JOURNAL OF VIROLOGY, JUlY 1990, p. 3527-3531
Vol. 64, No. 7
0022-538X/90/073527-05$02.00/0 Copyright © 1990, American Society for Microbiology
Development of a Retroviral Vector for Inducible Expression of Transforming Growth Factor ,13 M. L. McGEADY,t* P. M. ARTHUR, AND M. SEIDMAN Otsuka America Pharmaceutical, Inc., Research Laboratories, 9900 Medical Center Drive, Rockville, Maryland 20850 Received 2 January 1990/Accepted 5 April 1990
A retroviral vector system for the expression of exogenous genes under the control of an inducible promoter developed. By utilizing this system, the cDNA for human transforming growth factor 01 (TGF-I1) was inserted into a retroviral vector under the control of an internal mouse metallothionein promoter and introduced via infection into normal rat kidney fibroblasts (NRK-49F) and epithelial cells (NRK-52E), Chinese hamster ovary cells (CHO), and the human monocytic cell line U937. Control of TGF-131 expression, achieved by Cd2+ induction of vector-encoded TGF-,l1 mRNA, was cel line specific and resulted in a concomitant increase in neutralizable TGF-pl production by the cells. Autocrine stimulation of vector-containing ceUls by vector-encoded TGF-01 was detected by an increase in soft-agar colony formation of NRK-49F infectants compared with that of the control ceUls. In addition, the use of a second internal promoter in a retroviral vector of similar design allowed isolation of stable infectants from a cel line (CHO) in which the viral long terminal repeat does not function efficiently. was
Retroviral vector systems have been useful for the introduction and expression of genes in a variety of cell types, including those which are not readily transfectable with expression plasmids (36). Amphotropic-packaging cell lines constructed with the env gene from the 4070A murine retrovirus have been used to encapsidate retroviral vector RNA (5, 24, 34), yielding viral stocks with a wide host range (4, 17, 28). The control of gene expression via transcription may be accomplished in these retroviral vector systems by using the viral long terminal repeat (LTR) (41). Alternatively, internal promoters located within the proviral sequences have been successfully exploited (27, 41). By this mechanism, several genes can be transcribed independently from a single integrated proviral genome, allowing introduction of multiple genes into a single cell. In addition, the use of retroviral vectors containing internal promoters allows efficient transcription of an introduced gene in cells in which the LTR does not function efficiently (16, 32, 40). The Moloney murine leukemia virus-derived LTR, while active in many cells, does not function well in nondifferentiated F9 cells (15, 18, 20, 40) or in some hematopoietic cells (23, 42). This appears to be due in part to the absence of necessary transcription factors (35) and possibly to the presence of negative regulatory factors (11). Introduction of an inducible metallothionein (MT) promoter (10) into a retroviral vector provides an additional level of transcriptional control. The MT promoter has been used successfully in self-inactivating vectors (43, 44), but attempts to use it in a retroviral vector with a functioning LTR have failed to show induction (23). In this study, the mouse MT-1 promoter was introduced as an internal retroviral vector promoter to control transcription of the transforming growth factor ,13 (TGF-p1) gene. TGF-P, a product of transformed as well as normal cells (2, 6, 25, 31, 37), is a multifunctional regulator of cell growth. Corresponding author. t Present address: Laboratory of Tumor Immunology and Biology, Building 10, 5B52, National Cancer Institute, Bethesda, MD *
20892.
It can induce anchorage-independent growth of fibroblasts alone or in combination with epidermal growth factor (EGF) (25, 31, 37) and can also function as a potent inhibitor of cell growth (19, 26, 29, 38). We were interested in studying the biology of cells expressing TGF-P. Our concern about the inhibitory effects of this molecule led us to place the gene for TGF-p1 under the control of the inducible mouse MT-1 promoter to prevent constitutive production of the potentially growth-inhibitory TGF-1l by infected cells, which might preclude their isolation as stable cell lines. This approach did permit the construction of cell lines expressing TGF-P, but the degree of regulation depended on the cell type into which the vector was introduced. Vector construction infection of cells, and establishment of cell lines. A cDNA clone containing the sequence for the pre-pro-human TGF-131 was isolated from human erythroleukemia cells by using specific priming. DNA sequence analysis revealed it to be essentially the same as the previously reported human TGF-1l gene (8) (data not shown). A 1.3-kilobase (kb) EcoRI-NcoI fragment of this clone was introduced into Moloney sarcoma virus-derived retroviral vector 1521 (22) immediately 3' to an internal MT-1 promoter (Fig. 1); the resulting plasmid will be referred to as 1525. A second TGF-,1l retroviral vector (1528) was constructed. It was identical to vector 1525, except that it contained a 350-base-pair fragment which carried the simian virus 40 early-region promoter (the PvuII-to-HindIII fragment) inserted 5' to the G418 resistance gene. This vector was designed to initiate a transcript internally at the simian virus 40 promoter, which would contain the G418 resistance gene as the first open reading frame on the RNA. Following transfection of plasmids 1525 and 1528 into i2 cells (21) and G418 selection, ecotropic vector-packaging lines were isolated. Amphotropic stocks of the viral vector were produced by PA317 cells (24) following infection with ecotropic-vector stocks and selection in G418 media. (Titers of ecotropic and amphotropic vector stocks on NIH 3T3 cells ranged from 1 x 105 to 5 x 105 G418-resistant colonies per ml.) Ecotropic and amphotropic stocks of retroviral vector 1525 were used to infect several established cell lines. The 3527
3528
J. VIROL.
NOTES A.
A.
1
2
3 4 5 6 7 8 9 10 11 12
!9
7 Kb
4
3 Kb40
B. [
3
SD
LTR
1525
0
LTR
pBR on
G418
MT-1j TGF,B cDNA
B.
