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.

Loss of metastatic and primary tumor factor X activator capabilities by Lewis lung carcinoma cells cultured in vitamin K-dependent protein deficient serum.

A highly metastatic line of Lewis lung tumor cells established in fetal bovine serum (10%) was subcultured into normal rodent (mouse or rat) serum or rodent serum made deficient in functional vitamin K-dependent proteins (barium sulfate adsorption or warfarin treatment of animals). Following injection of cells cultured in normal rodent serum into C57BL/6 mice, Factor X activator activity in the primary tumors increased at a near linear rate per gram tumor and attained 5- to 8-fold higher levels than did cells grown in either of the deficient sera. Secondary lung foci were also visible in all mice of the normal-rodent serum groups within 10 days after injection, and by 21 days extensive tumor growth in the lungs had developed. No secondary lung foci were apparent in any mice of the deficient serum groups throughout 21 days of tumor burden. Cells cultured in nonrodent serum (fetal bovine serum) were less proficient than cells grown in normal mouse serum in developing primary tumor Factor X activator activity and producing secondary tumors. Exposure of cells cultured in barium sulfate-treated mouse serum to normal mouse serum for 3 h and 3 weeks prior to injection partially restored primary tumor Factor X activator and metastatic competence. These data strongly suggest that in Lewis lung tumor cells at least one species selective, plasma/serum vitamin K-dependent protein plays a major role in the regulation of metastatic events and demonstrate that there is a positive correlation between primary tumor Factor X activation activity and metastasis.

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.

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.

A new warfarin metabolite: structure and function.

The metabolism of the clinically utilized, anticoagulant warfarin [4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one] by rat liver microsomes has been investigated. The structure of a new warfarin metabolite [4-hydroxy-3-(3-oxo-1-phenyl-1-butenyl)-2H-1-benzopyran-2-one] (dehydrowarfarin) has been determined by mass spectral comparison with the chemically synthesized compound. The formation of dehydrowarfarin is catalyzed by cytochrome P-450 and is unusual in that the final product is effectively dehydrogenated warfarin.

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.

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.

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.

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.

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.

Reappraisal of thromboplastin.

Proficiency testing surveys in the state of New York indicate that despite increased sophistication in instrumentation, there has been no real improvement in interlaboratory reproducibility in prothrombin-time determinations over the last 10 years. This lack of improvement most pronounced in the therapeutic range of 20 to 30 sec. One reason may be that between types produced by the same manufacturer. While it has been possible in several laboratories to synthesize experimentally a thromboplastin with known content of lipid and active protein, no efforts have been made to make such a product by the same manufactures. While it has been made to make such a product commercially available. Blood levels of of warfarin were measured but cannot be reliably used to monitor anticoagulation. In a preliminary study, factor Xa activity was measured using chromogenic substrate S2222. Factor Xa activity gave a positive correlation with prothrombin times of patients receiving warfarin therapy. Chromogenic substrate factor assays may represent a future method of choice for controlling anticoagulant therapy.

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.