Empowering rice seedling growth by endophytic Bradyrhizobium sp. SUTN9-2.

Bradyrhizobium sp. strain SUTN9-2 was confirmed as rice endophytic bacteria and also as rice growth promotion agent. SUTN9-2 showed the capability of plant growth promotion characteristics, such as indole-3-acetic acid (IAA) and 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase productions and nitrogen fixation. In this study, the ability of SUTN9-2 to stimulate rice growth was investigated at different stages with N-free and NH4 NO3 under in vivo condition. The rice dry weight and chlorophyll content could be enhanced when SUTN9-2 was inoculated in N-free, especially at seedling stage (7 and 14 dai). The rice dry weight was also increased when SUTN9-2 was inoculated with NH4 NO3 at 7 and14 dai. The results of quantitative analysis of IAA and ACC deaminase were inconsistent with the expression of genes involved in IAA (nit) and ACC deaminase (acdS) productions. This inconsistently could implied that IAA and ACC deaminase produced from SUTN9-2 do not directly affect rice growth, but other factors resulting from the production of IAA and ACC deaminase could be involved. Moreover, the expression of genes involved in nitrogen fixation (nifH and nifV) of SUTN9-2 was also induced in rice tissues. This finding suggested that rice growth promotion may be supported by NH4 NO3 together with nitrogen fixation by SUTN9-2. SIGNIFICANCE AND IMPACT OF THE STUDY: Indole-3-acetic acid, 1-amino-cyclopropane-1-carboxylic acid deaminase productions and nitrogen fixation may play important roles in rice growth promotion by endophytic SUTN9-2, especially at early rice seedling growth stage, which has the potential to be used as rice seedling growth promoter in the system of rice intensification.

Gene polymorphisms in antioxidant enzymes correlate with the efficacy of androgen-deprivation therapy for prostate cancer with implications of oxidative stress.

Background: Oxidative stress mitigated by antioxidant enzymes is thought to be involved in the progression to castration-resistant prostate cancer (CRPC) during androgen-deprivation therapy (ADT). This study investigated the association between genetic variations in antioxidant enzymes and the efficacy of ADT as well as its biological background. Patients and methods: The non-synonymous or promoter-locating polymorphisms of antioxidant enzymes were examined as well as the time to CRPC progression and overall survival in 104 and 92 patients treated with ADT for metastatic and non-metastatic prostate cancer, respectively. In addition, intracellular reactive oxygen species and expression levels of antioxidant enzymes were examined in castration-resistant and enzalutamide-resistant cells. Results: In metastatic prostate cancer, the AG/GG allele in GSTM3 rs7483 and CT/TT allele in CAT rs564250 were associated with a significantly lower risk of progression to CRPC and all-cause death compared with homozygotes of the major AA allele (hazard ratio [HR]; [95% confidence interval (CI)], 0.55 [0.34-0.86], P = 0.0086) and CC allele (HR; [95% CI], 0.48 [0.24-0.88], P = 0.016), respectively. On multivariate analyses, only GSTM3 rs7483 was associated with significant progression risk (AG/GG versus AA; HR; [95% CI], 0.45 [0.25-0.79], P = 0.0047) even after Bonferroni adjustment. In non-metastatic prostate cancer, the AG/GG allele in GSTM3 rs7483 was associated with a significantly lower risk of progression to CRPC (HR; [95% CI], 0.35 [0.10-0.93], P = 0.034) and all-cause death (HR; [95% CI], 0.26 [0.041-0.96], P = 0.043) compared with the AA allele. Intracellular reactive oxygen species levels were increased, accompanied with augmented GSTM3 expression in both castration-resistant and enzalutamide-resistant cells. Conclusions: Differential activity of antioxidant enzymes caused by the polymorphism in GSTM3 may contribute to resistance to hormonal therapy through oxidative stress. The GSTM3 rs7483 polymorphism may be a promising biomarker for prostate cancer patients treated with ADT.