1 2 3 4 5 6 7
[] 7
1 Kb
FIG. 1. Construction and proviral structure of plasmid vector 1525. (A) The 1.3-kb EcoRI-to-NcoI fragment from human TGF-Pl cDNA was inserted into the EcoRI site of vector 1521 following treatment of the fragment with Klenow fragment and attachment of EcoRI linkers. S.D., Splice donor; tp, packaging signal; pBR0n, pBR322 origin of replication; G418, G418 resistance gene; MT-1, mouse MT-1 promoter. (B) Proviral structure of retrovirus 1525 following infection and stable integration. The plasmid vector, referred to as 1525, now contains the gene for pre-pro-TGF-,B under the transcriptional control of the MT promoter.
normal rat kidney fibroblastic line NRK-49F and the epithelial line NRK-52E were infected with the ecotropic packaged vector 1525 stocks (7), while CHO cells were infected with amphotropic stocks of vectors 1525 and 1528. Colonies of G418-resistant cells were isolated and expanded into cell lines for further characterization. The human monocytic cell line U937 was infected with an amphotropic stock of vector 1525, and following selection of a stable population of G418-resistant cells, single-cell cloning by terminal dilution was used to generate cell lines. Induction of TGF-13-specific transcription from the MT-1 promoter. Previous studies utilizing retroviral vectors have not demonstrated induction of the MT promoters in vectors with functioning LTRs (23). However, because the TGF-,B gene is under the transcriptional control of the mouse MT-1 promoter in vectors 1525 and 1528, induciblity of transcription with heavy metals was tested. The predicted size of a TGF-1l transcript initiating at this promoter and terminating in the proviral LTR was approximately 3 kb, a size easily distinguished from other predicted transcripts by Northern (RNA) blotting. NRK-52E, NRK-49F, and U937-1525 infectants were incubated for 16 to 24 h with and without CdSO4. Northern blots of TGF-f3-specific RNA from these cells are shown in Fig. 2. In three individual NRK-52E infectants (a, b, and c), transcription from this promoter was readily induced by 5 ,uM CdSO4, as evidenced by the appearance of a 3-kb RNA (Fig. 2A). In addition, there was a shift away from transcription of the 6.5-kb full-length proviral transcript, and there was possible autoinduction of the 2.5-kb endogenous rat TGF-pl transcript and a novel 1.5-kb transcript from the rat locus (39). While 5 ,uM CdSO4 worked well as a transcriptional inducer, it was toxic to cells, and therefore induction regimens which preserved cell viability were tested. Both 1 ,uM CdSO4 and 10 mM sodium butyrate worked well as inducers of transcription from the MT-1 promoter, and they exhibit little or no toxicity for the cells (Fig. 2A, lanes 8 through 12). CdSO4 (1 ,uM) was subsequently used for induction of all 1525 infectant cell lines.
KbS-_
C. 45678910P
C.
1
7Kb _-
2 3 4 5 6 7 8 9 10 11 12
_
*-
i
-
7 Kh
3K t- -
FIG. 2. Northern blot of 1525 infectant cell line RNA. Total RNA (10 ,ug) was isolated, electrophoresed, transferred to nitrocellulose filters (22), and hybridized with a TGF-p1-specific nicktranslated probe. (A) NRK-52E cell RNA. Lane 1, NRK-52E cell; lanes 2 and 3, NRK-52E-1525(a) infectant; lanes 4 and 5, NRK52E-1525(b) infectant; lanes 6 through 12, NRK-52E-1525(c) infectant. Lanes 1, 2, 4, 6, and 8, RNA from untreated cells. Results for RNA from cells treated with 5 ,uM CdSO4 (lanes 3, 5, and 7), EGF (lane 9), 1 ,uM CdSO4 (lane 10), 5 mM sodium butyrate (lane 11), and 10 mM sodium butyrate (lane 12) are also shown. (B) NRK-49F cell RNA. Lane 1, 1525(a) infectant; lane 2, 1525(b) infectant; lane 3, 1525(c) infectant; lane 4, uninfected NRK-49F cells; lane 5, 1525(c) infectant treated with 5 mM sodium butyrate; lane 6, 1525(c) infectant treated with 1 ,uM CdSO4 for 20 h; lane 7, 1525(c) infectant treated with 1 ,iM CdSO4 for 70 h. (C) U937 cell RNA. Lanes 1 and 2, 1525(a) infectant; lanes 3 and 4, 1525(b) infectant; lanes 5 and 6, 1525(c) infectant; lanes 7 and 8, 1525(d) infectant; lanes 9 and 10, 1525(e) infectant. Lanes 1, 3, 5, 7, and 9, untreated cell RNA; lanes 2, 4, 6, 8, and 10, 1 ,uM CdSO4-treated cell RNA; lane 11, uninfected U937 cell RNA; lane 12, untreated 1525 infectant.
MT-1 TGF-,1-specific transcription was not as readily inducible in the NRK-49F-1525 infectants. Three NRK49F-1525 infectants (a, b, and c) showed constitutive transcription from this promoter (Fig. 2B, lanes 1 through 3), and one showed modest induction when incubated in CdSO4 (1 puM) for 72 h but not when incubated for 24 h (lanes 4 through 7). Hence, for all subsequent experiments utilizing this cell line, growth conditions included 1 ,uM CdSO4. In contrast, none of the five U937-1525 infectants (a, b, c, d, and e) tested exhibited constitutive transcription from the MT-1 promoter, and three of the five clones tested were inducible (Fig. 2C, lanes 1 through 10). However, all U937 infectants had a high level of the 2.5-kb endogenous TGF-pl transcript. This represents an elevated level compared with that of the uninfected control cells and may be caused by autoinduction
VOL. 64, 1990
NOTES
A.
3529
TABLE 1. TGF-P activity in serum-free mediaa s1 LTR
pBR 1528
I
_- -
V
G418
o ,1
o
418
LTR
-IY MTT II TGF. cDNA
TGF-1 Source of cells media
Incubated without CdSO4
Incubated with 1 ,uM CdSO4
Neutralization
NRK-49F NRK-49F-1525(c) NRK-52E NRK-52E-1525(c) U937 U937-1525(1)
NT NT NT 450 35 92
82.5 375 16 1,250 NT 140
NT 97.8 NT 92.1 NT 97.5
Kb
B. 1
2
3 4
5 6 7
A* 7.5 -5
A
3
Kb Kb
Kb
ft
FIG. 3. Northern blot of vector 1528-infected CHO cell RNA. (A) Structure of integrated proviral vector 1528. SV40 ori, Simian virus 40 origin of replication and early region promoter. Other abbreviations are as in Fig. 1 legend. (B) Total cellular RNA (10 ,ug) was isolated, electrophoresed, transferred to nitrocellulose filters, and hybridized with a TGF-pl-specific probe. Lane 1, Untreated 1525-U937 infectant (control for size); lane 2, 1525-U937 infectant treated with 1 ,uM CdSO4; lane 3, untreated 1525-CHO infectant; lane 4, 1525-CHO infectant treated with 1 ,uM CdSO4; lane 5, untreated 1528-CHO infectant; lane 6, 1528-CHO infectant treated with 1 p.M CdSO4; lane 7, 1528-CHO infectant treated with 5 ,uM CdSO4.
