Multifunctional bioscaffolds for 3D culture of melanoma cells reveal increased MMP activity and migration with BRAF kinase inhibition.

Matrix metalloproteinases (MMPs) are important for many different types of cancer-related processes, including metastasis. Understanding the functional impact of changes in MMP activity during cancer treatment is an important facet not typically evaluated as part of preclinical research. With MMP activity being a critical component of the metastatic cascade, we designed a 3D hydrogel system to probe whether pharmacological inhibition affected human melanoma cell proteolytic activity; metastatic melanoma is a highly aggressive and drug-resistant form of skin cancer. The relationship between MMP activity and drug treatment is unknown, and therefore we used an in situ fluorogenic MMP sensor peptide to determine how drug treatment affects melanoma cell MMP activity in three dimensions. We encapsulated melanoma cells from varying stages of progression within PEG-based hydrogels to examine the relationship between drug treatment and MMP activity. From these results, a metastatic melanoma cell line (A375) and two inhibitors that inhibit RAF (PLX4032 and sorafenib) were studied further to determine whether changes in MMP activity led to a functional change in cell behavior. A375 cells exhibited increased MMP activity despite an overall decrease in metabolic activity with PLX4032 treatment. The changes in proteolytic activity correlated with increased cell elongation and increased single-cell migration. In contrast, sorafenib did not alter MMP activity or cell motility, showing that the changes induced by PLX4032 were not a universal response to small-molecule inhibition. Therefore, we argue the importance of studying MMP activity with drug treatment and its possible implications for unwanted side effects.

A randomized phase III trial comparing S-1 versus UFT as adjuvant chemotherapy for stage II/III rectal cancer (JFMC35-C1: ACTS-RC).

BACKGROUNDS: Preventing distant recurrence and achieving local control are important challenges in rectal cancer treatment, and use of adjuvant chemotherapy has been studied. However, no phase III study comparing adjuvant chemotherapy regimens for rectal cancer has demonstrated superiority of a specific regimen. We therefore conducted a phase III study to evaluate the superiority of S-1 to tegafur-uracil (UFT), a standard adjuvant chemotherapy regimen for curatively resected stage II/III rectal cancer in Japan, in the adjuvant setting for rectal cancer. PATIENTS AND METHODS: The ACTS-RC trial was an open-label, randomized, phase III superiority trial conducted at 222 sites in Japan. Patients aged 20-80 with stage II/III rectal cancer undergoing curative surgery without preoperative therapy were randomly assigned to receive UFT (500-600 mg/day on days 1-5, followed by 2 days rest) or S-1 (80-120 mg/day on days 1-28, followed by 14 days rest) for 1 year. The primary end point was relapse-free survival (RFS), and the secondary end points were overall survival and adverse events. RESULTS: In total, 961 patients were enrolled from April 2006 to March 2009. The primary analysis was conducted in 480 assigned to receive UFT and 479 assigned to receive S-1. Five-year RFS was 61.7% [95% confidence interval (CI) 57.1% to 65.9%] for UFT and 66.4% (95% CI 61.9% to 70.5%) for S-1 [P = 0.0165, hazard ratio (HR): 0.77, 95% CI 0.63-0.96]. Five-year survival was 80.2% (95% CI 76.3% to 83.5%) for UFT and 82.0% (95% CI 78.3% to 85.2%) for S-1. The main grade 3 or higher adverse events were increased alanine aminotransferase and diarrhea (each 2.3%) in the UFT arm and anorexia, diarrhea (each 2.6%), and fatigue (2.1%) in the S-1 arm. CONCLUSION: One-year S-1 treatment is superior to UFT with respect to RFS and has therefore become a standard adjuvant chemotherapy regimen for stage II/III rectal cancer following curative resection.

Microwave spectroscopy of platinum monofluoride and platinum monochloride in the X 2Π(3/2) states.

