ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 294, No. 1, April, pp. 168-172, 1992
Quantification of Cytochrome P450 Reductase Gene Expression in Human Tissues’ Elizabeth
A. Shephard, **2 Colin N. A. Palmer,tp3 H. J. Segall,t14 and Ian R. Phillips?
*Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WClE 6BT, United Kingdom; and fDepartment of Biochemistry, Queen Mary and Westfield College, University of London, Mile End Road, London E!l4NS, United Kingdom
Received August 21, 1991, and in revised form November
26,199l
We have isolated and sequenced cDNA clones that code for a variant of human cytochrome P450 reductase. An RNase protection assay was used to quantify the corresponding mRNA in adult and fetal tissues. The results demonstrate that, in the samples analyzed, the cytochrome P450 reductase gene displays very little interindividual variation in its expression in adult liver and is subject to little developmental or tissue-specific regulation.
0 1992 Academic Press, Inc.
The flavoprotein NADPH-dependent cytochrome P450 reductase (EC 1.6.2.4) plays an essential role in the cytochrome P450-mediated monooxygenase system of microsomal membranes, where it transfers electrons from NADPH to cytochromes P450 (1,2). The monooxygenase system is present in virtually all mammalian tissues and is involved in the synthesis of endogenous compounds such as steroids, fatty acids, and prostaglandins and in the activation and detoxification of foreign hydrophobic compounds, including many therapeutic drugs, environmental pollutants, and chemical carcinogens (3). Cytochrome P450 reductase has also been implicated in the NADPH-linked peroxidation of microsomal lipids (4) and the oxidative degradation of heme (5-7). The ratio of cytochrome P450 reductase to total cytochromes P450 in rat liver microsomal membranes is 1:15 (8). Some inducers of cytochrome P450, such as pheno1 Supported by grants from the Cancer Research Campaign and the City and Hackney Health Authority. ’ To whom correspondence should be addressed. 3 Present address: Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, CA 92037. 4 Permanent address: Department of Veterinary Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, CA 95616.
168
barbital, can increase the amount of cytochrome P450 reductase and its mRNA by about 2.5-fold, thus maintaining the ratio (8-10). But others such as P-naphthoflavone, do not induce the reductase (8, 10). Cloned cDNAs coding for cytochrome P450 reductase have been isolated from several species including rat (ll), rabbit (12), and yeast (13). We have previously demonstrated, through the use of a partial-length human cytochrome P450 reductase clone, that in man the protein is encoded by a single gene located on chromosome 7q11.2 (14). In this paper we report the isolation and sequence of a full-length cDNA clone coding for an allelic variant of human P450 reductase, and the quantification by an RNase protection assay of the corresponding mRNA in adult and fetal liver and in adult extrahepatic tissues. MATERIALS AND METHODS cDNA library screening. A human placental cDNA library (in Xgtll) (Clontech) was screened with a 263-bp EcoRI/PstI DNA fragment derived from the 5’ end of a truncated 1950-bp human cytochrome P450 reductase cDNA clone (XHP450Rl) isolated previously (14). DNA sequencing. cDNAs were inserted into pUC19 and restriction mapped. Restriction fragments were subcloned into M13mp19 or pUC19 and sequenced by the single-stranded (15) or double-stranded (16) dideoxy chain termination methods, respectively, using the Klenow fragment of Eschrichia coli DNA polymerase or modified T7 polymerase (“Sequenase,” United States Biochemicals) for the former method and Sequenase for the latter. When necessary, a deaza dGTP sequencing kit (Pharmacia) was used to sequence through compressions. RNA isolation. Adult liver samples were obtained from Dr. Urs Meyer (Base1 Biocenter), Dr. Alan Boobis (Hammersmith Hospital, London), and colleagues at the Addenbrookes Hospital, Cambridge. One sample was obtained from a lobactomy performed at University College Hospital, London. The fetal liver samples were from the Medical Research Council Tissue Bank (Royal Marsden Hospital, London). Extrahepatic tissues were from St. Bartholomew’s Hospital, London. All tissues were frozen in liquid nitrogen and stored as l-g pieces at -78°C. Frozen tissue was placed in a coffee grinder with pellets of frozen COs and ground to a powder. RNA was extracted by the guanidinium thiocyanate/LiCl method (17). 0003-9861/92 $3.00 Copyright 0 1992 by Academic Press, IIX. All rights of reproduction in any form reserved.
