Retinoid target genes in acute promyelocytic leukemia.

All-trans-retinoic acid (RA)-based differentiation therapy induces clinical remissions in acute promyelocytic leukemia (APL). This has propelled interest in elucidating the molecular mechanisms responsible for these remissions. The t(15;17) rearrangement results in the expression of the PML/RARalpha fusion transcript that is paradoxically linked to the etiology and clinical retinoid response in APL. PML/RARalpha expression blocks terminal myeloid differentiation in APL. Treatment with pharmacological RA dosages overcomes the dominant-negative effects of PML/RARalpha to activate transcription of retinoid target genes. This regulation is linked directly to RA effects in APL, including PML/RARalpha degradation and induction of differentiation. Identifying retinoid target genes is an important step in developing a mechanistic understanding of RA effects in APL. RA target genes have been uncovered through the use of molecular genetic approaches as well as unique cellular and transgenic APL models. Recent developments in the proteomic and functional genomic fields are providing useful tools for elucidating mechanisms of RA response or resistance in APL. These target genes represent potential therapeutic targets in APL and other retinoid-responsive diseases. Previous spotlights in Leukemia have highlighted the importance of cytokine effects and signal transduction crosstalk in retinoid response in APL and in normal hematopoiesis. This review builds on prior work by addressing the role of retinoid target genes in mediating retinoid response or resistance in APL.

4HPR triggers apoptosis but not differentiation in retinoid sensitive and resistant human embryonal carcinoma cells through an RARgamma independent pathway.

Retinoids signal biological effects through retinoic acid receptors (RAR) and retinoid X receptors (RXR) and their co-regulators. We previously reported that all-trans retinoic acid (RA) triggers terminal differentiation in the human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1), through an RARgamma dependent pathway. RARgamma repression in NT2/D1-R1 cells accounts for RA resistance in this line. This report finds RARgamma repression is due to selective repression of RARgamma but not RARbeta transcription in NT2/D1-R1 cells. The repression is neither due to mutations in RARgamma nor its promoter containing the RA response element. Prior work was confirmed and extended by demonstrating that an RARgamma selective agonist preferentially signals differentiation of NT2/D1 cells, while RARalpha/beta, RARbeta, RXR agonists and an RAR pan-antagonist do not even when NT2/D1 cells are treated with these retinoids at 10 microM dosages. None of these examined retinoids induced differentiation of the RA resistant NT2/D1-R1 cells. In contrast, N-(4-hydroxyphenyl)retinamide (4HPR), a reported transcriptional activator of RARgamma was shown to potently induce growth inhibition and apoptosis in both NT2/D1 and NT2/D1-R1 cells. 4HPR-induced apoptosis was unaffected by co-treatment of both cell lines with equimolar RAR antagonist. Semi-quantitative reverse transcription-polymerase chain reaction (RT - PCR) assays of total RNA from 4HPR-treated NT2/D1 and NT2/D1-R1 cells did not reveal RARgamma induction. Since 4HPR signals in RA-resistant NT2/D1-R1 cells having an RARgamma transcriptional block, these results indicate that 4HPR triggers apoptosis but not differentiation through an RARgamma independent pathway. Taken together, these findings implicate a therapeutic role for 4HPR mediated apoptosis in germ cell tumors even when a maturation block is present.

Interpreting patterns of gene expression with self-organizing maps: methods and application to hematopoietic differentiation.

Array technologies have made it straightforward to monitor simultaneously the expression pattern of thousands of genes. The challenge now is to interpret such massive data sets. The first step is to extract the fundamental patterns of gene expression inherent in the data. This paper describes the application of self-organizing maps, a type of mathematical cluster analysis that is particularly well suited for recognizing and classifying features in complex, multidimensional data. The method has been implemented in a publicly available computer package, GENECLUSTER, that performs the analytical calculations and provides easy data visualization. To illustrate the value of such analysis, the approach is applied to hematopoietic differentiation in four well studied models (HL-60, U937, Jurkat, and NB4 cells). Expression patterns of some 6,000 human genes were assayed, and an online database was created. GENECLUSTER was used to organize the genes into biologically relevant clusters that suggest novel hypotheses about hematopoietic differentiation-for example, highlighting certain genes and pathways involved in "differentiation therapy" used in the treatment of acute promyelocytic leukemia.

Specific retinoid receptors cooperate to signal growth suppression and maturation of human embryonal carcinoma cells.

