Molecular and Cellular Endocrinology, 89 (1992) 19-24 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/92/$05.00
19
MOLCEL 02843
Activity of the CYPI7 promoter in bovine adrenocortical before and after phenotypic switching Deepak S. Lala Department
’
cells
and Peter J. Hornsby
of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA (Received 27 May 1992; accepted 6 July 1992)
Key words: Adrenocortical
cell; Steroid 17a-hydroxylase; Senescence; Phenotypic switching; Gene expression; Transfection
Summary Cultured bovine adrenocortical cells reach replicative senescence after loo-120 population doublings in culture. Before reaching senescence, cells undergo high frequency phenotypic switching from CYP17 + to CYP17 - , where ‘ + ’ and ‘ - ’ refer to the ability of intracellular cyclic AMP to induce expression of CYP17 (steroid 17&-hydroxylase). We used luciferase reporter constructs to assess the activity of the CY?‘l7 promoter in bovine adrenocortical cells before and after phenotypic switching. We constructed two plasmids containing - 2544 to + 29 and - 488 to + 29 of the 5’ region of cYPl7 linked to a promoterless luciferase gene. Because of technical difficulties with transient transfection of late-passage bovine adrenocortical cells, these experiments were performed using stable transfection. Cells at early passage (PDL 10) and late passage (PDL 55) were cotransfected with either of these two plasmids ligated to pSV3neo, and G418-resistant pools of clones were derived. The activity of the 07’17 promoter in these transfectants was tested by growing cells in complete medium until semiconfluent and then transferring them into defined medium with cholera toxin and insulin-like growth factor I for 6 h. Luciferase activity was consistently induced by cholera toxin/IGF-I over five passages in pooled clones derived by transfection of early passage cells with the -488 construct. Despite the lack of expression of the endogenous CYP17 gene in transfectants from late-passage cells, induced luciferase activity was higher in late-passage transfectants than early-passage transfectants for both the -2544 and -488 constructs. However, the -2544 construct had a lower response to cholera toxin than the -488 construct in cells from both PDLs, indicating the potential existence of a negative regulatory element. The lower expression of the -2544 construct was confirmed in assays on individual clones of cells as well as pooled clones. These data indicate that the loss of cyclic AMP-inducible CYP17 expression during bovine adrenocortical cell senescence likely does not result from a loss of positive regulatory factors that act within 2.5 kb upstream of the CYP17 promoter.
Introduction Cultured bovine adrenocortical cells are capable of continuous proliferation for loo-120 population doublings before reaching replicative senescence (Hornsby et al., 1989, 1990). During long-term growth there are
Correspondence to: Peter J. Hornsby, Huffington Center on Aging, M320, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030; USA. This work was supported by grant AG 06108 from the National Institute on Aging. ’ Present address: Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
changes in the expression of differentiated function genes, particularly CYP17 (steroid 17a-hydroxylase). Cells undergo high frequency phenotypic switching from CYP17 + to CYP17 - , where ‘ +’ and ‘ -’ refer to the ability of intracellular cyclic AMP to induce CYP17 expression. Phenotypic switching is dependent on cell division (Yang and Hornsby, 1989) and computer modelling of the phenomenon is consistent with a frequency of switching of 3% to 6% per generation (Hornsby and Yang, 1991). Other work shows that phenotypic switching can be dissociated on a cell-by-cell basis from replicative senescence (Yang and Hornsby, 1989). Transcription of CYPl7 is cell-type specific and shows an absolute dependence on prior activation of
cyclic AMP-dependent protein kinase (John et al., 1986; Zuber et al., 1986; Brentano et al., 1990; Lund et al., 1990; Zanger et al.. 1991). Analysis of the CYP17 promoter shows that at least two regions of the gene, - 243/ - 225 and -8O/ - 40, can confer a tissucspecific cyclic AMP response to a reporter gene in mouse adrenal tumor Yl cells (Lund et al., 1990; Zanger et al., 1991). These two regions show little sequence similarity to each other or to cyclic AMP response elements found in other genes. Gel mobility shift assays indicate that the -243/225 sequence binds a novel uncharacterized protein that is presumably involved in the cyclic AMP response of the gene (Lund et al., 1990; Zanger et al., 1991). Changes in the amount or activity of such required regulatory proteins could account for the loss of CYP17 expression in phenotypic switching. Such changes would be expected to be reflected in a lower activity of the CYP17 promoter after phenotypic switching. Thus in order to investigate the possibility of changes in such factors we constructed plasmids in which the CYPZ7 promoter is linked to a reporter gene, luciferase. Although no more than 300 bp of 5’ sequence is adequate for cyclic AMP-regulated reporter gene expression and the expression of such promoter constructs is restricted to steroidogenic cells, the possibility of more distal 5’ information being necessary for cell specificity or developmental timing of CYPZ7 expression is open, particularly since the analysis of the promoter has been carried out in a cell line (Yl) which does not express its endogenous CYP17 gene. Suggestive evidence for the existence of more distal regulatory elements in CYPl7 is provided by the finding that there are tissue-specific differences in DNA methylation of the - 1800 to -2500 bp region of the gene and that specific sites in this region show demethylation in bovine adrenocortical cells when the cells are placed in culture (Hornsby et al., 1992). Because of these considerations, two fusion gene constructs were made by placing either - 488 to + 29 or - 2544 to +- 29 bp of the 5’ regulatory region of the CYP17 gene in front of a promoterless firefly luciferase gene. These constructs were then used to assess the activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching by transfection into CYP17 + and CYP17 - cells and assaying cyclic AMP-dependent luciferase activity. Materials
and methods
CYPl74uciferase constructs Plasmids containing fragments of CYP17 5’ region linked to a promoterless luciferase gene were constructed as follows. As previously described (Hornsby et al., 1992), oligonucleotides corresponding to -488 to - 462 and + 29 to + 2 of bovine CYPl7Al (Bhasker et al., 1989) were used to amplify a 520 bp fragment by