Platinum monofluoride (PtF) and platinum monochloride (PtCl) were detected in the gas phase using a source-modulated microwave spectrometer. The PtF and PtCl radicals were generated in a free space cell using the sputtering reaction from a platinum sheet placed on the inner surface of a stainless steel cathode through a dc glow discharge plasma of CF(4) and Cl(2), respectively, diluted with Ar. Rotational transitions were measured in the region between 150 and 313 GHz. Rotational, centrifugal distortion, and several fine- and hyperfine-structure constants were determined by a least-squares analysis. The observed fine-structure spectral patterns indicate that both PtF and PtCl radicals have the (2)Π(3/2) electronic ground states, while the related cyanide PtCN and hydride PtH radicals have the (2)Δ(5/2) electronic ground states.

Low-energy vibrations of the group 10 metal monocarbonyl MCO (M = Ni, Pd, and Pt): rotational spectroscopy and force field analysis.

The rotational spectra of NiCO and PdCO in the ground and ν(2) excited vibrational states were observed by employing a source-modulated microwave spectrometer. The NiCO and PdCO molecules were generated in a free space cell by the sputtering reaction of nickel and palladium sheets, respectively, lining the inner surface of a stainless steel cathode with a dc glow plasma of CO and Ar. The molecular constants of NiCO and PdCO were determined by least-squares analysis. By force field analysis for the molecular constants of not only NiCO and PdCO but also of PtCO as previously reported, the harmonic force constants were determined for these three group 10 metal monocarbonyls. The vibrational wavenumbers derived for the lower M-C stretching vibrations were in good agreement with those obtained from the IR spectra in noble gas matrices and those predicted by several quantum chemical calculations published in the past. The bending vibrational wavenumbers derived by the force field analysis were also consistent with most quantum chemical calculations previously reported, but showed systematic discrepancies from the matrix IR values by about 40 cm(-1), even after reassignment (ν(2) band → 2ν(2) band) of the matrix IR spectra of PdCO and PtCO.

Effects of an elemental diet to reduce adverse events in patients with esophageal cancer receiving docetaxel/cisplatin/5-fluorouracil: a phase III randomized controlled trial-EPOC 2 (JFMC49-1601-C5).

BACKGROUND: Oral mucositis (OM) is an unpleasant adverse event in patients receiving chemotherapy. A prospective feasibility study showed that elemental diet (ED), an oral supplement that does not require digestion, may prevent OM. Based on this, we established a central review system for oral cavity assessment by dental oncology specialists blinded to background data. We used this system to elucidate the preventive effect of an ED against OM in patients with esophageal cancer receiving docetaxel, cisplatin, and 5-fluorouracil (DCF) therapy. PATIENTS AND METHODS: In this phase III, multicenter, parallel-group, controlled trial, patients consuming a normal diet orally were randomly assigned (1 : 1) to receive two cycles of DCF with (group A) or without (group B) an ED (Elental® 160 g/day). We assessed the incidence of grade ≥2 OM evaluated by two reviewers, changes in body weight, prealbumin, C-reactive protein, and DCF completion rate based on ED compliance. RESULTS: Of the 117 patients randomly assigned to treatment, four failed to start treatment and were excluded from the primary analysis; thus, groups A and B comprised 55 and 58 patients, respectively. There were no significant differences in background characteristics. Grade ≥2 OM was observed in eight (15%) and 20 (34%) patients in groups A and B, respectively (P = 0.0141). Changes in body weight and prealbumin during the two DCF cycles were significantly higher in group A than B (P = 0.0022 and 0.0203, respectively). During the first cycle, changes in C-reactive protein were significantly lower in group A than B (P = 0.0338). In group A (receiving ED), the DCF completion rate was 100% in patients with 100% ED compliance and 70% in patients failing ED completion (P = 0.0046). CONCLUSIONS: The study findings demonstrate that an ED can prevent OM in patients with esophageal cancer receiving chemotherapy.

Detection of free monomeric silver(I) and gold(I) cyanides, AgCN and AuCN: microwave spectra and molecular structure.

