Cytotechnology 9: 69-75, 1992. 9 1992KluwerAcademic Publishers. Printedh~the Netherlands.
Novel cell lines derived from transgenic mice expressing recombinant human proteins T r a n s g e n i c h e p a t o m a - d e r i v e d cell lines
Fr6d6ric Perraud, Wilfried Dalemans, Dalila Ali-Hadji and Andrea Pavirani Department of Molecular and Celhdar Biology, Transgene S.A., 11 Rue de Molsheim, 67082 Strasbourg Cedex, France Received 28 February 1992; accepted in revised form 7 July 1992
Key words: transgenic mice, trans-immortalization, hepatocyte, cq-antitrypsin, factor IX, onc gene Abstract We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human Ctl-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human Ctl-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.
Introduction Mammalian ceils are the most suited host system for the production of complex human recombinant proteins since they are capable of ensuring posttranslational modifications (e.g., glycosylation, sulfation on tyrosine residues, 7-carboxylation of glutamic acid residues) which may be necessary for full biological activity. However, in certain cases, where high expression levels of recombinant human proteins have been obtained by transfecting available cell lines like CHO, BH_K, or HepG2, only partial biological activity has been observed (Kaufmann et al., 1986). This is partly due to incomplete posttranslation processing by the heterologous host cell. It would
therefore be desirable to express such complex recombinant proteins in more specialised cells. Unfortunately, such cells are not always available as established cell lines. To overcome this problem, we have investigated the potential of transgenic mice as a source of novel cell lines for the expression of complex proteins by employing a procedure that we have previously termed transimmortalization (Pavirani et al., 1989). This approach consists of co-expressing an onc gene and a human protein of interest in target cell tissues of transgenic mice, in which tumours subsequently arise. The neoplastic ceils are then established and cultured as permanent cell lines producing the human proteins. We have used liver specific regulatory DNA
70 sequences to co-express onc genes, such as c - m y c or simian virus 40 T antigen (SV40 TAg), together with model h u m a n hepatic proteins of therapeutic interest. These latter were oq-antitrypsin (oqAT), a major antiprotease present in the lung (Crystal, 1989), and coagulation factor IX (FIX) whose lack or impaired function is responsible for haemophilia B (Furie and Furie, 1988). These novel expression cell lines were extensively characterized by electron microscopy, immunohistochemistry, m R N A expression pattern of m a j o r hepatic markers, and expression and secretion of the respective human recombinant proteins which were shown to be fully active. Moreover, studies were conducted on the activity of the endogenous mouse c y t o c h r o m e P450 en-
zymes, which are responsible for the oxidation and biotransformation of drugs and e n v i r o n m e n tal pollutants in the liver (Morel et al., 1990).
Materials and m e t h o d s D N A constructions
p T G 2 9 8 4 (Dalemans et al., 1990) (Fig. 1A) was constructed by fusing a 1.8 kilobase (kb) promoter K p n I fragment of the h u m a n c~lAT (hoqAT) gene (Long et al., 1984) to the second and third coding exons of the routine c - m y c gene (Bernard et al., 1983). In addition, a 16 kb SalI fragment containing the entire h o q A T gene including 1.5
Fig. 1. Schematic representation of the DNA constructions used to generate transgenic mice. A, B, C: microinjected transgenes excised from pTG2984, pTG4912 and pTG3960, respectively. Hatched boxes represent 5' flankin~promoter (p) regions: solid boxes numbered in romans designate exons (black boxes: human (h) OtlAT; white boxes: mouse (m) c-nzycand SV40 TAg). Black triangles (A) indicate deletions present in introns of the hFIX gene. Thin lines represent introns/not coding regions. Representations are not on scale.
