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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 9 2956-2961
Copyright © 1997 by The Endocrine Society

Original Studies

Progesterone Regulated Expression of Flavin-Containing Monooxygenase 5 by the B-Isoform of Progesterone Receptors: Implications for Tamoxifen Carcinogenicity

Marilyn M. Miller1, R. Andrew James, Jennifer K. Richer, David F. Gordon2, William M. Wood2 and Kathryn B. Horwitz3

Department of Medicine, University of Colorado Health Sciences Center (M.M.M., R.A.J., J.K.R., D.F.G., W.M.W., K.B.H.), Denver, Colorado 80262; Departments of Obstetrics and Gynecology and Anatomy, McGill University (M.M.M.), Montreal, Quebec, Canada H3A 1A1; and Department of Medicine, University of Newcastle-Upon-Tyne (R.A.J.), Newcastle, NE2 4HH, United Kingdom

Address all correspondence and requests for reprints to: Kathryn B. Horwitz, Department of Medicine, Division of Endocrinology, Box B-151, University of Colorado Health Sciences Center, Denver, Colorado 80262.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progesterone is a key developmental, proliferative, and differentiative hormone in the breast and endometrium, and it can accelerate carcinogenesis in the mammary gland epithelium. In the breast and uterus, progesterone acts through two coexpressed isoforms of progesterone receptors, the B- and A-receptors. To study the function of each isoform in isolation, we previously constructed two breast cancer cell lines that stably and independently express either B-receptors (YB cells) or A-receptors (YA cells). In the present study, YA or YB cells were left untreated, or were treated with the synthetic progestin R5020, and the messages present in each cell line under the two conditions were analyzed by differential display. Two message species are described that are regulated only by B-receptors. One of these is regulated in a ligand-independent manner. A third set of messages, encoding flavin-containing monooxygenase 5 (FMO5), was induced by R5020 only in YB cells. A-receptors appear to be inhibitory. FMOs are involved in the metabolic activation of drugs and xenobiotic compounds, including the antiestrogen tamoxifen, to carcinogenic intermediates. It is possible, therefore, that by upregulating the levels of FMO5, progesterone enhances the carcinogenicity of tamoxifen in target tissues that overexpress progesterone B-receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROGESTERONE is a key developmental, growth, and differentiative hormone in the breast and uterus (1, 2), and under appropriate conditions, progesterone can accelerate carcinogenesis in mammary gland epithelium (Ref. 3 and references therein). However, only a few progesterone-regulated genes have been defined in any tissue (4, 5, 6). To understand the role of progesterone in normal and malignant cell processes, the present study sought to define the subset of proteins regulated by progesterone, using human breast cancer cells as models.

Analysis of progesterone action is complicated by the fact that two progesterone receptor (PR) isoforms are coexpressed in human target cells (7): 120-kilodalton B-receptors, and N-terminally truncated 94-kilodalton A-receptors. Thus, when equimolar levels of the two isoforms are expressed, A:A, A:B, or B:B homo- and heterodimers, form at 1:2:1 molar ratios. This is important, as the two receptor isoforms regulate transcription unequally when occupied by progesterone agonists (8). with A:A homodimers usually weaker than B:B homodimers. Additionally, in the A:B heterodimer, the inhibitory transcriptional phenotype of A-receptors is dominant. On the other hand, only B-receptors can paradoxically activate transcription when bound by antagonists (9, 10, 11). All known progesterone-dependent target tissues and cells express both PR isoforms, but recent data in breast cancers (12) and uteri (13) suggest that the ratio of B- to A-receptors can flucuate widely. This structural variability would influence the responsiveness of tissues to progestational signals. To study the function of the two PR isoforms independently, we isolated a PR-negative subline (Y cells) of the A- plus B-receptor-positive T47D breast cancer cell line, and then stably reintroduced expression vectors encoding either A- or B-receptors into Y cells to create, respectively, YA and YB cells (14). The protein levels of each PR isoform expressed in these new cell lines is equivalent to the levels of that isoform present in wild-type T47D cells (14). We have used these new cells to study isoform-specific regulation of PR target genes by the method of differential display (15).