The chemistry of the group 11 metal cyanide system has been of considerable interest because of the commercial importance of some of the complexes formed in this system. These metal cyanides contain one-dimensional linear -M-CN-M-CN-M- chains in the solid state; however, they have not been observed as free monomeric species. This Article reports the first detection of monomeric AgCN and AuCN in the gas phase by using rotational spectroscopy. The sputtering reaction of silver and/or gold sheets placed on a stainless steel cathode with CH3CN diluted in Ar resulted in the production of AgCN and AuCN spectra. Spectroscopic observation of the parent and several rare isotopic species allowed the determination of r(0), r(s), r(Iepsilon), r(m)(1), and r(m)(2) structures in the case of AgCN and r(0) and r(s) structures in the case of AuCN. All data indicate that these two species have a linear MCN geometry with a low-energy bending vibration.

Synthesis of microgel sensors for spatial and temporal monitoring of protease activity.

Proteases are involved in almost every important cellular activity, from embryonic morphogenesis to apoptosis. To study protease activity in situ, hydrogels provide a synthetic mimic of the extracellular matrix (ECM) and have utility as a platform to study activity, such as those related to cell migration, in three-dimensions. While 3-dimensional visualization of protease activity could prove quite useful to elucidate the proteolytic interaction at the interface between cells and their surrounding environment, there has been no versatile tool to visualize local proteolytic activity in real time. Here, micron-sized gels were synthesized by inverse suspension polymerization using thiolene photo-click chemistry. The size distribution was selected to avoid cellular uptake and to lower cytotoxicity, while simultaneously allowing the integration of peptide-based FRET sensors of local cell activity. Proteolytic activity of collagenase was detected within an hour via changes in fluorescence of embedded microgels; incubation of microgel sensors with A375 melanoma cells showed upregulated MMP activity in the presence of soluble fibronectins in media. The microgel sensors were readily incorporated into both gelatin and poly(ethylene glycol) (PEG) hydrogels and used to successfully detect spatiotemporal proteolytic activity of A375 melanoma cells. Finally, a tumor model was constructed from a hydrogel microwell array that was used to aggregate A375 melanoma cells, and local variations in proteolytic activity were monitored as a function of distance from the cell aggregate center.

PEG-peptide hydrogels reveal differential effects of matrix microenvironmental cues on melanoma drug sensitivity.

Metastatic melanoma is highly drug resistant, though the exact mechanisms of this resistance are not completely understood. One method to study melanoma drug responsiveness in vitro is through the use of multicellular spheroids, which have been found to exhibit decreased drug sensitivity compared to traditional 2D culture on various substrates. Because it is unclear whether dimensionality, cell-matrix interactions, and/or cell-cell contacts may influence melanoma drug responsiveness, we utilized a synthetic PEG-based hydrogel to compare the responses of cells cultured on top of or encapsulated within matrices with the same adhesive ligand density, polymer density, and material properties. We found that depending on the stage of progression at which the melanoma cells were derived, the cells responded differently to PLX4032 treatment, a commercially available melanoma drug. In particular, early stage WM35 cells were insensitive to dimensionality (i.e., 2D versus 3D culture), while metastatic A375 cells exhibited decreased responsiveness in 3D compared to 2D. To further understand the role of the microenvironment in early stage melanoma cells, we tested single WM35 cells and multicellular WM35 spheroids in 3D. The results revealed that the spheroids were similarly sensitive to PLX4032 treatment compared to single cell encapsulations. Collectively, this study implicates the role that 3D microenvironments (i.e., dimensionality) may play in observed melanoma drug responsiveness, and the potential lack of influence of cell-matrix interactions over cell-cell contacts in early stages of melanoma resistance to PLX4032-induced apoptosis.

Evaluation of resectability after neoadjuvant chemotherapy for primary non-resectable colorectal liver metastases: A multicenter study.