71 and 4 kb of 5' and 3' flanking sequences, respectively, was inserted downstream of the c-myc expression cassette. pTG4912 (Perraud et al., 1991) (Fig. 1B) was constructed by replacing the SV40 early promoter/ enhancer of pTG 173 (Jallat et al., 1990b) by the human otlAT promoter, whereby the SV40 TAg comes under control of this latter promoter. pTG3960 (Jallat et al., 1990a) (Fig. 1C) consisted of the human FIX genomic sequences including 5 kb and 0.3 kb of 5' and 3' flanking sequences, respectively; these genomic sequences were isolated from a XEMBL3 library derived from a 4XY chromosome lymphoblastoid cell line (Pavirani et al., 1987). This construct contains the majority of the FIX gene with the exception of 4.8 kb and 7.1 kb of internal sequences in intron A and F, respectively (Anson et al., 1985). Generation of transgenic mice
Purified transgenes devoid of vector DNA were microinjected into the male pronucleus of fertilized eggs from C57B1/6 • SJL hybrid crosses (Hogan et al., 1986). Transgenic mice were detected by blot hybridization of tail DNA (Palmiter et al., 1982). Development of hepatomas was detected by palpation of the abdomen. Cell culture and biological characterization
Hepatomas were surgically removed from tumourbearing mice and were minced with scissors under sterile conditions. Tissue fragments were washed with Hanks' balanced salt solution to eliminate red blood cells and incubated twice for 30 min in William's E basal medium containing 0.05% collagenase. Cells were collected by centrifugation, plated in Primaria or standard tissue culture flasks and than cultured in William's E medium supplemented with 5% fetal calf serum (Institut J. Boy, Reims, France), hydrocortisone (300 ng/ml), epidermal growth factor (EGF) (50 ng/ml), insulin (10 gg/ml) and transferrin (10 gg]ml) (all from Sigma, St Louis, MO, USA). At confluency, cells were detached by treat-
ment with 0.05% (w/v) trypsin made in phosphatebuffered saline; cells were subsequently plated in culture flasks at appropriate dilutions. Determination and analysis of marker proteins
Levels of human or mouse proteins were measured by enzyme-linked immunoadsorbent assay (ELISA) using antisera against human o~AT (Cappel, Malvem, PA, USA), human FIX (Kit diagnostica Stago, Asnieres, France) or mouse albumin (Nordic Immunology, Tilburg, The Netherlands). FIX procoagulant activity was determined using a Cephalin Kaolin one stage assay (Austen and Rhyme, 1975). mRNA analysis
Expression of the mRNA for major hepatic markers was studied by dot blot analysis (Costanzi and Gillespie, 1987). Sequences of the specific oligonucleotides used as hybridization probes are described in Dalemans et al. (1990) and Jallat et al. (1990a). Preparation of microsomes and P450 activity
Microsomes were prepared from cell cultures according to Morel et al. (1990). Total cytochrome P450 activity was assayed by differential spectrophotometry (Omura and Sato, 1964).
Results Generation of o'ansgenic mice prone to hepatoma development
Two DNA constructions were used to generate transgenic mice prone to the development of liver tumours (hepatomas) (Fig. IA and B); pTG2984 and pTG4912 both contain a 1.5 kb fragment of the promoter region of the human OqAT which directs the expression of murine c-myc and SV40 TAg, respectively. A complete copy of the human cqAT gene is moreover present in pTG2984. Two and four transgenic mouse lines were established
72 Table I. Levels of mouse albumin and human recombinant proteins secreted in the supernatants of trans-immortalized hepatic cell lines
Cell line
Number of passages
Marker protein secretion p.~lO6 cells/24 ha Mouse albumin
TMHepTG2984 2 TMHepTG4912 10 TMHepTG 3960 x 2984 x 4912 39 TMHepTG 3960 x 2984 x 4912 48
40 18 12 23
p.~ml/2-4hb Human ~IAT
Human FIX
5
5
-
7
-
-
4 8
0.35 0.80
15 10
aAmounts were determined by ELISA and corresponded to those measured at the indicated passage; bproduction was measured at high cell density. f r o m the f o r m e r a n d latter c o n s t r u c t , respectively. All six m o u s e lines were p r o n e to h e p a t o m a d e v e l o p m e n t , a p h e n o t y p e w h i c h was t r a n s m i t t e d to the p r o g e n y . T h e t i m e o f t u m o u r o n s e t was r e l a t e d to the n a t u r e o f the o n c g e n e (on a v e r a g e , 12 a n d 6 m o n t h s for p T G 2 9 8 4 a n d p T G 4 9 1 2 transgenic mice, respectively). Histological e x a m i n a t i o n of t u m o u r s f r o m T M T G 2 9 8 4 a n d T M T G 4 9 1 2 m i c e s h o w e d that both k i n d of m a l i g -
n a n t cells were of h e p a t o c y t i c origin. N o pathological c h a n g e s were o b s e r v e d in other o r g a n s or tissues. Transgenic mice ( T M T G 3 9 6 0 ) containing hum a n F I X s e q u e n c e s (Fig. 1C) i n t e g r a t e d in their g e n o m e were g e n e r a t e d a n d s h o w n to e x p r e s s the r e c o m b i n a n t p r o t e i n in the c i r c u l a t i o n at levels o f up to 40 p.g/ml. M i c e then were s u c c e s s i v e l y c r o s s e d with t r a n s g e n i c T M T G 2 9 8 4 a n d T M T G -
Fig. 2. Phase contrast micrograph of trans-immortalized cell line TMHepTG3960 x 2984 x 4912 39 after 4 weeks in culture (four passages). Bar: 100 p.m.