Characterization of anti-P monoclonal antibodies directed against the ribosomal protein-RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice.

Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus.

Characterization of putative toxin/antitoxin systems in Vibrio parahaemolyticus.

AIM: To obtain more information about the toxin/antitoxin (TA) systems in the Vibrio genus and also to examine their involvement in the induction of a viable but nonculturable (VBNC) state, we searched homologues of the Escherichia coli TA systems in the Vibrio parahaemolyticus genome. METHODS AND RESULTS: We found that a gene cluster, vp1842/vp1843, in the V. parahaemolyticus genome database has homology to that encoding the E. coli TA proteins, DinJ/YafQ. Expression of the putative toxin gene vp1843 in E. coli cells strongly inhibited the cell growth, while coexpression with the putative antitoxin gene vp1842 neutralized this effect. Mutational analysis identified Lys37 and Pro45 in the gene product VP1843 of vp1843 as crucial residues for the growth retardation of E. coli cells. VP1843, unlike the E. coli toxin YafQ, has no protein synthesis inhibitory activity, and that instead the expression of vp1843 in E. coli caused morphological change of the cells. CONCLUSIONS: The gene cluster vp1842/vp1843 encodes the V. parahaemolyticus TA system; VP1843 inhibits cell growth, whereas VP1842 serves as an antitoxin by forming a stable complex with VP1843. SIGNIFICANCE AND IMPACT OF THE STUDY: The putative toxin, VP1843, may be involved in the induction of the VBNC state in V. parahaemolyticus by inhibiting cell division.

ZNF143 interacts with p73 and is involved in cisplatin resistance through the transcriptional regulation of DNA repair genes.

Zinc-finger protein 143 (ZNF143) is a human homolog of Xenopus transcriptional activator staf that is involved in selenocystyl tRNA transcription. We previously showed that ZNF143 expression is induced by treatment with DNA-damaging agents and that it preferentially binds to cisplatin-modified DNA. In this study, the potential function of ZNF143 was investigated. ZNF143 was overexpressed in cisplatin-resistant cells. ZNF143 knockdown in prostate cancer caused increased sensitivity for cisplatin, but not for oxaliplatin, etoposide and vincristine. We also showed that ZNF143 is associated with tumor suppressor gene product p73 but not with p53. p73 could stimulate the binding of ZNF143 to both ZNF143 binding site and cisplatin-modified DNA, and modulate the function of ZNF143. We provide a direct evidence that both Rad51 and flap endonuclease-1 are target genes of ZNF143 and overexpressed in cisplatin-resistant cells. Taken together, these experiments demonstrate that an interplay of ZNF143, p73 and ZNF143 target genes is involved in DNA repair gene expression and cisplatin resistance.

Clock and ATF4 transcription system regulates drug resistance in human cancer cell lines.

The mechanisms underlying cellular drug resistance have been extensively studied, but little is known about its regulation. We have previously reported that activating transcription factor 4 (ATF4) is upregulated in cisplatin-resistant cells and plays a role in cisplatin resistance. Here, we find out a novel relationship between the circadian transcription factor Clock and drug resistance. Clock drives the periodical expression of many genes that regulate hormone release, cell division, sleep-awake cycle and tumor growth. We demonstrate that ATF4 is a direct target of Clock, and that Clock is overexpressed in cisplatin-resistant cells. Furthermore, Clock expression significantly correlates with cisplatin sensitivity, and that the downregulation of either Clock or ATF4 confers sensitivity of A549 cells to cisplatin and etoposide. Notably, ATF4-overexpressing cells show multidrug resistance and marked elevation of intracellular glutathione. The microarray study reveals that genes for glutathione metabolism are generally downregulated by the knockdown of ATF4 expression. These results suggest that the Clock and ATF4 transcription system might play an important role in multidrug resistance through glutathione-dependent redox system, and also indicate that physiological potentials of Clock-controlled redox system might be important to better understand the oxidative stress-associated disorders including cancer and systemic chronotherapy.