BACKGROUND/AIM: The Kyushu Study Group of Clinical Cancer (KSCC) previously reported the safety and efficacy of neoadjuvant chemotherapy with mFOLFOX6 + bevacizumab for H2/H3 liver metastases of colorectal cancer. The aim of the current study was to evaluate the resectability of these metastases before and after chemotherapy as determined by independent liver surgeons. METHODS: Between May 2008 and April 2010, 40 patients were registered in a multicenter phase 2 trial of neoadjuvant chemotherapy (KSCC 0802). In Study 1, 5 independent liver surgeons from five different KSCC centers evaluated the resectability of liver metastases of colorectal cancer based on imaging studies performed before and after chemotherapy. Each surgeon was blinded to the other surgeons' evaluations. In addition, no information about the patients' characteristics was provided. In Study 2, 3 surgeons evaluated the resectability of these lesions based on imaging studies with discussion with each other, with the surgeons being provided with information on the patients' characteristics. RESULTS: In Study 1, 13 patients (36.1%) were evaluated to be resectable at baseline, whereas 17 patients (47.2%) were evaluated to be resectable after chemotherapy. In Study 2, 4 patients (11.1%) were evaluated to be resectable at baseline, compared to 23 patients (63.9%) after chemotherapy. CONCLUSION: Neoadjuvant chemotherapy with mFOLFOX6 + bevacizumab was confirmed to increase the resectability of non-resectable liver metastases of colorectal cancer according to the independent assessments of surgeons.

Biochemical and molecular aspects of genetic disorders of bilirubin metabolism.

Bilirubin, the oxidative product of heme in mammals, is excreted into the bile after its esterification with glucuronic acid to polar mono- and diconjugated derivatives. The accumulation of unconjugated and conjugated bilirubin in the serum is caused by several types of hereditary disorder. The Crigler-Najjar syndrome is caused by a defect in the gene which encodes bilirubin UDP-glucuronosyltransferase (UGT), whereas the Dubin-Johnson syndrome is characterized by a defect in the gene which encodes the canalicular bilirubin conjugate export pump of hepatocytes. Animal models such as the unconjugated hyperbilirubinemic Gunn rat, the conjugated hyperbilirubinemic GY/TR-, and the Eisai hyperbilirubinemic rat, have contributed to the understanding of the molecular basis of hyperbilirubinemia in humans. Elucidation of both the structure of the UGT1 gene complex, and the Mrp2 (cMoat) gene which encodes the canalicular conjugate export pump, has led to a greater understanding of the genetic basis of hyperbilirubinemia.

cDNA cloning of a putative G protein-coupled receptor from brain.

Using degenerate oligonucleotide primers corresponding to conserved regions of the G-protein coupled receptor superfamily, we carried out a low-stringency polymerase chain reaction and obtained two novel partial-length clones from a rat brain cDNA library. We used one of these clones for conventional library screening and isolated a longer cDNA clone, designated as RBU-15, from another rat brain library. Although RBU-15 was truncated at its 5' end, Northern blot analysis revealed that the gene was expressed in the brain and spleen. Next, we isolated a full-length cDNA clone, designated as HB-954, from a human fetal brain library, using RBU-15 as a probe. The deduced amino acid sequence of HB-954 contained four putative glycosylation sites in the N-terminal part, seven transmembrane domains, and a large cytosolic domain in the C-terminal part. The protein products of RBU-15 and HB-954 likely belong to a distinctive subfamily, because no receptors in the superfamily were found to be closely related to them.

Chemical modification of rat hepatic microsomes with N-ethylmaleimide results in inactivation of both UDP-N-acetylglucosamine-dependent stimulation of glucuronidation and UDP-glucuronic acid uptake.

Chemical modification of rat hepatic microsomes with N-ethylmaleimide (NEM) resulted in inactivation of UDP-N-acetylglucosamine (UDP-GlcNAc)-dependent stimulation of glucuronidation of p-nitrophenol. Inactivation kinetics and pH dependence were in agreement with the modification of a single sulfhydryl group. NEM also inactivated the uptake of UDP-glucuronic acid (UDP-GlcUA) but not UDP-glucose. With various sulfhydryl-modifying reagents, the inactivation of UDP-GlcUA uptake was linked to that of glucuronidation. UDP-GlcUA protected against NEM-sensitive inactivation of both UDP-GlcNAc-dependent stimulation of glucuronidation and UDP-GlcUA uptake, suggesting that the sulfhydryl group is located within or near the UDP-GlcUA binding site of the microsomal protein involved in the stimulation. Using microsomes labeled with biotin-conjugated maleimide and immunopurification with anti-peptide antibody against UDP-glucuronosyltransferase family 1 (UGT1) isozymes, immunopurified UGT1s were found to be labeled with the maleimide and UDP-GlcUA protected against the labeling as it did with the NEM-sensitive inactivation. These data suggest the involvement of a sulfhydryl residue of microsomal protein in the UDP-GlcNAc-dependent stimulation mechanism via the stimulation of UDP-GlcUA uptake into microsomal vesicles.