73 4912 mice, which are both prone to hepatoma development. The resulting triple transgenic mice developed hepatomas as expected after a few months. H e p a t o m a - d e r i v e d cell lines
Continuous cell lines could be established from neoplastic cells derived from these transgenic hepatomas (Table 1). Protocols had initially to be optimized in order to maintain a differentiated phenotype of the hepatocytes during cell culturing. The use of the medium described in Material and Methods resulted in the maintenance of a hepatocyte-like morphology as confirmed by electron microscopy (not shown). Cell lines secreted mouse albumin at similar levels (5 p.g/ml/24 h), a secretion stable for at least 40 passages. In the case of TMHepTG2984 (line 2), the concentration of human cqAT secreted in the culture supernatant was 7 I.tg/ml/24 h. The recombinant molecule was glycosylated and shown to be active. Moreover, a comparative study between two independent cell lines each derived from the respective onc genes, showed that TMHepTG2984 (line 2) was more responsive to EGF than TMHepTG4912 (line 10) (Perraud et al., 1991), indicating that TMHepTG2984 cell lines were more differentiated than TMHepTG4912 cell lines. In the case of triple transgenic mice, expressing human CtlAT and FIX in the circulation, hepatic cell lines could also be established (TMHepTG3960 x 2984 x 4912) (Fig. 2). These cell lines secrete mouse albumin, human oqAT and human FIX in their supematants (Table 1). Cell lines 39 and 48 expressed murine albumin at levels between 15 and 20 ktg/106 cells/24 h while human CtlAT levels averaged 6 ~tg/106 cells/24 h for the two cell lines. Productivity of human FIX measured at passage 12 and 23, respectively, was 0.15 and 0.25 ktg/106 cells/24 h for line 39 and 48. When cells were maintained at high cell density, cell lines expressed -0.35 and 0.8 ~tg/ml/24 h of human FIX respectively. The human FIX protein was fully active, the
clotting activity being close to 100%, thus similar to the activity of plasma-derived FIX. The recombinant protein was also comparable to plasmaderived FIX in terms of electrophoretic migration (with a relative mass of 70 Kd), and immunorecognition. Correct processing of the pro peptide was demonstrated by N-terminal amino acid sequencing of the first 10 residues. At present, the potential of this novel expression system of human FIX in terms of industrial exploitation is under evaluation. Characterization o f the differentiation status o f hepatoma-derived cell lines
mRNA levels for mouse albumin, txjAT and transferrin were studied in the cells trans-immortalized by either one or two of the onc genes used; TMHepTG2984 (line 2), TMHepTG4912 (line 10) and TMHepTG3960 x 2984 x 4912 (lines 39 and 48) had comparable levels of the respective mRNAs (Dalemans et al., 1990; Jallat et al., 1990a; Perraud et al., 1991). Cell line TMHepTG4912 (line 1O) also expressed t~ fetoprotein (a fetal hepatic marker) and acute phase protein (a stress marker) and thus seemed to be less differentiated than the two other cell lines. Analysis of total cytochrome P450 activity from microsomes showed that TMHepTG3960 x 2984 x 4912 (line 48) had attained P450 levels of 87 picomoles/mg of total microsomal protein. This represents 1/10 of the level found in control primary cultures of mouse hepatocytes but was 6-fold higher than the P450 levels in the commonly used rat hepatic cell line FAO (Deschatrette et al., 1974). Total P450 levels in other trans-immortalized hepatic cell lines were lower.