This study addresses the contributions of specific retinoid receptors during all-trans-retinoic acid (RA)-mediated differentiation and growth suppression of human embryonal carcinoma cells. The pleiotropic effects of RA are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), members of the nuclear receptor family of transcription factors. After RA-treatment the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) displays limited proliferative potential, reduced tumorigenicity, and morphologic and immunophenotypic neuronal maturation. RARgamma over-expression in NT2/D1 cells signals mesenchymal NT2/D1 terminal differentiation while RARalpha and RARbeta do not and RARgamma overcomes retinoid resistance in an NT2/D1 clone (NT2/D1-RI) having deregulated RARgamma expression. Since RARgamma transfectants do not display neuronal maturation, this study sought to identify cooperating retinoid receptors engaged in NT2/D1 differentiation. Through gain of function experiments, this report highlights RXRbeta as playing an important role along with RARgamma in signaling differentiation of NT2/D1 cells. Stable over-expression of RXRbeta, but not RXRalpha or RXRgamma, was found to signal NT2/D1 growth suppression and to induce a non-neuronal morphology and immunophenotype. Notably, co-transfection of RARgamma and RXRbeta resulted in marked growth suppression and for the first time, expression of typical neuronal markers of NT2/D1 differentiation. To clarify the role of RXRbeta and RARgamma in this differentiation program, a modified transient fibroblast growth factor-4 (FGF4) promoter-enhancer reporter assay that reflects effective RA-mediated differentiation of NT2/D1 cells was employed. Transfection of RARgamma or RXRbeta in NT2/D1 cells augments transcriptional repression of the FGF4 reporter and RARgamma and RXRbeta co-transfection markedly repressed reporter activity, indicating the combined role of these receptors in RA-induced NT2/D1 differentiation. Taken together, these findings reveal specific retinoid receptors must cooperate to signal terminal growth suppression and maturation of NT2/D1 cells. Since the transcriptional repression of FGF4 is coupled to the effective maturation of human embryonal carcinoma cells, the described co-transfection strategy should prove useful to identify genes with positive or negative effects on the differentiation program of these tumor cells.

Validation of precision-cut liver slices in dynamic organ culture as an in vitro model for studying CYP1A1 and CYP1A2 induction.

The utilization of precision-cut liver slices in dynamic organ culture as an in vitro model was validated by comparing the induction of the biomarker responses following in vitro (rat liver slice) and in vivo exposure of rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The biomarker responses investigated were cytochrome P450s 1A1 and 1A2 (CYP1A1 and CYP1A2) mRNA, protein, and activities. Precision-cut rat liver slices were incubated in dynamic organ culture for 24 hr with medium containing 0.001-10 nM TCDD or medium without TCDD (control). The resultant mean TCDD concentration in the slices ranged from 19 to 80,925 ppt (wet wt), respectively. A concentration-dependent induction of CYP1A1 mRNA, protein, and activities and a more modest induction of CYP1A2 mRNA was observed in liver slices at all medium concentrations of TCDD. The O-demethylation of 7-methoxyresorufin, a marker for CYP1A2 activity, was induced at TCDD medium levels of 0.01 nM and greater, whereas a detectable increase in CYP1A2 protein occurred only at the higher concentrations. Comparable liver concentrations of TCDD (8-64,698 ppt wet wt) were achieved at 24 hr following a single in vivo exposure of rats to TCDD at doses ranging from 0.002 to 5 microg/kg po. Concentration-effect and dose-response relationships for induction of CYP1A1 and CYP1A2 were similar following in vitro and in vivo exposure to TCDD, although the magnitude of induction was greater for in vivo exposure. The data support the use of liver slices in dynamic organ culture for assessing the relative in vivo potency of a compound to induce CYP1A1 and CYP1A2. Human tissue can also be readily utilized in this in vitro model to predict the biological and toxicological effects of a given in vivo exposure to TCDD.

Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR.

RXR is a nuclear receptor that plays a central role in cell signaling by pairing with a host of other receptors. Previously, 9-cis-retinoic acid (9cRA) was defined as a potent RXR activator. Here we describe a unique RXR effector identified from organic extracts of bovine serum by following RXR-dependent transcriptional activity. Structural analyses of material in active fractions pointed to the saturated diterpenoid phytanic acid, which induced RXR-dependent transcription at concentrations between 4 and 64 microM. Although 200 times more potent than phytanic acid, 9cRA was undetectable in equivalent amounts of extract and cannot be present at a concentration that could account for the activity. Phytanic acid, another phytol metabolite, was synthesized and stimulated RXR with a potency and efficacy similar to phytanic acid. These metabolites specifically displaced [3H]-9cRA from RXR with Ki values of 4 microM, indicating that their transcriptional effects are mediated by direct receptor interactions. Phytol metabolites are compelling candidates for physiological effectors, because their RXR binding affinities and activation potencies match their micromolar circulating concentrations. Given their exclusive dietary origin, these chlorophyll metabolites may represent essential nutrients that coordinate cellular metabolism through RXR-dependent signaling pathways.