Systematic mutations of highly conserved His49 and carboxyl-terminal of recombinant porcine liver NADH-cytochrome b5 reductase solubilized domain.

The cDNA encoding solubilized porcine liver NADH-cytochrome b5 reductase catalytic domain (Pb5R) was cloned and overexpressed in Escherichia coli. A highly conserved His49 and a C-terminal Phe272 of Pb5R, which are located near the isoalloxazine moiety of the FAD, were systematically modulated by site-directed mutagenesis. Large structural change was not detected on the absorption and circular dichroism spectra of mutant proteins. Drastic changes in enzymatic properties were not observed, but the apparent Km value for soluble form of porcine liver cytochrome b5 (Pb5) was affected by the substitutions of His49 with glutamic acid and with lysine, deletion of C-terminal Phe272, and addition of Gly273. The values of the catalytic constant (kcat) were obviously decreased by the substitution of His49 with glutamic acid or the addition of Gly273. In these two mutants, the rate for reduction of FAD was decreased, and the rate for autoxidation of reduced FAD was increased. These results showed that His49 and C-terminal carboxyl group in Pb5R are not critical for the electron transfer to Pb5, but the electrostatic environmental changes at these positions could affect the recognition of Pb5 and modulate the catalytic function of the enzyme by changing the stability of reduced FAD.

Role of transforming growth factor-beta 1 in invasion and metastasis in gastric carcinoma.

PURPOSE: Transforming growth factor-beta1 (TGF-beta1) is a major modulator of cellular proliferation and extracellular matrix formation. We determined the role of TGF-beta1 in invasion and metastasis in gastric cancer. MATERIALS AND METHODS: We detected TGF-beta1 expression in primary and lymph node metastatic lesions of gastric cancer, using an antibody and in situ hybridization. The plasma TGF-beta1 levels in the peripheral vein and in the tumor drainage vein were assayed. RESULTS: In the cytoplasm of cancer cells, TGF- beta1 was immunostained in 35.9% (78 of 217) of primary gastric carcinomas, and this expression was confirmed by in situ hybridization. Of 59 gastric carcinomas with a TGF-beta1-negative primary tumor, metastatic lymph nodes were positive for TGF-beta1 staining in 32 cases (54.2%). Positive staining of TGF-beta1 in gastric cancer tissues was closely related to serosal invasion, infiltrative growth, and lymph node metastasis. Multivariate analysis showed that the expression of TGF-beta1 was an independent risk factor for serosal invasion and infiltrative growth of the tumor. The plasma level of TGF-beta1 did not differ between TGF-beta1-negative and -positive groups. There were also no differences in plasma TGF-beta1 levels among each tumor stage, between the peripheral and the tumor drainage veins, and between preoperative and postoperative testings. CONCLUSION: Transforming growth factor-beta1 is closely related to the invasion and metastasis of gastric cancer, and production of TGF-beta1 in the tumor does not contribute to the total amount of TGF-beta1 in the blood circulation. We interpret our observations to mean that in a tumor microenvironment, TGF-beta1 alters the biologic behavior of the tumor.

Effects of chrysotile asbestos on lung and pleural carcinogenesis of N-bis(2-hydroxypropyl)nitrosamine in rats.