Discussion In this study, we have shown the feasibility of establishing cell lines secreting recombinant human proteins from onc gene-induced transgenic hepatomas. Cells showed an adult-like differentiation status as confirmed by electron microscopy and by expression of adult hepatic markers pro-
74 teins, e.g., a l b u m i n , ot1AT a n d t r a n s f e r r i n . T h e s e c e l l l i n e s w e r e u s e d for the p r o d u c t i o n o f h u m a n ~IAT and a more complex protein, human coagulation F I X . In the c a s e o f F I X , these n o v e l h e p a t i c c e l l lines w e r e c a p a b l e o f c o m p l e x p o s t t r a n s l a t i o n a l m o d i f i c a t i o n s n e c e s s a r y for full b i o l o g i c a l a c t i v i t y o f the protein. T h e s e c r e t i o n o f h u m a n OtlAT o r h u m a n F I X was s t a b l e d u r i n g the entire t i m e c o u r s e o f the s t u d y a n d at p r e s e n t the p o t e n tial o f the t r a n s - i m m o r t a l i z e d cell lines to be e x p l o i t e d for h u m a n F I X i n d u s t r i a l p r o d u c t i o n is under evaluation. A n o t h e r p o t e n t i a l a p p l i c a t i o n o f t h e s e cell lines is r e l a t e d to the use o f h e p a t o c y t e c u l t u r e s in the s a f e t y t e s t i n g o f d r u g s a n d o t h e r c h e m i c a l s . In this r e s p e c t , it is i m p o r t a n t that the cell lines e x p r e s s a v a r i e t y o f c y t o c h r o m e P 4 5 0 s , w h i c h are the m a j o r e n z y m e s i n v o l v e d in d r u g b i o t r a n s f o r m a t i o n p r o c e s s e s . W e a n a l y z e d o u r cell lines for the p r e s e n c e o f total c y t o c h r o m e P 4 5 0 a c t i v i t y , a n d o b s e r v e d that c e l l lines e x p r e s s i n g b o t h the t w o o n c g e n e s ( c - m y c a n d S V 4 0 T A g ) h a d a h i g h e r total c y t o c h r o m e P 4 5 0 a c t i v i t y than cell lines w h e r e o n l y one o n c g e n e w a s e x p r e s s e d . T o c o m p l e t e t h e s e results, f u r t h e r s t u d i e s w i t h d i f f e r e n t c y t o c h r o m e P 4 5 0 e n z y m e s ( P 4 5 0 I A , IIB, I I C a n d I I I A ) are in progress.
Acknowledgements W e are d e e p l y i n d e b t e d to o u r f r i e n d , J e a n - P i e r r e L e c o c q , w h o s e i n t e l l i g e n t a d v i c e and e n c o u r a g e m e n t h a v e b e e n i n s t r u m e n t a l in c o n d u c t i n g this w o r k . W e t h a n k M. C o u r t n e y for c r i t i c a l r e a d i n g o f the m a n u s c r i p t , R . G . C r y s t a l for the c~IAT g e n e , all o u r c o l l a b o r a t o r s a n d t e c h n i c i a n s w h o s e s k i l l f u l w o r k has b e e n g r e a t l y a p p r e c i a t e d , N. M o n f r i n i f o r e d i t o r i a l h e l p a n d B. H e l l e r for a r t w o r k . W o r k on F I X w a s s u p p o r t e d b y Institut Merieux, France.
References Anson DS, Austen DEG and Brownlee GG (1985) Expression of human clotting factor IX from recombinant DNA clones in mammalian cells. Nature 315: 683-685. Austen DEG and Rhymes IL (1975) A Laboratory Manual of Blood Coagulation (Blackwell Scientific). Bernard O, Cory S, Gerondakis S, Webb E and Adams JM (1983) Sequence of the murine and human cellular myc oncogenes and two modes of myc transcription resulting from chromosome translocation in B lymphoid tumours. EMBO J 2: 2375-2383. Costanzi C and Gillespie D (1987) Fast blots: immobilization of DNA and RNA from cells. In: Berger SL, Kimmel AR (eds) Guide to Molecular Cloning Techniques, Vol 152. Orlando: Academic Press, pp. 582-587. Crystal RG (1989) The ~l-antitrypsin gene and its deficiency states. Trends Genet 5:411-417. Dalemans W, Perraud F, Le Meur M, Gerlinger P, Courmey M and Pavirani A (1990) Heterologous protein expression by trans-immortalised differentiated liver cell lines derived from transgenic mice. Biologicals 18: 191-198. Deschatrette J and Weiss MC (1974) Characterization of differentiated and dedifferentiated clones of a rat hepatoma. Biochimie 56:1603-1611. Furie B and Furie BC (1988) The molecular basis of blood coagulation. Cell 53: 505-518. Hogan B, Costantini F and Lacy E (1986) Manipulating the mouse embryo, Cold Spring Harbor Laborator3, Press, Cold Spring Harbor, NY, pp. 332. Jaliat S, Perraud F, Dalemans W, Balland A, Dieterle A, Faure T, Meulien P and Pavirani A (1990a) Characterization of recombinant human Factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines. EMBO J 9: 3295-3301. Jallat S, Meulien P, Pavirani A and Perraud F (1990b) Patent Cooperation Treaty (PCT)/FR90/00606. Kaufman RJ, Wasley LC, Furie BC, Furie B and Shoemaker CB (1986) Expression, purification and characterization of recombinant 7-carboxylated Factor IX synthesized in chinese hamster ovary cells. J Biol Chem 261: 9622-9628. Long GL, Chandra T, Woo SLC, Davie EW and Kurachi K (1984) Complete sequence of the cDNA for human Ctl-antitrypsin and the gene for the S variant. Biochemistry 23: 4828-4837. Morel F, Beaune PH, Ratanasavanh D, Flinois JP, Yang CS, Guengerich FP and Guillouzo A (1990) Expression of cytochrome P450 enzymes in cultured human hepatocytes. Eur J Biochem 191: 437-444. Omura T and Sato R (1964) The carbon monoxide-building pigment of liver microsomes. II Solubilization, purification and properties. J Biol Chem 239: 2379-2385. Palmiter RD, Chen HY and Brinster RL (1982) Differential regulation of metallothionein-thymidine kinase fusion genes in transgenic mice and their offspring. Cell 29: 701-710.