due to vector 1525 infection (Fig. 2C, lanes 11 through 12) (39). Transcription of vector 1528 in CHO cells. CHO cells were infected with amphotropic viral stocks of vector 1525. However, following G418 selection, the titer, measured as G418resistant colonies, was only 0.1% of that of the same stock plated on NRK-49F cells. In addition, the colonies isolated were unstable in G418-containing media and contained no 7-kb proviral RNA by Northern blot analysis (Fig. 3). Therefore, it appears that the block to infection was not at the level of receptors; rather, it appears that the Moloney sarcoma virus LTR did not function as a strong enhancerpromoter in these cells. Because transcription of the G418 resistance gene depends on initiation at this promoter, no cells stably producing the gene product could be isolated. In contrast to these results, infection of CHO cells with amphotropic stocks of vector 1528 resulted in the isolation of stable G418-resistant colonies, and characterization of the viral RNA in these cells revealed several TGF-31-specific species (Fig. 3). There was some 7.4-kb proviral RNA, but the amount was considerably reduced compared with that of U937-1525 infectants, suggesting less-efficient functioning of the LTR in CHO cells. In addition, there was a 5-kb internally promoted transcript as well as a 3-kb MT-1promoted transcript. The latter transcript, while constitutively present in this clone, showed a modest induction over control levels following treatment of the cells with Cd2+. Induction of TGF-,1-specific 3-kb RNA correlates with
equivalents (ng/107 cells) in samples:
a Cells were incubated for 20 h in serum-free media (previously described for NRK cells [22]; for U937 cells, RPMI 1640 with 1% Nutridoma SP [Boehringer Mannheim]) with or without 1 ,uM CdSO4. Media were dialyzed extensively against 0.2 M acetic acid, lyophilized, and suspended in phosphate-buffered saline. Samples were tested for ability to replace TGF-P in the NRK-49F agar assay (2) in the presence of 1 ng of EGF per ml and compared with a standard curve derived by using known amounts of human-plateletderived TGF-pl. Acidified, dialyzed, lyophilized serum-free conditioned media from CdSO4-induced cell lines were added to media containing EGF and 50 ,ug of anti-TGF-31 IgG or control IgG. Following incubation at 37°C, 104 NRK49F indicator cells were added and the mixture was adjusted to 0.3% Noble agar. After 10 days, the number of colonies per plate was determined and the percent neutralization was calculated. In these experiments, 93 to 99% of the human platelet-derived TGF-p1 used as a control was neutralized by this antibody. NT, Not tested.
increased TGF-01 protein production. In two of the three cell lines into which retroviral vector 1525 was introduced, transcription of TGF-,1-specific RNA could be easily induced by activation of the MT-1 promoter with CdSO4. To determine if induction of the 3-kb TGF-pl RNA resulted in an increase in the level of the TGF-pl protein produced by the cells, the amount of TGF-P activity with and without induction was measured. The biological assay used to determine the activity measures the amount of acid-activated TGF-f in serum-free conditioned media collected from infectant cells that replaces the TGF-3 added to NRK-49F cell soft-agar colony-forming assay (1). In this assay, NRK-49F cells form colonies in soft agar only when both TGF-1 and EGF are added in a dose-dependent manner. The addition of 1 ,uM CdSO4 to NRK-52E-1525 infectants resulted in an approximately threefold increase in the amount of TGF-, activity produced (Table 1). This correlates well with the increase in the level of 3-kb TGF-pl RNA in the cells. The amount of TGF-P activity present without induction presumably results from NRK-52E-1525 infectant having a lower but detectable constitutive level of transcription of the 3-kb vector RNA (Fig. 2). In the case of the U937 infectants, while the increase in TGF-P activity correlates with RNA levels prior to and following induction, the cells generally appear to be poor producers of TGF-P protein. All three cell lines produce some acid-activatable TGF-, from transcription of the endogenous gene, as indicated by the level of TGF-P produced by the uninfected control cells. However, in these experiments, the level of TGF-13 transcription from the internal MT-1 promoter is clearly reflected by the level of TGF-,B activity produced by the cells. To determine if the agar colony-promoting activity was due to TGF-pl, the acidified conditioned media collected from all the 1525 infectants following induction were tested for neutralization by antibody directed against TGF-pi (R & D Systems). This antibody, prepared as an immunoglobulin G (IgG) fraction, was made against porcine TGF-i1 and has neutralizing activity for human, rodent, and porcine TGFp1. Samples of acidified conditioned media from 1525 infec-
3530
J. VIROL.
NOTES
TABLE 2. Growth of 1525-NRK-49F cells in agar No. of % of total Cell type' Addition(s)b colonies
NRK-49F
1525-NRK-49F
colonies
EGF EGF + TGF-P
5,880
0 3.2 100.0
EGF EGF + TGF-P
0 520 6,120
0 8.4 100.0
0 192
a 104 control or vector infectant cells seeded per plate (2) in the presence of 1 F.M CdSO4. b EGF and TGF-3 were each added at 1 ng/ml. -, No addition.
tants were tested for promotion of NRK-49F cell agar colony formation in the presence of EGF following preincubation with 50 ,ug of anti-TGF-pl IgG or nonimmune IgG. The results in Table 1 indicate a high level of neutralization of the TGF-P activity produced by NRK and U937-1525 infectant cell lines, suggesting that almost all the activity produced by the infectants following induction is due to TGF-p1 production.