QUANTIFICATION
OF HUMAN
P450 REDUCTASE
GENE
EXPRESSION
169
RNme protection assay. A 180-bp fragment of clone 27, extending from the 5’-most PstI site of this clone to the EcoRI cloning site (see Fig. 1) was inserted into pBluescript KS which had been digested with GGAGACl!l’Z-ACACCC~TC~AGGCGGQlvAs&srHisValAs@rhrSsrSaxThrValSsxGl"AlaValAlaGl"GluValSsr
AAGTATCT 60
252180-
AcGAGcAcxlT OT 1uHisPhe Asz%suSarGlWalLysPhaAlaValPheGlyLsuGlyAsnLYsThrTYYG AAiUCCATWkAAGTi~ACAA--km AsnAlaMstGlyLysTyrValAspLysArgLe"GluJX.nLeuGlyAlaQlnArgIlaPhs D660 GluLsuG1yLs"GlyAspAspAspGlyAs~Gl"Gl"AspPhsIle'IhrTrpArgDl" CAGTrCTA~CAGC720 GlnPhsTrpProAlaValCysGl"HisPh&lyValGl"AlaThr?ZlyGluGl"SsrSsr WXACACCGACATAGA-TACAAlTZGC!CAGTAmIlaArgGlnTyrGluLeuValValHisThrAspIlsAspAlaAlaLysValTyx%btGly rZCXTlUAmAAGAACG840 GAGA--ACGAGAACCAGAAGCCC
1"SsrDlyAepHisValAla His~GluteuAapIleSerAepGerLyeIleRrgTyrO GlGTACCC~CCA-E--C! ValTyr~oAlaAsnAs~erAlaLeuValAsnGlnLe"GlyLysIleLsuGlyAlaAsp CCA~ C LeuAsnAsnLeuAepOluOluSerAenLysLyeHisProPa LeuAepValValI4stSar CcoMcCCTA CGTCCTACCGCA~CCTTACl'ACCEGACA~CCAACCCXXGll40 ~IB"Asp11eTkAsnP?.-0Pr0 ProCysPrdIhrserTyrA~AlaLs" C--ArgThrAenValLeuTyrOluLeuAlaalnTyrAlaserO -~AAAaGAOCPGTACCTCAGCTOOO c.rixGcAAGA'FX'lE Le~LyeMetAla8erGs~s~l~l~lyL~lu~~~~s~ValVal GAC!CAC&~-~~A~A&~~C1380 AepHisLeuCyeOluLeuIauPmArgL.m.GlnAl~ er1leAlaSerSer lY!-CCCCAA-CA--MT~ CTAAG SsrLysValHisProAsnSarValHisIleCysAlaValValValGluTyrOluThrLys c3cC RlaOlyArgIloAQ1Ly~l~alAla~~~~~~~lu~o -===vF= a Gly GA~'NX!CCA~~CCAGTrA~l'2CCC1560 GluA8nGlyGly~AlaLeuValPr&stPhsVaULrgLy TK!AMW!CACCA-lTAlGQW3GCCC-TA PhsLysAlaThrPhrProVa1IloMetVa1G1yPrcGlyl%rGlyTrPHisProPheIle aOCTPCATCCROOPCXaOOC -GlyPhsIlaGlnGluArgAlaTrp~"Ar$~GlnGlyLysGl"ValGlyGluThr~" Clt3TACTA~Cl740 LeuTyrTyrDlyC'yslsr~~l~p~~u~~~luGlu~~laGln T GTCCCAC PhsHis~p(lly~~~LouThrOlnLouAsnValAla~~sr~luGl~srHis AAG3WTAWRXAGCACX! WWAAAGCAAGACffiAGAGCAC~ TmAmAA LysVal~alGlnHisLs~"LysGlnAspArgZl"HisLeuTrPLysLs"IleGlu -rcxxcAmTGmAGAAc W GlyGlyAlaHisIleTyrValCysGlyAspAlaArgAsnMstAlaAzyAspValGl.nAsn ACC BACTAC ‘l%rPhaTy?cAspIleValAl~luLeualyAlaMstGl"HisAlaGlnAlaValAspTyr ATCAAGAAACNA~A CpocC IlsLysLysLeuWetlhrL~ly~~r~~pVal~sr*** lGccccAcccA~C CTQGCTC~CCGTAGT ~TGGGCGCAGGCCCAGTZACAAAG&?KXK2160 C!CKXK!A lYXA~G'l?3TMATAATl.lTAMTAA-~ GTA
FIG.
1.
540 600
780
FIG. 2. Analysis of cytochrome P450 reductase mRNA by RNase protection. Molecular weight markers (1-kb ladder, Bethesda Research Laboratories) (a), undigested antisense RNAprob (b), RNase protection analysis of 10 fig of total RNA isolated from different adult human liver samples (c-l). Each assay contained 1 X lo5 cpm of antisense RNA probe.
1020 1080
1200 1260
1440 l5O0 1620 1680
1800 1860 1920 1980 2040 2100 2220 2400
The cDNA and deduced amino acid sequences of human cy-
tochrome P450reductase.The underlinedsequencerepresentstheregion of the mRNA that is complementary to the antisense RNA probe used
for the quantification experiments. The dots indicate the position of the polymorphic sites. The boxed sequence represents the poly(A) addition site signal sequence.