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'-->5' exonuclease activity.

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

Enhanced expression of the DNA topoisomerase II gene in response to heat shock stress in human epidermoid cancer KB cells.

Type II DNA topoisomerase breaks both DNA strands, and many anticancer agents including etoposide (VP-16) and teniposide (VM-26) have been developed by targeting topoisomerase II molecules. In this study we examined whether expression of the topoisomerase II gene is regulated in response to heat shock stress in human epidermoid cancer KB cells. Exposure of KB cells to 42 degrees C for 3 to 24 h permitted cell growth at a slightly reduced rate but still at an exponential rate, in comparison with that at 37 degrees C, whereas exposure to 45 degrees C for 15 to 120 min caused the almost complete cessation of exponential growth. There appeared 5-fold or higher increases in mRNA levels of both topoisomerase II and a heat shock protein, hsp-70, after exposure to 42 degrees C for 3 h, but only a slight, if any, increase in topoisomerase I mRNA. Nuclear run-on assays showed increased transcription of topoisomerase II and the hsp-70 gene after exposure to 42 degrees C. By contrast, KB cells induced a rapid and transient increase of topoisomerase II mRNA after exposure to 45 degrees C for 15 to 30 min, whereas the cellular level of hsp-70 mRNA was dramatically enhanced 60 min after exposure to 45 degrees C. The immunoblot assay also demonstrated increased expression of topoisomerase II in KB cells exposed to 42 degrees C. Decatenation activity of the nuclear extracts from KB cells was increased 1.5-fold by exposure to 42 degrees C, but there appeared no increase in topoisomerase I activity. Prior exposure of KB cells to 42 degrees C enhanced the cytotoxicity of VP-16, but not that of a topoisomerase I-targeting agent, a camptothecin analogue, CPT-11. However, exposure of KB cells to 42 degrees C after treatment with VP-16 did not enhance the cytotoxicity induced by the drug. The formation of cleavable DNA-topoisomerase II-VP-16 complexes was also greatly increased by prior exposure to 42 degrees C. Our present study proposes the hypothesis that the topoisomerase II gene might be one of the heat-shock-inducible genes and that hyperthermic anticancer therapy with topoisomerase II-targeting antitumor agents can be improved.

A canalicular multispecific organic anion transporter (cMOAT) antisense cDNA enhances drug sensitivity in human hepatic cancer cells.

The human cMOAT gene encodes a membrane protein involved in the ATP-dependent transport of hydrophobic compounds. To determine whether cMOAT is associated with drug sensitivity, we transfected an expression vector containing cMOAT antisense cDNA into the HepG2 human hepatic cancer cell line. We observed a reduction in cMOAT protein, as well as an enhanced level of glutathione, in the antisense transfectants. The transfectants displayed an increased sensitivity to cisplatin, vincristine, doxorubicin, and the camptothecin derivatives, (4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino)carbonyl oxy]dione hydrochloride triethydrate and 7-ethyl-10-hydroxycamptothecin, but not to etoposide, 3-[4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosoure a, 5-fluorouracil, and mitomycin C. Results suggest that cMOAT levels are inversely correlated with those of glutathione, and that cMOAT and its related genes may be involved in the sensitivity of cells to certain anticancer agents.

Transcription factor Y-box binding protein 1 binds preferentially to cisplatin-modified DNA and interacts with proliferating cell nuclear antigen.

