Interferon regulatory factor 7 mediates obesity-associated MCP-1 transcription.

Hypertrophy, associated with adipocyte dysfunction, causes increased pro-inflammatory adipokine, and abnormal glucose and lipid metabolism, leading to insulin resistance and obesity-related-health problems. By combining DNA microarray and genomic data analyses to predict DNA binding motifs, we identified the transcription factor Interferon Regulatory Factor 7 (IRF7) as a possible regulator of genes related to adipocyte hypertrophy. To investigate the role of IRF7 in adipocytes, we examined gene expression patterns in 3T3-L1 cells infected with a retrovirus carrying the IRF7 gene and found that enforced IRF7 expression induced the expression of monocyte chemoattractant protein-1 (MCP-1), a key initial adipokine in the chronic inflammation of obesity. CRISPR/Cas9 mediated-suppression of IRF7 significantly reduced MCP-1 mRNA. Luciferase assays, chromatin immunoprecipitation PCR analysis and gel shift assay showed that IRF7 transactivates the MCP-1 gene by binding to its proximal Interferon Stimulation Response Element (ISRE), a putative IRF7 binding motif. IRF7 knockout mice exhibited lower expression of MCP-1 in epidydimal white adipose tissue under high-fat feeding conditions, suggesting the transcription factor is physiologically important for inducing MCP-1. Taken together, our results suggest that IRF7 transactivates MCP-1 mRNA in adipocytes, and it may be involved in the adipose tissue inflammation associated with obesity.

Estimation of Irradiation Doses of Raw Beef Liver Samples using 5,6-Dihydrothymidine as an Irradiation Marker.

The quantities of (5S)- and (5R)-5,6-dihydrothymidine (total DHdThd) in frozen beef liver samples were compared between those irradiated by an electron beam (EB) and those subjected to γ-irradiation. DNA extracted from the samples was enzymatically digested to nucleosides and analyzed by liquid chromatography-tandem mass spectrometry for total DHdThd and 2'-deoxythymidine (dThd). Total DHdThd was formed radiospecifically and dose-dependently and the dose-response curves of the ratio of total DHdThd to dThd (total DHdThd/dThd) were similar for both the EB- and γ-irradiated samples. The total DHdThd/dThd was stable after long-term storage (4 months) at -20 °C and the followed heat treatment in a microwave oven. The total DHdThd/dThd could be a robust marker and is equally effective at quantitating both EB- and γ-ray irradiation history. The irradiation doses of raw beef liver samples were estimated using the dose-response curves of the total DHdThd/dThd of other irradiated samples. The ratio of the estimated dose to the actual dose was 0.74-1.30 in the irradiation range of 4.67-7.62 kGy.

Rapid and Reliable Method for Determining Irradiation Histories of Ground Beef and Prawns by Measuring 5,6-Dihydrothymidine.

A rapid and reliable method for determining irradiation histories of ground beef and prawns was developed on the basis of a method for determining the irradiation history of beef liver by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of 5,6-dihydrothymidine (DHdThd). Improvements in the method included the following: (1) 50% ethanol precipitation in the DNA extraction step was conducted before the RNase step, (2) snake venom phosphodiesterase I was used for DNA digestion to boost liberation of DHdThd, and (3) a matrix-matched calibration curve was used for determining DHdThd by LC-MS/MS analysis. This method successfully determined irradiation histories of ground beef and prawns. Furthermore, a close correlation between the formation of DHdThd and 2-alkylcyclobutanones, which are an established index of irradiation histories, was observed in ground beef. DHdThd in DNA could be a promising candidate for a new index of irradiation histories of various foods.

Determination of irradiation histories of raw beef livers using liquid chromatography-tandem mass spectrometry of 5,6-dihydrothymidine.