75 Pavirani A, Meulien P, Harrer H, Schamber F, Dott K, Villeval D, Cordier Y, Wiesel ML0 Mazurier C, Van de Pol H, Piquet Y, Cazenave JP and Lecocq JP (1987) Choosing a host cell for active recombinant factor VIII production using vaccinia virus. Bio/Technology 5: 389-392. Pavirani A, Skern T, Le Meur M, Lutz Y, Lathe R, Crystal RG, Fuchs JP, Gerlinger P and Courtney M (1989) Recombinant proteins of therapeutic interest expressed by lymphoid cell lines derived from transgenic mice. Bio/Technology 7: 10491054.
Perraud F, Dalemans W, Gendrault JL, Dreyer D, Ali-Hadji D, Faure T and Pavirani A (1991) Characterization of transimmortalised hepatic cell lines established from transgenic mice. Exp Cell Res 195: 59-65.
Address lbr offprims: Andrea Pavirani, Department of Molecular and Cellular Biology, Transgene S.A., 1 I Rue de Molsheim, 67082 Strasbourg Cedex, France.
Novel cell lines derived from transgenic mice expressing recombinant human proteins T r a n s g e n i c h e p a t o m a - d e r i v e d cell lines
Fr6d6ric Perraud, Wilfried Dalemans, Dalila Ali-Hadji and Andrea Pavirani Department of Molecular and Celhdar Biology, Transgene S.A., 11 Rue de Molsheim, 67082 Strasbourg Cedex, France Received 28 February 1992; accepted in revised form 7 July 1992
Key words: transgenic mice, trans-immortalization, hepatocyte, cq-antitrypsin, factor IX, onc gene Abstract We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human Ctl-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human Ctl-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.
Introduction Mammalian ceils are the most suited host system for the production of complex human recombinant proteins since they are capable of ensuring posttranslational modifications (e.g., glycosylation, sulfation on tyrosine residues, 7-carboxylation of glutamic acid residues) which may be necessary for full biological activity. However, in certain cases, where high expression levels of recombinant human proteins have been obtained by transfecting available cell lines like CHO, BH_K, or HepG2, only partial biological activity has been observed (Kaufmann et al., 1986). This is partly due to incomplete posttranslation processing by the heterologous host cell. It would
therefore be desirable to express such complex recombinant proteins in more specialised cells. Unfortunately, such cells are not always available as established cell lines. To overcome this problem, we have investigated the potential of transgenic mice as a source of novel cell lines for the expression of complex proteins by employing a procedure that we have previously termed transimmortalization (Pavirani et al., 1989). This approach consists of co-expressing an onc gene and a human protein of interest in target cell tissues of transgenic mice, in which tumours subsequently arise. The neoplastic ceils are then established and cultured as permanent cell lines producing the human proteins. We have used liver specific regulatory DNA
70 sequences to co-express onc genes, such as c - m y c or simian virus 40 T antigen (SV40 TAg), together with model h u m a n hepatic proteins of therapeutic interest. These latter were oq-antitrypsin (oqAT), a major antiprotease present in the lung (Crystal, 1989), and coagulation factor IX (FIX) whose lack or impaired function is responsible for haemophilia B (Furie and Furie, 1988). These novel expression cell lines were extensively characterized by electron microscopy, immunohistochemistry, m R N A expression pattern of m a j o r hepatic markers, and expression and secretion of the respective human recombinant proteins which were shown to be fully active. Moreover, studies were conducted on the activity of the endogenous mouse c y t o c h r o m e P450 en-
zymes, which are responsible for the oxidation and biotransformation of drugs and e n v i r o n m e n tal pollutants in the liver (Morel et al., 1990).
