Estrogen receptor alpha increases basal and cigarette smoke extract-induced expression of CYP1A1 and CYP1B1, but not GSTP1, in normal human bronchial epithelial cells.

Gender-specific estrogen receptor alpha (ERalpha) expression may plausibly influence lung carcinogenesis in females. Initial genome-wide microarray studies confirmed that carcinogen metabolism genes (CYP1A1, CYP1B1) were those most responsive to cigarette smoke extract (CSE) in normal bronchial epithelial (NHBE) cells. These two genes encoding phase I bioactivating enzymes and the GSTP1 gene encoding a phase II deactivating enzyme were then tested for induction by ERalpha. NHBE cells (native ERalpha-) were transfected with wild-type ERalpha-adenoviral constructs, and then exposed to CSE, 17beta-estradiol (E2), and/or the ERalpha inhibitor, ICI 182,780. The expression levels of CYP1A1, CYP1B1, and GSTP1 were then determined by RNA-specific quantitative RT-PCR and immunoassay. ERalpha increased the basal expression of CYP1B1 4.04-fold (P < 0.01) at the mRNA level and 6.5-fold at the protein level. ERalpha also increased the CSE-induced mRNA expression of CYP1B1 2.26-fold (P < 0.01), but not the protein expression. ERalpha did not alter the CYP1A1 mRNA levels, but did increase protein expression 2.0-fold (P < 0.01) on CSE exposure, and 6.2-fold (P < 0.01) upon E2 exposure. These effects could be inhibited by ICI 182,780. ERalpha did not alter the expression of GSTP1. Chromatin immunoprecipitation assay (ChIP) assay confirmed ERalpha binding to CYP1B1 promoter near the transcription start site. These results suggest that ERalpha regulates the CYP1B1 expression at a transcriptional level, and CYP1A1 expression at a translational level. These data raise the possibility that inter-gender differences in expression of ERalpha that are known to exist in human lung may contribute to inter-individual expression differences in CYP1A1 and CYP1B1, and to differences in carcinogen metabolism and mutation.

Gene regulation in an MCF-7 cell line that naturally expresses an estrogen receptor unable to directly bind DNA.

In the described studies, we have developed a variant of the MCF-7 cell line, M-ERd3/g8, for analysis of 17-beta-estradiol (E2)-action without direct DNA interaction by E2-receptors. M-ERd3/g8 cells principally express the estrogen receptor alpha (ER) form ERDelta3 which, due to skipping of exon 3, lacks the second zinc finger of ER that is required for direct DNA interaction. This was achieved by introduction of siRNA targeting exon 3 to a Tamoxifen-selected MCF-7 variant, TMX 2-11, expressing approximately equal amount of full-length ER and ERDelta3 proteins. M-ERd3/g8 cells exhibited a normal nuclear ER localization, and ERDelta3 expression levels were similar to those for full-length ER protein in MCF-7 cells. Ser 118 phosphorylation of the ERDelta3 was triggered by E2 treatment. The expression of several well characterized E2-responsive markers was strongly modified in the M-ERd3/g8 cells. The E2-induction of progesterone receptor (PR) and HEM45 mRNAs was abolished. The effect on pS2 mRNA expression was complex: the pS2 mRNA levels fell approximately 50-fold in control M-ERd3/g8 cells. There was E2-induction of pS2-expression but with an altered temporal pattern. This was blocked by inhibitors of the p42/44 mitogen activated protein (MAP) kinase and inositol triphosphate (PI3) kinase pathways suggesting a role for cytoplasmic signaling pathways. Gene array analysis and real-time polymerase chain reaction (PCR) studies identified several genes whose expressions were induced in E2-treated M-ERd3/g8 cells. These included A-Myb, a homolog to the avian myoblastosis virus oncogene, carbonic anhydrase XII (CAXII), chemokine ligand 12 (CXCL-12), early growth response 3 (EGR 3), fibrinogen B beta (FibBbeta), along with serine protease 23 (SPUVE). The responses fell into several temporal patterns. A-Myb, CAXII, CXCL-12 and EGR 3 were E2-induced within 2 h. The expression of CXCL-12 and EGR 3 was persistent to 24 h, while that of A-Myb and CAXII was not persistent in M-ERd3/g8 cells. FibBbeta and SPUVE expression was not induced until times later than 6 h. Expression of none of the genes was elevated prior to 2 h, but the utilization of a 24 h time point for the gene array analysis may have eliminated the most transiently responsive genes. Immediate early 3 (IE3) was down-regulated by E2 in the M-ERd3/g8 cells but was transiently up-regulated during the 2-6 h period in MCF-7 cells. Basal levels of several of the genes were strongly reduced in M-ERd3/g8, compared to MCF-7. The studies suggest that M-ERd3/g8 cells provide a new model for studies of E2-action without direct ER binding to DNA and where E2-action must be via alternate pathways.