A method for detecting irradiation histories of raw beef livers was developed by measuring 5,6-dihydrothymidine (DHdThd) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liver DNA was extracted using phenol-chloroform extraction followed by precipitation in 50% ethanol. DNA was then enzymatically digested and nucleosides were purified using an OASIS MCX column. DHdThd and thymidine (dThd) contents of resulting test solutions were analyzed using LC-MS/MS. DHdThd was detected specifically after γ-irradiation. Concentration ratios of DHdThd to dThd in the test solutions increased dose-dependently after irradiation at 1.0-11.3kGy, which included the practical dose for sterilization of 2-7kGy. Dose-response curves from beef livers of individual animals almost overlapped. Thus, this method is a candidate for the detection of irradiation histories of foods from which DNA can be extracted.

Pilot study of MDR1 gene transfer into hematopoietic stem cells and chemoprotection in metastatic breast cancer patients.

A major problem in high-dose chemotherapy with autologous hematopoietic stem cell transplantation is insufficient function of reconstituted bone marrow that limits the efficacy of post-transplantation chemotherapy. Because transduction of hematopoietic stem cells with the multidrug resistance 1 (MDR1) gene might circumvent this problem, we conducted a pilot study of MDR1 gene therapy against metastatic breast cancer. Peripheral blood stem cells were harvested, and one-third of the cells were transduced with MDR1 retrovirus. After the reconstitution of bone marrow function, the patients received high-dose chemotherapy with transplantation of both MDR1-transduced and unprocessed peripheral blood stem cells. The patients then received docetaxel chemotherapy. Two patients received transplantation of the MDR1-transduced cells in 2001. Peripheral blood MDR1-transduced leukocytes were 3-5% of the total cells after transplantation, but decreased gradually. During docetaxel chemotherapy, an increase in the rate of MDR1-transduced leukocytes (up to 10%) was observed. Comparison of docetaxel-induced granulocytopenia in the two patients suggested a bone marrow-protective effect of the MDR1-transduced cells. No serious side-effect was observed, and the patients were in complete remission for more than 3 years. The MDR1-transduced cells gradually decreased and disappeared almost entirely by the end of 2004. Results of linear amplification-mediated polymerase chain reaction of the MDR1-transduced leukocytes suggested no sign of abnormal amplification of the transduced cells. A third patient received transplantation of the MDR1-transduced cells in 2004. These results suggest the feasibility of our MDR1 gene therapy against metastatic breast cancer, and follow-up is ongoing.

A T3587G germ-line mutation of the MDR1 gene encodes a nonfunctional P-glycoprotein.

The human multidrug resistance gene 1 (MDR1) encodes a plasma membrane P-glycoprotein (P-gp) that functions as an efflux pump for various structurally unrelated anticancer agents. We have identified two nonsynonymous germ-line mutations of the MDR1 gene, C3583T MDR1 and T3587G MDR1, in peripheral blood cell samples from Japanese cancer patients. Two patients carried the C3583T MDR1 allele that encodes H1195Y P-gp, whereas a further two carried T3587G MDR1 that encodes I1196S P-gp. Murine NIH3T3 cells were transfected with pCAL-MDR-IRES-ZEO constructs carrying either wild-type (WT), C3583T, or T3587G MDR1 cDNA and selected with zeocin. The resulting zeocin-resistant mixed populations of transfected cells were designated as 3T3/WT, 3T3/H1195Y, and 3T3/I1196S, respectively. The cell surface expression of I1196S P-gp in 3T3/I1196S cells could not be detected by fluorescence-activated cell sorting, although low expression of I1196S P-gp was found by Western blotting. H1195Y P-gp expression levels in 3T3/H1195Y cells were slightly lower than the corresponding WT P-gp levels in 3T3/WT cells. By immunoblotting analysis, both WT P-gp and H1195Y P-gp were detectable as a 145-kDa protein, whereas I1196S P-gp was visualized as a 140-kDa protein. 3T3/I1196S cells did not show any drug resistance unlike 3T3/H1195Y cells. Moreover, a vanadate-trap assay showed that the I1196S P-gp species lacks ATP-binding activity. Taken together, we conclude from these data that T3587G MDR1 expresses a nonfunctional P-gp and this is therefore the first description of such a germ-line mutation. We contend that the T3587G MDR1 mutation may affect the pharmacokinetics of MDR1-related anticancer agents in patients carrying this allele.