The present study demonstrates that the overall pattern of expressed messenger RNAs (mRNAs) in YA and YB cells is remarkably similar, that some messages may be regulated in a ligand-independent manner, and that the progestin agonist R5020 modulates transcription of a small but unique subset of messages. Among the messages regulated by R5020, but only in cells expressing the B-isoform, are those encoding flavin-containing monooxygenase 5 (FMO5). In the liver, studies suggest that FMOs can metabolize drugs and xenobiotics to reactive intermediates that bind covalently with microsomal proteins and DNA (16, 17). Tamoxifen, for example, can be metabolized to a reactive intermediate by these enzymes (16), and such tamoxifen adducts have been described in the uterine DNA of breast cancer patients (18). Our data suggest the interesting hypothesis, that in target cells that overexpress PR B-receptors, progesterone can induce FMO5 and enhance the carcinogenicity of tamoxifen, and therefore, that an excess of B-receptors may serve as a risk marker.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

Construction and characterization of YA and YB breast cancer cell lines that stably express either PR A-receptors or B-receptors, respectively, has been previously described (14). Cells were plated at 1 x 107 cells/75 cm2 tissue culture flask and allowed to grow for 6 days in MEM supplemented with 5% FCS until they were approximately 75% confluent. YA and YB cells were then treated with 20 nM of the progestin R5020 (New England Nuclear, Dupont, Boston, MA) or vehicle alone for 18 h, before harvesting as previously described (14).

Isolation and purification of total RNA and poly A+ mRNA

Total cellular RNA was isolated with guanidinium isothiocyanate followed by centrifugation through a CsCl cushion. DNA was eliminated from total RNA using RNase-free DNase I (Message Clean, GenHunter, Brookline, MA). Poly(A)+ RNA was separated from total RNA by affinity chromatography on oligo(dT)-cellulose (type 7; Pharmacia; Piscataway, NJ) according to our previously published procedures (19).

Differential display of mRNA

We evaluated approximately half (~5000 mRNAs) of the mRNA species estimated to be present in eukaryotic cells (15). Ten arbitrary primers were examined with each of four anchored primers (RNAmap kit II, GenHunter, Brookline, MA). RT-PCR was performed on purified total RNA as described, using Moloney Murine leukemia virus reverse transcriptase. Complementary DNAs (cDNAs) were then selectively amplified by PCR with the appropriate primer pairs in the presence of [35S]deoxycytidine ATP. Aliquots of separate duplicate PCR reactions containing amplified cDNA fragments were then electrophoretically resolved on adjacent lanes of a 6% polyacrylamide/urea denaturing gel. Following transfer, drying, and autoradiography, bands were evaluated for differentially displayed candidate messages.

Recovery, reamplification, and cloning of cDNAs

The autoradiographed film was accurately aligned with the dried gel and differentially regulated PCR fragments were excised, eluted by boiling, ethanol precipitated, and reamplified by PCR with the original primer pairs. The PCR product of the expected size was excised from a 1% agarose gel, and the DNA was purified by silica gel adsorption (QIAEX II kit, Qiagen, Chatsworth, CA). PCR products were ligated into pCRII and transformed into competent INF F’ E. coli (One Shot Invitrogen TA Cloning, Invitrogen, San Diego, CA). Following blue/white selection, appropriate clones were grown and plasmid DNA was isolated. Sequencing was performed by the Cancer Center Core Laboratory at the University of Colorado Health Sciences Center, Denver, CO. Sequences were searched against the NIH GenBANK database using a BLAST algorithm.

Northern blotting

Total RNA (20 µg) was separated on a 1% agarose/6% formaldehyde gel according to our published protocol (19). The RNA was transferred overnight to a nylon membrane (TurboBlotter; Schleicher and Schuell, Keene, NH) and fixed by UV cross-linking (Stratalinker; Stratagene, La Jolla, CA). Blots were probed with 2.0 x 107 cpm [32P]cDNA labeled by nick translation from the cloned candidate PCR fragments. Northern blots were subsequently probed with 32P-labeled ß-actin to assess loading uniformity.

Rapid amplication of cDNA ends (RACE) was used to obtain further 5' sequence information from the 360-bp cDNA fragment regulated by R5020 in YB cells (Fig. 3Go). A 26-bp primer complementary to a region of the 360-bp sequence was used for first-strand synthesis using Moloney Murine leukemia virus reverse transcriptase (Clontech Marathon cDNA Amplification Kit; Clontech Laboratories, Palo Alto, CA) using poly(A)+ RNA from YB cells treated with R5020. Following second-stand synthesis, double-stranded cDNA was blunt ended with T4 DNA polymerase I and ligated to an adapter primer supplied with the Marathon Kit. Adapter ligated cDNAs were amplified by long-distance PCR (Boehringer Mannheim, Indianapolis, IN). The PCR conditions consisted of 30 sec at 94 C for denaturation, 30 cycles at 68 C, and 30 sec for annealing and extension. Amplifed PCR products were size separated on a 1% agarose gel, transferred to a Nytran membrane, and hybridized to a 36-bp oligonucleotide just 5' of the 26 nucleotide RT primer to verify the specificity of the amplification. Positively hybridizing PCR products were subcloned into PCR 2.1 (Invitrogen), and miniprep DNAs were screened for internal sequences by Southern blot analysis (20) using the radiolabeled 36 mer. An 800-bp positively hybridizing insert was sequenced from both ends using M13 forward and reverse primers, and searched against the GenBANK database. To reconfirm its differential expression pattern, an approximately 300-bp EcoRI/BamHI fragment from the 5' end of the 800-bp clone was labeled and used for Northern blotting.