Biological consequence of vector 1525-induced TGF-pi1 production by NRK-49F cells. The majority of the TGF-pl produced and secreted by many cell types is an inactive precursor that is complexed to another protein(s) and that can be activated by several mechanisms to produce the mature molecule. The biologically active form of human TGF-,1l appears to be a homodimer of 12,000-dalton polypeptides linked by disulfide bonds to form the 24,000-dalton, biologically-active "mature form" of the protein (3, 12, 29). DNA sequence analysis of TGF-pi cDNA clones predicts that the 12,000-dalton subunit consists of a 112-amino-acid polypeptide cleaved from the C terminus of a 390-amino-acid precursor, pre-pro-TGF-pl (8, 9, 33). Introduction of the cDNA for pre-pro-TGF-pl into CHO cells by transfection resulted in the production of a 95- to 120-kilodalton precursor complex comprising mature TGF-pl, pro-TGF-f31, and the pro region of the precursor (13, 14). The majority of material produced by CHO cells appears to be identical to that isolated from human platelets, the most concentrated source of human TGF-P (3). However, a small fraction of the TGF-pl produced by these cells is in the form of the biologically active 24-kilodalton mature protein (13, 14). The NRK-49F-1525 infectants were further analyzed to determine if the TGF-pl protein produced is biologically active in an autocrine fashion. As stated above, NRK-49F cells form colonies in soft agar in the presence of exogenously added EGF and TGF-P (1). Therefore, to determine if the TGF-pl produced by the NRK-49F infectants could function in an autocrine fashion and replace the requirement for exogenously added TGF-P in this assay, Cd2+-induced infectants and control cells were seeded in soft agar in the presence of EGF only. The results indicate that approximately 8% of the cells seeded in soft agar in the presence of EGF formed colonies, compared with 3% of the uninfected NRK-49F cells (Table 2). While the 1525 infectants produce approximately four times as much TGF-pi (Table 1) as the control cells, the majority of it is in an inactive form. However, the ability of the 1525 infectants to respond in this assay suggests that there is a sufficient amount of active material to drive a low-level autocrine stimulation. In conclusion, insertion of TGF-pl cDNA into a retroviral vector under the control of an inducible promoter has allowed introduction of the gene in a regulatable state into a
wide variety of cell types (i.e., fibroblastic, epithelial, and monocytic). While previous studies have indicated that MT promoters function in retroviral vectors in which the LTR is transcriptionally inactivated (43, 44), our experiments demonstrate that the MT-1 promoter can be regulated in vectors with functional LTRs as well. Thus, the LTR can be used to drive transcription of the selectable marker gene whose constitutive production is required, while the regulatable promoter drives transcription of a gene whose expression should be tightly controlled. The regulatability of the MT-1 promoter varied with the cell type but could be well controlled in two of the four cell types. While the data presented in this paper represent only a relatively small number of individual clones, overall we found that the MT-1 promoter was tightly regulated by Cd2" in >50% of the U937 infectants and 70% of the epithelial infectants analyzed. In the other two cell lines, CHO and NRK-49F, transcription from this promoter was increased by physiologically tolerated levels of Cd2". The efficiency of translation and secretion of the protein varied independently of promoter regulation. In one cell line, NRK-49F, in which a biological effect by TGF-P production could be readily measured, the results of autocrine stimulation were detectable. Recently this vector system has been used to study regulated gene expression in rodent and human mammary epithelial cells (M. L. McGeady, unpublished data). Hence, it appears that a vector of this nature, able to infect many cell types and containing a regulatable promoter, should be useful for the study of growth factors and other proteins whose constitutive expression alters cell growth in such a way as to preclude isolation of stable producer cells. LITERATURE CITED 1. Anzano, M., A. Roberts, C. Myers, A. Komoriya, L. Lamb, J. Smith, and M. Sporn. 1982. Synergistic interaction of transforming growth factors from murine sarcoma cells. Cancer Res. 42:4776-4778. 2. Anzano, M., A. Roberts, J. Smith, M. Sporn, and J. DeLarco. 1983. Sarcoma growth factor from conditioned medium of virally transformed cells is composed of both type a and type ,B transforming growth factors. Proc. Natl. Acad. Sci. USA 80: 6264-6268. 3. Assoian, R., A. Komoriya, C. Meyers, and M. Sporn. 1983. Transforming growth factor , in human platelets: identification of a major storage site, purification, and characterization. J. Biol. Chem. 258:7155-7160. 4. Cloyd, M., M. Thompson, and J. Hartley. 1985. Host range of mink cell focus-inducing viruses. Virology 140:239-248. 5. Cone, R. D., and R. C. Mulligan. 1984. High efficiency gene transfer into mammalian cells: generation of helper free recombinant retrovirus with broad mammalian host range. Proc. Natl. Acad. Sci. USA 81:6349-6353. 6. DeLarco, J., and G. Todaro. 1978. Growth factors from murine sarcoma virus transformed cells. Proc. Natl. Acad. Sci. USA 75:4001-4005. 7. DeLarco, J., and G. Todaro. 1978. Epithelioid and fibroblastic rat kidney cell clones: epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation. Journal of Cell. Physiol. 94:335-342. 8. Derynck, R., J. A. Jarrett, E. Y. Chen, D. H. Eaton, J. R. Bell, R. K. Assoian, A. B. Roberts, M. B. Sporn, and D. V. Goeddel. 1985. Human transforming growth factor-p complementary DNA sequence and expression in normal and transformed cells. Nature (London) 316:701-705. 9. Derynck, R., J. A. Jarrett, E. Y. Chen, and D. V. Goeddel. 1986. The murine transforming growth factor p precursor. J. Biol. Chem. 261:4377-4379. 10. Durnam, D. M., F. Perrin, F. Gannon, and R. D. Palmiter. 1980.
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ated by epidermal growth factor: isolation from non-neoplastic tissue. Proc. Natl. Acad. Sci. USA 78:5339-5343. Rubenstein, J. L. R., J. F. Nicholas, and F. Jacob. 1984. Construction of a retrovirus capable of transducing and expressing genes in multipotential embryonic cells. Proc. Natl. Acad. Sci. USA 81:7137-7140. Sharples, K., G. D. Plowman, T. M. Rose, D. R. Twardzik, and A. F. Purchio. 1987. Cloning and sequence analysis of simian transforming growth factor-p cDNA. DNA 6:239-244. Sorge, J., D. Wright, V. D. Erdman, and A. E. Cutting. 1984. Amphotropic retrovirus vector system for human cell gene transfer. Mol. Cell. Biol. 4:1730-1737. Speck, N. A., and D. Baltimore. 1987. Six distinct nuclear factors interact with the 75-base-pair repeat of the Moloney murine leukemia virus enhancer. Mol. Cell. Biol. 7:1101-1110. Temin, H. 1986. Retrovirus vectors for gene transfer: efficient integration into and expression of exogenous DNA in vertebrate cell genomes, p. 149-187. In R. Kucherlaptai (ed.), Gene transfer. Plenum Publishing Corp., New York. Todaro, G. J. 1982. Autocrine secretion of peptide growth factors by tumor cells. Natl. Cancer Inst. Monogr. 10:139-147. Tucher, R. F., G. D. Shipley, H. L. Moses, and R. W. Holley. 1984. Growth inhibitor from BSC-1 cells is closely related to the platelet type p transforming growth factor. Science 226:705-
707. 39. van Obberghen-Schilling, E., N. Roche, K. Flanders, M. Sporn, and A. Roberts. 1988. Transforming growth factor p-1 positively regulates its own expression in normal and transformed cells. J. Biol. Chem. 263:7741-7746. 40. Wagner, E. F., M. Vanek, and B. Vennstrom. 1985. Transfer of
41.