Upstream regions of the estrogen receptor alpha proximal promoter transcript regulate ER protein expression through a translational mechanism.

Estrogen receptor alpha (ER) mRNA is primarily transcribed from two promoters, the two transcripts share identical sequence encoding the same ER protein but differ in upstream regions. The 5' region of the two transcripts contain upstream open reading frames (uORFs) encoding potential peptides of 20 and 18 amino acids. The peptides have five C-terminal residues in common. These studies were undertaken to determine if the uORFs and encoded peptides differentially affected expression of ER. Expression of green fluorescent protein (GFP) reporter constructs containing upstream proximal promoter transcript sequences with the first 18 codons of ER fused to GFP was tested in HeLa cells. The cells expressed reduced levels of GFP as compared to the pEGFP-N1 parent vector; the effect was dependent on the presence of an intact proximal ER transcript uORF. Similar regulation by the uORF was seen in transfected MCF-7, MDA MB-231 and Ishikawa cells. Only protein expression was affected by eliminating the uORF; RNA levels were unchanged. This indicates the mechanism is translational rather than being an effect of the introduced point mutations on either mRNA stability or transcription. Eliminating the uORF did not significantly increase expression from similar distal promoter transcript ER-GFP constructs. However, study of in-frame fusions of GFP to the entire proximal and distal uORFs and to their translational start motifs showed that the translational start region of the distal uORF was inherently better at initiating translation than the AUG environment of the proximal promoter transcript uORF. The data indicate there are regulatory properties suppressing expression from the ER translation start which are specific to the unique regions of the ER proximal promoter transcript and these are likely associated with the proximal transcript uORF peptide product.

Expression of an estrogen receptor alpha variant protein in cell lines and tumors.

Human estrogen receptor alpha (ER) mRNA is a mixture of wild type and alternatively spliced variants. Many studies have examined the potential of ER mRNA profiles to serve as diagnostic/prognostic cancer biomarkers, but only a few have attempted to correlate ER mRNA profiles with protein expression. Representative ER mRNA pools were reproduced from the cDNAs of MCF-7 cells, a human breast tumor and human uterus and translated in a protease-free environment by reticulocyte lysates to determine relative translation efficiencies between the various ER mRNA transcripts and to facilitate identification of translated proteins. Cell line and tumor extracts were then examined for expression of the ER variant proteins identified in reticulocyte lysate translations. Each of the ER mRNA pools were translated by reticulocyte lysates into two ER proteins with molecular weights of approximately 60 and 52 kD. Western immunoblotting with various C- and N-terminal-directed, anti-ER antibodies and comparison with expressed ER protein standards established that the 52 kD protein (ERDelta7P) was translated from the predominant splice variant mRNA in each pool, which is missing exon 7. The 60 kD protein contained wild type ER sequence minus 61 C-terminal amino acids lost due to an intentional run off truncation. ERDelta7P expression was subsequently demonstrated in MCF-7 cells by Western immunoblotting with the site-directed antibodies. A protein corresponding to ERDelta7P was also detected in other ER positive breast tumor cell lines, and extracts of ER positive breast and uterine tumors. This widespread expression of ERDelta7P in vivo suggests that it may have some biological function. ERDelta7P may also affect immunohistochemical evaluation of ER positivity in tumors depending upon the level of its expression and the antibody used.

Expression of an estrogen receptor alpha variant protein in cell lines and tumors.