Breast cancer resistance protein: molecular target for anticancer drug resistance and pharmacokinetics/pharmacodynamics.

Breast cancer resistance protein (BCRP) is a half-molecule ATP-binding cassette transporter that forms a functional homodimer and pumps out various anticancer agents, such as 7-ethyl-10-hydroxycamptothecin, topotecan, mitoxantrone and flavopiridol, from cells. Estrogens, such as estrone and 17beta-estradiol, have been found to restore drug sensitivity levels in BCRP-transduced cells by increasing the cellular accumulation of such agents. Furthermore, synthetic estrogens, tamoxifen derivatives and phytoestrogens/flavonoids have now been identified that can effectively circumvent BCRP-mediated drug resistance. Transcellular transport experiments have shown that BCRP transports sulfated estrogens and various sulfated steroidal compounds, but not free estrogens. The kinase inhibitor gefitinib inhibited the transporter function of BCRP and reversed BCRP-mediated drug resistance both in vitro and in vivo. BCRP-transduced human epidermoid carcinoma A431 (A431/BCRP) and BCRP-transduced human non-small cell lung cancer PC-9 (PC-9/BCRP) cells showed gefitinib resistance. Physiological concentrations of estrogens (10-100 pM) reduced BCRP protein expression without affecting its mRNA levels. Two functional polymorphisms of the BCRP gene have been identified. The C376T (Q126Stop) polymorphism has a dramatic phenotype as active BCRP protein cannot be expressed from a C376T allele. The C421A (Q141K) polymorphism is also significant as Q141K-BCRP-transfected cells show markedly low protein expression levels and low-level drug resistance. Hence, individuals with C376T or C421A polymorphisms may express low levels of BCRP or none at all, resulting in hypersensitivity of normal cells to BCRP-substrate anticancer agents. In summary, both modulators of BCRP and functional single nucleotide polymorphisms within the BCRP gene affect the transporter function of the protein and thus can modulate drug sensitivity and substrate pharmacokinetics and pharmacodynamics in affected cells and individuals.

Estrogen-mediated post transcriptional down-regulation of breast cancer resistance protein/ABCG2.

Breast cancer resistance protein (BCRP)/ABCG2 mediates concurrent resistance to chemotherapeutic agents, such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan, by pumping them out of cells. We previously reported that BCRP transports sulfated estrogens. In the present study, we show that at physiologic levels, estrogens markedly decrease endogenous BCRP expression in the estrogen-responsive and estrogen receptor alpha (ERalpha)-positive human breast cancer MCF-7 cells, but not in estrogen-nonresponsive human cancer cells. 17 beta-Estradiol (E(2)) also significantly reduces exogenous BCRP expression, driven by a constitutive promoter, in BCRP-transduced estrogen-responsive and ERalpha-positive MCF-7 (MCF-7/BCRP) and T-47D cells, but not in BCRP-transduced estrogen-nonresponsive MDA-MB-231 and SKOV-3 cells. E(2) potentiates the cytotoxicity of SN-38, but not vincristine, in MCF-7/BCRP cells significantly, and increases cellular topotecan uptake in MCF-7 and MCF-7/BCRP cells. Antiestrogen tamoxifen partially reverses E(2)-mediated BCRP down-regulation in MCF-7 and MCF-7/BCRP cells and treatment of MCF-7/BCRP cells with an ERalpha small interfering RNA abolished E(2)-mediated BCRP down-regulation, suggesting that interaction of E(2) and ERalpha is necessary for BCRP down-regulation. E(2) does not affect endogenous BCRP mRNA levels in MCF-7 cells or exogenous BCRP mRNA levels in MCF-7/BCRP cells. The results from pulse-chase labeling experiments with MCF-7/BCRP cells suggest that decreased protein biosynthesis and maturation, but not alterations in protein turnover, might underlie E(2)-mediated BCRP down-regulation. These data indicate that estrogen down-regulates BCRP expression by novel posttranscriptional mechanisms. This is the first report of small molecules that can affect BCRP protein expression in cells and may therefore assist in establishing new strategies for regulating BCRP expression.