42.
43.
44.
genes into early embryonal carcinoma cells by retrovirus infections: efficient expression from an internal promoter. EMBO J. 4:663-666. Wei, C.-M., M. Gibson, P. G. Spear, and E. M. Scolnick. 1981. Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1. J. Virol. 39:935-944. Williams, D. A., J. H. Orkin, R. C. Mulligan. 1986. Retrovirus mediated transfer of human adenosine deaminase sequences into cells in culture and into murine haematopoietic cells in vivo. Proc. Natl. Acad. Sci. USA 83:2566-2570. Yee, J. K., J. Moores, D. Jolly, J. Wolff, J. Respess, and T. Friedmann. 1987. Gene expression from transcriptionally disabled retroviral vectors. Proc. Natl. Acad. Sci. USA 84:51995201. Yu, S. F., T. Von Ruden, P. Kanhoff, C. Garber, M. Jeiberg, U. Ruther, W. F. Anderson, E. Wagner, and E. Gilboa. 1986. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc. Natl. Acad. Sci. USA 83:3194-3198.
Vol. 64, No. 7
0022-538X/90/073527-05$02.00/0 Copyright © 1990, American Society for Microbiology
Development of a Retroviral Vector for Inducible Expression of Transforming Growth Factor ,13 M. L. McGEADY,t* P. M. ARTHUR, AND M. SEIDMAN Otsuka America Pharmaceutical, Inc., Research Laboratories, 9900 Medical Center Drive, Rockville, Maryland 20850 Received 2 January 1990/Accepted 5 April 1990
A retroviral vector system for the expression of exogenous genes under the control of an inducible promoter developed. By utilizing this system, the cDNA for human transforming growth factor 01 (TGF-I1) was inserted into a retroviral vector under the control of an internal mouse metallothionein promoter and introduced via infection into normal rat kidney fibroblasts (NRK-49F) and epithelial cells (NRK-52E), Chinese hamster ovary cells (CHO), and the human monocytic cell line U937. Control of TGF-131 expression, achieved by Cd2+ induction of vector-encoded TGF-,l1 mRNA, was cel line specific and resulted in a concomitant increase in neutralizable TGF-pl production by the cells. Autocrine stimulation of vector-containing ceUls by vector-encoded TGF-01 was detected by an increase in soft-agar colony formation of NRK-49F infectants compared with that of the control ceUls. In addition, the use of a second internal promoter in a retroviral vector of similar design allowed isolation of stable infectants from a cel line (CHO) in which the viral long terminal repeat does not function efficiently. was
Retroviral vector systems have been useful for the introduction and expression of genes in a variety of cell types, including those which are not readily transfectable with expression plasmids (36). Amphotropic-packaging cell lines constructed with the env gene from the 4070A murine retrovirus have been used to encapsidate retroviral vector RNA (5, 24, 34), yielding viral stocks with a wide host range (4, 17, 28). The control of gene expression via transcription may be accomplished in these retroviral vector systems by using the viral long terminal repeat (LTR) (41). Alternatively, internal promoters located within the proviral sequences have been successfully exploited (27, 41). By this mechanism, several genes can be transcribed independently from a single integrated proviral genome, allowing introduction of multiple genes into a single cell. In addition, the use of retroviral vectors containing internal promoters allows efficient transcription of an introduced gene in cells in which the LTR does not function efficiently (16, 32, 40). The Moloney murine leukemia virus-derived LTR, while active in many cells, does not function well in nondifferentiated F9 cells (15, 18, 20, 40) or in some hematopoietic cells (23, 42). This appears to be due in part to the absence of necessary transcription factors (35) and possibly to the presence of negative regulatory factors (11). Introduction of an inducible metallothionein (MT) promoter (10) into a retroviral vector provides an additional level of transcriptional control. The MT promoter has been used successfully in self-inactivating vectors (43, 44), but attempts to use it in a retroviral vector with a functioning LTR have failed to show induction (23). In this study, the mouse MT-1 promoter was introduced as an internal retroviral vector promoter to control transcription of the transforming growth factor ,13 (TGF-p1) gene. TGF-P, a product of transformed as well as normal cells (2, 6, 25, 31, 37), is a multifunctional regulator of cell growth. Corresponding author. t Present address: Laboratory of Tumor Immunology and Biology, Building 10, 5B52, National Cancer Institute, Bethesda, MD *
20892.
It can induce anchorage-independent growth of fibroblasts alone or in combination with epidermal growth factor (EGF) (25, 31, 37) and can also function as a potent inhibitor of cell growth (19, 26, 29, 38). We were interested in studying the biology of cells expressing TGF-P. Our concern about the inhibitory effects of this molecule led us to place the gene for TGF-p1 under the control of the inducible mouse MT-1 promoter to prevent constitutive production of the potentially growth-inhibitory TGF-1l by infected cells, which might preclude their isolation as stable cell lines. This approach did permit the construction of cell lines expressing TGF-P, but the degree of regulation depended on the cell type into which the vector was introduced. Vector construction infection of cells, and establishment of cell lines. A cDNA clone containing the sequence for the pre-pro-human TGF-131 was isolated from human erythroleukemia cells by using specific priming. DNA sequence analysis revealed it to be essentially the same as the previously reported human TGF-1l gene (8) (data not shown). A 1.3-kilobase (kb) EcoRI-NcoI fragment of this clone was introduced into Moloney sarcoma virus-derived retroviral vector 1521 (22) immediately 3' to an internal MT-1 promoter (Fig. 1); the resulting plasmid will be referred to as 1525. A second TGF-,1l retroviral vector (1528) was constructed. It was identical to vector 1525, except that it contained a 350-base-pair fragment which carried the simian virus 40 early-region promoter (the PvuII-to-HindIII fragment) inserted 5' to the G418 resistance gene. This vector was designed to initiate a transcript internally at the simian virus 40 promoter, which would contain the G418 resistance gene as the first open reading frame on the RNA. Following transfection of plasmids 1525 and 1528 into i2 cells (21) and G418 selection, ecotropic vector-packaging lines were isolated. Amphotropic stocks of the viral vector were produced by PA317 cells (24) following infection with ecotropic-vector stocks and selection in G418 media. (Titers of ecotropic and amphotropic vector stocks on NIH 3T3 cells ranged from 1 x 105 to 5 x 105 G418-resistant colonies per ml.) Ecotropic and amphotropic stocks of retroviral vector 1525 were used to infect several established cell lines. The 3527
3528
J. VIROL.
NOTES A.