Human estrogen receptor alpha (ER) mRNA is a mixture of wild type and alternatively spliced variants. Many studies have examined the potential of ER mRNA profiles to serve as diagnostic/prognostic cancer biomarkers, but only a few have attempted to correlate ER mRNA profiles with protein expression. Representative ER mRNA pools were reproduced from the cDNAs of MCF-7 cells, a human breast tumor and human uterus and translated in a protease-free environment by reticulocyte lysates to determine relative translation efficiencies between the various ER mRNA transcripts and to facilitate identification of translated proteins. Cell line and tumor extracts were then examined for expression of the ER variant proteins identified in reticulocyte lysate translations. Each of the ER mRNA pools were translated by reticulocyte lysates into two ER proteins with molecular weights of approximately 60 and 52 kD. Western immunoblotting with various C- and N-terminal-directed, anti-ER antibodies and comparison with expressed ER protein standards established that the 52 kD protein (ERDelta7P) was translated from the predominant splice variant mRNA in each pool, which is missing exon 7. The 60 kD protein contained wild type ER sequence minus 61 C-terminal amino acids lost due to an intentional run off truncation. ERDelta7P expression was subsequently demonstrated in MCF-7 cells by Western immunoblotting with the site-directed antibodies. A protein corresponding to ERDelta7P was also detected in other ER positive breast tumor cell lines, and extracts of ER positive breast and uterine tumors. This widespread expression of ERDelta7P in vivo suggests that it may have some biological function. ERDelta7P may also affect immunohistochemical evaluation of ER positivity in tumors depending upon the level of its expression and the antibody used.

Analysis of amplified DNA molecules by capillary electrophoresis and laser induced fluorescence.

The polymerase chain reaction (PCR) has revolutionized molecular biology. Portions of single-copy per cell genes (and cDNAs) prepared from very small tissue or cell samples can be specifically amplified for use in sequence determination, gene identification, and quantitation. Improvements to the method, such as the introduction of genetically engineered, thermostable polymerases, more precise thermocyclers and more efficient reverse transcriptases for mRNA conversion to cDNA, have combined to make RNA-PCR (also called reverse transcriptase, or RT-PCR) and PCR more reproducible and specific. Coupled with the high sensitivity of the reactions, RT-PCR and PCR are increasingly used as quantitative bio-analytical techniques.

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells.

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17beta-estradiol (E2) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor alpha (ERalpha) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERalpha, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERalpha, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERalpha mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERalpha or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERalpha mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E2 metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis.

Estrogen receptor mRNA splice variants produced from the distal and proximal promoter transcripts.

Relative proportions of the estrogen receptor (ER) alternatively spliced mRNA variants from the proximal (A) and distal (B) promoter pre-mRNA transcripts were measured in normal human uterus, an endometrial tumor, and in T47D, MCF-7, and BT-20 breast tumor cell lines. A single tube RNA-PCR method was developed to determine the proportions of the individual transcripts and a nested, competitive RNA-PCR method to determine the proportions of the alternatively spliced variants. Except for the BT-20 cells, the patterns of splice variants produced from each transcript were very similar. In BT-20 cells no splice variants were detected for the minor (< or = 1%) A promoter transcript, although the B promoter transcript was alternatively spliced similarly to the other samples, with the exon 7 variant as the major mRNA form. These results indicate that the mRNA spliced variant patterns in most tissues and tumors will be essentially unaffected by any changes in the A and B promoter ER mRNA transcript ratios that may occur. At least one exception does exist, however, and only more comprehensive studies can determine whether the BT-20 cells are unique or part of a larger subgroup.

Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells.

Human cytochromes P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) catalyze the metabolic activation of a number of procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. The aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a marked effect on estrogen metabolism in MCF-7 breast-tumor cells by induction of these two enzymes. To investigate whether induction of CYP1A1 and CYP1B1 by AhR agonists and the associated increase in E2 metabolism are common to all breast epithelial cells and breast-tumor cells, we determined the effects of TCDD on E2 metabolism, and CYP1A1 and CYP1B1 mRNA levels in a series of non-tumor-derived breast epithelial (184A1 and MCF-10A) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cell lines. In 184A1 cells, which did not express detectable estrogen receptor (ER) alpha mRNA, CYP1A1 mRNA and activity were induced by TCDD, and enhanced E2 metabolism in TCDD-treated cells was predominantly E2 2-hydroxylation. In MCF-10A, MCF-7, T-47D, ZR-75-1 and BT-20 cells, which expressed varying levels of ER alpha mRNA, both CYP1A1 and CYP1B1 mRNA levels and rates of both E2 2- and 4-hydroxylation were highly elevated following exposure to TCDD. In MDA-MB-157, MDA-MB-231 and MDA-MB-436 cells, which did not express detectable ER alpha mRNA and generally displayed fibroblastic or mesenchymal rather than epithelial morphology, CYP1B1 induction was favored, and the rate of E2 4-hydroxylation exceeded that of 2-hydroxylation in TCDD-treated cells. These results show that breast epithelial cells and tumor cells vary widely with regard to AhR-mediated CYP1A1 and CYP1B1 induction, suggesting that factors in addition to the AhR regulate CYP1A1 and CYP1B1 gene expression. In these cell lines, significant CYP1A1 inducibility was restricted to cultures displaying epithelial morphology, whereas CYP1B1 inducibility was observed in cells of both epithelial and mesenchymal morphology.