Materials and m e t h o d s D N A constructions
p T G 2 9 8 4 (Dalemans et al., 1990) (Fig. 1A) was constructed by fusing a 1.8 kilobase (kb) promoter K p n I fragment of the h u m a n c~lAT (hoqAT) gene (Long et al., 1984) to the second and third coding exons of the routine c - m y c gene (Bernard et al., 1983). In addition, a 16 kb SalI fragment containing the entire h o q A T gene including 1.5
Fig. 1. Schematic representation of the DNA constructions used to generate transgenic mice. A, B, C: microinjected transgenes excised from pTG2984, pTG4912 and pTG3960, respectively. Hatched boxes represent 5' flankin~promoter (p) regions: solid boxes numbered in romans designate exons (black boxes: human (h) OtlAT; white boxes: mouse (m) c-nzycand SV40 TAg). Black triangles (A) indicate deletions present in introns of the hFIX gene. Thin lines represent introns/not coding regions. Representations are not on scale.
71 and 4 kb of 5' and 3' flanking sequences, respectively, was inserted downstream of the c-myc expression cassette. pTG4912 (Perraud et al., 1991) (Fig. 1B) was constructed by replacing the SV40 early promoter/ enhancer of pTG 173 (Jallat et al., 1990b) by the human otlAT promoter, whereby the SV40 TAg comes under control of this latter promoter. pTG3960 (Jallat et al., 1990a) (Fig. 1C) consisted of the human FIX genomic sequences including 5 kb and 0.3 kb of 5' and 3' flanking sequences, respectively; these genomic sequences were isolated from a XEMBL3 library derived from a 4XY chromosome lymphoblastoid cell line (Pavirani et al., 1987). This construct contains the majority of the FIX gene with the exception of 4.8 kb and 7.1 kb of internal sequences in intron A and F, respectively (Anson et al., 1985). Generation of transgenic mice
Purified transgenes devoid of vector DNA were microinjected into the male pronucleus of fertilized eggs from C57B1/6 • SJL hybrid crosses (Hogan et al., 1986). Transgenic mice were detected by blot hybridization of tail DNA (Palmiter et al., 1982). Development of hepatomas was detected by palpation of the abdomen. Cell culture and biological characterization
Hepatomas were surgically removed from tumourbearing mice and were minced with scissors under sterile conditions. Tissue fragments were washed with Hanks' balanced salt solution to eliminate red blood cells and incubated twice for 30 min in William's E basal medium containing 0.05% collagenase. Cells were collected by centrifugation, plated in Primaria or standard tissue culture flasks and than cultured in William's E medium supplemented with 5% fetal calf serum (Institut J. Boy, Reims, France), hydrocortisone (300 ng/ml), epidermal growth factor (EGF) (50 ng/ml), insulin (10 gg/ml) and transferrin (10 gg]ml) (all from Sigma, St Louis, MO, USA). At confluency, cells were detached by treat-
ment with 0.05% (w/v) trypsin made in phosphatebuffered saline; cells were subsequently plated in culture flasks at appropriate dilutions. Determination and analysis of marker proteins
Levels of human or mouse proteins were measured by enzyme-linked immunoadsorbent assay (ELISA) using antisera against human o~AT (Cappel, Malvem, PA, USA), human FIX (Kit diagnostica Stago, Asnieres, France) or mouse albumin (Nordic Immunology, Tilburg, The Netherlands). FIX procoagulant activity was determined using a Cephalin Kaolin one stage assay (Austen and Rhyme, 1975). mRNA analysis
Expression of the mRNA for major hepatic markers was studied by dot blot analysis (Costanzi and Gillespie, 1987). Sequences of the specific oligonucleotides used as hybridization probes are described in Dalemans et al. (1990) and Jallat et al. (1990a). Preparation of microsomes and P450 activity
Microsomes were prepared from cell cultures according to Morel et al. (1990). Total cytochrome P450 activity was assayed by differential spectrophotometry (Omura and Sato, 1964).