PsB and EcoRI. The resulting construct (pBSOR& was linearized by digestion with Hind111 (which cuts in the polylinker on the 5’ side of the EcoRI site). The sample was then incubated with proteinase K (50 fig/ml) and 10% SDS5 for 10 min at 37”C, extracted once with phenol/ chloroform (l:l, v/v), and ethanol precipitated. A 253-nucleotide-long radiolabeled “antisense” RNA probe was produced from the linearized Bluescript construct through the use of an in vitro transcription kit (Stratagene) according to the supplier’s recommendations. The reaction contained 10 units of T7 RNA polymerase and 50 /.&i [w~‘P]CTP (800 Ci/mmol, ICN). The DNA template was removed by digestion with RNase-free DNaseI (Stratagene). The RNA transcript was precipitated, resuspended, and purified by electrophoresis through an 8 M urea/6% polyacrylamide gel. Gel-purified RNA was resuspended in hybridization buffer (80% formamide, 40 mM Pipes, pH 6.4,0.4 M NaCl, 1 mM EDTA) and stored in aliquots at -20°C. All hybridizations were performed in a total volume of 30 pl containing 10 or 20 fig of sample RNA. The total amount of RNA was made up to 30 pg by addition of calf liver tRNA (Boehringer). An antisense RNA probe (0.4-1.0 X 10’ cpm) was included in each hybridization reaction. The hybridization mix was heated at 80°C for 10 min and then incubated at 45’C overnight. After hybridization, unprotected RNA was digested with RNase A. The mixture was then treated with proteinase K and extracted with phenol/chloroform (l:l, v/v). Protected RNA was precipitated with ethanol, resuspended, and electrophoresed through an 8 M urea/6% polyacrylamide gel. The gel was washed in 10% methanol and 10% acetic acid, rinsed in water, and dried under vacuum. The protected RNA probe was visualized by autoradiography at -78°C using an intensifying screen and quantified by scanning densitometry using an Ultrascan laser densitometer (LKB). Comparison with a standard curve of undigested radiolabeled probe permitted the absolute quantification of the corresponding mRNA in terms of molecules/pg of total RNA. This was converted to molecules/cell by using the average RNA content of a mammalian cell (5 pg).
5 Abbreviations used: Pipes, 1,4-piperazinediethanesulfonic dodecyl sulfate.
sodium
acid; SDS,
170 RESULTS
SHEPHARD AND
DISCUSSION
Isolation and sequence of cytochrorne P450 reductase cDNAs. Despite exhaustive rescreening of two human liver cDNA libraries, we were unable to isolate a cytochrome P450 reductase cDNA longer than the 1.95kb cDNA reported previously (14). To obtain a full-length cDNA for this protein we screened a human placental cDNA library which contained cDNA inserts that ranged in size from 0.8 to 3.6 kb with an average size of 1.8 kb. A screen of approximately 80,000 plaques of the placental cDNA library with a fragment derived from the 5’ end of a partial-length human cytochrome P450 reductase cDNA clone isolated from a liver library (14) yielded three positives (clones 27, 28, and 35). The cDNAs isolated from these clones were about 2100,2400, and 1900 nucleotides long, respectively. Their restriction maps overlapped with each other and with that of the cDNA isolated from the liver library. The sequence derived from these clones (Fig. 1) extends from the third base of the initiation codon and includes an open reading frame of 2028 bp followed by a stop codon and a 3’ noncoding region of 372 bp that contains a polyadenylation signal sequence (AATAAA) located 14 bp from a poly(A) tail. No differences in sequence were found in the overlapping regions of the liver and placental cDNAs. The deduced amino acid sequence has 93% similarity to that of rat cytochrome P450 re-
ET AL.
ductase (11). There is also a high degree of nucleotide similarity between the 3’ noncoding regions of cytochrome P450 reductase mRNAs of man, rat (ll), and rabbit (12). The evolutionary conservation of sequences within this region suggests that they may have an important functional role. The amino acid sequence derived from the human cDNAs is identical to that determined by microsequence analysis and mass spectrometry of peptide fragments (18). Hanui et al. (18) found evidence for two polymorphic sites within the protein sequence: val or ala at position 499 and g/n or arg at position 551. The sequence we report has a val at position 499 and a g/n at position 551 (Fig. 1). However, Yamano et al. (19) reported a cytochrome P450 reductase sequence that contained ala at position 499 and g/n at position 551. Taken together, these results indicate that there must be at least three alleles for cytochrome P450 reductase within the human population. Quuntifiation of cytochrome P450 reductase gene expression. We wished to determine the extent to which cytochrome P450 reductase gene expression varied between individuals and whether it was subject to developmental and/or tissue-specific regulation. This required an assay that was accurate, precise, and sensitive. Our approach was to develop an assay based on RNase protection. The antisense RNA probe used in these experi-
prostate MCF-7 cell line colon placenra Foetal liver 3 Foe&l liver 2 Foetal liver 1
tr
12
$ B
11 10
2
9
I
7654328
1
. mRNAs/cell FIG. 3. Quantification of cytochrome pg of total RNA from each sample.
