Acta Neuropathol (1991) 81:641 - 648

Acta Heuropathologm (~)Springer-Verlag 1991

Spontaneous multidrug transport in human glioma cells is regulated by transforming growth factors type 13" H. J. Schluesener 1 and R. Meyermann 2

Abteilung ffir Neurologie, Neuroimmunologie, Universit~it W/Jrzburg, Josef-Schneider-StraBe 11, W-8700 Wiirzburg, Federal Republic of Germany 2 Institut ftir Hirnforschung, Universit~it Tiibingen, Federal Republic of Germany Received August 13, 1990/RevisedDecember 10, 1990/Revised,accepted December 28, 1990 Summary. The multidrug transporting cell membrane

molecule P-glycoprotein can be spontaneously expressed in human glioma cells. Transcripts of mdr genes were detected in glial tumor cells by polymerase chain reaction and Northern blotting, expression of P-glycoprotein was analyzed by immunocytochemistry and functional activity by cytofluorometry of fluorescent probe transport. In vitro treatment of glioma cells with vincristine induced coordinate over-expression of both mdrl and mdr3 genes associated with very high Pglycoprotein-mediated multidrug transport, resistant to the inhibitory activity of chemosensitizers like verapamil. The physiological modulators of multidrug transport are as yet unknown. We therefore initiated a screening program to analyze the effects of cytokines on multidrug transport. We observed, that transforming growth factors (TGF)-~I, -~2, and -~1.2 - but not the related bone morphogenetic protein (BMP) 2 - inhibited multidrug transport. Interestingly, BMP 2 antagonized the TGF-~ induced inhibition of multidrug transport. Key words: Multidrug resistance - Glial tumors Transforming growth factor type ~ - Bone morphogenetic protein

17, 44]. Chemotherapy can select for multidrug-resistant tumor cells over-expressing mdr genes and it has been suggested that multidrug resistance poses a major problem in chemotherapy [2, 3, 10, 20, 23, 36]. A heterogenous group of agents, the 'chemosensitizers' interfere with function of the P-glycoprotein [10, 18, 19, 21, 41], generally by competitive inhibition of multidrug transport. Physiological regulators of mdr-gene expression or P-glycoprotein function are unknown. There is only limited information available on multidrug transport in human glioma cells. We, therefore, analyzed the expression of mdr genes in human glioma cell lines by diagnostic polymerase chain reaction (PCR) and Northern blotting. Expression of P-glycoprotein was demonstrated by immunocytochemistry and multidrug transport was corroborated by cytofluorometry of fluorescent-dye transport [34, 43]. In addition, we analyzed whether multidrug transport can be modulated by cytokines, notably the transforming growth factors (TGF) type ~, that have been implicated repeatedly in the pathogenesis of human CNS tumors [9, 12, 50].

Material and methods

Culture of human glioma cells The multidrug transporting cell membrane molecule P-glycoprotein mediates cross-resistance (multidrug resistance, mdr) to a variety of chemotherapeutic drugs like vinca alkaloids, anthracyclines, cyclosporines, or epipodophyllotoxins [14, 22, 24]. A small gene family encodes the P-glycoproteins [28, 35, 47], which are homologous to bacterial transport proteins [5]. mdr genes are constitutively and physiologically expressed in a small number of specific sites [8, 11, * Supported by a grant from the Bundesministerium far Forschung und Technologie Offprint requests to: H. J. Schluesener (address see above)

Cell lines were established from stereotactic biopsies of glial tumors. The histology of brain tumors was studied by routine staining (hematoxylin/eosin, Elastica van Giesen, periodic acidicshift reaction) and immunocytochemistry for intermediate filament proteins [glial fibrillary acidic protein (GFAP), vimentin, neurofilament] and P-glycoprotein.The protocol for immunocytochemistry is given below. Brain tissue was minced and cultured in Dulbecco's modified Eagle's medium (DMEM) 10 % fetal calf serum (FCM). After 4-6 weeks, tumor cells were subcultured in DMEM/FCS or in DMEM/FCS supplemented with vincristine sulfate (Sigma, Deisenhofen, FRG). Multidrug resistance was tested by growing cells in medium containing vincristine, vinblastine, actinomycinD, puromycin, mitomycinC, or ethidiumbromide (all reagents were from Sigma). For analysisof the effects of cytokines, glioma cellswere seeded at low density into six-welltissue culture plates (1 • 10 4 cells/wellin