Gefitinib reverses breast cancer resistance protein-mediated drug resistance.

Breast cancer resistance protein (BCRP) is an ATP binding cassette transporter that confers resistance to a series of anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), topotecan, and mitoxantrone. In this study, we evaluated the possible interaction of gefitinib, a selective epidermal growth factor receptor tyrosine kinase inhibitor, with BCRP. BCRP-transduced human epidermoid carcinoma A431 (A431/BCRP) cells acquired cellular resistance to gefitinib, suggesting that BCRP could be one of the determinants of gefitinib sensitivity in a certain sort of cells. Next, the effect of gefitinib on BCRP-mediated drug resistance was examined. Gefitinib reversed SN-38 resistance in BCRP-transduced human myelogenous leukemia K562 (K562/BCRP) or BCRP-transduced murine lymphocytic leukemia P388 (P388/BCRP) cells but not in these parental cells. In addition, gefitinib sensitized human colon cancer HT-29 cells, which endogenously express BCRP, to SN-38. Gefitinib increased intracellular accumulation of topotecan in K562/BCRP cells and suppressed ATP-dependent transport of estrone 3-sulfate, a substrate of BCRP, in membrane vesicles from K562/BCRP cells. These results suggest that gefitinib may overcome BCRP-mediated drug resistance by inhibiting the pump function of BCRP. Furthermore, P388/BCRP-transplanted mice treated with combination of irinotecan and gefitinib survived significantly longer than those treated with irinotecan alone or gefitinib alone. In conclusion, gefitinib is shown to interact with BCRP. BCRP expression in a certain sort of cells is supposed to be one of the determinants of gefitinib sensitivity. Gefitinib inhibits the transporter function of BCRP and reverses BCRP-mediated drug resistance both in vitro and in vivo.

Single amino acid substitutions in the transmembrane domains of breast cancer resistance protein (BCRP) alter cross resistance patterns in transfectants.

Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette transporters that has an N-terminal ATP binding domain and a C-terminal transmembrane domain (TM). Expression of wild-type BCRP confers resistance to multiple chemotherapeutic agents such as mitoxantrone, SN-38 and topotecan, but not to doxorubicin. We made 32 BCRP mutants with an amino acid substitution in the TMs (7 E446-mutants in TM2, 15 R482-mutants in TM3, 4 N557-mutants in TM5 and 6 H630-mutants in TM6) and examined the effect of the substitutions on cellular drug resistance. PA317 cells transfected with any one of the 7 E446-mutant BCRP cDNAs did not show drug resistance. Cells transfected with any one of the 13 R482X2-BCRP cDNAs (X2 = N, C, M, S, T, V, A, G, E, W, D, Q and H, but not Y and K) showed higher resistance to mitoxantrone and doxorubicin than the wild-type BCRP-transfected cells. Cells transfected with N557D-BCRP cDNA showed similar resistance to mitoxantrone but lower resistance to SN-38 than the wild-type BCRP-transfected cells. Cells transfected with N557E-, H630E- or H630L-BCRP cDNA showed similar degrees of resistance to mitoxantrone and SN-38. Estrone and fumitremorgin C reversed the drug resistance of cells transfected with R482-, N557- or H630-mutant BCRP cDNA. Cells transfected with R482G- or R482S-BCRP cDNA showed less intracellular accumulation of [3H]mitoxantrone than the wild-type BCRP-transfected cells. These results suggest that E446 in TM2, R482 in TM3, N557 in TM5 and H630 in TM6 play important roles in drug recognition of BCRP.