P450 reductase mRNA in human tissues. Data were obtained from RNase protection
assays of 10 and 20
QUANTIFICATION
OF HUMAN
P450 REDUCTASE
abcdef
FIG. 4. RNase protection analysis of cytochrome P450 mRNA in human fetal liver samples. Undigested antisense RNA probe (a). RNase protection analysis of tRNA (b), and 10 pg of total RNA isolated from adult liver (c) and from different human fetal liver samples (d-f). Each assay contained 4 X 10’ cpm of antisense RNA probe.
ments contained a region complementary to a section extending from position 297 to 465 of the coding sequence of the cytochrome P450 reductase mRNA (Fig. 1). This region was chosen because it contained neither of the polymorphic sites that has been identified in this gene (18; this paper). The RNase protection assay revealed that the cytochrome P450 reductase mRNA was present in similar amounts in all 20 adult liver samples analyzed (Fig. 2 and data not shown). For quantification purposes, 10 and 20 pugof total RNA from each liver sample were assayed independently. After correction for the amount of total RNA, the two assays gave identical results, thus indicating that the technique was reproducible and gave a linear response. The linearity of the assay extended from 0.1 to over 100 molecules/cell (data not shown). The use of a pure mRNA transcript derived by in vitro transcription of a cloned cDNA confirmed the accuracy of the assay. The results demonstrated that the concentration of the cytochrome P450 reductase mRNA displayed an interindividual variation of no more than threefold (from 1 to 3 molecules/liver cell) (Fig. 3). The accurate quantification obtained by the RNase protection assay confirms and extends our preliminary data derived from Northern blot hybridization analysis of a limited number of adult liver samples (14). Cytochrome P450 reductase mRNA was found to be present in extrahepatic tissues such as prostate, adrenal, colon, and placenta and in the human breast cancer cell line MCF-7 (Fig. 3 and data not shown). The concentration of the mRNA in these tissues (about 1 molecule/cell)
GENE
171
EXPRESSION
was similar to that of the lowest values found in adult liver samples and was about half the average for adult liver. Immunohistochemical studies of human tissues indicate that cytochrome P450 reductase is present in all hepatocytes, but in extrahepatic tissues such as the adrenal it is confined to specific regions (20). Thus, it is likely that the mRNA also is not uniformly distributed in the cells of extrahepatic tissues, and consequently, in the cells in which it is expressed, it may be present in concentrations very similar to that found in adult hepatocytes. The mRNA is also present in fetal liver samples ranging from 12 to 20 weeks gestational age (Figs. 3 and 4), but at concentrations lower than those found in adult liver (between 0.3 and 0.5 molecules/cell) (Fig. 3). Taking into account the fact that a large proportion of fetal liver is composed of hemopoietic cells (21), it is apparent that the concentration of cytochrome P450 reductase mRNA in fetal hepatocytes cannot be much less than that present in adult hepatocytes. We have demonstrated that there is very little interindividual variation in the expression of the cytochrome P450 reductase gene in adult liver. In addition, the gene is subject to little developmental or tissue-specific regulation and thus is an example of a constitutively regulated gene. This is in marked contrast to many cytochrome P450 genes, which exhibit large interindividual variations in their expression in adult liver (ranging from 10s to 1000s of fold) and are subject to marked developmental and tissue-specific regulation (22,23; C. Palmer, E. Shephard, and I. Phillips, unpublished). ACKNOWLEDGMENTS We thank Dr. Urs Meyer, Dr. Alan Boobis, and colleagues at the Addenbrookes Hospital, Cambridge, and St. Bartholomew’s Hospital, London, for adult human tissue samples, and the MRC Tissue Bank, The Royal Marsden Hospital, for human fetal liver samples. The work was supported by grants from the Cancer Research Campaign and the City and Hackney Health Authority.
REFERENCES 1. Lu, A. Y. H., and Coon, M. J. (1968) J. Biol. Chm. 1332.
243,
1331-
2. Lu, A. Y. H., Junk, K. W., and Coon, M. J. (1969) J. Btil. 244,3714-3721.
Chem.
3. Ortiz de Montellano, P. R. (Ed.) (1986) Cytochrome P-450: Structure, Mechanism, and Biochemistry, Plenum, New York. 4. Buege, J. A., and Aust, S. D. (1978) in Methods in Enzymology (Fleischer, S., and Packer, L., Eds.), Vol. 52, pp. 302-310, Academic Press, San Diego. 5. Masters, B. S., and Schacter, B. A. (1976) Ann. Clin. Res. 17, 1827. 6. Guengerich, F. P., and Strickland, T. W. (1977) Mol. Pharmacol. 13,993-1004. 7. Guengerich, F. P. (1978) Biochemistu 17, 3633-3639. 8. Shephard, E. A., Phillips, I. R., Bayney, R. M., Pike, S. F., and Rabin, B. R. (1983) Biochem. J. 21, 333-340.
172
SHEPHARD
9. Gonzalez, F. J., and Kasper, C. B. (1980) Biochemistry 19, 17901796. 10. Shephard, E. A., Phillips, I. R., Pike, S. F., Ashworth, A., and Rabin, B. R. (1982) FEBS L&t. 160,375-380. 11. Porter, T. D., and Kasper, C. B. (1985) Proc. Natl. Acad. Sci. USA
82,973-977. 12. Katagiri, M., Murakami,
ET AL. 17. Cathala, G., Savouret, J.-F., Mendes, B., West, B. L., Karin, Martial, J. A., and Baxter, J. D. (1983) DNA 2,329-334.