642 1 ml medium). After 24 h serially diluted cytokines were added [maximal concentrations of TGF-~: 300 ng/ml; maximal concentration of bone morphogenetic protein (BMP) 2:10 ag/ml]. Porcine and human TGF-~I, -~2, and -~.2 were obtained from R&D Research (Minneapolis, Minn.). Human recombinant BMP 2 was a gift from Dr. John Wozney (Genetics Institute, Boston, Mass.).

(4091-4250) of the human mdrl gene. The reaction was performed with an Intelligent Heating Block (Biometra, Goettingen, FRG) using a thermocouple to monitor the reaction temperature within the Eppendorf tube. The PCR profile used was: 10 s, 94 ~ 20 s 55 ~ 30 s 72~ Expression of the mdr gene was demonstrated by agarose gel electrophoresis of the PCR amplified reaction product obtained after 30 cycles of PCR.

Effects o f T G F - ~ o n cell proliferation Cells (1 • 103) were seeded into flat-bottom microtiter plates in a final volume of 100 ~tl DMEM with 1% FCS. Serially diluted TGF-~ was added starting from a maximal concentration of 30 ng/ml. After 28 h, cultures were pulsed with 0.5 ~Ci [3Hmethyl]thymidine (Amersham-Buchler, Braunschweig, FRG). After 24 h, incorporated radioactivity was determined by liquid scintillation counting.

N o r t h e r n blotting Northern blotting was done by a standard procedure [32]. Poly(A) + RNA (20 ~tg) was resolved by gel electrophoresis (1% agarose) and blotted onto nitrocellulose filters (AmershamBuchler). Blots were probed with a radiolabelled (random primer kit, Pharmacia-LKB) human mdr cDNA obtained from American Type Culture Collection (Rockville, Md.).

Staining of cell m o n o l a y e r s C y t o f l u o r o m e t r y of multidrug t r a n s p o r t Monolayers were fixed and stained after decanting the medium from tissue culture plates by incubation for 30 s in a solution of i g/1 Coomassie blue in 40 % methanol, 10 % acetic acid and 50 % water.

I m m u n o c y t o c h e m i s t r y and ultrastructural analysis Frozen sections or paraffin sections were used for immunocytochemistry of the original tumor material. After deparaffinization/dehydration the sections were incubated with porcine serum (Biochrom, Berlin, FRG) for 30 rain. The primary antibodies (anti-GFAE 1:200, Dakopatts, Hamburg, FRG; anti-Vimentin, 1:200, Dakopatts; anti-neurofilament, 1:200, Dakopatts; JSB-1, 1:10, Biochrom) were applied for 1 h. Sections were then washed in Tris buffer and the endogeneous peroxidase blocked by 100 % methanol containing 0.5% H202. Biotinylated Fab fragments directed against either rabbit or mouse immunoglobulins (Amersham-Buchler) were used as secondary antibodies. After extensive washing of the sections avidin-peroxidase complex (Sigma) was applied. Diaminobenzidine was used as a substrate. Immunocytochemistry with human.glioma cells was performed by initially seeding cells into chamber glass slides (8 chambers/slide, I x 104 cells/chamber) in 200 gl medium. After 24 h TGF-~3 was added at different concentrations and 48 h later cells were fixed in 4 % paraformaldehyd/PBS (pH 7.4) for 30 min and processed for immunocytochemistry [27, 28]. The cells were studied for expression fo GFAR vimentin and P-glycoprotein by immunocytochemistry as described above. For ultrastructural analysis, glioma cells were fixed for 4 h. in 2 % glutaraldehyde (in cacodylate buffer, pH 7.4) and postfixed with 1% osmium tetroxide diluted in cacodylate buffer (pH 7.4). After dehydration of cells in ethanol the monolayer was embedded in Araldite. Ultrathin sections were counterstained with lead citrate and uranyl acetate and analyzed with a Zeiss EM 10CR electron microscope.