Results Generation of o'ansgenic mice prone to hepatoma development
Two DNA constructions were used to generate transgenic mice prone to the development of liver tumours (hepatomas) (Fig. IA and B); pTG2984 and pTG4912 both contain a 1.5 kb fragment of the promoter region of the human OqAT which directs the expression of murine c-myc and SV40 TAg, respectively. A complete copy of the human cqAT gene is moreover present in pTG2984. Two and four transgenic mouse lines were established
72 Table I. Levels of mouse albumin and human recombinant proteins secreted in the supernatants of trans-immortalized hepatic cell lines
Cell line
Number of passages
Marker protein secretion p.~lO6 cells/24 ha Mouse albumin
TMHepTG2984 2 TMHepTG4912 10 TMHepTG 3960 x 2984 x 4912 39 TMHepTG 3960 x 2984 x 4912 48
40 18 12 23
p.~ml/2-4hb Human ~IAT
Human FIX
5
5
-
7
-
-
4 8
0.35 0.80
15 10
aAmounts were determined by ELISA and corresponded to those measured at the indicated passage; bproduction was measured at high cell density. f r o m the f o r m e r a n d latter c o n s t r u c t , respectively. All six m o u s e lines were p r o n e to h e p a t o m a d e v e l o p m e n t , a p h e n o t y p e w h i c h was t r a n s m i t t e d to the p r o g e n y . T h e t i m e o f t u m o u r o n s e t was r e l a t e d to the n a t u r e o f the o n c g e n e (on a v e r a g e , 12 a n d 6 m o n t h s for p T G 2 9 8 4 a n d p T G 4 9 1 2 transgenic mice, respectively). Histological e x a m i n a t i o n of t u m o u r s f r o m T M T G 2 9 8 4 a n d T M T G 4 9 1 2 m i c e s h o w e d that both k i n d of m a l i g -
n a n t cells were of h e p a t o c y t i c origin. N o pathological c h a n g e s were o b s e r v e d in other o r g a n s or tissues. Transgenic mice ( T M T G 3 9 6 0 ) containing hum a n F I X s e q u e n c e s (Fig. 1C) i n t e g r a t e d in their g e n o m e were g e n e r a t e d a n d s h o w n to e x p r e s s the r e c o m b i n a n t p r o t e i n in the c i r c u l a t i o n at levels o f up to 40 p.g/ml. M i c e then were s u c c e s s i v e l y c r o s s e d with t r a n s g e n i c T M T G 2 9 8 4 a n d T M T G -
Fig. 2. Phase contrast micrograph of trans-immortalized cell line TMHepTG3960 x 2984 x 4912 39 after 4 weeks in culture (four passages). Bar: 100 p.m.
73 4912 mice, which are both prone to hepatoma development. The resulting triple transgenic mice developed hepatomas as expected after a few months. H e p a t o m a - d e r i v e d cell lines
Continuous cell lines could be established from neoplastic cells derived from these transgenic hepatomas (Table 1). Protocols had initially to be optimized in order to maintain a differentiated phenotype of the hepatocytes during cell culturing. The use of the medium described in Material and Methods resulted in the maintenance of a hepatocyte-like morphology as confirmed by electron microscopy (not shown). Cell lines secreted mouse albumin at similar levels (5 p.g/ml/24 h), a secretion stable for at least 40 passages. In the case of TMHepTG2984 (line 2), the concentration of human cqAT secreted in the culture supernatant was 7 I.tg/ml/24 h. The recombinant molecule was glycosylated and shown to be active. Moreover, a comparative study between two independent cell lines each derived from the respective onc genes, showed that TMHepTG2984 (line 2) was more responsive to EGF than TMHepTG4912 (line 10) (Perraud et al., 1991), indicating that TMHepTG2984 cell lines were more differentiated than TMHepTG4912 cell lines. In the case of triple transgenic mice, expressing human CtlAT and FIX in the circulation, hepatic cell lines could also be established (TMHepTG3960 x 2984 x 4912) (Fig. 2). These cell lines secrete mouse albumin, human oqAT and human FIX in their supematants (Table 1). Cell lines 39 and 48 expressed murine albumin at levels between 15 and 20 ktg/106 cells/24 h while human CtlAT levels averaged 6 ~tg/106 cells/24 h for the two cell lines. Productivity of human FIX measured at passage 12 and 23, respectively, was 0.15 and 0.25 ktg/106 cells/24 h for line 39 and 48. When cells were maintained at high cell density, cell lines expressed -0.35 and 0.8 ~tg/ml/24 h of human FIX respectively. The human FIX protein was fully active, the
clotting activity being close to 100%, thus similar to the activity of plasma-derived FIX. The recombinant protein was also comparable to plasmaderived FIX in terms of electrophoretic migration (with a relative mass of 70 Kd), and immunorecognition. Correct processing of the pro peptide was demonstrated by N-terminal amino acid sequencing of the first 10 residues. At present, the potential of this novel expression system of human FIX in terms of industrial exploitation is under evaluation. Characterization o f the differentiation status o f hepatoma-derived cell lines
mRNA levels for mouse albumin, txjAT and transferrin were studied in the cells trans-immortalized by either one or two of the onc genes used; TMHepTG2984 (line 2), TMHepTG4912 (line 10) and TMHepTG3960 x 2984 x 4912 (lines 39 and 48) had comparable levels of the respective mRNAs (Dalemans et al., 1990; Jallat et al., 1990a; Perraud et al., 1991). Cell line TMHepTG4912 (line 1O) also expressed t~ fetoprotein (a fetal hepatic marker) and acute phase protein (a stress marker) and thus seemed to be less differentiated than the two other cell lines. Analysis of total cytochrome P450 activity from microsomes showed that TMHepTG3960 x 2984 x 4912 (line 48) had attained P450 levels of 87 picomoles/mg of total microsomal protein. This represents 1/10 of the level found in control primary cultures of mouse hepatocytes but was 6-fold higher than the P450 levels in the commonly used rat hepatic cell line FAO (Deschatrette et al., 1974). Total P450 levels in other trans-immortalized hepatic cell lines were lower.