The Y-box binding protein (YB-1) binds to inverted CCAAT box sequences that are present in the promoter region of many genes. We previously showed that YB-1 is overexpressed in human cancer cell lines that are resistant to cisplatin and that the depletion of YB-1 by transfection of a vector expressing YB-1 antisense RNA increases the sensitivity of human cancer cells to cisplatin. To determine whether YB-1 can bind to cisplatin-modified DNA, we fused YB-1 cDNA to glutathione S-transferase (GST) cDNA and purified the resulting GST fusion protein. When we tested the fusion protein with unmodified or cisplatin-modified oligonucleotides, we found that GST-YB-1 bound more strongly to cisplatin-modified oligonucleotides, as did GST fusion proteins of high mobility group 1 (HMG1), HMG2, and xeroderma pigmentosum group A protein. When we assayed the ability of proliferating cell nuclear antigen (PCNA) to interact with the GST fusion proteins, we observed binding to YB-1 but not to HMG1, HMG2, or xeroderma pigmentosum group A. Subsequent experiments demonstrated that YB-1 and PCNA interact directly via the COOH-terminal region of YB-1. Using immunochemical coprecipitation methods, we observed binding of YB-1 and PCNA in vivo. These results suggest that YB-1 can function as a recognition protein for cisplatin-damaged DNA and that it may be important in DNA repair or in directing the cellular response to DNA damage.

Direct interaction of p53 with the Y-box binding protein, YB-1: a mechanism for regulation of human gene expression.

The Y-box binding protein, YB-1, belongs to a family of multifunctional proteins which regulate gene expression on both transcriptional and translational levels. The tumor suppressor gene p53 displays growth suppressive properties by regulating gene expression through transcriptional regulation. We now demonstrate that YB-1 directly interacts with p53 using an in vitro pull-down assay. Using immunochemical co-precipitation methods, we also found that the two proteins are bound in vivo. Deletion analysis showed that three independent domains of YB-1, one at the N-terminal and two at the C-terminal, interact with p53. Conversely, a 14 amino acid sequence at the C-terminal of p53 was required for its interaction with YB-1. Gel mobility shift assays showed that the interaction of YB-1 with p53 stimulated the sequence-specific DNA binding of p53 to its consensus sequence. By contrast, this interaction inhibited the binding of YB-1. Using a p53-responsive p21 promoter linked to a reporter gene, it can be shown that antisense expression of YB-1 inhibits the induction of this promoter by p53 in transient transfection assays. These findings delineate a straightforward mechanism for gene expression through p53-YB-1 interaction.

Identification of the protein cross-linked to 3'-terminus of 5 S RNA in rat liver ribosomal 60 S subunits.

To cross-link the 3'-terminus of 5 S RNA to its neighbouring proteins, ribosomal 60 S subunits of rat liver were oxidized with sodium periodate and reduced with sodium borohydride. 5 S RNP was then isolated by EDTA treatment followed by sucrose density-gradient centrifugation and subjected to SDS-polyacrylamide gel electrophoresis. The protein with a slower mobility than the L5 protein, which was thought to be cross-linked 5 S RNP, was labeled with 125I, treated with RNAase, and analyzed by two-dimensional polyacrylamide gel electrophoresis, followed by radioautography. A radioactive spot located anodically from L5 protein was observed, suggesting that it is the L5 protein-oligonucleotide complex. When analyzed by SDS slab polyacrylamide gel electrophoresis followed by radioautography, the peptide pattern of the alpha-chymotrypsin digest of this 125I-labeled protein-oligonucleotide complex was similar to that of the digest of 125I-labeled L5 protein. The results indicate that L5 protein binds to the 3'-terminal region of 5 S RNA in rat liver 60 S subunits.

Cross-linking of L5 protein to 5 S RNA in rat liver 60-S subunits by ultraviolet irradiation.