the polymcrase chain reaction (PCR) with bovine DNA as template. The primers wcrc synthesized to contain sites for restriction enzymes to facilitate subcloning of the PCR-amplified product. The PCR-amplified product was digested with &WI and Snll and subcloned into plBI31. The insert in this plasmid had a sequence consistent with that previously reported (Bhasker ct al., 1989). The insert was removed using Sac.1 and XCroII and was cloned into the lucifcrase expression plasmid. pXP2 (Nordeen, 1988). The thymidine kinase promoter was removed from the pXP2 derivative pT109luc with Sac1 and &III and replaced with the 517 bp CYPI7 promoter to give pXP2-CYP17( -48X). In order to investigate the potential role of elements upstream of -488 in the bovine CYPl7 gene, a CYPl7-luciferase plasmid with - 2544 to + 29 of the 5’ region was constructed as follows. As previously described (Hornsby et al., 1992). a bovine genomic library was screened using CYPZ7 cDNA. The - 11 kb Sal IEcoRI insert of a hybridizing A clone, containing the entire coding region and 2.5 kb of 5’ flanking region of CYPZ7A2, was subcloned into pBluescript II KS. The 5’ region was isolated as a SalI-StuI fragment ( - 2544 to - 19) and was used to replace the sequence -488 to - 19 in pXP2-CYP17( - 4881, which was removed by digestion with Sal1 and StclI, giving pXP2-CYP17( - 2544). Growth of hor*ine adrenocortical cells Adrenocortical cells were prepared from adrenal cortex tissue (zona fasciculata-reticularis) from 2-yearold steers by collagenase/DNase digestion, as previously described (Gospodarowicz et al., 1977). Cells were stored frozen in 5% dimethyl sulfoxide until required for experiments. Frozen cells were thawed and plated in culture dishes coated with fibronectin. Cells were grown in a 1 : 1 mixture of Dulbecco’s Eagle’s medium (DME) and Ham’s F-12 (F-12) medium with 2% fetal bovine serum (Irvine Scientific, Irvine, CA, USA), 10% horse serum (Sigma Chemical Co., St. Louis, MO, USA), 1 ng/ml of recombinant basic fibroblast growth factor (Imcera), 20 nM selcnite, 1 PM a-tocopherol, and antibiotics. The gas phase used was 5% 02, 90% N,, and 5% CO,. Subculturing was performed by incubation with Pronase E (neutral protease type XIV, Sigma) in serum-containing medium using a 1 : 5 split ratio. Cumulative population doubling levels (PDL) were calculated as described previously (Hornsby and Harris, 1987). Cotransfection with CYPI 7-luciferase constructs and pS V3neo CYPl7-luciferase constructs were cotransfected with plasmid pSV3neo (Southern and Berg, 1982), containing part of the early region of SV40 virus and the bacterial neo gene. Clones derived by transfection of
bovine adrenocortical cells with this plasmid have been described previously (Cheng et al., 1989). Plasmids to be cotransfected were first ligated to each other to form long concatemers. Both plasmids were linearized in the p-lactamase region with blunt-end producing restriction enzymes and were ligated (in a 3 : 1 ratio of luciferase construct to pSV3neo) using T4 DNA ligase (2 units/pug DNA) in the presence of 15% polyethylene glycol 8000 for l-2 h at room temperature. The reaction was stopped by precipitation with ammonium acetate/ethanol and the ligated material was pelleted by centrifugation. The resultant high molecular weight DNA was introduced into bovine adrenocortical cells grown in complete medium to 50-60% confluence by electroporation (Cheng et al., 1989) or Lipofectin (Gibco/BRL) (Hornsby and Salmons, 1992) using 20 pg DNA per 10 cm dish. Cells were maintained in normal complete medium for 24 h before transfer into medium containing 200 pg/ml G418 (Gibco/BRL). After 14 days incubation with G418, surviving cells were replated and grown in mass culture (1: 5 split ratio) in the continuous presence of G418. Alternatively, after 14 days incubation with G418, surviving cells were replated at low density. Clones were allowed to grow for 10 days, isolated with cloning rings, and grown up to an approximate area of 50 cm2 (equivalent to 25 population doublings). Clones were studied within the next six passages beyond this point. Luciferase assay
Cells were grown in 35 mm plates in complete medium with G418 until almost confluent and then changed to defined serum-free medium comprising DME/F-12 1: 1, 50 pg/ml bovine serum albumin (BSA), 2 mM ascorbic acid, 20 nM selenite, 1 PM cy-tocopherol, with optional addition of 10 nM recombinant insulin-like growth factor I (IGF-I) (Imceral and/or 1 nM cholera toxin. After a 6 h incubation, cells were harvested for assay of luciferase using scintillation counting as previously described (Nguyen et al., 1988). Cells were washed twice with cold phosphatebuffered saline and scraped in 200 ~1 of lysis buffer comprising 25 mM potassium phosphate pH 7.8, 8 mM Mg ‘+ 7 1 mM dithiothreitol, 1 mM EDTA, 1% Triton X-100, 1% BSA, and 15% glycerol. The suspension was centrifuged at 10,000 rpm for 5 min. 50 ~1 of the supernatant was then added to 200 ~1 of reaction mixture containing 0.2 mM luciferin and 0.8 mM ATPMg2+ in 100 mM potassium phosphate buffer pH 7.8. Light emission was determined by placing the tube in a glass vial in a scintillation counter used in normal counting mode. Light production as (cpm)1/2 was converted to pg luciferase using a standard curve constructed with various amounts of pure luciferase (Sigma). DNA was measured by Hoechst 33258 fluo-
rescence (Labarca and Paigen, 19801 on the nuclear pellet dissolved in 50 mM NaOH. 17mHydroxylase assay
For induction of the endogenous CYPl7 gene, cells were grown to semi-confluence in complete medium with G418. Cells were then incubated in defined medium containing 10 nM IGF-I with or without 1 nM cholera toxin for 72 h. At the end of this period, cells were incubated with 10 PM progesterone substrate in defined medium for 2 h. Steroids were extracted from the medium with dichloromethane. Product 17cY-hydroxyprogesterone was separated from progesterone by high performance liquid chromatography using elution with a linear methanol : water gradient (20%-100% over 10 min). Detection was by absorbance at 240 nm. The identity of ultraviolet -absorbing peaks was established by comparison of retention times of authentic standards. Conversion rates were calculated by comparing peak areas for substrate and product with peak areas of known amounts of standards, calculating percent conversion to product, and converting percent conversion to pmol from the known amount of precursor added. Conversion rates were expressed as pmol/105 cells/h. Results
An overview of these experiments is shown in Fig. 1. Because of technical difficulties with transient transfection of later-passage bovine adrenocortical cells, these experiments were performed using stable transfection and pools of clones. Previously, we showed that this cell type was amenable to stable transfection with relatively high efficiency (Cheng et al., 1989). Cells at
Fig. 1. Overview of experiments on the activity of the cyp17 promoter in cultured bovine adrenocortical cells before and after phenotypic switching. The decline in the expression of GYP17 is shown with respect to the population doubling level and diagrammatically represents both the total population 07’17 mRNA level after cyclic AMP induction and also the percentage of CYP17+ cells in the population assessed by in situ hybridization (data from Hornsby et al., 1987; Ryan et al., 1989). Transfection of CYP17-luciferase constructs with pSV3neo, selection of pooled clones in G418, and assay of cholera toxin-induced luciferase was performed at PDL 10 and PDL 55, when most cells are CYP17 + and CYP17 - respectively.
