u g h its R A R receptors remains to be investigated. Nevertheless the binding

o f the typical a n t i - n e u r o e c t o d e r m a l antibodies specific for N C A M ( U J 1 3 A and 5.1.H.11) and for L1 (UJ127.11 and U J 181.4) cell adhesion molecules indicates that these cells have a n e u r o e c t o d e r m a l origin.

Cytogenetic analysis and DNA index A b n o r m a l k a r y o t y p e s were f o u n d in the cultured cells (Fig. 4a). Cytogenetic analysis o f the cell line s h o w e d the presence in 50 metaphases o f a m o d a l c h r o m o s o m e n u m ber o f 63, ranging f r o m 58 to 69 c h r o m o s o m e s , w i t h o u t dms. The D N A index by flow c y t o m e t r y was 1.6, in the aneuploid range (Fig. 4b). The 10 metaphases that were k a r y o t y p e d did n o t all show the same pattern. A c c o r d i n g to the m e a n n u m b e r o f c h r o m o s o m e s per m e t a p h a s e (61.2 per sample), each c h r o m o s o m e should be present in a m e a n n u m b e r o f 2.66. As s h o w n in Table 2 some c h r o m o s o m e s were often overrepresented, particularly c h r o m o s o m e 3 but also 1, 2, 5, 7, 11 and 17. Others tended to be underrepresented, such as 8, 13, 14, 15, 22 and Y. A m o n g the various m a r k e r s present, two, s h o w n as M a r 1 a n d M a r 2 in Fig. 4a, were respectively f o u n d in 70% and 80% o f the k a r y o t y p e d metaphases. It was not possible to find the origin o f these markers. Other rearranged c h r o m o s o m e s were present: 1q - (50 % o f the metaphases), 2 q - (60%) and 3q + (20%).

412 B

10o

I/i

E e--

o

0

iiii

0

1O0

200

Channel number

Fig. 4. A A karyotype of LI cells. B DNA index of LI cells. The first peak shows the reference diploid Go/G1 peak of unstimulated peripheral blood leukocytes; in comparison the G0/G~ peak of

aneuploid LI cells yields higher fluorescence, second peak. The DNA index, expressed as ratio of the two peaks is 1.6. The third peak shows the LI cells in G2/M phase

Table 2. Cytogenetic findings in LI cells. Frequency and mean of normal and marker chromosomes in 10 methaphases

Expression of GHRH, IGF-II, POMC and TG mRNA

Chromosome number

Cell karyotype number 1 2 3 4 5 6 7

8

9 10

1

3

3

4

4

3

4

4

4

4

4

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y M

2 6 2 4 2 2 1 2 2 3 3 2 1 2 2 3 3 3 4 2 2 3 0 3

2 3 3 3 3 4 2 2 2 3 3 2 1 2 3 3 3 3 2 3 2 3 0 4

4 4 3 3 3 3 2 3 2 2 2 2 1 3 2 3 3 2 2 2 1 3 0 5

3 4 2 2 3 3 3 2 2 3 2 0 2 1 3 3 2 2 3 1 1 2 1 1

5 6 2 3 2 3 2 3 2 2 2 1 2 1 3 3 2 2 2 2 2 2 1 3

2 3 2 4 3 3 2 2 2 2 2 1 2 2 2 2 2 2 2 2 2 3 0 3

3 5 2 4 2 2 2 2 3 4 2 2 2 2 2 4 3 1 2 2 2 2 0 1

3 4 4 4 3 3 1 2 2 3 3 1 1 1 2 3 3 2 2 2 2 4 1 4

3 4 3 4 2 3 2 2 2 3 3 2 1 1 2 3 4 2 3 2 2 2 1 2

3 4 3 4 4 3 2 2 2 2 3 1 1 1 4 4 4 2 1 2 2 2 1 3

No

Mean

3.7 3.0 4.3 2.7 3.5 2.7 2.9 1.9 2.2 2.1 2.7 2.5 1.4 1.4 1.6 2.5 3.1 2.9 2.1 2.3 2.0 1.8 2.6 0.5 2.9

62 64 64 55 60 56 61 64 62 64 61.2

M = markers; No = chromosomes number Over 50 metaphases were counted and the 10 karyotyped are reported in the present table