Diagnostic PCR Total RNA was prepared from glioma cells with RNAzol (Cinna/Biotecx Lab., Fiendswood,Tex.). Poly(A) + RNA was prepared on oligo(dT)-cellulose (mRNA purification kit, Pharmacia-LKB, Freiburg, FRG) as directed by the supplier, cDNA was synthesized from 0.2 ~tg poly(A) + RNA with recombinant M-MLV (Gibco/BRL, Eggenstein, FRG) using the anti-sense primer. PCR was performed on the reaction product with Taq polymerase (Amersham-Buchler) and 27mer amplimeres spanning a 160-bp region

Glioblastoma cells were stained for 15 rain with rhodamine 123 (R123; 1 x 106 cells/ml, 5 ~tg/ml R123 in DMEM, 5 % FCS and 25 mM Hepes). Cells were then washed with 5 ml dye-free medium, resuspended in 5 ml 37 ~ dye-free medium (containing 10 ~tg/ml verapamil (Isoptin, Knoll, Darmstadt, FRG) or other chemosensitiziers to block P-glycoprotein-mediated R123 transport). After 1 h, cells were centrifuged, washed and processed for cytofluorography with a FACScan cytofluorometer (Becton Dickinson, Munich, N.D.).

Results Brain tissue f r o m patients with suspected brain t u m o r s was stereotactically r e m o v e d and characterized by standard histological m e t h o d s . B i o p s y tissue f r o m six glial t u m o r s was used to establish cell lines (Table 1). Tissue was m i n c e d into pieces smaller t h a n 1 m m 3 and the resulting tissue f r a g m e n t s cultured in f l a t - b o t t o m microtiter plates for 4 - 6 weeks. E x p l a n t e d t u m o r cells were t h e n r e c o v e r e d by trypsinization and established as cell lines by r e p e a t e d subcultivation, T h e i m m u n o s t a i n i n g of cell cultures d e m o n s t r a t e d that all of the cells express G F A P (Table 1). We n o t e d , that cells f r o m o n e line, p a t - l , were s p o n t a n e o u s l y resistant to m i t o m y c i n C, vincristine, vinblastine and o t h e r cytostatic agents. F u r t h e r m o r e cells rapidly a d a p t e d to increasing doses of vincristine (subline p a t - l . m d r ) . A s this type of pleiotropic resistance to various c h e m o t h e r a p e u t i c agents was suggestive of multidrug resistance, we d e v e l o p e d a diagnostic P C R with amplimers flanking a c o n s e r v e d region of the h u m a n m d r genes to d e m o n s t r a t e m d r gene transcripts in cellular m R N A . W i t h P C R , m d r gene transcripts were identified (Fig. 1 a) in m R N A f r o m pat-1 and p a t - l . m d r cells b u t n o t in o t h e r line cells. To f u r t h e r verify expression of m d r genes we analyzed transcripts by N o r t h e r n blotting (Fig. 1 b). Two transcripts were seen with m R N A f r o m pat-1 and p a t - l . m d r cells, a larger (4.5 kb) c o r r e s p o n d i n g to the h u m a n m d r l and a smaller (4.1 kb) c o r r e s p o n d i n g to t h e h u m a n mdr3 gene transcripts [47].

643 Table 1. Characterization of human gliomas and cell lines Gliomas Patient code

Age

Sex

Type of tumor

GFAP

pat-1 pat-2 pat-3 pat-4 pat-5 pat-6

66 76 65 23 50 38

f m f f f m

glioblastoma glioblastoma glioblastoma ganglioglioma astrocytoma grade III oligoastrocytoma

++ + ++ + + +

P-glycoprotein expression Patient code

Immunocytochemistry

pat-1 pat-2 pat-3 pat-4 pat-5 pat-6

+ . n.d. . . .

PCR

Northern blots

+ .

.

. . .

. . .

Multidrug transport

mdrl, mdr3 + .

-

n.d.

-

. . .