22
early passage (PDL 10) and a later passage (PDL 55) were cotransfected with CYP17-luciferase constructs ligated to pSV3neo and transfectants were selected in G418. At PDL 10 most cells are CYP17 + and at PDL 55 almost all cells are CYP17 - . Prior work established that clones of bovine adrenocortical cells produced by transfection of pSV3neo show vigorous growth and that such clones express CYP17 if the cells of origin were capable of expression of CYPZ7 or do not express the gene if the precursor cells had undergone phenotypic switching prior to transfection (Cheng et al., 1989). Preliminary experiments were performed to assess the characteristics of bovine adrenocortical cells transfected with CYPI 7-luciferase constructs. After transfection of the - 488 CYP17-luciferase construct in cells at PDL 10, the culture was incubated with 200 pg/ml of G418 for 10 days. The cells were then subcultured; no nontransfected cells remained at this time. The pooled transfectant clones were grown in mass culture in the continued presence of G418 and the induction of luciferase was assayed. Luciferase activity was increased by agents that increase intracellular cyclic AMP, such as cholera toxin, and by cyclic AMP analogs. Cell type specificity was demonstrated by transfecting bovine kidney fibroblasts, which do not normally express CYPZ7, with the same construct. In this case, luciferase activity was very low and unresponsive to cholera toxin (data not shown). Other preliminary experiments showed that IGF-I was required for efficient induction of transfected luciferase in defined medium. IGF-I increased the level of luciferase activity in bovine adrenocortical cells transfected with the - 488 CYPZ7luciferase construct. However, it had similar effects in bovine adrenocortical cells stably transfected with luciferase under the control of other promoters (herpes simplex thymidine kinase and SV40) whereas these promoters did not respond to cyclic AMP (data not shown). Thus the IGF-I effect probably represents a non-promoter-specific effect on luciferase activity, perhaps by increasing the half-life of luciferase, which is known to be very short in mammalian cells (Thompson et al., 1991). In the presence of IGF-I, maximum induction of luciferase by cholera toxin was observed 6 h after the addition of cholera toxin. Having established the cell-type specificity of the cyclic AMP response for the -488 CYP17-luciferase construct, and having established standard conditions for testing promoter activity (defined medium, IGF-I, cholera toxin), we performed additional experiments to assess the reproducibility of induction of the CYPI 7 promoter by cholera toxin. Pooled transfected cells were grown in mass culture with a 1: 5 split ratio at subcultures. At each passage, cholera toxin induction of luciferase was assayed. Fig. 2 shows that although absolute levels of luciferase activity showed a tendency an
-
-
passage 1
passage 2
passage 3
passage 4
passage 5
Fig. 2. Stability of expression from the transfected C’YPI7 promoter in bovine adrenocortical cells. Cells at PDL 10 were cotransfected with pXP2-CYP17(-488) and pSV3neo as described in Materials and methods. Passage 1 indicates cells subcultured after the initial 14-day selection in G418. Passages 2-5 were made by continued growth of the cells in complete medium with G418 with I : 5 subcultures. At each passage, semi-confluent cells were incubated in defined medium with addition of 10 nM IGF-I and 1 nM cholera toxin. After 6 h, cells were harvested for assay of luciferase activity.
to vary between passages, at each passage cholera toxin increased luciferase activity 2- to 4-fold above the level observed with IGF-I alone. The promoter activity of CYP17-luciferase constructs in cells transfected at PDL 10 was then compared with that in cells transfected at PDL 55. Fig. 3 shows that cells at PDL 10 and PDL 55 transfected with the -488 construct demonstrated induction of luciferase by 2- to 3-fold and 7- to %fold, respectively, when intracellular levels of cyclic AMP were raised. Furthermore, there was a 2-fold induction of the - 2544 construct in cells transfected at PDL 55 whereas in PDL 10 cells, although luciferase was expressed from this construct, it was not cholera toxin responsive. In cells transfected with CYPI7-luciferase constructs expression of luciferase was compared with expression of endogenous CYPI 7. As anticipated from previous data from cells at these PDLs, cells transfected at PDL 10 had cholera toxin-inducible 17a-hydroxylase whereas cells transfected at PDL 55 did not (Fig. 4). An additional experiment was performed to confirm that the apparently lower expression of the -2544 construct versus the -488 construct was reflected in individual clones of cells as well as pools. Southern
23
1
.’ PDL 10 \
PDL 55 ,
PDL 10 L
” pxPz-cYP17(-488)
PDL 55 I
Y pxP2-CYP17(-2544)
Fig. 3. Promoter activity of fusion genes containing different lengths of the 5’ flanking region of ClTl7 linked to luciferase in bovine adrenocortical cells before and after loss of endogenous 07’17 expression by phenotypic switching. Luciferase activity was measured following a 6 h incubation in defined medium with 10 nM IGF-I and optionally 1 nM cholera toxin. Data are derived from triplicate plates of each pool (mean* SE). Two separate transfections to yield two separate pools of clones were performed at each PDL with the -488 construct (indicated as ‘1’ and ‘2’).