Preliminary reports from our laboratory indicated that at least s o m e n e u r o e c t o d e r m a l t u m o u r s , especially n e u r o b l a s t o m a s , express m R N A s c o d i n g for n e u r o p e p tides such as P O M C a n d C R F ( S t e p h a n o u et al. 1990, 1991). I n L I cells, N o r t h e r n b l o t analysis d e m o n s t r a t e d the p r e s e n c e o f P O M C , I G F - I I a n d G H R H m R N A t r a n s c r i p t s (Fig. 5). O n l y a 9.5 k b P O M C t r a n s c r i p t was d e t e c t e d , similar in size to t h a t d e s c r i b e d in n e u r o b l a s t o m a (Fig. 5, p a n e l A). I G F - I I m R N A w a s expressed as a 4.9 k b b a n d (Fig. 5, p a n e l C). T w o G H R H m R N A t r a n s c r i p t s were f o u n d , a m o r e p r e d o m i n a n t 0.75 k b species similar to the h y p o t h a l a m i c G H R H m R N A a n d a larger, less a b u n d a n t , 10 k b m R N A species (Fig. 5, P a n e l B). T h e f u n c t i o n a l significance o f b o t h n o r m a l a n d a b normal GHRH, IGF-II and POMC messages adds new interest to the L I cell line. L I cells also express t r a n s g l u t a m i n a s e - C ( t y p e II o r '"tissue") m R N A (Fig. 5, p a n e l D). I t is n o t e w o r t h y t h a t T G s h o w e d t h e p r e s e n c e o f extra-species o f m R N A , as well as the 3.7 k b message. T h e s e t r a n s c r i p t s are i d e n t i c a l in size to t h o s e f o u n d in three h u m a n n e u r o b l a s t o m a cell lines ( M e l i n o G . a n d P i a c e n t i n i M., u n p u b l i s h e d o b s e r v a t i o n ) a n d in a h u m a n e r y t h r o l e u k a e m i a cell line ( S u e d h o f f et al. 1990). T h e f u n c t i o n o f these e x t r a - b a n d s f o r T G is u n k n o w n , even t h o u g h K n i g h t et al. (1990) r e p o r t e d a b e r r a n t p r o t e i c f o r m s o f T G in t u m o u r cell lines, p o s s i b l y in r e l a t i o n to cell adhesiveness a n d m e t a s tasising p o t e n t i a l . H o w e v e r , T G h a s b e e n d e m o n s t r a t e d to be i n v o l v e d in the p r o g r a m m e d cell d e a t h , a p o p t o s i s , i n d u c e d b y r e t i n o i c a c i d in differentiating t u m o u r cell lines e x p r e s s i n g i d e n t i c a l T G m R N A b a n d s f o u n d in L I cells ( M e l i n o et al. 1988; P i a c e n t i n i et al. 1991).

413

Fig. 5A-D. Northern blot analysis with the POMC probe A showing the 9.5 kb mRNA transcript. B Shows the expression of a 0.75 kb GHRH mRNA message by LI cells (lane 2) as compared to the 0.75 kb message expressed by normal hypothalamic tissue (lane 1) (the mark around 2 kb is an artifact present on the original film, indeed the normal hypothalamic tissue shows only one 0.75 kb

mRNA transcript in our hands, as in the literature); LI cells expressed also a less abundant 10 kb large transcript (not shown). C Shows the expression of the 4.9 kb IGF-II mRNA message. D Shows a Northern analysis on LI cells expressing the 3.7 kb transglutaminase-C message, as well as other abnormal mRNA transcripts (described in the text)