A.
1
2
3 4 5 6 7 8 9 10 11 12
!9
7 Kb
4
3 Kb40
B. [
3
SD
LTR
1525
0
LTR
pBR on
G418
MT-1j TGF,B cDNA
B.
1 2 3 4 5 6 7
[] 7
1 Kb
FIG. 1. Construction and proviral structure of plasmid vector 1525. (A) The 1.3-kb EcoRI-to-NcoI fragment from human TGF-Pl cDNA was inserted into the EcoRI site of vector 1521 following treatment of the fragment with Klenow fragment and attachment of EcoRI linkers. S.D., Splice donor; tp, packaging signal; pBR0n, pBR322 origin of replication; G418, G418 resistance gene; MT-1, mouse MT-1 promoter. (B) Proviral structure of retrovirus 1525 following infection and stable integration. The plasmid vector, referred to as 1525, now contains the gene for pre-pro-TGF-,B under the transcriptional control of the MT promoter.
normal rat kidney fibroblastic line NRK-49F and the epithelial line NRK-52E were infected with the ecotropic packaged vector 1525 stocks (7), while CHO cells were infected with amphotropic stocks of vectors 1525 and 1528. Colonies of G418-resistant cells were isolated and expanded into cell lines for further characterization. The human monocytic cell line U937 was infected with an amphotropic stock of vector 1525, and following selection of a stable population of G418-resistant cells, single-cell cloning by terminal dilution was used to generate cell lines. Induction of TGF-13-specific transcription from the MT-1 promoter. Previous studies utilizing retroviral vectors have not demonstrated induction of the MT promoters in vectors with functioning LTRs (23). However, because the TGF-,B gene is under the transcriptional control of the mouse MT-1 promoter in vectors 1525 and 1528, induciblity of transcription with heavy metals was tested. The predicted size of a TGF-1l transcript initiating at this promoter and terminating in the proviral LTR was approximately 3 kb, a size easily distinguished from other predicted transcripts by Northern (RNA) blotting. NRK-52E, NRK-49F, and U937-1525 infectants were incubated for 16 to 24 h with and without CdSO4. Northern blots of TGF-f3-specific RNA from these cells are shown in Fig. 2. In three individual NRK-52E infectants (a, b, and c), transcription from this promoter was readily induced by 5 ,uM CdSO4, as evidenced by the appearance of a 3-kb RNA (Fig. 2A). In addition, there was a shift away from transcription of the 6.5-kb full-length proviral transcript, and there was possible autoinduction of the 2.5-kb endogenous rat TGF-pl transcript and a novel 1.5-kb transcript from the rat locus (39). While 5 ,uM CdSO4 worked well as a transcriptional inducer, it was toxic to cells, and therefore induction regimens which preserved cell viability were tested. Both 1 ,uM CdSO4 and 10 mM sodium butyrate worked well as inducers of transcription from the MT-1 promoter, and they exhibit little or no toxicity for the cells (Fig. 2A, lanes 8 through 12). CdSO4 (1 ,uM) was subsequently used for induction of all 1525 infectant cell lines.
KbS-_
C. 45678910P
C.
1
7Kb _-
2 3 4 5 6 7 8 9 10 11 12
_
*-
i
-
7 Kh
3K t- -
FIG. 2. Northern blot of 1525 infectant cell line RNA. Total RNA (10 ,ug) was isolated, electrophoresed, transferred to nitrocellulose filters (22), and hybridized with a TGF-p1-specific nicktranslated probe. (A) NRK-52E cell RNA. Lane 1, NRK-52E cell; lanes 2 and 3, NRK-52E-1525(a) infectant; lanes 4 and 5, NRK52E-1525(b) infectant; lanes 6 through 12, NRK-52E-1525(c) infectant. Lanes 1, 2, 4, 6, and 8, RNA from untreated cells. Results for RNA from cells treated with 5 ,uM CdSO4 (lanes 3, 5, and 7), EGF (lane 9), 1 ,uM CdSO4 (lane 10), 5 mM sodium butyrate (lane 11), and 10 mM sodium butyrate (lane 12) are also shown. (B) NRK-49F cell RNA. Lane 1, 1525(a) infectant; lane 2, 1525(b) infectant; lane 3, 1525(c) infectant; lane 4, uninfected NRK-49F cells; lane 5, 1525(c) infectant treated with 5 mM sodium butyrate; lane 6, 1525(c) infectant treated with 1 ,uM CdSO4 for 20 h; lane 7, 1525(c) infectant treated with 1 ,iM CdSO4 for 70 h. (C) U937 cell RNA. Lanes 1 and 2, 1525(a) infectant; lanes 3 and 4, 1525(b) infectant; lanes 5 and 6, 1525(c) infectant; lanes 7 and 8, 1525(d) infectant; lanes 9 and 10, 1525(e) infectant. Lanes 1, 3, 5, 7, and 9, untreated cell RNA; lanes 2, 4, 6, 8, and 10, 1 ,uM CdSO4-treated cell RNA; lane 11, uninfected U937 cell RNA; lane 12, untreated 1525 infectant.
MT-1 TGF-,1-specific transcription was not as readily inducible in the NRK-49F-1525 infectants. Three NRK49F-1525 infectants (a, b, and c) showed constitutive transcription from this promoter (Fig. 2B, lanes 1 through 3), and one showed modest induction when incubated in CdSO4 (1 puM) for 72 h but not when incubated for 24 h (lanes 4 through 7). Hence, for all subsequent experiments utilizing this cell line, growth conditions included 1 ,uM CdSO4. In contrast, none of the five U937-1525 infectants (a, b, c, d, and e) tested exhibited constitutive transcription from the MT-1 promoter, and three of the five clones tested were inducible (Fig. 2C, lanes 1 through 10). However, all U937 infectants had a high level of the 2.5-kb endogenous TGF-pl transcript. This represents an elevated level compared with that of the uninfected control cells and may be caused by autoinduction
VOL. 64, 1990
NOTES
A.