Alternative splicing of CYP2D mRNA in human breast tissue.

The human cytochrome P450 (CYP) 2D subfamily comprises the CYP2D6 gene and four pseudogenes, CYP2D7P1 and 2 and CYP2D8P1 and 2. The CYP2D6 gene product is a prominent drug-metabolizing enzyme, which is probably constitutive and has no known inducing agents. Alternative splicing of the pre-mRNAs of these genes has been detected in human liver and breast tissue. RNA-PCR, competitive RNA-PCR, Southern blotting, cDNA sequencing, and gene-specific PCR have been used to fully characterize the alternatively spliced forms of CYP2D mRNA in human breast tissue in the region of exon 5 to 8. Such alternative splicing could regulate the expression of CYP2D6 protein. A full-length mRNA (exons 5 to 8), and variants c (exon 6 deleted), b' (3' portion of exon 6 deleted), e (3' portion of exon 6 deleted, 3' 57-bp portion of intron 6 included), d (3' 57-bp portion of intron 6 included), and b (intron 6 included) were characterized and quantitated. Variant c was derived from CYP2D6, variants d, e, and b were from CYP2D7P, and variant b' and full-length mRNA were derived from both CYP2D6 and 2D7P. Full-length mRNA was a minor form in human breast tissue where variants b' and c predominated. Human breast tumor MCF-7 cells had CYP2D mRNA splice variant patterns similar to those of human breast tissue, while human liver tumor HepG2 cells had wild-type mRNA predominating. These results suggest that CYP2D6 could be regulated tissue specifically using tissue-specific alternative mRNA splicing.

Comparisons of CYP2D messenger RNA splice variant profiles in human lung tumors and normal tissues.

Allelic variants of the CYP2D6 gene, a member of the cytochrome P450 gene superfamily, have been implicated in susceptibility to lung carcinogenesis. Human breast CYP2D6 and CYP2D7P (from a pseudogene) mRNAs were previously reported to be expressed as a series of splice variants. In this study, the expression of full-length and splice variants of these mRNAs in human lung tissue and tumors are reported for the first time and are compared in order to probe the potential for differential CYP2D6 regulation in lung normal tissue and tumors. The splice variant profiles differed within the same individual, but no consistent differences were detected.

The molecular epidemiology of lung cancer.

One in ten tobacco smokers develops bronchogenic carcinoma over a lifetime. The study of susceptibility of an individual and a population to lung cancer traditionally has been limited to the study of tobacco smoke dose and family history of cancer. New insights into lung carcinogenesis have made the study of molecular markers of risk possible in human populations in the emerging field of molecular epidemiology. This review summarizes data addressing the relationships of human lung cancer to polymorphisms of phase I procarcinogen-activating and phase II-deactivating enzymes and intermediate biomarkers of DNA mutation, such as DNA adducts, oncogene and tumor suppressor gene mutation, and polymorphisms. These parameters are reviewed as they relate to tobacco smoke exposure, procarcinogen metabolizing polymorphisms, and the presence of lung cancer. Problem areas in biomarker validation, such as cross-sectional data interpretation; tissue source, race, statistical power, and ethical implications are addressed.

Quantitation of estrogen receptor mRNA and its alternatively spliced mRNAs in breast tumor cells and tissues.

Estrogen receptor (ER) mRNA exists as wild-type (full-length) and alternatively spliced variants in cell lines, normal tissues, and tumors. Most of the alternatively spliced variants discovered so far are missing one or more complete exons. RNase protection and RNA-PCR assays used previously to determine the relative concentration of a particular ER spliced-variant mRNA to wild-type mRNA have produced equivocal results because the probes/primers targeted only small regions within the nucleotide sequence. Variant ER mRNAs missing an exon outside the probe/primer region will react as if they were wild-type and any alternatively spliced variants containing a deletion at the probe/primer annealing site(s) will not be detected. A highly sensitive, competitive RNA-PCR assay has been developed that is quantitative with respect to the relative composition of wild-type ER and its alternatively spliced-mRNA forms, and semiquantitative with respect to their concentrations in cells and tissues. Separation and quantitation of the products are rapidly and accurately achieved by, respectively, capillary electrophoresis and laser-induced fluorescence. Wild-type ER mRNA concentration can be measured independently of all the reported exon deletion forms in a single PCR assay. Specific exon deletion forms can be measured by ER cDNA amplification with overlapping primer sets. Results obtained with RNAs isolated from two MCF-7 cell lines, a T-47D cell line, and five breast tumor tissues are presented.