M.,
18. Hanui, M., McManus, M. E., Birkett, D. J., Lee, T. D., and Shively, J. E. (1989) Biochemistry 28,8639-8645. 19. Yamano, S., Aoyama, T., McBride, 0. W., Hardwick, J. P., Gelboin, H. V., and Gonzalez, F. J. (1989) Mol. Pharmacol. 36.83-88.
H., Yabusaki, Y., Sugiyama, T., Okamoto, M., Yamano, T., and Ohkawa, H. (1986) J. Biochem. 100,945-954. 13. Yabusaki, Y., Murakami, H., and Ohkawa, H. (1988) J. Biochem. 103,1994-1010.
20. McManus, M. E., de la Hall, P., Stupans, I., Burgess, W., Brennan, J., and Birkett, D. J. (1988) in Microsomes and Drug Oxidations (Miners, J. O., Birkett, D. J., Drew, R., May, B. K., and McManus, M. E., Eds.), pp. 29-28, Taylor & Francis, London.
14. Shephard, E. A., Phillips, I. R., Santisteban, I., West, L. F., Palmer, C. N. A., Ashworth, A., and Povey, S. (1989) Ann. Hum. Genet. 63, 291-301.
21. Weatherall, D. J., and Clegg, J. B. (Eds.) (1981) The Thalassaemia Syndrome, Blackwell Scientific, Oxford.
15. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., and Roe, B. A. (1980) J. Mol. Bid. 143,161-178. 16. Mierendorf, R. C., and Pfeffer, D. (1987) in Methods in Enzymology (Berger, S. L., and Kimmel, A. R., Eds.), Vol. 152, pp. 556-562, Academic Press, San Diego.
22. Palmer, C. N. A., Shephard, E. A., and Phillips, I. R. (1990) B&hem.
Sot. Trans. l&615-616. 23. Palmer, C. N. A., Shephard, E. A., and Phillips, I. R. (1990) in Drug Metabolizing Enzymes: Genetics, Regulation and Toxicology (Ingelman-Sundberg, M., Gust&son, J.-A., and Orrenius, S., Eds.), p. 86, Karolinska Institute, Stockholm.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 294, No. 1, April, pp. 168-172, 1992
Quantification of Cytochrome P450 Reductase Gene Expression in Human Tissues’ Elizabeth
A. Shephard, **2 Colin N. A. Palmer,tp3 H. J. Segall,t14 and Ian R. Phillips?
*Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WClE 6BT, United Kingdom; and fDepartment of Biochemistry, Queen Mary and Westfield College, University of London, Mile End Road, London E!l4NS, United Kingdom
Received August 21, 1991, and in revised form November
26,199l
We have isolated and sequenced cDNA clones that code for a variant of human cytochrome P450 reductase. An RNase protection assay was used to quantify the corresponding mRNA in adult and fetal tissues. The results demonstrate that, in the samples analyzed, the cytochrome P450 reductase gene displays very little interindividual variation in its expression in adult liver and is subject to little developmental or tissue-specific regulation.
0 1992 Academic Press, Inc.
The flavoprotein NADPH-dependent cytochrome P450 reductase (EC 1.6.2.4) plays an essential role in the cytochrome P450-mediated monooxygenase system of microsomal membranes, where it transfers electrons from NADPH to cytochromes P450 (1,2). The monooxygenase system is present in virtually all mammalian tissues and is involved in the synthesis of endogenous compounds such as steroids, fatty acids, and prostaglandins and in the activation and detoxification of foreign hydrophobic compounds, including many therapeutic drugs, environmental pollutants, and chemical carcinogens (3). Cytochrome P450 reductase has also been implicated in the NADPH-linked peroxidation of microsomal lipids (4) and the oxidative degradation of heme (5-7). The ratio of cytochrome P450 reductase to total cytochromes P450 in rat liver microsomal membranes is 1:15 (8). Some inducers of cytochrome P450, such as pheno1 Supported by grants from the Cancer Research Campaign and the City and Hackney Health Authority. ’ To whom correspondence should be addressed. 3 Present address: Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, CA 92037. 4 Permanent address: Department of Veterinary Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, CA 95616.