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  		Figure 3. Constitutively regulated B-receptor-specific message. A, Similar mRNAs were probed with a 450-bp 32P-labeled fragment isolated by differential display. Arrow, Indicates an ~4000-bp message. B, Same Northern blot hybridized with a labeled ß-actin probe to demonstrate equal RNA loading.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Four sets of cells were analyzed for constitutive and progesterone-regulated mRNA expression. Untreated YA and YB cells were compared with each other (YA- vs. YB-) and to messages in YA and YB cells following 18 h of treatment (YA+ vs. YB+) with the progestin R5020. As expected, the vast majority of the message fragments were neither modulated by the PR isoform present in the cell nor by the presence of hormone. Figure 1Go shows an example of the differential display patterns obtained. Two bands, one marked by a star, the other labeled FMO5, are discussed below.



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  		Figure 1. An example of differentially displayed mRNA fragments derived from breast cancer cell lines expressing either B- or A-receptors in the presence or absence of R5020. Total RNA from YA (A) and YB (B) cells, either untreated (-) or treated (+) with R5020, was reverse transcribed with T12 MA. Product cDNAs were PCR amplified with a T12MA primer/anchor primer 13 combination in the presence of [35S]deoxycytidine ATP. Products were resolved on a 6% urea denaturing gel. Arrow, Indicates a ~360-bp band present only in hormone- treated YB cells (YB+). An (~520-bp) band (star) was present in all cell types except hormone-untreated YB cells. The vast majority of bands are identical in two cell types. Duplicate sets are shown.

 
Ligand-free regulation by B-receptors

The Northern blot in Fig. 2AGo shows a 3200-bp RNA transcript labeled by a 380-bp band excised from a differential display gel. The same Northern blot hybridized with a labeled ß-actin cDNA demonstrates the relative amounts of RNA loaded (Fig. 2BGo). A BLAST search with the sequencing data indicates that this message encodes a novel protein. This Northern blot confirms an unusual ligand-independent up-regulation restricted to B-receptor-containing cells. Gene regulation by ligand-free steroid receptors has recently been described (21). The authors speculate that the receptors are activated by means of cross-talk with other signaling pathways. A second example of this type of regulation by ligand-free B-receptors is demonstrated in Fig. 1Go (starred product) in which the product is down-regulated (lanes 5 and 6). It has not been characterized further.



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  		Figure 2. B-receptor specific message regulated by ligand-independent mechanisms. A, mRNAs from YA (A) or (YB) (B) cells either untreated (-) or treated with (+) R5020 were resolved on agarose gels, transferred to a nylon membrane, and probed with a 380-bp 32P-labeled fragment isolated by differential display. Arrow, Indicates an ~3200-bp message. B, Same Northern blot probed with 32P-labeled ß-actin demonstrating relative amounts of RNA loaded from YA or YB cells either untreated (-) or treated (+) with R5020.

 
Figure 3AGo shows a 4000-kilobase (kb) transcript that hybridized to a radiolabeled 450-bp band excised from a differential display gel that was seen in YB cells whether or not they received hormone, but was absent in YA cells irrespective of hormone treatment. Equal loading of RNA was demonstrated by hybridization to a labeled ß-actin cDNA (Fig. 3BGo). This message is either constitutively regulated and represents an extremely rare message that is unique to YB cells, or it is a message that is regulated by B-receptors through both ligand-dependent and -independent mechanisms (21). Sequence analysis shows that the protein encoded by this message is also novel.