Breast cancer resistance protein exports sulfated estrogens but not free estrogens.

Breast cancer resistance protein (BCRP), an ATP-binding cassette transporter, confers resistance to a series of anticancer reagents such as mitoxantrone, 7-ethyl-10-hydroxycamptothecin, and topotecan. We reported previously that estrone and 17beta-estradiol reverse BCRP-mediated multidrug resistance. In the present study, we demonstrate that BCRP exports estrogen metabolites. First, we generated BCRP-transduced LLC-PK1 (LLC/BCRP) cells, in which exogenous BCRP is expressed in the apical membrane, and investigated transcellular transport of 3H-labeled compounds using cells plated on microporous filter membranes. The basal-to-apical transport (excretion) of mitoxantrone, estrone, and 17beta-estradiol was greater in LLC/BCRP cells than in LLC-PK1 cells. Thin-layer chromatography of transported steroids revealed that the transport of estrone and 17beta-estradiol was independent of BCRP expression. Alternatively, increased excretion of estrone sulfate and 17beta-estradiol sulfate was observed in LLC/BCRP cells. BCRP inhibitors completely inhibited the increased excretion of sulfated estrogens across the apical membrane. Conversion of estrogens into their sulfate conjugates was similar between LLC/BCRP and LLC-PK1 cells, suggesting that the increased excretion of estrogen sulfates was attributable to BCRP-mediated transport. Next, the uptake of 3H-labeled compounds in membrane vesicles from BCRP-transduced K562 (K562/BCRP) cells was investigated. 3H-labeled estrone sulfate, but not 3H-labeled estrone or 17beta-estradiol, was taken up by membrane vesicles from K562/BCRP cells, and this was ATP-dependent. Additionally, BCRP inhibitors suppressed the transport of estrone sulfate in membrane vesicles from K562/BCRP cells. These results suggest that BCRP does not transport either free estrone or 17beta-estradiol but exports sulfate conjugates of these estrogens.

C421A polymorphism in the human breast cancer resistance protein gene is associated with low expression of Q141K protein and low-level drug resistance.

Breast cancer resistance protein (BCRP) confers multidrug resistance to cancer cells against agents such as SN-38 (an active metabolite of irinotecan), mitoxantrone, and topotecan. Among 59 human tumor cell lines tested, 6 cell lines, A549, NCI-H460, KM-12, HT-29, OVCAR-5, and RPMI8226, showed high BCRP expression. BCRP cDNA was isolated from 11 cancer cell lines and three variant cDNAs [G34A substituting Met for Val-12 (V12M), C421A substituting Lys for Gln-141 (Q141K), and 944-949 deletion lacking Ala-315 and Thr-316 (delta315-6)] were identified. G34A and C421A variants were polymorphisms, and 944-949 deletion was a splicing variant. C421A BCRP-transfected PA317 cells showed markedly decreased protein expression and low-level drug resistance compared with wild-type BCRP-transfected cells when transfectants expressed similar levels of BCRP mRNA. G34A or 944-949-deleted BCRP-transfected PA317 cells showed similar or somewhat lower protein expression and drug resistance compared with wild-type BCRP-transfected cells. Of 124 healthy Japanese volunteers, 67 were wild-type, 48 were heterozygous, and 9 were homozygous for the C421A allele. These results suggest that some people possess the C421A polymorphic BCRP gene and express low amounts of Q141K BCRP. In addition to that, C376T polymorphism in exon 4 substituting stop codon for Gln-126 was found in 3 of the 124 general Japanese population. This C376T polymorphism may also have high impact because active BCRP protein will not be expressed from the C376T allele. Therefore, people with C376T and/or C421A polymorphisms may express low amounts of BCRP, and this low BCRP expression might result in hypersensitivity of normal cells to such anticancer drugs as irinotecan and mitoxantrone.