Vimentin

NF

(+)

-

(+) + BV (+) BV

-

+ -

-

P-glycoprotein sc,

BV

sc, BV BV BV BV BV

was i n c u b a t e d at 37 ~ in the p r e s e n c e of verapamil, an agent that c o m p e t e s with R123 for cellular export. This control clearly d e m o n s t r a t e d , t h a t R123 transport was genuinely m e d i a t e d by P-glycoprotein (Fig. 3a). Pat-1 cells rapidly t r a n s p o r t e d R123 and dye efflux could be b l o c k e d by verapamil, a c h e m o s e n s i t i z e r inhibiting P-glycoprotein f u n c t i o n (Fig. 3 a). I n addition, it could be d e m o n s t r a t e d that various cytostatic agents k n o w n to be t r a n s p o r t e d by Pglycoprotein, like vinbtastine, vincristine, doxorubicin, and p u r o m y c i n (data n o t shown), effectively c o m p e t e d

Effects of TGF-[3 Patient code

Inhibition of multidrug transport

Increase in cell size

Change in monolayer formation

pat-1 pat-2 pat-3 pat-4 pat-5 pat-6

+ -

+ + + + -

+ -

f: Female; m: male; GFAP: glial fibrillary acidic protein; NF. neurofilament; (+): weak staining; +: most of cells are positive; + + : most tumor cells are strongly stained; sc: single cells are stained; BV: blood vessels are stained; n.d.: not done

E x p r e s s i o n o f the m u l t i d r u g transporting cell m e m b r a n e m o l e c u l e P-glycoprotein was d e m o n s t r a t e d by i m m u n o c y t o c h e m i s t r y with m o n o c l o n a l a n t i b o d y JSB1, specific for a c o n s e r v e d cytoplasmic p a r t of Pg l y c o p r o t e i n [4, 37]. P-glycoprotein expression could be d e m o n s t r a t e d in pat-1 cells and p a t - l . m d r cells (Fig. 2), but n o t in the o t h e r glioma cell lines. T h e d a t a are s u m m a r i z e d in Table 1. For d e m o n s t r a t i o n of m u l t i d r u g t r a n s p o r t (Fig. 3), we used a fluorescent p r o b e , r h o d a m i n e 123 (R123). This vital dye is r e m o v e d f r o m the cell b y m u l t i d r u g t r a n s p o r t via P - g l y c o p r o t e i n and p r o b e e x p o r t can be competitively inhibited b y various agents binding to P-glycoprotein. Cells were stained for 15 m i n with R123 and o n e sample was k e p t on ice for 1 h to block e x p o r t o f the fluorescent dye (multidrug t r a n s p o r t is A T P d e p e n d e n t and is, therefore, b l o c k e d on ice and in the p r e s e n c e o f azide). A n o t h e r sample was i n c u b a t e d for I h at 37 ~ ( e x p o r t of fluoresecent p r o b e ) . A s a control, an additional sample

Fig. 1 a, b. Expression of mdr genes demonstrated by diagnostic polymerase chain reaction (PCR) and Northern blotting. Total RNA was prepared from pat-1 glioblastoma cells with RNAzol. Poly(A) + RNA was prepared on oligo(dT)-cellulose (mRNA purification kit) as directed by the manufacturer, a PCR. cDNA was synthesized from 0.2 ~g poly(A) + RNA with recombinant M-MLV using the anti-sense primer. PCR was performed on the reaction product with Taq-polymerase and 27mer amplimeres spanning a 160-bp region (4091-4250) of the human mdrl gene. The reaction was performed with an Intelligent heating block using a thermocouple to monitor the reaction temperature within the Eppendorf tube. The PCR profile used was: 10 s, 94 ~ 20 s 55 ~ 30s 72 ~ Expression of the mdr gene was demonstrated by agarose gel electrophoresis of the PCR amplified reaction product obtained after 30 cycles of PCR: lane 1:123 bp size marker (Gibco-BRL); lane 2: PCR with RNA from pat-4 cells (no multidrug transport); lane 3: PCR with mRNA from pat-1 cells; lane 4: PCR with mRNA from pat-l.mdr cells, b Nothern blotting was done by standard procedure [29] with 20 ~g poly(A) + RNA of pat-1 and pat-l.mdr cells. Blots were probed with radiolabelled mdr cDNA, detecting mdrl and mdr3 gene transcipts. Stringency washes were done as follows: 2 x 5 min, 2 • SSC, room temperature; 3 x 10 min, 2 x SSC, 0.5 % SDS, 50~ 1 • 20 min, 2 x SSC, 0.5 % SDS, 63~