After rat liver 60-S ribosomal subunits were irradiated with ultraviolet light at 254 nm, they were treated with EDTA and then subjected to sucrose density-gradient centrifugation to isolate 5 S RNA-protein complex. When 5 S RNA-protein was analyzed by SDS-acrylamide gel electrophoresis which dissociated noncovalent 5 S RNA-protein, two protein bands were observed. The one showed a slower mobility than the protein band (L5) of 5 S RNA-protein from non-irradiated 60 S subunit and the other showed the same mobility as L5 protein. Since the former band was shown to be specific to ultraviolet-irradiation, it was considered as cross-linked 5 S RNA-protein. After the two protein bands were iodinated with 125 I, labeled protein was extracted and treated with RNAase. Thereafter, it was analyzed by two-dimensional acrylamide gel electrophoresis, followed by autoradiography. The results indicate that the protein component of cross-linked 5 S RNA-protein is L5 protein (ribosomal protein; these proteins are designated according to the proposed uniform nomenclature. The correlation between that and our nomenclature was reported by McConkey et al. (1979) Mol. Gen. Genet. 169, 1-6. They also confirm the results previously reported (Terao, K., Takahashi, Y. and O(gata, K. (1975) Biochim. Biophys. Acta 402, 230-237).

Studies on the negative circular dichroic bands around 297 nm of ribosomes from bacterial cells.

The small negative CD bands around 297 nm of isolated 30-S and 50-S ribosomal subunits were precisely measured for three bacteria, Bacillus stearothermophilus, Bacillus subtilis and Escherichia coli Q 13. The intensities of the negative CD bands of 30-S subunits were always much greater than those of 50-S subunits irrespective of the bacterial strains, which may be related to the difference in comformations of rRNAs and proteins in the complexes between these subribosomal particles. The dissociation of 70-S ribosomes into two subunits by lowering Mg2+ concentration caused evident enhancement of intensity of the 297 nm CD band, which was completely reversed by the association of the two subunits into 70-S particles. The melting profiles of CD spectra 3 B. stearothermophilus and E. coli were compared and both subunits of the former were found to be more heat stable than those of the latter. It was found that 5 M urea and 0.5% sodium dodecyl sulfate (SDS) treatment caused considerable reduction of the negative CD intensity around 297 nm of 30-S subunits but no significant change of 50-S subunits, while no significant change was observed for the CD spectra of isolated 16-S and 23-S rRNAs by the same treatment. Effects of EDTA treatment and then addition of Mg2+ on the CD spectra and fluorescence emission spectra of the subunits were also observed and the contribution by the interaction between rRNA s and proteins in ribosomes to the small negative band around 297 nm was discussed.

Identification of neighbouring protein pairs in the rat liver 40-S ribosomal subunits cross-linked with dimethyl suberimidate.

(1) The 40-S ribosomal subunits of rat liver were treated with a bifunctional cross-linking reagent, dimethyl suberimidate. Cross-linked protein-protein dimers were separated by two-dimensional acrylamide gel electrophoresis. The stained cross-linked complexes within the gel were radioiodinated without the elution of proteins from the gel and were cloven into the original monomeric protein constituents by ammonolysis. The proteins in each dimer were finally identified by two-dimensional acrylamide gel electrophoresis of the cloven monomeric proteins, followed by radioautography of the stained gel. (2) The molecular weights of cross-linked complexes were determined by SDS-polyacrylamide gel electrophoresis and were compared with those of their constituent proteins. (3) The following dimers were proposed from these results: S3-S12 (S3 or S3a-S11), S4-S12 (S3b-S11, S5-S7 (S4-S6), S5-S22 (S4-S23 or S24), S6-S8 (S5-S7), S8-S16 (S7-S18), S17-S21 (S16--S19) and S22A-S22B (S23-S24), designated according to our numbering system [1]. The designations according to the proposed uniform nomenclature [2] are described in parentheses.

Enhanced production of tissue-type plasminogen activator by estradiol in a novel type variant of human breast cancer MCF-7 cell line.