blotting showed that -2544 transfectants did not have a lower copy number of the CYPl7-luciferase plasmid than the -488 transfectants (not shown). There was
1 + h
c
4
Cl IGIJ IGF choleratoxin
ekme 1 \
II clone 2
clone
3 I
pXWCYP17(-488)
Cl”“F 1 L
clone 2 Y
clone 3 I
pXp2-CYP17(-2544)
Fig. 5. Activity of the CYPl7 promoter in individual clones of PDL 10 cells transfected with pXP2CYP17( - 488) or pXP2-CYP17(-2544). Three clones from each transfected pool were isolated at random. The induction of luciferase by cholera toxin was investigated as described in the legend to Fig. 3. Data are derived from triplicate plates of each clone (mean f SE).
consistent cholera toxin induction of luciferase activity in individual clones derived by transfection of earlypassage cells with the -488 construct and relatively weak or no induction in clones with the -2544 construct (Fig. 5). Clones carrying the -2544 construct showed the expected induction of 17cy-hydroxylase activity by cholera toxin (not shown). Moreover, all clones examined, with either construct, showed luciferase activity, demonstrating the efficiency of the plasmid ligation technique used for cotransfection. Discussion
PDL 10
PDL 55
PDL 10
PDL 55
WV pXP2-CYP17(-488)
pXP2-CYP17(-2544)
Fig. 4. 17a-Hydroxylase activity in different transfected cell populations. Cells at PDL 10 and PDL 55 were transfected with CYPl~luciferase constructs as described in the legend to Fig. 3. 17cu-Hydroxylase activity was assayed under the conditions used in that experiment, except that the incubation with cholera toxin was extended to 72 h. The individual values and means from duplicate plates are shown.
These data indicate that phenotypic switching from CYP17 - to CYP17 + in bovine adrenocortical cells as they senesce in culture does not result from a simple loss of regulatory factors that act within 2.5 kb of the CW17 promoter. For both the - 488 and - 2544 CYP17-luciferase constructs, induced luciferase activity was actually higher in later-passage than in early-passage cells. Apparently, positive transcription factors acting within - 2544 to + 29 of the CYP17 gene must be present in cells that are no longer able to express endogenous CYH7. CYP17 - bovine adrenocortical cells thus resemble the Yl mouse adrenocortical tumor cell line, which expresses CYP17 reporter constructs but not its endogenous Cyp17 gene. The Cyp17 con-
‘4
structs used in the Y 1 cell studies, like those constructed for these experiments, show cell type specificity and induction by cyclic AMP (Brentano et al., 1990; Lund et al., 1990). Other events, such as changes in DNA methylation, could be responsible for phenotypic switching. Although loss of promoter activity might be expected to be associated with hypermethylation, the CYP17Al and CYPl7A2 genes show specific demethylation in bovine adrenocortical cells soon after being placed in culture (Hornsby et al., 1992). The methylation status of the CYP17 genes was assessed by Southern blotting of DNA isolated at various times after placing the cells in culture and digested with methylation-sensitive enzymes. The data showed that whereas one CpG site at - 1800 is always methylated in adrenocortical cells, two other sites at - 2150 and - 2300 which are methylated in the adrenal cortex in vivo become demethylated when adrenocortical cells are placed in culture (Hornsby et al., 1992). Moreover, the site at - 1800 is nonmethylated in fibroblasts, which do not express endogenous CYPl7 or CYPl74uciferase constructs. The present data suggest that the region -500 to - 2500 of CYPI 7 contains a negative regulatory element. Sequence analysis indicates that the region between - 300 to - 1800 consists of several of repeats that are common in bovine genes, whereas the region that shows tissue-specific hypomethylation does not (Hornsby et al., 1992). Further analysis of the -500 to - 2500 region will be required to confirm the existence
of a negative regulatory element, its potential role in phenotypic switching, and the significance of the specific changes in DNA methylation of CYP17 in cultured bovine adrenocortical cells.