Estrone and 17beta-estradiol reverse breast cancer resistance protein-mediated multidrug resistance.

Breast cancer resistance protein (BCRP), an adenosine triphosphate-binding cassette transporter, confers resistance to a series of anticancer reagents, including mitoxantrone, SN-38 and topotecan. In the present study, we found that estrone and 17beta-estradiol potentiated the cytotoxicity of mitoxantrone, SN-38 and topotecan in BCRP-transduced K562 cells (K562 / BCRP). These estrogens showed only a marginal effect, or none, in parental K562 cells. Estrone and 17beta-estradiol increased the cellular accumulation of topotecan in K562 / BCRP cells, but not in K562 cells, suggesting that these estrogens inhibit the BCRP-mediated drug efflux and overcome drug resistance.

Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization.

Breast cancer resistance protein (BCRP) is a half-molecule ABC transporter highly expressed in mitoxantrone-resistant cells. In our study we established PA317 transfectants expressing Myc-tagged BCRP (MycBCRP) or HA-tagged BCRP (HABCRP). The exogenous BCRP protein migrated as a 70-kDa protein in SDS-PAGE under reducing condition, but migrated as a 140-kDa complex in the absence of reducing agents. The 140-kDa BCRP complex was heat-stable but dissociated into 70-kDa BCRP with the addition of 2-mercaptoethanol. The 140-kDa BCRP complex was immunoprecipitated with anti-Myc antibody from the lysates of PA317 cells double-transfected with MycBCRP and HABCRP. The 140-kDa complex reacted with anti-HA and anti-BCRP antibodies and after the addition of reducing agents, a 70-kDa protein reacting with anti-Myc, anti-HA and anti-BCRP antibodies was detected. These results clearly indicate that BCRP forms a homodimer bridged by disulfide bonds. To assess the possible dominant-negative inhibition of BCRP drug efflux pump, various mutant BCRP cDNAs were isolated by PCR mutagenesis. First, mutant BCRP cDNAs were introduced to parental PA317 cells and tested for their function as drug-resistance genes. Next, inactive BCRP cDNA clones were introduced to MycBCRP-transfected cells and tested for the ability to lower drug resistance. Among the 8 inactive mutant cDNA clones tested, HABCRP cDNA clone 15 with an amino acid change from Leu to Pro at residue 554 in the fifth transmembrane domain of BCRP partially reversed the drug resistance of MycBCRP-transfected cells. These results suggest that homodimer formation is essential for BCRP drug resistance, implicating this dominant-negative inhibition as a new strategy to circumvent drug resistance.

Tissue- and Stage-Specific Expression of Sericin Genes in the Middle Silk Gland of Bombyx mori: (sericin mRNA/northern blotting/gene regulation).

Sericin gene expression in the middle silk glands during Bombyx mori larval development was analyzed using probes from a genomic DNA clone for 10.5 kb sericin mRNA. The 10.5 kb mRNA, the most abundant in the fifth instar, is not detected in the third feeding, fourth feeding and fourth moulting stages. It becomes detectable at 2 days of the fifth instar, and accumulates rapidly. The second major mRNA in the late fifth instar, a 9.0 kb component having a similar sequence to the 10.5 kb mRNA, becomes detectable only at 6 and 7 days of the instar by use of the repetitious coding sequence probe of the sericin clone. Using the same probe about 20 kb RNAs with a fainter intensity than that of the major mRNAs are detected. They are present extremely faintly in the third and fourth feeding stages, disappear in the fourth moulting stage, and increase in the fifth instar. Two other faint poly(A)+ RNA components are detected by a DNA probe containing the 5' end sequence of the sericin clone. One is 4.3 kb, and appears in the third, fourth and fifth feeding stages but not in the fourth moulting stage. The other is 3.0 kb, and it becomes detectable after 1 day of the fifth instar.