ES-1 cells, which showed a higher sensitivity to the cytocidal action of estradiol were isolated from a human breast cancer MCF-7 cell line. Growth of ES-1 cells was inhibited by a dose of 17-beta estradiol that stimulated the growth of the parental MCF-7 cells. Proteins secreted from MCF-7 and ES-1 cells when cultured with 17-beta estradiol were compared by sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis (SDS-PAGE). Addition of estradiol to culture medium enhanced secretion of a protein of molecular mass of 52 kDa in media for both MCF-7 and ES-1 cell lines, but the secretion of a second 67 kDa protein was enhanced about 10-fold only in ES-1 cells. The analysis by SDS-PAGE of culture medium immunoprecipitated with anti-tissue-type plasminogen activator (t-PA) antibody demonstrated that the band of 67 kDa protein specifically secreted from estradiol-treated ES-1 cells contained t-PA. Zymography assays, quantitative immunoreactive assays, and Northern analysis showed about 5-fold specific increase by estradiol of t-PA with molecular mass of 65-70 kDa in ES-1 but not in its parental MCF-7 cells. Cellular level of the plasminogen activity was also specifically enhanced in ES-1 cells by estradiol, but only a slightly in MCF-7 cells. By contrast, another urokinase-type PA (u-PA) with molecular weight of 55 kDa showed very low level activity in both MCF-7 and ES-1 cell lines in the presence of estradiol. Formation of t-PA mRNA was specifically enhanced in ES-1 cells when ES-1 cells were treated for more than 12 h with 10(-8) M 17-beta estradiol. Estradiol did not elongate the lifetime of t-PA mRNA in ES-1 cells. A unique phenotype of ES-1 cells in response to estradiol is discussed in relation to activating expression of the t-PA gene.

Cellular balance of glutathione levels through the expression of gamma-glutamylcysteine synthetase and glutathione thiol transferase genes in human hepatic cells resistant to a glutathione poison.

Buthionine sulfoximine (BSO) is a synthetic amino acid that irreversibly inhibits glutathione biosynthesis and deranges reduced glutathione (GSH) metabolism in liver cells. We isolated two BSO-resistant lines, HLE/BSO2-1 and HLE/BSO2-2, from human hepatic HLE/WT cells. Cellular levels of the Pi class glutathione thiol transferase (GSTP1) were 3-fold lower in BSO-resistant lines than in HLE/WT cells. By contrast, gamma-glutamylcysteine synthetase (GCS) heavy subunit (GCSh) mRNA levels were markedly decreased in HLE/BSO2-1 and HLE/BSO2-2 as compared with HLE/WT. The expression of a dominant-negative mutant of c-Jun inhibited the GCSh promoter activity in HLE/WT, but not in HLE/BSO2-1. Cellular levels of AP-1, however, were not decreased in either BSO-resistant cell line. Transfection of GCSh promoter of various lengths driven reporter constructs showed no sequence-specific increase in the promoter activities in HLE/BSO2-1. However, transfection of GSTP1 cDNA into HLE/BSO2-1 and HLE/BSO2-2 restored the levels of GCSh mRNA and the GCSh promoter activity to those of HLE/WT. Sequences between -315 and -241 bp of the 5' region contained an AP-1 site responsible for the enhanced GCSh promoter activity in GSTP1 transfectants of HLE/BSO2-1. In vivo footprint analysis showed a specific protection of the AP-1 site on GCSh promoter in GSTP1 transfected HLE/BSO2-1. GSH homeostasis thus appears to be maintained by an interaction between GSTP1 and GCS in human hepatic cells resistant to the GSH poison.

Increased expression of an ATP-binding cassette superfamily transporter, multidrug resistance protein 2, in human colorectal carcinomas.