References Bhasker, C.R., Adler. B.S., Dec. A., John, M.F_, Kagimoto, M., Zuber, M.X.. Ahlgren. R., Wang, X.. Simpson. E.R. and Waterman, M.R. (1989) Arch. B&hem. Biophys. 271, 479-487. Brentano, ST., Picado-Leonard, J.. Mellon, S.H.. Moore. C.C. and Miller, W.L. (1990) Mol. Endocrinol. 4, lY722 1974. Cheng, C.Y., Ryan, R.F., Vo. T.P. and Hornsby. P.J. tlYX9) Exp. Cell Res. 180. 49-62. Gospodarowicz, D., Ill, C.R. Hornsby. P.J. and Gill. G.N. (1977) Endocrinology 100, 10X0-1089. Hornsby, P.J. and Harris, SE. (1987) Exp. Cell Res. 168, 2033217. Hornsby, P.J. and Salmons, B. (1992) in Cell and Tissue Culture: Laboratory Procedures (Griffiths, J.B., Doyle. A. and Newell. D.G., eds.), Wiley, Chichester (in press). Hornsby, P.J. and Yang, L. (1991) Mech. Ageing Dev. 58, l-12. Hornsby. P.J., Hancock, J.P., Vo, T.P., Nason, L.M., Ryan, R.F. and McAllister. J.M. (1987) Proc. Natl. Acad. Sci. USA 84, 1580-1584. Hornsby, P.J., Ryan, R.F. and Chcng, C.Y. (1989) Exp. Gerontol. 24. 539-558. Hornsby, P.J., Cheng, C.Y., Ryan. R.F. and Yang, L. (lY90) in Molecular Biology of Aging (Finch, C.E. and Johnson, T.E., eds.), pp. 249-263, Wiley-Liss, New York. Hornsby, P.J., Yang, L., Raju, S.G., Maghsoudlou, S.S., Lala, D.S. and Nallaseth, F.S. (1992) DNA Cell Biol. II, 385-395. John, M.E., John, M.C., Boggaram, V.. Simpson, E.R. and Waterman, M.R. (1986) Proc. Natl. Acad. Sci. USA 83, 47154719. Labarca, C. and Paigen, K. (1980) Anal. Biochem. 102, 344-352. Lund, J., Ahlgren, R., Wu, D., Kagimoto, M., Simpson, E.R. and Waterman, M.R. (1990) J. Biol. Chem. 265, 3304-3312. Nguyen. V.T., Morange, M. and Bensaude, 0. (1988) Anal. Biochem. 171) 404-408. Nordeen, S.K. (1988) BioTechniques 6, 454-457. Ryan, R.F., Hancock, J.P., McDonald, J.J. and Hornsby, P.J. (1989) Exp. Cell Res. 180, 36-48. Southern, P.J. and Berg, P. (1982) J. Mol. Appl. Genet. I, 327-341. Thompson, J.F., Hayes, L.S. and Lloyd, D.B. (1991) Gene 103, 171-177. Yang, L. and Hornsby, P.J. (1989) J. Cell Sci. 94, 757-768. Zanger, U.M., Lund, J., Simpson, E.R. and Waterman, M.R. (1991) J. Biol. Chem. 266, 11417-11420. Zuber, M.X., John, M.E., Dkamura, T., Simpson, E.R. and Waterman, M.R. (1986) J. Biol. Chem. 261, 2475-2482.
19
MOLCEL 02843
Activity of the CYPI7 promoter in bovine adrenocortical before and after phenotypic switching Deepak S. Lala Department
’
cells
and Peter J. Hornsby
of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA (Received 27 May 1992; accepted 6 July 1992)
Key words: Adrenocortical
cell; Steroid 17a-hydroxylase; Senescence; Phenotypic switching; Gene expression; Transfection
Summary Cultured bovine adrenocortical cells reach replicative senescence after loo-120 population doublings in culture. Before reaching senescence, cells undergo high frequency phenotypic switching from CYP17 + to CYP17 - , where ‘ + ’ and ‘ - ’ refer to the ability of intracellular cyclic AMP to induce expression of CYP17 (steroid 17&-hydroxylase). We used luciferase reporter constructs to assess the activity of the CY?‘l7 promoter in bovine adrenocortical cells before and after phenotypic switching. We constructed two plasmids containing - 2544 to + 29 and - 488 to + 29 of the 5’ region of cYPl7 linked to a promoterless luciferase gene. Because of technical difficulties with transient transfection of late-passage bovine adrenocortical cells, these experiments were performed using stable transfection. Cells at early passage (PDL 10) and late passage (PDL 55) were cotransfected with either of these two plasmids ligated to pSV3neo, and G418-resistant pools of clones were derived. The activity of the 07’17 promoter in these transfectants was tested by growing cells in complete medium until semiconfluent and then transferring them into defined medium with cholera toxin and insulin-like growth factor I for 6 h. Luciferase activity was consistently induced by cholera toxin/IGF-I over five passages in pooled clones derived by transfection of early passage cells with the -488 construct. Despite the lack of expression of the endogenous CYP17 gene in transfectants from late-passage cells, induced luciferase activity was higher in late-passage transfectants than early-passage transfectants for both the -2544 and -488 constructs. However, the -2544 construct had a lower response to cholera toxin than the -488 construct in cells from both PDLs, indicating the potential existence of a negative regulatory element. The lower expression of the -2544 construct was confirmed in assays on individual clones of cells as well as pooled clones. These data indicate that the loss of cyclic AMP-inducible CYP17 expression during bovine adrenocortical cell senescence likely does not result from a loss of positive regulatory factors that act within 2.5 kb upstream of the CYP17 promoter.
Introduction Cultured bovine adrenocortical cells are capable of continuous proliferation for loo-120 population doublings before reaching replicative senescence (Hornsby et al., 1989, 1990). During long-term growth there are
Correspondence to: Peter J. Hornsby, Huffington Center on Aging, M320, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030; USA. This work was supported by grant AG 06108 from the National Institute on Aging. ’ Present address: Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
changes in the expression of differentiated function genes, particularly CYP17 (steroid 17a-hydroxylase). Cells undergo high frequency phenotypic switching from CYP17 + to CYP17 - , where ‘ +’ and ‘ -’ refer to the ability of intracellular cyclic AMP to induce CYP17 expression. Phenotypic switching is dependent on cell division (Yang and Hornsby, 1989) and computer modelling of the phenomenon is consistent with a frequency of switching of 3% to 6% per generation (Hornsby and Yang, 1991). Other work shows that phenotypic switching can be dissociated on a cell-by-cell basis from replicative senescence (Yang and Hornsby, 1989). Transcription of CYPl7 is cell-type specific and shows an absolute dependence on prior activation of