168
barbital, can increase the amount of cytochrome P450 reductase and its mRNA by about 2.5-fold, thus maintaining the ratio (8-10). But others such as P-naphthoflavone, do not induce the reductase (8, 10). Cloned cDNAs coding for cytochrome P450 reductase have been isolated from several species including rat (ll), rabbit (12), and yeast (13). We have previously demonstrated, through the use of a partial-length human cytochrome P450 reductase clone, that in man the protein is encoded by a single gene located on chromosome 7q11.2 (14). In this paper we report the isolation and sequence of a full-length cDNA clone coding for an allelic variant of human P450 reductase, and the quantification by an RNase protection assay of the corresponding mRNA in adult and fetal liver and in adult extrahepatic tissues. MATERIALS AND METHODS cDNA library screening. A human placental cDNA library (in Xgtll) (Clontech) was screened with a 263-bp EcoRI/PstI DNA fragment derived from the 5’ end of a truncated 1950-bp human cytochrome P450 reductase cDNA clone (XHP450Rl) isolated previously (14). DNA sequencing. cDNAs were inserted into pUC19 and restriction mapped. Restriction fragments were subcloned into M13mp19 or pUC19 and sequenced by the single-stranded (15) or double-stranded (16) dideoxy chain termination methods, respectively, using the Klenow fragment of Eschrichia coli DNA polymerase or modified T7 polymerase (“Sequenase,” United States Biochemicals) for the former method and Sequenase for the latter. When necessary, a deaza dGTP sequencing kit (Pharmacia) was used to sequence through compressions. RNA isolation. Adult liver samples were obtained from Dr. Urs Meyer (Base1 Biocenter), Dr. Alan Boobis (Hammersmith Hospital, London), and colleagues at the Addenbrookes Hospital, Cambridge. One sample was obtained from a lobactomy performed at University College Hospital, London. The fetal liver samples were from the Medical Research Council Tissue Bank (Royal Marsden Hospital, London). Extrahepatic tissues were from St. Bartholomew’s Hospital, London. All tissues were frozen in liquid nitrogen and stored as l-g pieces at -78°C. Frozen tissue was placed in a coffee grinder with pellets of frozen COs and ground to a powder. RNA was extracted by the guanidinium thiocyanate/LiCl method (17). 0003-9861/92 $3.00 Copyright 0 1992 by Academic Press, IIX. All rights of reproduction in any form reserved.
QUANTIFICATION
OF HUMAN
P450 REDUCTASE
GENE
EXPRESSION
169
RNme protection assay. A 180-bp fragment of clone 27, extending from the 5’-most PstI site of this clone to the EcoRI cloning site (see Fig. 1) was inserted into pBluescript KS which had been digested with GGAGACl!l’Z-ACACCC~TC~AGGCGGQlvAs&srHisValAs@rhrSsrSaxThrValSsxGl"AlaValAlaGl"GluValSsr
AAGTATCT 60
252180-
AcGAGcAcxlT OT 1uHisPhe Asz%suSarGlWalLysPhaAlaValPheGlyLsuGlyAsnLYsThrTYYG AAiUCCATWkAAGTi~ACAA--km AsnAlaMstGlyLysTyrValAspLysArgLe"GluJX.nLeuGlyAlaQlnArgIlaPhs D660 GluLsuG1yLs"GlyAspAspAspGlyAs~Gl"Gl"AspPhsIle'IhrTrpArgDl" CAGTrCTA~CAGC720 GlnPhsTrpProAlaValCysGl"HisPh&lyValGl"AlaThr?ZlyGluGl"SsrSsr WXACACCGACATAGA-TACAAlTZGC!CAGTAmIlaArgGlnTyrGluLeuValValHisThrAspIlsAspAlaAlaLysValTyx%btGly rZCXTlUAmAAGAACG840 GAGA--ACGAGAACCAGAAGCCC
1"SsrDlyAepHisValAla His~GluteuAapIleSerAepGerLyeIleRrgTyrO GlGTACCC~CCA-E--C! ValTyr~oAlaAsnAs~erAlaLeuValAsnGlnLe"GlyLysIleLsuGlyAlaAsp CCA~ C LeuAsnAsnLeuAepOluOluSerAenLysLyeHisProPa LeuAepValValI4stSar CcoMcCCTA CGTCCTACCGCA~CCTTACl'ACCEGACA~CCAACCCXXGll40 ~IB"Asp11eTkAsnP?.-0Pr0 ProCysPrdIhrserTyrA~AlaLs" C--ArgThrAenValLeuTyrOluLeuAlaalnTyrAlaserO -~AAAaGAOCPGTACCTCAGCTOOO c.rixGcAAGA'FX'lE Le~LyeMetAla8erGs~s~l~l~lyL~lu~~~~s~ValVal GAC!CAC&~-~~A~A&~~C1380 AepHisLeuCyeOluLeuIauPmArgL.m.GlnAl~ er1leAlaSerSer lY!-CCCCAA-CA--MT~ CTAAG SsrLysValHisProAsnSarValHisIleCysAlaValValValGluTyrOluThrLys c3cC RlaOlyArgIloAQ1Ly~l~alAla~~~~~~~lu~o -===vF= a Gly GA~'NX!CCA~~CCAGTrA~l'2CCC1560 GluA8nGlyGly~AlaLeuValPr&stPhsVaULrgLy TK!AMW!CACCA-lTAlGQW3GCCC-TA PhsLysAlaThrPhrProVa1IloMetVa1G1yPrcGlyl%rGlyTrPHisProPheIle aOCTPCATCCROOPCXaOOC -GlyPhsIlaGlnGluArgAlaTrp~"Ar$~GlnGlyLysGl"ValGlyGluThr~" Clt3TACTA~Cl740 LeuTyrTyrDlyC'yslsr~~l~p~~u~~~luGlu~~laGln T GTCCCAC PhsHis~p(lly~~~LouThrOlnLouAsnValAla~~sr~luGl~srHis AAG3WTAWRXAGCACX! WWAAAGCAAGACffiAGAGCAC~ TmAmAA LysVal~alGlnHisLs~"LysGlnAspArgZl"HisLeuTrPLysLs"IleGlu -rcxxcAmTGmAGAAc W GlyGlyAlaHisIleTyrValCysGlyAspAlaArgAsnMstAlaAzyAspValGl.nAsn ACC BACTAC ‘l%rPhaTy?cAspIleValAl~luLeualyAlaMstGl"HisAlaGlnAlaValAspTyr ATCAAGAAACNA~A CpocC IlsLysLysLeuWetlhrL~ly~~r~~pVal~sr*** lGccccAcccA~C CTQGCTC~CCGTAGT ~TGGGCGCAGGCCCAGTZACAAAG&?KXK2160 C!CKXK!A lYXA~G'l?3TMATAATl.lTAMTAA-~ GTA
FIG.