The expression of ATP-binding cassette superfamily transporter genes, such as P-glycoprotein/multidrug resistance (MDR) 1 and MDR protein (MRP) 1, is often up-regulated in various tumor types and is involved in responses to some anticancer chemotherapeutic agents. Five human MRP subfamily members (MRP2-6) with structural similarities to MRP1 have been identified. The relationships between MRP2-6 mRNA levels and drug resistance are not well understood. Data on 45 patients with colorectal cancer were analyzed. Of the ATP-binding cassette superfamily genes, we asked whether mRNA levels of MDR1, MRP1, MRP2, and MRP3 correlated with drug resistance to anticancer agents. For this analysis, we used quantitative reverse transcription-PCR, and the sensitivity to anticancer agents in surgically resected colon carcinomas was determined using the in vitro succinate dehydrogenase inhibition test. MDR1, MRP1, and MRP3 were highly expressed in normal colorectal mucosa, and the relative mRNA levels of MDR1, MRP1, and MRP3 in cancerous tissues compared with noncancerous tissues were decreased or unchanged. By contrast, MRP2 mRNA expression was low in normal colorectal mucosa and specifically increased in cancer regions compared with noncancerous regions. Of the anticancer agents prescribed for patients with colorectal cancers, including doxorubicin, mitomycin C, cisplatin, 5-fluorouracil, etoposide, and a camptothecin derivative, mRNA expression of MRP2 was significantly associated with resistance to cisplatin. MRP2 may be important for resistance to cisplatin treatment in colorectal cancer.

Nuclear expression of the Y-box binding protein, YB-1, as a novel marker of disease progression in non-small cell lung cancer.

Transcription factor Y-box binding protein 1 (YB-1) that binds to the inverted CCAAT box is involved not only in transcription of various genes but also in cell proliferation and DNA repair. We determined whether localization of YB-1 in either the nucleus or cytoplasm could serve as a prognostic marker for patients with non-small cell lung cancer (NSCLC). In 196 NSCLC patients, expression of YB-1 protein in the nucleus or cytoplasm was immunohistochemically evaluated. Of the 196 tumors examined, 88 (44.9%) were positive for YB-1 expression in the nucleus. Nuclear YB-1 expression significantly correlated with T factor, lymph node metastasis, and stage of the disease. Patients with a nuclear YB-1 tumor had a poorer prognosis than did those with a cytoplasmic YB-1 tumor in all of the NSCLC patients (P = 0.0494) and in patients with squamous cell carcinoma (P = 0.0313) but not in patients with adenocarcinomas. Nuclear localization of the YB-1 protein may prove to be an important factor of disease progression for patients with NSCLC, in particular, in cases of squamous cell carcinoma.

Nuclear expression of YB-1 protein correlates with P-glycoprotein expression in human osteosarcoma.

The Y-box-binding protein, YB-1, is a member of the DNA-binding protein family. It binds to the Y-box, an inverted CCAAT box, in the promoter region of the human multidrug resistance 1 gene, which encodes P-glycoprotein (P-gp). Nuclear localization of YB-1 protein has been reported to be associated with the intrinsic expression of P-gp in human breast cancer. We studied the immunohistochemical expression of YB-1 protein in 69 untreated biopsy specimens of conventional osteosarcomas and compared it with the expression of P-gp. Furthermore, cell proliferation, as determined by the MIB-1-labeling index (MIB-1-LI), was measured by immunohistochemistry. In all 69 untreated osteosarcomas, YB-1 protein was expressed in the cytoplasm. In 32 of 69 (46%) cases, YB-1 was also localized in the nucleus. The expression of P-gp was evident in 23 of these 32 cases, and there was a significant correlation between the nuclear expression of YB-1 and P-gp expression (P < 0.0001). Chondroblastic osteosarcoma expressed YB-1 in the nucleus more frequently (eight of nine cases) than did other types of osteosarcoma, whereas P-gp was also frequently expressed in chondroblastic subtype. There was no correlation between the nuclear expression of YB-1 and histological grade. The MIB-1-LI was significantly higher in cases showing the nuclear expression of YB-1 (MIB-1-LI averaged 22.56 in cases with only cytoplasmic expression of YB-1 but averaged 28.20 in cases with cytoplasmic and nuclear expression of YB-1; P = 0.0477). In human osteosarcoma, nuclear localization of YB-1 protein was associated with the expression of P-gp, suggesting that YB-1 could be a prognostic marker for multidrug resistance in osteosarcoma.