The effects of chrysotile asbestos on lung and pleural carcinogenesis by N-bis(2-hydroxypropyl)nitrosamine (DHPN) in male Wistar rats were studied. Chrysotile, 30 mg per rat, was injected into the left pleural cavity and 3 g/kg body wt. DHPN was injected once into the abdominal cavity. Lung tumors (adenoma, adenocarcinoma, squamous cell carcinoma, and combined carcinoma) occurred at the highest incidence (100%). Adenocarcinoma was seen in 4 of 11 (36%) rats killed at 35 weeks and in 6 of 12 (50%) rats killed at 52 weeks, squamous cell carcinoma occurred in 1 of 11 (9%) rats killed at 35 weeks and 3 of 12 (25%) rats killed at 52 weeks, and mixed carcinoma was seen in 1 of 12 (8%) rats killed at 52 weeks, which received chrysotile and DHPN. Adenocarcinoma was seen in 9 of 11 (82%) rats which received DHPN only and killed at 52 weeks. Mesotheliomas were seen in 2 of 11 (18%) rats, killed at 35 weeks, and 3 of 12 (25%) rats, killed at 52 weeks, which received chrysotile and DHPN. Hyaline thickening of the pleura was seen in 100% of rats receiving chrysotile. Mesothelial cell hyperplasia and adenomatous and/or fibromatous growth of the mesothelium were seen in the pleura on both sides, ranging from 36% to 50% and 31% to 64% in rats receiving chrysotile and DHPN, respectively. Asbestos bodies were seen in the pleura on both sides and in the lung.

Effect of DNA conformation on cisplatin adduct formation.

The anticancer drug cis-diamminedichloroplatinum(II) (cisplatin) has been shown previously to form adducts preferentially within internucleosomal or linker DNA rather than to DNA within the nucleosome. To determine whether other "open" regions of chromatin have an increased affinity for cisplatin, adduct formation within specific chromatin domains was analyzed. There was a significant increase in cisplatin-DNA adduct formation for DNA associated with the nuclear matrix (NM) compared with other chromatin domains and total unfractionated DNA. In contrast, treatment of the same cells with trans-diamminedichloroplatinum(II) (transplatin) did not result in preferential adduct formation. These findings led to the hypothesis that it might be possible to alter DNA to make it a more favorable target for cisplatin. The effect of arginine butyrate on cisplatin-DNA adduct formation was analyzed in human cancer cells. The combination of arginine butyrate and cisplatin resulted in a concentration-responsive increase in cisplatin-DNA adduct formation in PC-3 cells and an overall increase in cisplatin-DNA adduct formation in three other human cancer cell lines. The same combination also resulted in a significant increase in drug-induced cytotoxicity at a low concentration of cisplatin. These results suggest that chromatin configuration can affect cisplatin adduct formation.

L-histidinol potentiates hyperthermic cell-killing in-vitro.

To clarify the effect of L-histidinol on hyperthermic killing of cells, HeLa and mouse sarcoma-180 (S-180) cells were exposed to heat in vitro in the presence of L-histidinol and the clonogenic surviving fraction of the cells was examined. After pretreating the cells with L-histidinol for 4 h, exposure of the cells to the combination of heat at 43-degrees-C and L-histidinol for various times (30 min to 4 h) reduced the surviving fraction more prominently than heat alone. Optimal concentrations to confer effective enhancement of heat on HeLa and S-180 cells were 9 mM and 3 mM, respectively. Those concentrations showed little toxicity of L-histidinol alone. Enhancement of the effect of heat by L-histidinol was observed only at 43-degrees-C, but not at 41 and 42-degrees-C. Flow cytometric DNA analysis was used to examine the cell cycle transit effect of L-histidinol. L-histidinol alone arrested the HeLa cells in G1-phase. Heat treatment at 43-degrees-C led to an accumulation of the cells in G2/M-phase and a decrease in the G1-phase fraction. The effect of combined treatment with heat and L-histidinol was complementary, in which L-histidinol attenuated the accumulation of the cells in G2/M-phase and prevented a decrease in the G1-phase fraction. Thus, L-histidinol has the capacity to potentiate hyperthermic cell killing in vitro by a mechanism that may be related to the cell cycle transit effect.