Inhibition of estrone sulfatase in human liver microsomes by quercetin and other flavonoids.

Inhibition of estrone sulfatase activity offers the potential for breast cancer prevention therapy by blocking a route to estrogen synthesis. We have investigated the inhibition of this activity by natural flavonoids in a human hepatic microsomal preparation in vitro. The majority of studies were performed with a male liver, but male and female livers exhibited comparable estrone sulfatase activities. The natural flavonoids, quercetin, kaempferol, and naringenin, significantly inhibited estrone sulfatase activity with I50 < 10 microM for the most potent, quercetin. Estrone sulfatase activity in the liver microsomes was biphasic, with a high affinity, low capacity, low concentration activity (Km 14.3 microM, Vmax 0.5 nmol/min/mg protein), probably steroid sulfatase-catalysed, and a low affinity, high capacity, high concentration activity (Km 1.5 mM, Vmax 21.5 nmol/min/mg protein), probably arylsulfatase C or E-catalysed. The former activity was inhibited uncompetitively by quercetin, the latter competitively. Quercetin, a natural dietary constituent, is a potent inhibitor of estrone sulfatase in vitro, and thus has the potential to express antiestrogenic activity in vivo.

Characterization of purified human recombinant cytochrome P4501A1-Ile462 and -Val462: assessment of a role for the rare allele in carcinogenesis.

Human cytochrome P4501A1 (CYP1A1) occurs extrahepatically and is polymorphic, the common form having Ile at position 462 and the rare form having Val. The rare allele has been associated with enhanced susceptibility to lung cancer. To resolve its role in cancer we have constructed CYP1A1-Val462 cDNA by site-directed mutagenesis from CYP1A1-Ile462, as confirmed by sequencing and allele-specific PCR. Both alleles were expressed in Escherichia coli, and CYP1A1-Ile462 and -Val462 were purified to electrophoretic homogeneity. The secondary structures of both forms were virtually identical, with high alpha helix content, as assessed by circular dichroism. The P450s stereoselectively and regioselectively catalyzed the metabolism of (R)- and (S)- warfarin, in reconstituted systems, with very similar profiles. Both P450s produced (R)-6- and 8-hydroxy-warfarin with Km values of 0.40 +/- 0.06 and 0.43 +/- 0.05 mM, respectively, and Vmax values of 84.0 +/- 6.8 and 137.7 +/- 8.9 pmol/min/nmol CYP1A1-Val462, respectively, 1.0 +/- 0.1 and 1.0 +/- 0.1 mM, respectively, and 46.7 +/- 2.5 and 80.0 +/- 4.4 pmol/min/nmol CYP1A1-Ile462, respectively. Reconstituted CYP1A1-Val462 catalyzed ethoxyresorufin metabolism at a slightly but significantly higher rate than did CYP1A1-Ile462; Vmax values were 4.4 +/- 0.6 and 3.1 +/- 0.3 nmol/min/nmol CYP1A1, respectively. However, with the carcinogen benzo(a) pyrene as substrate, reconstituted CYP1A1-Ile462 together with epoxide hydrolase produced 7,8- and 9,10-dihydrodiols at comparable rates than did CYP1A1-Val462. Thus, the apparently greater susceptibility of the CYP1A1-Val462 genotype to lung cancer is probably not related to greater extents of carcinogen bioactivation.

Expression of cytochromes P450 in human breast tissue and tumors.

In an effort to determine which members of the cytochrome P450 (CYP) superfamily are expressed in human breast tissue and tumors, RNA-polymerase chain reaction studies have been undertaken. Detection of expressed CYP mRNAs identifies those forms of the enzyme that are capable of expression in breast tissue, and provides insight into the potential for in situ xenobiotic and therapeutic drug metabolism. CYP1A1 mRNA was present in (5/11) breast tissues and (6/13) tumors. When normal and tumor tissues were from the same individuals, higher amplification occurred in normal tissues. CYP1B1 mRNA was present in all but one tissue, and CYP2C mRNA forms were present in all of the tissues. CYP3A4 mRNA was present in (8/11) normal breast tissues and (2/13) tumor tissues, and CYP3A5 mRNA was present in (9/11) normal tissues and (2/13) tumor tissues. The expression of the CYP3A mRNA forms was not coincident, suggesting differential regulation. CYP2D6 mRNA was present in (10/11) normal breast tissue and (10/13) tumors. Two splice variants of CYP2D6 mRNA were also detected; one with a 207 bp intron spliced in was detected in all of the normal tissue samples and (11/13) tumors, whereas another (which lacks a 3'-portion of exon 6) was detected in (9/11) normal breast tissues and (7/13) tumors. Thus, examples of each of the xenobiotic-metabolizing CYP1, CYP2, and CYP3 subfamilies were detected in low levels in human normal breast tissue and tumors. The machinery for possible in situ bioactivation of xenobiotics and modification of therapeutic drugs is thus present in human breast tissue.

Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR.

In competitive RNA-PCR studies, contaminating DNA can produce incorrect results because of its potential to act as a second competitor. Preliminary studies using published methods for DNase I digestion of DNA as a contaminant of RNA, followed by thermal inactivation of the enzyme at 95 degrees C for 5 min before reverse transcription and PCR, suggested that the mRNA was also affected by these treatments. This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation. Competitive RNA-PCR of DT-diaphorase transcript was used to quantitate the effects of the various treatments. Other transcripts with varying initial concentrations were visually compared to ensure that the effects observed were not unique to specific mRNAs. With 1 U of DNase I/microgram RNA, thermal denaturation of the enzyme at 75 degrees C for 5 min preserved nearly all of the mRNA. Thermal denaturation at 95 degrees C for 5 min inactivated approximately 80% of the mRNA, whereas heating at 55 degrees C for 10 min did not completely denature the DNase I. For RNA-PCR of every transcript investigated, incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs. This treatment is highly recommended as a routine step in RNA-PCR and particularly with competitive RNA-PCR with human breast tissue samples (and presumably other human tissues), which are often contaminated with small amounts of genomic DNA.

Human cytochromes P4501A1 and P4501A2: R-warfarin metabolism as a probe.

Two forms of the cytochrome P450 enzyme superfamily, P4501A1 and P4501A2, that are heterogeneously distributed in populations and are induced in response to environmental factors are important because of their capacity to bioactivate procarcinogens. Phenotyping P4501A1 and P4501A2 in individuals will thus provide assessments of those individuals' susceptibility to procarcinogens. The anticoagulant drug warfarin is metabolized by human P4501A1 and P4501A2, and we have characterized this metabolism for the R-warfarin enantiomer as a potential in vivo probe. cDNA-expressed human P4501A1 and P4501A2 are regioselective for R-warfarin 6- and 8-hydroxylation with very similar KM values: 1.4 mM (6-hydroxylation), 1.2 mM (8-hydroxylation), 1.6 mM (6-hydroxylation), and 1.4 mM (8-hydroxylation), respectively, indicating possible binding competition for R-warfarin between the two forms. However, when comparing 6- and 8-hydroxylation, P4501A1 showed weak regioselectivity for 8-hydroxylation, whereas P4501A2 exhibited strong regioselectivity for 6-hydroxylation, with 6-hydroxylation/8-hydroxylation ratios of 0.6 and 5.0, respectively. These findings were confirmed by using microsomes from 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated HepG2 and MCF-7 cells expressing only P4501A1 (ratios of 0.7), and from human hepatic microsomal preparations containing only P4501A2 (average ratios of 4.0). P4501A2 levels in the liver preparations, as assessed by densitometry of immunoblots, correlated with R-warfarin 6-hydroxylation rates (r2 = 0.83) and caffeine 3-demethylation rates (r2 = 0.67), but not with R-warfarin 8-hydroxylation rates. P450s 2A6, 2B6, 2C9, 2D6, 2E1, and 3A4 did not yield either 6- or 8-hydroxy-warfarin from R-warfarin. We conclude that R-warfarin 6-hydroxylation rates are markers for human hepatic P4501A2, whereas ratios of 6-hydroxylation/8-hydroxylation could be used in vitro as a marker for P4501A1.

Determination of theophylline and its metabolites in rat liver microsomes and human urine by capillary electrophoresis.

A capillary electrophoretic (CE) method has been developed for the determination of theophylline and all of its identified and potential metabolites. The method is rapid, resolves all metabolites to baseline, and requires extraction of only some biological fluids. It has been applied to the analysis of theophylline metabolism by hepatic microsomes from rats treated with a variety of inducing agents for different forms of P450 enzymes which metabolize theophylline, and to human urine spiked with theophylline and its metabolites, and concentrated by solid-phase extraction.