cyclic AMP-dependent protein kinase (John et al., 1986; Zuber et al., 1986; Brentano et al., 1990; Lund et al., 1990; Zanger et al.. 1991). Analysis of the CYP17 promoter shows that at least two regions of the gene, - 243/ - 225 and -8O/ - 40, can confer a tissucspecific cyclic AMP response to a reporter gene in mouse adrenal tumor Yl cells (Lund et al., 1990; Zanger et al., 1991). These two regions show little sequence similarity to each other or to cyclic AMP response elements found in other genes. Gel mobility shift assays indicate that the -243/225 sequence binds a novel uncharacterized protein that is presumably involved in the cyclic AMP response of the gene (Lund et al., 1990; Zanger et al., 1991). Changes in the amount or activity of such required regulatory proteins could account for the loss of CYP17 expression in phenotypic switching. Such changes would be expected to be reflected in a lower activity of the CYP17 promoter after phenotypic switching. Thus in order to investigate the possibility of changes in such factors we constructed plasmids in which the CYPZ7 promoter is linked to a reporter gene, luciferase. Although no more than 300 bp of 5’ sequence is adequate for cyclic AMP-regulated reporter gene expression and the expression of such promoter constructs is restricted to steroidogenic cells, the possibility of more distal 5’ information being necessary for cell specificity or developmental timing of CYPZ7 expression is open, particularly since the analysis of the promoter has been carried out in a cell line (Yl) which does not express its endogenous CYP17 gene. Suggestive evidence for the existence of more distal regulatory elements in CYPl7 is provided by the finding that there are tissue-specific differences in DNA methylation of the - 1800 to -2500 bp region of the gene and that specific sites in this region show demethylation in bovine adrenocortical cells when the cells are placed in culture (Hornsby et al., 1992). Because of these considerations, two fusion gene constructs were made by placing either - 488 to + 29 or - 2544 to +- 29 bp of the 5’ regulatory region of the CYP17 gene in front of a promoterless firefly luciferase gene. These constructs were then used to assess the activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching by transfection into CYP17 + and CYP17 - cells and assaying cyclic AMP-dependent luciferase activity. Materials
and methods
CYPl74uciferase constructs Plasmids containing fragments of CYP17 5’ region linked to a promoterless luciferase gene were constructed as follows. As previously described (Hornsby et al., 1992), oligonucleotides corresponding to -488 to - 462 and + 29 to + 2 of bovine CYPl7Al (Bhasker et al., 1989) were used to amplify a 520 bp fragment by
the polymcrase chain reaction (PCR) with bovine DNA as template. The primers wcrc synthesized to contain sites for restriction enzymes to facilitate subcloning of the PCR-amplified product. The PCR-amplified product was digested with &WI and Snll and subcloned into plBI31. The insert in this plasmid had a sequence consistent with that previously reported (Bhasker ct al., 1989). The insert was removed using Sac.1 and XCroII and was cloned into the lucifcrase expression plasmid. pXP2 (Nordeen, 1988). The thymidine kinase promoter was removed from the pXP2 derivative pT109luc with Sac1 and &III and replaced with the 517 bp CYPI7 promoter to give pXP2-CYP17( -48X). In order to investigate the potential role of elements upstream of -488 in the bovine CYPl7 gene, a CYPl7-luciferase plasmid with - 2544 to + 29 of the 5’ region was constructed as follows. As previously described (Hornsby et al., 1992). a bovine genomic library was screened using CYPZ7 cDNA. The - 11 kb Sal IEcoRI insert of a hybridizing A clone, containing the entire coding region and 2.5 kb of 5’ flanking region of CYPZ7A2, was subcloned into pBluescript II KS. The 5’ region was isolated as a SalI-StuI fragment ( - 2544 to - 19) and was used to replace the sequence -488 to - 19 in pXP2-CYP17( - 4881, which was removed by digestion with Sal1 and StclI, giving pXP2-CYP17( - 2544). Growth of hor*ine adrenocortical cells Adrenocortical cells were prepared from adrenal cortex tissue (zona fasciculata-reticularis) from 2-yearold steers by collagenase/DNase digestion, as previously described (Gospodarowicz et al., 1977). Cells were stored frozen in 5% dimethyl sulfoxide until required for experiments. Frozen cells were thawed and plated in culture dishes coated with fibronectin. Cells were grown in a 1 : 1 mixture of Dulbecco’s Eagle’s medium (DME) and Ham’s F-12 (F-12) medium with 2% fetal bovine serum (Irvine Scientific, Irvine, CA, USA), 10% horse serum (Sigma Chemical Co., St. Louis, MO, USA), 1 ng/ml of recombinant basic fibroblast growth factor (Imcera), 20 nM selcnite, 1 PM a-tocopherol, and antibiotics. The gas phase used was 5% 02, 90% N,, and 5% CO,. Subculturing was performed by incubation with Pronase E (neutral protease type XIV, Sigma) in serum-containing medium using a 1 : 5 split ratio. Cumulative population doubling levels (PDL) were calculated as described previously (Hornsby and Harris, 1987). Cotransfection with CYPI 7-luciferase constructs and pS V3neo CYPl7-luciferase constructs were cotransfected with plasmid pSV3neo (Southern and Berg, 1982), containing part of the early region of SV40 virus and the bacterial neo gene. Clones derived by transfection of
bovine adrenocortical cells with this plasmid have been described previously (Cheng et al., 1989). Plasmids to be cotransfected were first ligated to each other to form long concatemers. Both plasmids were linearized in the p-lactamase region with blunt-end producing restriction enzymes and were ligated (in a 3 : 1 ratio of luciferase construct to pSV3neo) using T4 DNA ligase (2 units/pug DNA) in the presence of 15% polyethylene glycol 8000 for l-2 h at room temperature. The reaction was stopped by precipitation with ammonium acetate/ethanol and the ligated material was pelleted by centrifugation. The resultant high molecular weight DNA was introduced into bovine adrenocortical cells grown in complete medium to 50-60% confluence by electroporation (Cheng et al., 1989) or Lipofectin (Gibco/BRL) (Hornsby and Salmons, 1992) using 20 pg DNA per 10 cm dish. Cells were maintained in normal complete medium for 24 h before transfer into medium containing 200 pg/ml G418 (Gibco/BRL). After 14 days incubation with G418, surviving cells were replated and grown in mass culture (1: 5 split ratio) in the continuous presence of G418. Alternatively, after 14 days incubation with G418, surviving cells were replated at low density. Clones were allowed to grow for 10 days, isolated with cloning rings, and grown up to an approximate area of 50 cm2 (equivalent to 25 population doublings). Clones were studied within the next six passages beyond this point. Luciferase assay