1.
540 600
780
FIG. 2. Analysis of cytochrome P450 reductase mRNA by RNase protection. Molecular weight markers (1-kb ladder, Bethesda Research Laboratories) (a), undigested antisense RNAprob (b), RNase protection analysis of 10 fig of total RNA isolated from different adult human liver samples (c-l). Each assay contained 1 X lo5 cpm of antisense RNA probe.
1020 1080
1200 1260
1440 l5O0 1620 1680
1800 1860 1920 1980 2040 2100 2220 2400
The cDNA and deduced amino acid sequences of human cy-
tochrome P450reductase.The underlinedsequencerepresentstheregion of the mRNA that is complementary to the antisense RNA probe used
for the quantification experiments. The dots indicate the position of the polymorphic sites. The boxed sequence represents the poly(A) addition site signal sequence.
PsB and EcoRI. The resulting construct (pBSOR& was linearized by digestion with Hind111 (which cuts in the polylinker on the 5’ side of the EcoRI site). The sample was then incubated with proteinase K (50 fig/ml) and 10% SDS5 for 10 min at 37”C, extracted once with phenol/ chloroform (l:l, v/v), and ethanol precipitated. A 253-nucleotide-long radiolabeled “antisense” RNA probe was produced from the linearized Bluescript construct through the use of an in vitro transcription kit (Stratagene) according to the supplier’s recommendations. The reaction contained 10 units of T7 RNA polymerase and 50 /.&i [w~‘P]CTP (800 Ci/mmol, ICN). The DNA template was removed by digestion with RNase-free DNaseI (Stratagene). The RNA transcript was precipitated, resuspended, and purified by electrophoresis through an 8 M urea/6% polyacrylamide gel. Gel-purified RNA was resuspended in hybridization buffer (80% formamide, 40 mM Pipes, pH 6.4,0.4 M NaCl, 1 mM EDTA) and stored in aliquots at -20°C. All hybridizations were performed in a total volume of 30 pl containing 10 or 20 fig of sample RNA. The total amount of RNA was made up to 30 pg by addition of calf liver tRNA (Boehringer). An antisense RNA probe (0.4-1.0 X 10’ cpm) was included in each hybridization reaction. The hybridization mix was heated at 80°C for 10 min and then incubated at 45’C overnight. After hybridization, unprotected RNA was digested with RNase A. The mixture was then treated with proteinase K and extracted with phenol/chloroform (l:l, v/v). Protected RNA was precipitated with ethanol, resuspended, and electrophoresed through an 8 M urea/6% polyacrylamide gel. The gel was washed in 10% methanol and 10% acetic acid, rinsed in water, and dried under vacuum. The protected RNA probe was visualized by autoradiography at -78°C using an intensifying screen and quantified by scanning densitometry using an Ultrascan laser densitometer (LKB). Comparison with a standard curve of undigested radiolabeled probe permitted the absolute quantification of the corresponding mRNA in terms of molecules/pg of total RNA. This was converted to molecules/cell by using the average RNA content of a mammalian cell (5 pg).
5 Abbreviations used: Pipes, 1,4-piperazinediethanesulfonic dodecyl sulfate.
sodium
acid; SDS,
170 RESULTS
SHEPHARD AND
DISCUSSION
Isolation and sequence of cytochrorne P450 reductase cDNAs. Despite exhaustive rescreening of two human liver cDNA libraries, we were unable to isolate a cytochrome P450 reductase cDNA longer than the 1.95kb cDNA reported previously (14). To obtain a full-length cDNA for this protein we screened a human placental cDNA library which contained cDNA inserts that ranged in size from 0.8 to 3.6 kb with an average size of 1.8 kb. A screen of approximately 80,000 plaques of the placental cDNA library with a fragment derived from the 5’ end of a partial-length human cytochrome P450 reductase cDNA clone isolated from a liver library (14) yielded three positives (clones 27, 28, and 35). The cDNAs isolated from these clones were about 2100,2400, and 1900 nucleotides long, respectively. Their restriction maps overlapped with each other and with that of the cDNA isolated from the liver library. The sequence derived from these clones (Fig. 1) extends from the third base of the initiation codon and includes an open reading frame of 2028 bp followed by a stop codon and a 3’ noncoding region of 372 bp that contains a polyadenylation signal sequence (AATAAA) located 14 bp from a poly(A) tail. No differences in sequence were found in the overlapping regions of the liver and placental cDNAs. The deduced amino acid sequence has 93% similarity to that of rat cytochrome P450 re-
ET AL.