644 with R123 for multidrug transport. I n p a t - l . m d r cells, selected for o v e r - e x p r e s s i o n of m d r genes, v e r a p a m i l (and o t h e r chemosensitizers) was inefficient in c o m p e titively inhibiting P-glycoprotein t r a n s p o r t f u n c t i o n (Fig. 3b). This clearly d e m o n s t r a t e s t h a t o v e r - e x p r e s sion of m d r genes can result in a considerable increase in m u l t i d r u g t r a n s p o r t resistant to inhibition by c h e m o s e n sitizers. Thus, o t h e r ways of inhibiting m u l t i d r u g transp o r t might be of relevance to the t h e r a p y of t u m o r s over-expressing m d r genes. To learn m o r e a b o u t the s p o n t a n e u o s expression of m u l t i d r u g t r a n s p o r t in glioma cells and its implications to t u m o r pathophysiology, it will be n e c e s s a r y to reveal at-1 a

i

10 0

i

ill.~

i

10 ~

~

~ II~H

I

I

I

Jll.l

10 2

I

I

IllUl

10 3

10 4

pat- 1 .mdr

b

,

10 0

Fig. 2a-c. Immunocytochemistry for P-glycoprotein expression demonstrated striking differences between cell line pat-1 (b), and cell line pat-l.mdr (c). Monoclonal mouse antibody JSB-1 against P-glycoprotein and a PAP-complex was used (control a: no JSB-1 antibody)

101

10 2

I'lul

I

10 3

'

IIlUl

10 4

Fig. 3a, b. Activity of P-glycoprotem membrane transport system. Glioblastoma cells were stained for 15 min with rhodamine 123 [1 • 106 cells/ml, 5 ,ag/ml R123 in Dulbecco's modified Eagle's medium (DMEM), 5 % fetal calf serum (FCS) and 25 mM Hepes]. Cells were then washed with 5 ml dye-free medium. One aliquot of cells was kept on ice in phosphate-buffered saline (pH 7.4) with 0.2 % sodium azide and 1% bovine serum albumin (to inhibit ATP-dependent R123 export via P-glycoprotein), one aliquot was resuspended in 5 ml 37~ dye-free medium (R123 export) and another aliquot was resuspended in 5 ml 37 ~ medium containing 10 ~g/ml verapamil to competitively inhibit P-glycoproteinmediated R123 transport. After 1 h, all cells were centrifuged, washed and processed for cytofluorography using a FACScan cytofluorometer, a Cytofluorographic profiles of pat-1 cells; (---) pat-1 cells stained by R123 and kept on ice (maximal staining), (-...-) multidrug transport of R123 inhibited by verapamil, b Cytofluorographic profiles of pat-l.mdr cells; (---) pat-l.mdr cells on ice, (-...-) multidrug transport of R123 could not be inhibited by verapamil, (-.-) multidrug transport of R123

645 Inhibition of multidrug transport (%)

100

80 60

40 20

0

transport

verapamll TQF-81

TGF-82

TQF-81,2

BMP 2 TQF-82/BMP 2

Fig. 4, Bone morphogenetic protein (BMP) 2 antagonizes transforming growth factor (TGF)-[3-induced inhibition of multidrug transport in human pat-l.mdr glioblastoma cells. Pat-l.mdr cells were cultured and processed for cytofluorography as described in Fig. 3. (-): Multidrug transport (cells kept at 37~ verapamih mutidrug transport of R123 could only be weakly inhibited by chemosensitizer verapamil. Treatment of pat-l.mdr glioma cells with TGF-[31, -[32, -~1.2 (1 ng/ml) for 5 days reduced multidrug transport capacity; BMP 2 (25 ng/ml) anatgonized the inhibitory effect of TGF-[32 [as well as inhibition by TGF-[31 or-[31.2(data not shown)]. The optimal concentrations for modulation of multidrug transport by TGF-[3 were chosen from a previously published report [38]. BMP 2 only antagonized TGF-[3-mediated inhibition of multidrug transport, but did not stimulate multidrug transport capacity in untreated pat-l.mdr cells