Treatment of advanced gastric cancer with 5-fluorouracil and cisplatin in combination with dipyridamole.

We investigated the efficacy of combination chemo-therapy using 5-fluorouracil (5-FU), cisplatin (CDDP), and dipyridamole (DP), which is based on the concept of double biochemical modulation. Twenty-eight patients with advanced gastric cancer were treated with the simultaneous continuous intravenous (i.v.) infusion of 5-FU (800 mg/m2/day) and DP (4 mg/kg/day), and i.v. infusion of CDDP (20 mg/m2/day) for 5 days. The cycles were repeated every 4 weeks. Twelve patients (43%) had a partial response (PR), while stable disease (NC) occurred in 13 patients (46%), and progression (PD) in 3 patients (11%). An improved performance status was observed in 20 patients (71%). The carcinoembryonic antigen (CEA) level was markedly decreased in 75% of the CEA-positive patients. Toxicity was acceptable. The mean steady state plasma concentration of total DP was 6.40.5 microM, which thus seemed adequate to potentiate the cytotoxicity of 5-FU. The treatment regimen described herein thus appears to be effective, safe and well tolerated by patients with advanced gastric cancer.

Synthesis of sex-specific forms of cytochrome P-450 in rat liver is transiently suppressed by hepatic monooxygenase inducers.

The effects of phenobarbital (PB), 3-methylcholanthrene (MC), and alpha-naphthoflavone (alpha-NF) on the synthesis of drug-inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), and sex-specific forms of cytochrome P-450, P-450(M-1), and P-450(F-1), in male and female rats were studied. Whereas P-450(PB-1) and P-450(MC-1) in liver microsomes were markedly induced in both sexes by treatment with PB and MC, respectively, the contents of P-450(M-1) and P-450(F-1) were significantly decreased by the treatments. alpha-NF, which is not a P-450 inducer, did not change the contents of sex-specific forms of cytochrome P-450. The translatable mRNAs of the P-450s were also determined by using an in vitro translation system. The mRNAs coding for P-450(PB-1) and P-450(MC-1) were increased by drug administrations. On the other hand, the mRNAs coding for P-450(M-1) and P-450(F-1) were transiently decreased by the drugs, and then returned to the normal levels. The time courses of the induction of the drug-inducible P-450s and the repression of the sex-specific P-450s showed no close correlation. alpha-NF had no effect on the synthesis of P-450(M-1) and P-450(F-1). We also found that the synthesis of P-450(M-1) in the livers of untreated rats showed no diurnal variations.

Drug-responsive and tissue-specific alternative expression of multiple first exons in rat UDP-glucuronosyltransferase family 1 (UGT1) gene complex.

Genomic clones of UDP-glucuronosyltransferase family 1 (UGT1) were isolated from wild-type Wistar rats. The UGT1 locus spans > 120 kb and forms a gene complex. In this locus nine unique first exons encoding NH2-terminal portions of each isoform were located at intervals of approximately 10 kb and followed by only one set of commonly used exons (exons II, III, IV, and V) encoding the COOH-terminal portion. From sequence analyses of the unique first exons, the amino acid sequences of the isoforms were deduced and they were divided into two groups: the Bilirubin cluster (B cluster) and the Phenol cluster (A cluster). A and B clusters consisted of four (A1-A4) and five (B1-B5) isoform-specific exons, respectively. A2, A3, B3, and B4 were identified as previously uncharacterized forms, while A4 and B4 were pseudogenes. Isoform B1 was a major component in hepatic microsomes of untreated rats and was induced in clofibrate- and dexamethasone-administered rats. A slight but a significant amount of B1 mRNA was also detected in various tissues such as intestine. mRNAs coding for isoform A1 and isoform A2 were induced in livers of methylcholanthrene (MC)-treated rats. Induction of A1 mRNA was also observed in kidneys of MC-treated rats. A genomic clone containing the commonly used exons was also isolated from Gunn rats and a single base deletion was identified in exon IV. Isoforms of the UGT1 family are made from the complex gene locus by an alternative combination of one of the unique first exons with the commonly used exons.