ductase (11). There is also a high degree of nucleotide similarity between the 3’ noncoding regions of cytochrome P450 reductase mRNAs of man, rat (ll), and rabbit (12). The evolutionary conservation of sequences within this region suggests that they may have an important functional role. The amino acid sequence derived from the human cDNAs is identical to that determined by microsequence analysis and mass spectrometry of peptide fragments (18). Hanui et al. (18) found evidence for two polymorphic sites within the protein sequence: val or ala at position 499 and g/n or arg at position 551. The sequence we report has a val at position 499 and a g/n at position 551 (Fig. 1). However, Yamano et al. (19) reported a cytochrome P450 reductase sequence that contained ala at position 499 and g/n at position 551. Taken together, these results indicate that there must be at least three alleles for cytochrome P450 reductase within the human population. Quuntifiation of cytochrome P450 reductase gene expression. We wished to determine the extent to which cytochrome P450 reductase gene expression varied between individuals and whether it was subject to developmental and/or tissue-specific regulation. This required an assay that was accurate, precise, and sensitive. Our approach was to develop an assay based on RNase protection. The antisense RNA probe used in these experi-
prostate MCF-7 cell line colon placenra Foetal liver 3 Foe&l liver 2 Foetal liver 1
tr
12
$ B
11 10
2
9
I
7654328
1
. mRNAs/cell FIG. 3. Quantification of cytochrome pg of total RNA from each sample.
P450 reductase mRNA in human tissues. Data were obtained from RNase protection
assays of 10 and 20
QUANTIFICATION
OF HUMAN
P450 REDUCTASE
abcdef
FIG. 4. RNase protection analysis of cytochrome P450 mRNA in human fetal liver samples. Undigested antisense RNA probe (a). RNase protection analysis of tRNA (b), and 10 pg of total RNA isolated from adult liver (c) and from different human fetal liver samples (d-f). Each assay contained 4 X 10’ cpm of antisense RNA probe.
ments contained a region complementary to a section extending from position 297 to 465 of the coding sequence of the cytochrome P450 reductase mRNA (Fig. 1). This region was chosen because it contained neither of the polymorphic sites that has been identified in this gene (18; this paper). The RNase protection assay revealed that the cytochrome P450 reductase mRNA was present in similar amounts in all 20 adult liver samples analyzed (Fig. 2 and data not shown). For quantification purposes, 10 and 20 pugof total RNA from each liver sample were assayed independently. After correction for the amount of total RNA, the two assays gave identical results, thus indicating that the technique was reproducible and gave a linear response. The linearity of the assay extended from 0.1 to over 100 molecules/cell (data not shown). The use of a pure mRNA transcript derived by in vitro transcription of a cloned cDNA confirmed the accuracy of the assay. The results demonstrated that the concentration of the cytochrome P450 reductase mRNA displayed an interindividual variation of no more than threefold (from 1 to 3 molecules/liver cell) (Fig. 3). The accurate quantification obtained by the RNase protection assay confirms and extends our preliminary data derived from Northern blot hybridization analysis of a limited number of adult liver samples (14). Cytochrome P450 reductase mRNA was found to be present in extrahepatic tissues such as prostate, adrenal, colon, and placenta and in the human breast cancer cell line MCF-7 (Fig. 3 and data not shown). The concentration of the mRNA in these tissues (about 1 molecule/cell)
GENE
171
EXPRESSION
was similar to that of the lowest values found in adult liver samples and was about half the average for adult liver. Immunohistochemical studies of human tissues indicate that cytochrome P450 reductase is present in all hepatocytes, but in extrahepatic tissues such as the adrenal it is confined to specific regions (20). Thus, it is likely that the mRNA also is not uniformly distributed in the cells of extrahepatic tissues, and consequently, in the cells in which it is expressed, it may be present in concentrations very similar to that found in adult hepatocytes. The mRNA is also present in fetal liver samples ranging from 12 to 20 weeks gestational age (Figs. 3 and 4), but at concentrations lower than those found in adult liver (between 0.3 and 0.5 molecules/cell) (Fig. 3). Taking into account the fact that a large proportion of fetal liver is composed of hemopoietic cells (21), it is apparent that the concentration of cytochrome P450 reductase mRNA in fetal hepatocytes cannot be much less than that present in adult hepatocytes. We have demonstrated that there is very little interindividual variation in the expression of the cytochrome P450 reductase gene in adult liver. In addition, the gene is subject to little developmental or tissue-specific regulation and thus is an example of a constitutively regulated gene. This is in marked contrast to many cytochrome P450 genes, which exhibit large interindividual variations in their expression in adult liver (ranging from 10s to 1000s of fold) and are subject to marked developmental and tissue-specific regulation (22,23; C. Palmer, E. Shephard, and I. Phillips, unpublished). ACKNOWLEDGMENTS We thank Dr. Urs Meyer, Dr. Alan Boobis, and colleagues at the Addenbrookes Hospital, Cambridge, and St. Bartholomew’s Hospital, London, for adult human tissue samples, and the MRC Tissue Bank, The Royal Marsden Hospital, for human fetal liver samples. The work was supported by grants from the Cancer Research Campaign and the City and Hackney Health Authority.
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