gene transcripts in TGF-6- treated pat-l.mdr cells (Fig. 5). Transcript levels were also much higher in cells treated with BMP 2 (Fig. 5). Thus, inhibition of multidrug transport by TGF-6 is mediated by mechanisms not directly correlating with major changes in mRNA or P-glycoprotein expression. In this regard it should be noted, that pat-1 and pat-l.mdr cells showed additional responses to TGF-6, but not to BMP 2. These effects might be epiphenomena but they should be briefly discussed. During logarithmic growth, TGF-61, -62, and -61.2 inhibited proliferation of pat-1 (Fig. 6) and pat-l.mdr cells (not shown). In addition, the TGF-~ induced a more flattened appearance of cells and a general increase in cell size (Fig. 7). This TGF-[~-induced glioma cell hyperplasia was not antagonized by BMP 2 (Fig. 7). This demonstrates that TGF-~ and BMP 2 have multiple effects on glial cells. Moreover, treatment of pat-1 or pat-1.mdr cultures with TGF-61, [32, and 61.2 - but not BMP 2 - induced a dose-dependent reorganization of the monolayer. Within 48 h pat-1 cells started to migrate to local centers,

% inhibition 1 O0

80

the molecular processes governing mdr gene expression [1, 32, 33]. We observed that multidrug transport in human pat-1 and pat-l.mdr, glioma cells can be modulated by cytokines. TGF-~I, -[31.2, and -82 inhibited multidrug transport (Fig. 4), while the related BMP 2 had no inhibitory effect. On the contrary, it antagonized the inhibitory effects of TGF-~ (Fig. 4). It is not surprising that the TGF-~ have identical effects, since it is assumed that they bind to the same receptors; however, this is the first report showing that a member of the decapentaplegic subfamily of TGF-~, the BMP 2, has effects on glial cells and is antagonizing the effects of TGF-13. We examined whether inhibition of multidrug transport correlated with detection of P-glycoprotein or specific m R N A (Fig. 5). Immunocytochemically Pglycoprotein expression was detected in TGF-~-treated cells (not shown). In addition, Northern blot analysis clearly demonstrated the presence of mdrl and mdr3

6o 40 20 o lOO

30

lO

3

1

0,3

ng

o.1

0,03

o,001

o

TGF-S2/ml

Fig. 6. Inhibition of pat-1 cell proliferation by TGF-[3. Cells (1 x 103) were seeded into flat-bottom microtiter plates in a final volume of 100 ,al DMEM with 1% FCS. Se>ially diluted TGF-[32 was added starting from a maximal concentration of 30 ng/ml. After 28 h, cultures were pulsed with 0.5 btCi [3H-methyl]thymidine. After 24 h incorporated radioactivity was determined by liquid scintillation counting

Forward s c a t t e r 500 r

400 300

200

Fig. 5 . Changes in mdrl and mdr3 gene transcript levels in response to TGF-[3 and BMP 2. Pat-l.mdr cells were cultured as described in Fig. 1 and incubated for 5 days with TGF-[32 (1 ng/ml) or BMP 2 (25 ng/ml). Northern blots were done as described in Fig. 1

100 0 TGF-B1

TQF-82

TQF-81.2

BMP 2

TGF-82/BMP 2

Fig. 7. BMP 2 did not modulate TGF-[3-induced glioma cell hyperplasia. TGF-[3 (10 ng/ml)-induced pat-Lmdr cell hyperplasia was analyzed by cytometry (increase in cell size is quantified by increased forward scatter). Cells were cultured as described in Fig. 3