3529
TABLE 1. TGF-P activity in serum-free mediaa s1 LTR
pBR 1528
I
_- -
V
G418
o ,1
o
418
LTR
-IY MTT II TGF. cDNA
TGF-1 Source of cells media
Incubated without CdSO4
Incubated with 1 ,uM CdSO4
Neutralization
NRK-49F NRK-49F-1525(c) NRK-52E NRK-52E-1525(c) U937 U937-1525(1)
NT NT NT 450 35 92
82.5 375 16 1,250 NT 140
NT 97.8 NT 92.1 NT 97.5
Kb
B. 1
2
3 4
5 6 7
A* 7.5 -5
A
3
Kb Kb
Kb
ft
FIG. 3. Northern blot of vector 1528-infected CHO cell RNA. (A) Structure of integrated proviral vector 1528. SV40 ori, Simian virus 40 origin of replication and early region promoter. Other abbreviations are as in Fig. 1 legend. (B) Total cellular RNA (10 ,ug) was isolated, electrophoresed, transferred to nitrocellulose filters, and hybridized with a TGF-pl-specific probe. Lane 1, Untreated 1525-U937 infectant (control for size); lane 2, 1525-U937 infectant treated with 1 ,uM CdSO4; lane 3, untreated 1525-CHO infectant; lane 4, 1525-CHO infectant treated with 1 ,uM CdSO4; lane 5, untreated 1528-CHO infectant; lane 6, 1528-CHO infectant treated with 1 p.M CdSO4; lane 7, 1528-CHO infectant treated with 5 ,uM CdSO4.
due to vector 1525 infection (Fig. 2C, lanes 11 through 12) (39). Transcription of vector 1528 in CHO cells. CHO cells were infected with amphotropic viral stocks of vector 1525. However, following G418 selection, the titer, measured as G418resistant colonies, was only 0.1% of that of the same stock plated on NRK-49F cells. In addition, the colonies isolated were unstable in G418-containing media and contained no 7-kb proviral RNA by Northern blot analysis (Fig. 3). Therefore, it appears that the block to infection was not at the level of receptors; rather, it appears that the Moloney sarcoma virus LTR did not function as a strong enhancerpromoter in these cells. Because transcription of the G418 resistance gene depends on initiation at this promoter, no cells stably producing the gene product could be isolated. In contrast to these results, infection of CHO cells with amphotropic stocks of vector 1528 resulted in the isolation of stable G418-resistant colonies, and characterization of the viral RNA in these cells revealed several TGF-31-specific species (Fig. 3). There was some 7.4-kb proviral RNA, but the amount was considerably reduced compared with that of U937-1525 infectants, suggesting less-efficient functioning of the LTR in CHO cells. In addition, there was a 5-kb internally promoted transcript as well as a 3-kb MT-1promoted transcript. The latter transcript, while constitutively present in this clone, showed a modest induction over control levels following treatment of the cells with Cd2+. Induction of TGF-,1-specific 3-kb RNA correlates with
equivalents (ng/107 cells) in samples:
a Cells were incubated for 20 h in serum-free media (previously described for NRK cells [22]; for U937 cells, RPMI 1640 with 1% Nutridoma SP [Boehringer Mannheim]) with or without 1 ,uM CdSO4. Media were dialyzed extensively against 0.2 M acetic acid, lyophilized, and suspended in phosphate-buffered saline. Samples were tested for ability to replace TGF-P in the NRK-49F agar assay (2) in the presence of 1 ng of EGF per ml and compared with a standard curve derived by using known amounts of human-plateletderived TGF-pl. Acidified, dialyzed, lyophilized serum-free conditioned media from CdSO4-induced cell lines were added to media containing EGF and 50 ,ug of anti-TGF-31 IgG or control IgG. Following incubation at 37°C, 104 NRK49F indicator cells were added and the mixture was adjusted to 0.3% Noble agar. After 10 days, the number of colonies per plate was determined and the percent neutralization was calculated. In these experiments, 93 to 99% of the human platelet-derived TGF-p1 used as a control was neutralized by this antibody. NT, Not tested.
increased TGF-01 protein production. In two of the three cell lines into which retroviral vector 1525 was introduced, transcription of TGF-,1-specific RNA could be easily induced by activation of the MT-1 promoter with CdSO4. To determine if induction of the 3-kb TGF-pl RNA resulted in an increase in the level of the TGF-pl protein produced by the cells, the amount of TGF-P activity with and without induction was measured. The biological assay used to determine the activity measures the amount of acid-activated TGF-f in serum-free conditioned media collected from infectant cells that replaces the TGF-3 added to NRK-49F cell soft-agar colony-forming assay (1). In this assay, NRK-49F cells form colonies in soft agar only when both TGF-1 and EGF are added in a dose-dependent manner. The addition of 1 ,uM CdSO4 to NRK-52E-1525 infectants resulted in an approximately threefold increase in the amount of TGF-, activity produced (Table 1). This correlates well with the increase in the level of 3-kb TGF-pl RNA in the cells. The amount of TGF-P activity present without induction presumably results from NRK-52E-1525 infectant having a lower but detectable constitutive level of transcription of the 3-kb vector RNA (Fig. 2). In the case of the U937 infectants, while the increase in TGF-P activity correlates with RNA levels prior to and following induction, the cells generally appear to be poor producers of TGF-P protein. All three cell lines produce some acid-activatable TGF-, from transcription of the endogenous gene, as indicated by the level of TGF-P produced by the uninfected control cells. However, in these experiments, the level of TGF-13 transcription from the internal MT-1 promoter is clearly reflected by the level of TGF-,B activity produced by the cells. To determine if the agar colony-promoting activity was due to TGF-pl, the acidified conditioned media collected from all the 1525 infectants following induction were tested for neutralization by antibody directed against TGF-pi (R & D Systems). This antibody, prepared as an immunoglobulin G (IgG) fraction, was made against porcine TGF-i1 and has neutralizing activity for human, rodent, and porcine TGFp1. Samples of acidified conditioned media from 1525 infec-
3530
J. VIROL.
NOTES
TABLE 2. Growth of 1525-NRK-49F cells in agar No. of % of total Cell type' Addition(s)b colonies
NRK-49F
1525-NRK-49F
colonies
EGF EGF + TGF-P
5,880
0 3.2 100.0
EGF EGF + TGF-P
0 520 6,120
0 8.4 100.0
0 192
a 104 control or vector infectant cells seeded per plate (2) in the presence of 1 F.M CdSO4. b EGF and TGF-3 were each added at 1 ng/ml. -, No addition.
tants were tested for promotion of NRK-49F cell agar colony formation in the presence of EGF following preincubation with 50 ,ug of anti-TGF-pl IgG or nonimmune IgG. The results in Table 1 indicate a high level of neutralization of the TGF-P activity produced by NRK and U937-1525 infectant cell lines, suggesting that almost all the activity produced by the infectants following induction is due to TGF-p1 production.