Discussion

and, to a lesser extent, also to D F M O . This was demonstrated by reduction of cell growth, modulation of surface antigens and ultrastructural changes. The morphology shows a low degree of glial differentiation with the presence of punctiform junctions, together with the expression of specific cell adhesion molecules. The differentiation agents caused a reduction in the number of nucleoli, with enlargment of the cytoplasm with larger amount of RER. It is noteworthy that RA induced Golgi-associated neurosecretory granules. Both RA and D F M O were able to reduce the cytoplasmic deposits of catalase crystals, enhancing the activity of the enzyme itself. The presence o f G H R H , I G F - I I , and P O M C transcripts suggests that the cells may also be able to secrete neuropeptides. It has been known for some time that both human and murine neuroectodermal tumour cells elaborate neuropeptides such as IGF, vasoactive intestinal polypeptide (VIP), POMC, corticotropin releasing factor (CRF) and prolactin (PRL). Clones of human neuroblastomas contain I G F - I I , VIP, P O M C or C R F m R N A , release immunoreactive neuropeptides, and may also express their receptors (El Badry et al. 1989; Muller et al. 1989; Stephanou et al. 1990; Washek et al. 1989). Because of the expression of both ligand and receptor, it has been suggested that, as for VIP and P O M C products, I G F - I I may function as an autocrine growth factor in neuroblastoma (El Badry et al. 1989, 1991). Again, by analogy with P O M C which is on the same chromosomal band as the N M Y C oncogene (2p23) (Schwab et al. 1984), the VIP gene is located on the same chromosomal band (6q22) as the oncogene CMYB that is expressed and modulated in differentiating neuroblastoma cells

A preliminary description of the glioblastoma cell line LI has been reported previously (Zupi et al. 1988). In the LI cell line we found a near triploid mean number of chromosomes per cell, as has been described in over 50% of the glioma cell lines. However, these data may fail to reflect the in vivo situation where up to 80 % o f the fresh tumours show a near diploid pattern. This may probably suggest an in vitro adaptation, selection or simply a high instability of the tumour cells. Unfortunately we could not examine the fresh tumour nor its early in vitro passages. Previous studies on primary tumours or cell lines derived from them have shown an increased number of some chromosomes, particularly number 7, with a decrease o f others such as 10 and 22 (Bigner et al. 1988; Ray et al. 1989). The LI cell line has 2 to 4 copies of chromosome 7 per cell. We also observed an increased number of chromosome 3 (3 to 6 copies). Both the lq deletion and the longer chromosome 3, present in LI, have also reported in other astrocytomas (Mitelman 1987). The cell line LI, like other glioblastoma cell lines, exhibits, heterogeneous in vitro responses to different chemotherapeutic agents, probably because o f concomitant occurence of proliferating and quiescent cells (Zupi et al. 1988). It is therefore important to understand the regulation o f growth and differentiation in these cells. To this end the present discussion will emphasise two aspects of the described cell line: i) the regulation of differentiation by retinoic acid and, ii) the expression of neuropeptides possibly related to growth. LI cells were able to differentiate in response to RA

414 (Gozes et al. 1987). Very little d a t a are available o n the expression o f other neuropeptides in other nervous tum o u r s . As a m a t t e r o f fact, recently various h u m a n C N S t u m o u r s , including gliomas, have been s h o w n to express b o t h I G F - I , - I I a n d type 1 I G F receptor (Glick et al. 1989; K u i h a r a et al. 1989) a n d different I G F - b i n d i n g proteins ( I G F B P ) ( U n t e r m a n et al. 1991). To o u r k n o w ledge this is the firs report describing the expression o f P O M C a n d G H R H m R N A s in a glioblastoma cell line. It is o f interest that the P O M C gene is expressed as a 9.5 kb transcript, larger t h a n the 1.2 kb pituitary m R N A but similar to that already described in some neuroblastomas ( S t e p h a n o u et al. 1990, 1991). Similarly, the G H R H transcript shows a n d a b n o r m a l l y high molecular weight o f 10 kb, as well as the n o r m a l h y p o t h a l a m i c 0.75 kb m R N A message. A l t h o u g h the functional significance o f neuropeptide expression b y glioblastoma cells is u n k n o w n , they m a y play a role in the a u t o c r i n e / p a r a crine regulation o f cell growth, as with n e u r o b l a s t o m a . A m o r e systematic investigation o f o n c o g e n e - n e u r o p e p tide relationship a n d their relation to cell g r o w t h and differentiation in glioblastoma w o u l d seem worthwhile.

Acknowledgements. This work was supported by grants from MURST and CNR (Progetto Finalizzato "Applicazioni ctiniche della ricerca oncologica"). The authors wish to thank Professor Stafford L. Lightman, Dr. B. Tedeschi and Dr. C. Leonelli for helpful discussions, and Mr G. Bonelli for technical help. DFMO was kindly supplied by Merrel Dow International Research Centre, Strasbourg, France.

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