Genetic and developmental diversity of hepatic cytochromes P450. Warfarin and progesterone metabolism by hepatic microsomes from four inbred strains of rat.

Hepatic microsomes from immature and sexually mature male and female ACI/SEGHsd, F344/NHsd, SHR/NHsd, and WKY/NCrl inbred rats were used to study cytochrome P450 (P450)-catalyzed oxidations of progesterone and both enantiomers of warfarin. Strains were selected on the basis of their different, homozygous allelic compositions for CYP2C11 and CYP2C13 (Rampersaud and Walz, 1992), but no correlations with the microsomal activities were observed. However, correlations were made regarding catalytic activities and the developmental control of CYP2A1 and CYP2C11 levels in microsomes. Other correlations were found for reactions of both warfarin enantiomers at the same atom or for a given enantiomer at different positions, and these appear to involve several P450 isozymes. Strain-dependent activity differences mainly involved the SHR/NHsd and WKY/NCrl strains. WKY/NCrl rats were the most unique strain, having low levels of CYP2C11 in adult males compared with the other inbreds but relatively high S-warfarin 4'- and 6-hydroxylase activities in immature animals of both sexes and adult females. These results suggest that the regulation and/or allozymic composition of hepatic P450s are different for WKY/NCrl rats, which makes them a poor choice as "normotensive controls" in comparison with hypertensive SHR rats.

Evidence that CYP2C19 is the major (S)-mephenytoin 4'-hydroxylase in humans.

The present study assesses the role of members of the human CYP2C subfamily in the 4'-hydroxylation of (S)-mephenytoin. When recombinant CYP2C proteins were expressed using a yeast cDNA expression system, 2C19 stereospecifically 4'-hydroxylated (S)-mephenytoin with a turnover number at least 10 times higher than that of human liver microsomes. 2C9 (both Ile359 and Leu359 alleles) and 2C18 (Thr385 and Met385 alleles) metabolized this substrate at a rate 100-fold lower than 2C19, and metabolism by these 2C proteins was not stereospecific for the S-enantiomer. 2C8 exhibited very little mephenytoin 4'-hydroxylase activity. In contrast, the Ile359 allele of 2C9 had a high turnover number for the hydroxylation of tolbutamide, while the Leu359 allele was less active toward this substrate. Immunoblot analysis of 16 human liver donor samples indicated that (S)-mephenytoin 4'-hydroxylase activity correlated with the hepatic CYP2C19 content, but it did not correlate with the hepatic content of CYP2C9. Moreover, direct sequencing of the polymerase chain reaction (PCR) products of 2C9 mRNA from six of these human livers through areas of known allelic variations indicated that the identity of the allele of 2C9 (Cys144 vs Arg, Tyr358 vs Cys, Ile359 vs Leu, or Gly417 vs Asp) did not appear to influence (S)-mephenytoin 4'-hydroxylase activity in these samples. These data indicate that 2C19 is the principal determinant of (S)-mephenytoin 4'-hydroxylase activity in human liver.

Expression of CYP1A1 and CYP1A2 genes in human liver.

Immunoblot analysis of human livers using a monospecific antibody to rat CYP1A2 section demonstrated that the expression of CYP1A2 protein is highly variable in human liver. Quantitative PCR analysis was then employed to examine the interindividual variability of both CYP1A1 and CYP1A2 mRNAs in human liver. Hepatic content of CYP1A2 mRNA correlated significantly with levels of CYP1A2 protein as analysed by immunoblot analysis (r = 0.58; p < 0.01). CYP1A2 mRNA content varied > 40-fold among individuals while CYP1A1 content varied > 20-fold. CYP1A2 mRNA was higher than CYP1A1 mRNA (approximately two to 30-fold) in livers of different individuals. The individual with the highest CYP1A1 and CYP1A2 mRNA amounts was a current smoker, but mRNA expression in two other smokers was within the range observed among nonsmokers. The expression of the two CYP1A mRNAs correlated highly (r = 0.72; p < 0.0005) when smokers were included, but the correlation was less significant (r = 0.62; p < 0.05) in nonsmokers. We amplified a full-length CYP1A2 cDNA clone by PCR from a liver which expressed extremely low amounts of CYP1A2 protein. Sequence analysis indicated that exon 4 was missing in this clone, but no other sequence changes were found. PCR analysis demonstrated that both the normally spliced mRNA and abnormally spliced mRNA could be detected in all human livers examined, but the normally spliced mRNA was more abundant than the splice variant. Therefore, sequence changes in the coding region of CYP1A2 did not account for the poor expression of CYP1A2 in this individual.