Biological consequence of vector 1525-induced TGF-pi1 production by NRK-49F cells. The majority of the TGF-pl produced and secreted by many cell types is an inactive precursor that is complexed to another protein(s) and that can be activated by several mechanisms to produce the mature molecule. The biologically active form of human TGF-,1l appears to be a homodimer of 12,000-dalton polypeptides linked by disulfide bonds to form the 24,000-dalton, biologically-active "mature form" of the protein (3, 12, 29). DNA sequence analysis of TGF-pi cDNA clones predicts that the 12,000-dalton subunit consists of a 112-amino-acid polypeptide cleaved from the C terminus of a 390-amino-acid precursor, pre-pro-TGF-pl (8, 9, 33). Introduction of the cDNA for pre-pro-TGF-pl into CHO cells by transfection resulted in the production of a 95- to 120-kilodalton precursor complex comprising mature TGF-pl, pro-TGF-f31, and the pro region of the precursor (13, 14). The majority of material produced by CHO cells appears to be identical to that isolated from human platelets, the most concentrated source of human TGF-P (3). However, a small fraction of the TGF-pl produced by these cells is in the form of the biologically active 24-kilodalton mature protein (13, 14). The NRK-49F-1525 infectants were further analyzed to determine if the TGF-pl protein produced is biologically active in an autocrine fashion. As stated above, NRK-49F cells form colonies in soft agar in the presence of exogenously added EGF and TGF-P (1). Therefore, to determine if the TGF-pl produced by the NRK-49F infectants could function in an autocrine fashion and replace the requirement for exogenously added TGF-P in this assay, Cd2+-induced infectants and control cells were seeded in soft agar in the presence of EGF only. The results indicate that approximately 8% of the cells seeded in soft agar in the presence of EGF formed colonies, compared with 3% of the uninfected NRK-49F cells (Table 2). While the 1525 infectants produce approximately four times as much TGF-pi (Table 1) as the control cells, the majority of it is in an inactive form. However, the ability of the 1525 infectants to respond in this assay suggests that there is a sufficient amount of active material to drive a low-level autocrine stimulation. In conclusion, insertion of TGF-pl cDNA into a retroviral vector under the control of an inducible promoter has allowed introduction of the gene in a regulatable state into a
wide variety of cell types (i.e., fibroblastic, epithelial, and monocytic). While previous studies have indicated that MT promoters function in retroviral vectors in which the LTR is transcriptionally inactivated (43, 44), our experiments demonstrate that the MT-1 promoter can be regulated in vectors with functional LTRs as well. Thus, the LTR can be used to drive transcription of the selectable marker gene whose constitutive production is required, while the regulatable promoter drives transcription of a gene whose expression should be tightly controlled. The regulatability of the MT-1 promoter varied with the cell type but could be well controlled in two of the four cell types. While the data presented in this paper represent only a relatively small number of individual clones, overall we found that the MT-1 promoter was tightly regulated by Cd2" in >50% of the U937 infectants and 70% of the epithelial infectants analyzed. In the other two cell lines, CHO and NRK-49F, transcription from this promoter was increased by physiologically tolerated levels of Cd2". The efficiency of translation and secretion of the protein varied independently of promoter regulation. In one cell line, NRK-49F, in which a biological effect by TGF-P production could be readily measured, the results of autocrine stimulation were detectable. Recently this vector system has been used to study regulated gene expression in rodent and human mammary epithelial cells (M. L. McGeady, unpublished data). Hence, it appears that a vector of this nature, able to infect many cell types and containing a regulatable promoter, should be useful for the study of growth factors and other proteins whose constitutive expression alters cell growth in such a way as to preclude isolation of stable producer cells. LITERATURE CITED 1. Anzano, M., A. Roberts, C. Myers, A. Komoriya, L. Lamb, J. Smith, and M. Sporn. 1982. Synergistic interaction of transforming growth factors from murine sarcoma cells. Cancer Res. 42:4776-4778. 2. Anzano, M., A. Roberts, J. Smith, M. Sporn, and J. DeLarco. 1983. Sarcoma growth factor from conditioned medium of virally transformed cells is composed of both type a and type ,B transforming growth factors. Proc. Natl. Acad. Sci. USA 80: 6264-6268. 3. Assoian, R., A. Komoriya, C. Meyers, and M. Sporn. 1983. Transforming growth factor , in human platelets: identification of a major storage site, purification, and characterization. J. Biol. Chem. 258:7155-7160. 4. Cloyd, M., M. Thompson, and J. Hartley. 1985. Host range of mink cell focus-inducing viruses. Virology 140:239-248. 5. Cone, R. D., and R. C. Mulligan. 1984. High efficiency gene transfer into mammalian cells: generation of helper free recombinant retrovirus with broad mammalian host range. Proc. Natl. Acad. Sci. USA 81:6349-6353. 6. DeLarco, J., and G. Todaro. 1978. Growth factors from murine sarcoma virus transformed cells. Proc. Natl. Acad. Sci. USA 75:4001-4005. 7. DeLarco, J., and G. Todaro. 1978. Epithelioid and fibroblastic rat kidney cell clones: epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation. Journal of Cell. Physiol. 94:335-342. 8. Derynck, R., J. A. Jarrett, E. Y. Chen, D. H. Eaton, J. R. Bell, R. K. Assoian, A. B. Roberts, M. B. Sporn, and D. V. Goeddel. 1985. Human transforming growth factor-p complementary DNA sequence and expression in normal and transformed cells. Nature (London) 316:701-705. 9. Derynck, R., J. A. Jarrett, E. Y. Chen, and D. V. Goeddel. 1986. The murine transforming growth factor p precursor. J. Biol. Chem. 261:4377-4379. 10. Durnam, D. M., F. Perrin, F. Gannon, and R. D. Palmiter. 1980.
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NOTES
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