B-receptor-specific and progestin-dependent regulation of FMO5

An approximately 360-bp mRNA fragment was amplified by the combination of arbitrary primer 13 and anchor primer T12 MA, from R5020-treated YB cells (Fig. 1Go, arrow, lanes 7 and 8) and represents a classically hormone-regulated message. Interestingly, it is only regulated by PR B-receptors. This cDNA fragment was excised from the duplicate lanes of the differential display gel, reamplified, and cloned. The insert was sequenced, radiolabeled, and used as a probe on a Northern blot (Fig. 4AGo) using total RNA from the basal and R5020-treated YA and YB cell lines. The labeled probe hybridized to a major transcript of approximately 3.8 kb and a minor one of approximately 2.6 kb that were expressed only in R5020-treated YB cells (Fig. 4AGo, lane 4). The 360-bp fragment was extended in the 5' direction by RACE using RNA from YB+ cells, to yield an 800-bp product. After the probe was removed, the Northern blot shown in Fig. 3AGo was reprobed with an approximate 300-bp EcoRI/BamHI fragment corresponding to the 5' end of the 800-bp RACE product. This Northern blot yielded the same two 3.8-kb and 2.6-kb transcripts, and an additional 1.8-kb species (Fig. 4BGo). The blot was then probed with a [32P]cDNA encoding ß-actin (Fig. 4CGo). This demonstrates relatively uniform RNA loading in lanes 1–3, with lane 4 somewhat underloaded. Thus, the extent of mRNA induction in lane 4 is probably underestimated.



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  		Figure 4. FMO5 transcripts are strongly up-regulated by R5020 in B-receptor-containing T47D breast cancer cells. A, mRNAs from YA (A) or (YB) (B) cells either untreated (-) or treated with (+) R5020 were resolved on agarose gels, transferred to a nylon membrane, and probed with a 360-bp 32P-labeled fragment isolated by differential display. B, Blot from A was stripped and reprobed with a 32P-labeled 300-bp fragment from the 5' end of the 800-bp RACE product derived from the original 360-bp fragment. C, Blot from B was stripped and reprobed with a 32P-labeled ß-actin probe. Mol wt of messages are shown on the right; mol wt of standards are shown on the left.

 
The 800-bp RACE product was sequenced, and a search of the DNA database showed that the first 197 nucleotides were 100% identical to the 3' noncoding region of an mRNA that encodes FMO5 (17). The remainder of the 800-bp fragment probably represents sequences further 3' in the untranslated region of the FMO5 message that have not been previously reported, but matched the sequence of the original 360-bp cDNA isolated from the differential display gel.

Figure 5Go shows a study with YA and YB cells, similar to that in Fig. 4Go, which also includes a Northern blot of wild-type T47D cells. These cells express approximately equimolar amounts of A- and B-receptors (7). As shown, FMO5 mRNAs are up-regulated 9- to 10-fold in R5020-treated YB cells, but not in YA cells. Interestingly, in wild-type T47D cells, this up-regulation is greatly attenuated. This is to be expected if only 25% of the receptors bind the FMO5 promoter as B:B homodimers. The remaining 75% bind either as A:A homodimers or A:B heterodimers, both of which would fail to activate transcription of this promoter (10, 11).



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  		Figure 5. Suppression of FMO5 up-regulation in T47D cells expressing equimolar levels of B- and A-receptors. YA, YB or wild-type T47D cells (that express equimolar levels of B- and A-receptors) were untreated (-) or treated (+) with R5020. mRNAs were resolved on agarose gels, then transferred to a nylon membrane and probed with the 360 bp 32P-labeled FMO5 fragment isolated by differential display. Numbers to the left indicate the position of {lambda} HindIII DNA size standards. The blot was stripped and reprobed with a 32P-labeled ß-actin probe.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
YA/YB cells and differential display

Because PR are important in breast cancers (22), and the A- and B-isoforms of PR are functionally different (8, 9), we have constructed model T47D breast cancer cell lines in which the physiological role of each isoform can be independently evaluated (14). These cells stably express each isoform separately, but at the same levels seen in wild-type T47D cells (14). Differential display of a subset of the messages expressed in these T47D cells suggests that, for the most part, the two stable cell lines, A-receptor containing YA cells and B-receptor containing YB cells, contain the same mRNA populations, confirming their value for analysis of isoform-specific effects. We report here data for only 50% of possible primer pairs and for only one progesterone treatment time. Thus it is clear that we have analyzed only a subset of the messages present in these cells that are capable of being regulated by progesterone and that can be displayed by the method used. This is supported by the fact that several messages known to be regulated by progesterone in breast cancer cells, including ones encoding lactate dehydrogenase (4), alkaline phosphatase (5), and fatty acid synthetase (6) were not identified in our analysis. Of course some of the differentially displayed bands among those that have not yet been sequenced could, in theory, encode these proteins. Also of interest is the fact that all three of the progestin-regulated messages we detected were under B-receptor control. This PR isoform is usually, but not always (8), the stronger transcriptional activator in experimental model systems. Nevertheless, there are conditions under which A-receptor effects predominate (9), and at least one gene, the multidrug resistance gene encoding P-glycoprotein, is under progesterone control by the A-isoform (23). However, our observation that only a low frequency of transcripts are progesterone-regulated is supported by earlier data (24).