Discussion In this study, we have shown the feasibility of establishing cell lines secreting recombinant human proteins from onc gene-induced transgenic hepatomas. Cells showed an adult-like differentiation status as confirmed by electron microscopy and by expression of adult hepatic markers pro-
74 teins, e.g., a l b u m i n , ot1AT a n d t r a n s f e r r i n . T h e s e c e l l l i n e s w e r e u s e d for the p r o d u c t i o n o f h u m a n ~IAT and a more complex protein, human coagulation F I X . In the c a s e o f F I X , these n o v e l h e p a t i c c e l l lines w e r e c a p a b l e o f c o m p l e x p o s t t r a n s l a t i o n a l m o d i f i c a t i o n s n e c e s s a r y for full b i o l o g i c a l a c t i v i t y o f the protein. T h e s e c r e t i o n o f h u m a n OtlAT o r h u m a n F I X was s t a b l e d u r i n g the entire t i m e c o u r s e o f the s t u d y a n d at p r e s e n t the p o t e n tial o f the t r a n s - i m m o r t a l i z e d cell lines to be e x p l o i t e d for h u m a n F I X i n d u s t r i a l p r o d u c t i o n is under evaluation. A n o t h e r p o t e n t i a l a p p l i c a t i o n o f t h e s e cell lines is r e l a t e d to the use o f h e p a t o c y t e c u l t u r e s in the s a f e t y t e s t i n g o f d r u g s a n d o t h e r c h e m i c a l s . In this r e s p e c t , it is i m p o r t a n t that the cell lines e x p r e s s a v a r i e t y o f c y t o c h r o m e P 4 5 0 s , w h i c h are the m a j o r e n z y m e s i n v o l v e d in d r u g b i o t r a n s f o r m a t i o n p r o c e s s e s . W e a n a l y z e d o u r cell lines for the p r e s e n c e o f total c y t o c h r o m e P 4 5 0 a c t i v i t y , a n d o b s e r v e d that c e l l lines e x p r e s s i n g b o t h the t w o o n c g e n e s ( c - m y c a n d S V 4 0 T A g ) h a d a h i g h e r total c y t o c h r o m e P 4 5 0 a c t i v i t y than cell lines w h e r e o n l y one o n c g e n e w a s e x p r e s s e d . T o c o m p l e t e t h e s e results, f u r t h e r s t u d i e s w i t h d i f f e r e n t c y t o c h r o m e P 4 5 0 e n z y m e s ( P 4 5 0 I A , IIB, I I C a n d I I I A ) are in progress.
Acknowledgements W e are d e e p l y i n d e b t e d to o u r f r i e n d , J e a n - P i e r r e L e c o c q , w h o s e i n t e l l i g e n t a d v i c e and e n c o u r a g e m e n t h a v e b e e n i n s t r u m e n t a l in c o n d u c t i n g this w o r k . W e t h a n k M. C o u r t n e y for c r i t i c a l r e a d i n g o f the m a n u s c r i p t , R . G . C r y s t a l for the c~IAT g e n e , all o u r c o l l a b o r a t o r s a n d t e c h n i c i a n s w h o s e s k i l l f u l w o r k has b e e n g r e a t l y a p p r e c i a t e d , N. M o n f r i n i f o r e d i t o r i a l h e l p a n d B. H e l l e r for a r t w o r k . W o r k on F I X w a s s u p p o r t e d b y Institut Merieux, France.
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Address lbr offprims: Andrea Pavirani, Department of Molecular and Cellular Biology, Transgene S.A., 1 I Rue de Molsheim, 67082 Strasbourg Cedex, France.