Cells were grown in 35 mm plates in complete medium with G418 until almost confluent and then changed to defined serum-free medium comprising DME/F-12 1: 1, 50 pg/ml bovine serum albumin (BSA), 2 mM ascorbic acid, 20 nM selenite, 1 PM cy-tocopherol, with optional addition of 10 nM recombinant insulin-like growth factor I (IGF-I) (Imceral and/or 1 nM cholera toxin. After a 6 h incubation, cells were harvested for assay of luciferase using scintillation counting as previously described (Nguyen et al., 1988). Cells were washed twice with cold phosphatebuffered saline and scraped in 200 ~1 of lysis buffer comprising 25 mM potassium phosphate pH 7.8, 8 mM Mg ‘+ 7 1 mM dithiothreitol, 1 mM EDTA, 1% Triton X-100, 1% BSA, and 15% glycerol. The suspension was centrifuged at 10,000 rpm for 5 min. 50 ~1 of the supernatant was then added to 200 ~1 of reaction mixture containing 0.2 mM luciferin and 0.8 mM ATPMg2+ in 100 mM potassium phosphate buffer pH 7.8. Light emission was determined by placing the tube in a glass vial in a scintillation counter used in normal counting mode. Light production as (cpm)1/2 was converted to pg luciferase using a standard curve constructed with various amounts of pure luciferase (Sigma). DNA was measured by Hoechst 33258 fluo-
rescence (Labarca and Paigen, 19801 on the nuclear pellet dissolved in 50 mM NaOH. 17mHydroxylase assay
For induction of the endogenous CYPl7 gene, cells were grown to semi-confluence in complete medium with G418. Cells were then incubated in defined medium containing 10 nM IGF-I with or without 1 nM cholera toxin for 72 h. At the end of this period, cells were incubated with 10 PM progesterone substrate in defined medium for 2 h. Steroids were extracted from the medium with dichloromethane. Product 17cY-hydroxyprogesterone was separated from progesterone by high performance liquid chromatography using elution with a linear methanol : water gradient (20%-100% over 10 min). Detection was by absorbance at 240 nm. The identity of ultraviolet -absorbing peaks was established by comparison of retention times of authentic standards. Conversion rates were calculated by comparing peak areas for substrate and product with peak areas of known amounts of standards, calculating percent conversion to product, and converting percent conversion to pmol from the known amount of precursor added. Conversion rates were expressed as pmol/105 cells/h. Results
An overview of these experiments is shown in Fig. 1. Because of technical difficulties with transient transfection of later-passage bovine adrenocortical cells, these experiments were performed using stable transfection and pools of clones. Previously, we showed that this cell type was amenable to stable transfection with relatively high efficiency (Cheng et al., 1989). Cells at
Fig. 1. Overview of experiments on the activity of the cyp17 promoter in cultured bovine adrenocortical cells before and after phenotypic switching. The decline in the expression of GYP17 is shown with respect to the population doubling level and diagrammatically represents both the total population 07’17 mRNA level after cyclic AMP induction and also the percentage of CYP17+ cells in the population assessed by in situ hybridization (data from Hornsby et al., 1987; Ryan et al., 1989). Transfection of CYP17-luciferase constructs with pSV3neo, selection of pooled clones in G418, and assay of cholera toxin-induced luciferase was performed at PDL 10 and PDL 55, when most cells are CYP17 + and CYP17 - respectively.
22
early passage (PDL 10) and a later passage (PDL 55) were cotransfected with CYP17-luciferase constructs ligated to pSV3neo and transfectants were selected in G418. At PDL 10 most cells are CYP17 + and at PDL 55 almost all cells are CYP17 - . Prior work established that clones of bovine adrenocortical cells produced by transfection of pSV3neo show vigorous growth and that such clones express CYP17 if the cells of origin were capable of expression of CYPZ7 or do not express the gene if the precursor cells had undergone phenotypic switching prior to transfection (Cheng et al., 1989). Preliminary experiments were performed to assess the characteristics of bovine adrenocortical cells transfected with CYPI 7-luciferase constructs. After transfection of the - 488 CYP17-luciferase construct in cells at PDL 10, the culture was incubated with 200 pg/ml of G418 for 10 days. The cells were then subcultured; no nontransfected cells remained at this time. The pooled transfectant clones were grown in mass culture in the continued presence of G418 and the induction of luciferase was assayed. Luciferase activity was increased by agents that increase intracellular cyclic AMP, such as cholera toxin, and by cyclic AMP analogs. Cell type specificity was demonstrated by transfecting bovine kidney fibroblasts, which do not normally express CYPZ7, with the same construct. In this case, luciferase activity was very low and unresponsive to cholera toxin (data not shown). Other preliminary experiments showed that IGF-I was required for efficient induction of transfected luciferase in defined medium. IGF-I increased the level of luciferase activity in bovine adrenocortical cells transfected with the - 488 CYPZ7luciferase construct. However, it had similar effects in bovine adrenocortical cells stably transfected with luciferase under the control of other promoters (herpes simplex thymidine kinase and SV40) whereas these promoters did not respond to cyclic AMP (data not shown). Thus the IGF-I effect probably represents a non-promoter-specific effect on luciferase activity, perhaps by increasing the half-life of luciferase, which is known to be very short in mammalian cells (Thompson et al., 1991). In the presence of IGF-I, maximum induction of luciferase by cholera toxin was observed 6 h after the addition of cholera toxin. Having established the cell-type specificity of the cyclic AMP response for the -488 CYP17-luciferase construct, and having established standard conditions for testing promoter activity (defined medium, IGF-I, cholera toxin), we performed additional experiments to assess the reproducibility of induction of the CYPI 7 promoter by cholera toxin. Pooled transfected cells were grown in mass culture with a 1: 5 split ratio at subcultures. At each passage, cholera toxin induction of luciferase was assayed. Fig. 2 shows that although absolute levels of luciferase activity showed a tendency an
-
-
passage 1
passage 2
passage 3
passage 4
passage 5
Fig. 2. Stability of expression from the transfected C’YPI7 promoter in bovine adrenocortical cells. Cells at PDL 10 were cotransfected with pXP2-CYP17(-488) and pSV3neo as described in Materials and methods. Passage 1 indicates cells subcultured after the initial 14-day selection in G418. Passages 2-5 were made by continued growth of the cells in complete medium with G418 with I : 5 subcultures. At each passage, semi-confluent cells were incubated in defined medium with addition of 10 nM IGF-I and 1 nM cholera toxin. After 6 h, cells were harvested for assay of luciferase activity.