Progesterone regulation of FMO5 in breast cancer cells

Our data unequivocally show that three mRNAs containing FMO5 sequences are progesterone regulated in breast cancer cells specifically under the control of the PR B-isoform. To our knowledge no other B-receptor-specific regulated gene has been previously described. Additionally, we found no previous reports showing that FMO5 messages are expressed in normal or malignant breast cells. However, evidence is accumulating for both developmental and progesterone regulation of the five known FMOs in several tissues. Early studies showed that the enzyme is induced in rabbit lung during pregnancy (25) and in hepatic microsomes of CD1 mice during late gestation (26). More recently, modulation of FMO isoform B levels has been demonstrated in rabbit lung and kidney during pregnancy and after progesterone administration (27). Similarly, elevated levels of plasma progesterone, but not of cortisol, correlate with elevated FMO2 enzyme levels in the maternal and fetal rabbit lung, both during gestation and postpartum (28). On the other hand, FMO2 is induced by both progesterone and cortisol in the rabbit kidney (28). Interestingly, the FMO2 isoform is also induced by 17ß-estradiol in the lung and by glucocorticoids in the liver (27), demonstrating tissue specific modulation of these enzymes by other steroid hormones. Regulation by progesterone and/or cortisol may be a direct consequence of the binding of PR B-receptors or glucocorticoid receptors to the FMO5 promoter, which contains a progesterone/glucocorticoid response element (R.M. Philpot, personal communication). Why PR A-receptors do not activate this promoter will be the subject of future studies.

We observed three transcripts (3.8 kb, 2.6 kb, and 1.8 kb) in poly(A)+ RNA isolated from human breast cancer cells that were labeled with the FMO5-specific probe. A recent study using human, rabbit, and guinea pig liver RNA demonstrated only two FMO5 products (3.8 kb and 2.6 kb) (17). The third, a 1.8-kb band that we observe may be caused by tissue-specific differences among FMO5 mRNAs produced in the breast compared with the liver, to the detection of a third FMO5 message that has not previously been reported, or to the presence of an unrelated mRNA that has a sequence similar to that of the FMO5 message. At present we cannot distinguish among these possibilities.

Our finding that FMO5 is progesterone regulated in breast cancer cells has interesting implications regarding drug metabolism in the breast and other progesterone target tissues. FMOs are flavin-containing microsomal monooxygenases that use NADPH as a cofactor. They catalyze the oxidation of a diverse array of substrates including hydrophobic foreign molecules and drugs (29, 30), and have a metabolic role akin to that of the cytochrome P450s (27). Indeed, FMOs as a group appear to be important in the detoxification of drugs, pesticides, and a variety of industrial, chemical, and other xenobiotics.

However, in addition to detoxification, under the influence of FMOs many xenobiotic compounds undergo metabolic activation and produce highly reactive and toxic intermediates that bind covalently to proteins or DNA (16, 31). For example, it has been suggested that the antiestrogen tamoxifen is a potential substrate for FMOs (16). The isoform of liver FMO that is responsible for the formation of the reactive tamoxifen intermediate has not yet been reported, and the FMO5 isoform of the enzyme was only recently cloned and sequenced from a human hepatic library (17). Although the antiestrogenic activity of tamoxifen is the major indicator for its current therapeutic and prophylactic use in breast cancer (32), concerns have been raised about site-specific second cancers arising after long-term therapy with this drug (33, 34). Specifically, tamoxifen-induced DNA adducts were recently described in five of seven endometrial samples of breast cancer patients (18). Our data offer the intriguing possibility that metabolic activation of a variety of drugs mediated by FMOs, including tamoxifen (16, 17), can be controlled by progesterone in target tissues like the breast and the uterus, that overexpress PR B-receptors. If so, in conditions of B-receptor excess, progesterone might accelerate tamoxifen-induced carcinogenesis. This also implies that heightened uterine B- to A-ratios might identify those women at greatest risk of developing tamoxifen-induced malignancies.


   Acknowledgments
 
We thank Roger Powell, MS for his careful handling of cell lines, Carol Sartorius, PhD, for construction of the YA and YB cells, and R.H. Philpot, PhD for helpful discussions.


   Footnotes
 
1 These studies were performed while on sabbatical leave at the University of Colorado. Back

2 Funded by grants NCI CA47411 (to W.M.W.) and NIH 1 R01 DK47407 (to D.F.G.). Back

3 Funded by NIH through Grants DK48238 and CA26869 and by Grant DAMD 17–94-J-4026 from the United States Army. Back

Received December 9, 1996.

Revised April 15, 1997.

Accepted June 16, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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