Exocyclic-keto reductase activities for progesterone and S-warfarin in hepatic microsomes from adult male rats.

Hepatic microsomes from adult male rats representing six inbred strains catalyzed quantitatively significant, NADPH dependent reductions of progesterone to the 20 beta (20R) alcohol and S-warfarin to its 11S-OH product. Microsomes from mature females and immature rats of both sexes were essentially devoid of these activities. Two strains of rat evidenced about 21% of these activities compared with the other strains and both activities were 25-81% repressed by treatment of rats with phenobarbital (PB). An excellent linear correlation was demonstrated for the two activities considering sex, age, NADPH much greater than NADH preference, PB-repression and strain differences. However, detergent latency (71%) and resistance to trypsinolysis were only observed for the keto-reductase activity with S-warfarin. Microsomes also catalyzed the reduction of progesterone to its 20 alpha-OH derivative but this activity preferred NADH greater than NADPH, was induced 2.7-fold by PB and was essentially independent of age, sex and animal strain. Furthermore, unlike the 20 beta-OH activity, this reduction was resistant to proteolytic inactivation.

Spermine stabilization of folded ribonuclease T1.

The interaction of ribonuclease T1 with tetraprotonated spermine (SPM4+), Mg2+, phosphate and other ionic ligands at pH 6.0 was investigated in binding experiments at 25 degrees C and/or by their effects on the midpoint temperature for thermal unfolding of the enzyme. SPM4+ binding with the native protein at 25 degrees C was characterized by an association constant of approximately 2 x 10(4) M-1. This ligand also binds to the unfolded protein but with a approximately 35-fold lower affinity. Phosphate binds at the active site whereas Mg2+ and SPM4+ cations compete for binding at a polyanionic locus that probably involves residues Glu-28, Asp-29, and Glu-31 at the C-terminal end of the alpha-helix. Steady-state kinetic studies using minimal RNA substrates demonstrated that SPM4+ binding with the enzyme does not affect its catalytic activity. SPM4+ also preferentially binds with the folded form of the disulfide-reduced enzyme which has the same or slightly enhanced catalytic properties compared with native ribonuclease T1. The unfolding rate for the native protein in 8 M urea was approximately 8-fold lower in the presence of 0.05 M SPM4+. SPM4+ appears to increase the amplitude of an unobserved fast phase(s) for refolding of the native enzyme. A single kinetic phase characterized refolding of the reduced enzyme which was slightly faster than the slowest refolding phase for the native form.

Potentiation of the antiviral activity of poly r(A-U) by riboflavin, FAD and FMN.

The role of riboflavin (RFN), FAD or FMN in modulating the antiviral activity of poly r(A-U) was examined by the human foreskin fibroblast-vesicular stomatitis virus bioassay in which the concentrations of poly r(A-U) was fixed at 0.1 mM or 0.2 mM while the riboflavin, FAD or FMN concentration was varied to produce variable RFN (or FAD or FMN)/ribonucleotide ratios ranging from 1/16 to 2/1. Riboflavin, FAD and FMN tested individually did not exhibit any antiviral activity, while poly r(A-U) alone exhibited antiviral activity. When poly r(A-U) was combined with riboflavin, FAD or FMN, the antiviral activity was potentiated seven- to twelve-fold at RFN (or FAD or FMN)/ribonucleotide ratios in the region of 1/4.

Enhancement of the antiviral and interferon-inducing activities of poly r(A-U) by carminic acid.

Experiments have been designed to systematically examine the effects of carminic acid (CAR) on the antiviral/interferon-inducing activity of poly r(A-U), using the human foreskin fibroblast-vesicular stomatitis virus bioassay system. Modulation of the antiviral/interferon-inducing activity of poly r(A-U) by carminic acid was examined at fixed poly r(A-U) concentrations of 0.05 mM or 0.2 mM while varying the carminic acid concentrations to produce variable CAR/ribonucleotide ratios ranging from 1:16 to 2:1. Carminic acid and poly r(A-U) were tested individually at the concentrations employed in the CAR/poly r(A-U) combinations. Neither the carminic acid alone nor poly r(A-U) alone were effective antiviral agents/interferon inducers. The antiviral/interferon-inducing activity of poly r(A-U) was potentiated twelve-fold at CAR/ribonucleotide ratios in the region of 1/6 to 1/4. These results suggest a synergism between the poly r(A-U) and the carminic acid at the concentrations employed in this study.