646 disrupting the monolayer structure. The monolayer sheets then contracted, leaving large areas devoid of cells and aggregates partially detached from the cell culture surface. This effect was reversible and removal of TGF-[3 from the cultures resulted into reestablishment of normal growth and monolayer morphology. TGF-13-treated pat-1 cells showed pronounced changes in the organization of the cytoplasm, whereas the chromatin pattern of the nuclei remained unchanged. The cytoplasm got more extended and contained increased numbers of organelles. The periphery of the cytoplasm showed dense accumulations of filaments. Such filaments were often found forming an extracellular network. We have recently observed identical effects of TGF-~ on fetal rat brain astrocytes [39]. The effects of TGF-13 were unique and could not be induced by TGF-c~, acidic or basic fibroblast growth factor, interferon-y, tumor necrosis factor-x or by interleukins (IL-1, IL-2, IL-3, IL-4, IL-6, IL-8) [38].

Discussion

P-glycoprotein is thought to function as an ATPdependent cellular transport system, removing certain hydrophobic substances, mostly derived from natural sources, like alkaloids or fungal toxins, from the cell. The pathophysiology of P-glycoprotein expression has stimulated much interest, since multidrug transport affects intracellular accumulation and activity of major classes of chemotherapeutic agents [2, 5, 10, 17, 20, 25, 28, 36, 41]. The expression of P-glycoprotein in tumor cells during chemotherapy or after in vitro exposure to antineoplastic agents is well documented [2, 3, 20, 36, 41] and gene transfer studies unequivocally demonstrated that the mdrl gene encoded P-glycoprotein is sufficient to cause multidrug resistance [10, 25]. The function of the human mdr3 gene has been less well studied but it might have similar properties [47]. It has been suggested that an early diagnosis of mdr might be of value in designing chemotherapeutic protocols because mdr in tumor cells may play a crucial role in determining the clinical response of cancer patients to chemotherapy. Multidrug transport is clearly not unique to tumor cells, and P-glycoprotein is physiologically expressed in a few specific sites, notably the excretory epithelium of liver, pancreas, kidney, colon, and jejunum [7, 8, 17]. Endothelial cells of the blood-brain barrier weakly express P-glycoprotein, which might function in protecting the central nervous system [7]. The physiological and pathophysiological factors regulating mdr gene expression in glial cells are unknown. We investigated mdr gene expression with a small panel of human glioma cell lines and detected spontaneous expression in one cell line, pat-1. Thus, P-glycoprotein can be expressed before therapy and without any exposure to chemotherapeutic drugs. This strongly argues for the existence of endogenous factors

inducing mdr gene expression. Spontaneous expression of mdr in tumor cells has been described [29]. Northern blot analysis of m R N A from pat-1 cells demonstrated expression of both the mdrl and mdr3 genes. Expression of mdrl and mdr3 genes has not been described previously in human brain cells but in mouse CNS transcripts of both mdrl and mdr3 genes have been detected [8]. Exposure of pat-1 cells to vincristine selected for cells with high levels of both mdrl and mdr3 transcripts. However, none of these genes appeared to be amplified (unpublished observation). In conjunction with other reports, these findings demonstrate that mdr gene over-expression is not directly correlated with, and is possibly preceding, gene amplification [40]. Thus, mdrl and mdr3 genes are constitutively and coordinately expressed in human pat-1 tumor cells and selection for multidrug resistance coordinately increases levels of mdrl and mdr3 gene transcripts. Such a coordinate over-expression of mdr genes has not as yet been described in glioblastoma cells. In general, expression of mdr genes is not regulated coordinately, and a differential over-expression of mdr genes has been observed in the murine macrophage-like cell line J774.2 [26, 31]. The molecular mechanisms regulating spontaneous expression of mdr genes in glial tumor cells are unknown. P-glycoprotein expression may be specifically induced as a mechanism for detoxifying cells. Alternatively, its activation may be part of a more complex, nonspecific response of the cell to metabolic or toxid stress, mdr genes can be activated by carcinogens and chemotherapeutics, possibly by directly activating the mdrl gene promotor [15, 16, 30]. In addition, there are two strong heat-shock consensus elements in the major mdrl gene promotor [6], which respond not only to heat shock but also to exposure of the cells to arsenite or cadmium salts [6]. Furthermore, mdr gene expression has been observed during tissue regeneration after surgical trauma and during pregnancy [1, 45]. Thus, not only xenobiotics but also physiological modulators regulate P-glycoprotein function [27]. It has been found that a variety of compounds inhibit the function of P-glycoprotein, rendering multidrugresistant tumor cells sensitive to drugs that would otherwise be ineffective. These compounds, referred to as "chemosensitiziers" [18, 21, 41], might be useful in the design of new chemotherapeutic protocols by making tumors more susceptible to chemotherapy. While effective multidrug transport can be blocked in pat-1 cells by verapamil, this chemosensitizer (and others) was ineffective in reducing multidrug transport in pat-l.mdr cells. The over-expression of mdr genes in this cell line resulted in very high rates of multidrug transport, resistant to inhibition by chemosensitizers. The characterization of physiological modulators of multidrug transport might be of great value in understanding physiological regulation of multidrug transport and might thereby provide a new pharmacological basis for altering P-glycoprotein expression and multidrug transport. However, the physiological mechanisms governing cellular multidrug transport are so far unknown. We observed that TGF-[31, -[32, and -[31.2 inhibit multi-