to vary between passages, at each passage cholera toxin increased luciferase activity 2- to 4-fold above the level observed with IGF-I alone. The promoter activity of CYP17-luciferase constructs in cells transfected at PDL 10 was then compared with that in cells transfected at PDL 55. Fig. 3 shows that cells at PDL 10 and PDL 55 transfected with the -488 construct demonstrated induction of luciferase by 2- to 3-fold and 7- to %fold, respectively, when intracellular levels of cyclic AMP were raised. Furthermore, there was a 2-fold induction of the - 2544 construct in cells transfected at PDL 55 whereas in PDL 10 cells, although luciferase was expressed from this construct, it was not cholera toxin responsive. In cells transfected with CYPI7-luciferase constructs expression of luciferase was compared with expression of endogenous CYPI 7. As anticipated from previous data from cells at these PDLs, cells transfected at PDL 10 had cholera toxin-inducible 17a-hydroxylase whereas cells transfected at PDL 55 did not (Fig. 4). An additional experiment was performed to confirm that the apparently lower expression of the -2544 construct versus the -488 construct was reflected in individual clones of cells as well as pools. Southern
23
1
.’ PDL 10 \
PDL 55 ,
PDL 10 L
” pxPz-cYP17(-488)
PDL 55 I
Y pxP2-CYP17(-2544)
Fig. 3. Promoter activity of fusion genes containing different lengths of the 5’ flanking region of ClTl7 linked to luciferase in bovine adrenocortical cells before and after loss of endogenous 07’17 expression by phenotypic switching. Luciferase activity was measured following a 6 h incubation in defined medium with 10 nM IGF-I and optionally 1 nM cholera toxin. Data are derived from triplicate plates of each pool (mean* SE). Two separate transfections to yield two separate pools of clones were performed at each PDL with the -488 construct (indicated as ‘1’ and ‘2’).
blotting showed that -2544 transfectants did not have a lower copy number of the CYPl7-luciferase plasmid than the -488 transfectants (not shown). There was
1 + h
c
4
Cl IGIJ IGF choleratoxin
ekme 1 \
II clone 2
clone
3 I
pXWCYP17(-488)
Cl”“F 1 L
clone 2 Y
clone 3 I
pXp2-CYP17(-2544)
Fig. 5. Activity of the CYPl7 promoter in individual clones of PDL 10 cells transfected with pXP2CYP17( - 488) or pXP2-CYP17(-2544). Three clones from each transfected pool were isolated at random. The induction of luciferase by cholera toxin was investigated as described in the legend to Fig. 3. Data are derived from triplicate plates of each clone (mean f SE).
consistent cholera toxin induction of luciferase activity in individual clones derived by transfection of earlypassage cells with the -488 construct and relatively weak or no induction in clones with the -2544 construct (Fig. 5). Clones carrying the -2544 construct showed the expected induction of 17cy-hydroxylase activity by cholera toxin (not shown). Moreover, all clones examined, with either construct, showed luciferase activity, demonstrating the efficiency of the plasmid ligation technique used for cotransfection. Discussion
PDL 10
PDL 55
PDL 10
PDL 55
WV pXP2-CYP17(-488)
pXP2-CYP17(-2544)
Fig. 4. 17a-Hydroxylase activity in different transfected cell populations. Cells at PDL 10 and PDL 55 were transfected with CYPl~luciferase constructs as described in the legend to Fig. 3. 17cu-Hydroxylase activity was assayed under the conditions used in that experiment, except that the incubation with cholera toxin was extended to 72 h. The individual values and means from duplicate plates are shown.
These data indicate that phenotypic switching from CYP17 - to CYP17 + in bovine adrenocortical cells as they senesce in culture does not result from a simple loss of regulatory factors that act within 2.5 kb of the CW17 promoter. For both the - 488 and - 2544 CYP17-luciferase constructs, induced luciferase activity was actually higher in later-passage than in early-passage cells. Apparently, positive transcription factors acting within - 2544 to + 29 of the CYP17 gene must be present in cells that are no longer able to express endogenous CYH7. CYP17 - bovine adrenocortical cells thus resemble the Yl mouse adrenocortical tumor cell line, which expresses CYP17 reporter constructs but not its endogenous Cyp17 gene. The Cyp17 con-
‘4
structs used in the Y 1 cell studies, like those constructed for these experiments, show cell type specificity and induction by cyclic AMP (Brentano et al., 1990; Lund et al., 1990). Other events, such as changes in DNA methylation, could be responsible for phenotypic switching. Although loss of promoter activity might be expected to be associated with hypermethylation, the CYP17Al and CYPl7A2 genes show specific demethylation in bovine adrenocortical cells soon after being placed in culture (Hornsby et al., 1992). The methylation status of the CYP17 genes was assessed by Southern blotting of DNA isolated at various times after placing the cells in culture and digested with methylation-sensitive enzymes. The data showed that whereas one CpG site at - 1800 is always methylated in adrenocortical cells, two other sites at - 2150 and - 2300 which are methylated in the adrenal cortex in vivo become demethylated when adrenocortical cells are placed in culture (Hornsby et al., 1992). Moreover, the site at - 1800 is nonmethylated in fibroblasts, which do not express endogenous CYPl7 or CYPl74uciferase constructs. The present data suggest that the region -500 to - 2500 of CYPI 7 contains a negative regulatory element. Sequence analysis indicates that the region between - 300 to - 1800 consists of several of repeats that are common in bovine genes, whereas the region that shows tissue-specific hypomethylation does not (Hornsby et al., 1992). Further analysis of the -500 to - 2500 region will be required to confirm the existence
of a negative regulatory element, its potential role in phenotypic switching, and the significance of the specific changes in DNA methylation of CYP17 in cultured bovine adrenocortical cells.
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