647 drug transport in pat-1 cells and even in glioma cells over-expressing the m d r l and mdr3 genes. Thus, the T G F - ~ are p o t e n t regulators of multidrug transport. T h e T G F - ~ are multifunctional regulators of growth and differentiation of a variety of normal and neoplastic cells with essential physiological functions during tissue f o r m a t i o n and repair. T h e y were originally detected in t u m o r cell culture supernatants and characterized by a bioassay of the induction of t r a n s f o r m e d growth in certain cell lines. The T G F - ~ are considered to be involved in the regulation of neoplastic processes [9, 12, 13, 50]. It has repeatedly b e e n observed that T G F - ~ is produced by t u m o r cells, notably by glioblastomas/astrocytomas, and defects of T G F - ~ synthesis or responsiveness have b e e n suggested as factors in t u m o r develo p m e n t [12]. A new subfamily of TGF-~, the decapentaplegic factors have b e e n described recently [49]. To this family belongs the BMP 2. Interestingly, BMP 2, although related to the T G F - ~ , did not inhibit multidrug transport but did antagonized the inhibitory effect of TGF-~. This is the first report that a m e m b e r of the decapentaplegic subgroup of the T G F - ~ has effects on glial cells. BMP 2 is k n o w n to induce ectopic cartilage f o r m a t i o n in vivo and is considered to be involved in b o n e f o r m a t i o n and remodeling. However, B M P 2 is present in a variety of tissue and it can be assumed that BMP 2 serves other biological functions in these tissues. In comparison to T G F - ~ , BMP 2 did not inhibit glioblastoma cell proliferation and did not induce (or antagonize) cell hyperplasia and changes in growth patterns. Interestingly, B M P 2 induces a coordinate increase in m d r l and mdr3 transcript levels in untreated glioblastoma cells and in glioblastoma cells treated with TGF-~. However, it should be n o t e d that decreases in multidrug transport capacity are not necessarily correlated with decreased m d r l and mdr3 gene transcript levels or i m m u n o c y t o chemically d e m o n s t r a b l e P-glycoprotein expression. Thus, our data further emphasize the point that m d r gene expression is complex with regulation on several levels [14]. O u r observation that multidrug transport can be m o d u l a t e d by cytokines like the T G F - ~ might o p e n a way to further i m p r o v e m e n t s in c h e m o t h e r a p y through a d e e p e r understanding of the physiological and pathophysiological regulation of multidrug transport.

Acknowledgements.We thank Drs. Krone and Grunert for making available tumor biopsies, Dr. John Wozney for human BMP 2, and Mrs. Alexandra Bunz for technical assistance.

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Spontaneous multidrug transport in human glioma cells is regulated by transforming growth factors type beta.

The multidrug transporting cell membrane molecule P-glycoprotein can be spontaneously expressed in human glioma cells. Transcripts of mdr genes were d...
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