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Supplementary Figure 1 from Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2′-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [125I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy. [Cancer Res 2007;67(14):6965–72]

Supplementary Table 1 from Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution

PURPOSE To develop an in vivo dosimeter system for stereotactic body radiation therapy (SBRT) that can perform accurate and precise real-time measurements, using a microsized amount of a photostimulable phosphor (PSP), BaFBr:Eu2+ . METHODS The sensitive volume of the PSP was 1.26 × 10-5 cm3 . The dosimeter system was designed to apply photostimulation to the PSP after the decay of noise signals, in synchronization with the photon beam pulse of a linear accelerator (LINAC), to eliminate the noise signals completely using a time separation technique. The noise signals included stem signals, and radioluminescence signals generated by the PSP. In addition, the dosimeter system was built on a storage-type dosimeter that could read out a signal after an arbitrary preset number of photon beam pulses were incident. First, the noise and photostimulated luminescence (PSL) signal decay times were measured. Subsequently, we confirmed that the PSL signals could be exclusively read out within the photon beam pulse interval. Finally, using a water phantom, the basic characteristics of the dosimeter system were demonstrated under SBRT conditions, and the feasibility for clinical application was investigated. The reproducibility, dose linearity, dose-rate dependence, temperature dependence, and angular dependence were evaluated. The feasibility was confirmed by measurements at various dose gradients and using a representative treatment plan for a metastatic liver tumor. A clinical plan was created with a two-arc beam volumetric modulated arc therapy using a 10 MV flattening filter-free photon beam. For the water phantom measurements, the clinical plan was compiled into a plan with a fixed gantry angle of 0°. To evaluate the energy dependence during SBRT, the percent depth dose (PDD) was measured and compared with those calculated via Monte Carlo (MC) simulations. RESULTS All the PSL signals could be read out while eliminating the noise signals within the minimum pulse interval of the LINAC. Stable real-time measurements could be performed with a time resolution of 56 ms (i.e., number of pulses = 20). The dose linearity was good in the dose range of 0.01-100 Gy. The measurements agreed within 1% at dose rates of 40-2400 cGy/min. The temperature and angular dependence were also acceptable since these dependencies had only a negligible effect on the measurements in SBRT. At a dose gradient of 2.21 Gy/mm, the measured dose agreed with that calculated using a treatment planning system (TPS) within the measurement uncertainties due to the probe position. For measurements using a representative treatment plan, the measured dose agreed with that calculated using the TPS within 0.5% at the center of the beam axis. The PDD measurements agreed with the MC calculations to within 1% for field sizes

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells., Highlights • Fucosyltransferase 8 gene expression is elevated in the tissues affected by endometrial endometrioid carcinoma. • Fucosyltransferase 8 protein is specifically detected in the glands affected by endometrial endometrioid carcinoma. • Silencing of fucosyltransferase 8 suppressed the proliferation of Ishikawa cells, an endometrial cancer cell line. • These results suggest that fucosyltransferase 8 might be involved in the proliferation of endometrial endometrioid carcinoma.

Adsorption and catalytic activation of the molecular oxygen on the hexagonal boron nitride (h-BN) monolayer doped with carbon atom have been studied theoretically using density functional theory. It is demonstrated that C doping in B position of h-BN (CB@h-BN) produces n-type semiconductor BN material with noticeable catalytic activity for O2 activation in the large area extended far away from the C impurity. The adsorption energy of O2 on CB@h-BN decreasing slowly with the increase in distance from the CB defect, while O2 remains highly activated. No such effect is observed for monolayer h-BN doped with different atoms of group III, IV and V and transition-metal elements, such as B, N, Al, Si, Ge, Ni, Pt, Pd, and Au, where O2 adsorbs only in the close vicinity of the dopant. It is shown that even small concentration of C dopants can functionalize the large surface area of monolayer BN making it promising catalytic material for oxygen activation and oxygen reduction reaction.

When antagonism is performed using sugammadex after continuous infusion of rocuronium, if the total amount of residual rocuronium can be esti- mated prior to performing antagonism, antagonism without excess or deficiency of sugammadex will be made possible. We therefore prepared a simple formula to predict residual amount of rocuronium in the body, which can be easily applied in clinical setting, and veri- fied it using Tivatrainer©.1. Pharmacokinetics of rocuronium was simulated, using a 3-compartment model. The following assumptions were made to derive the simple for- mula : when rocuronium is continuously infused to reach the steady state plasma concentration, an equal concentration in each compartment is reached. Only the amounts of rocuronium infused to the central com- partment and rocuronium excreted from there are thus considered, and these two amounts are in balance. For pharmacokinetic parameters, we referred to V. Saldien, Anesth Analg 2003 ; 97 : 44-9. 2. The prepared simple formula was verified using Tivatrainero. We considered a model in which initial boluses of 0.3, 0.6, 0.9, and 1.2 mg · kg(-1) were adminis- tered, and continuous infusion began at 30 minutes at the rate of 0.2, 0.3, 0.4, 0.5, 0.6, and 0.8 mg - kg-1 - hr-1. Patients with body weight of 50, 60, 70, and 80 kg were investigated.1. The derived simple formula was as fol- lows : Q=0.74 X R Q Total residual amount of rocuronium (mg) R Dose per hour (mg · hr(-1)) 2. The predicted value of the total residual amount obtained from the simple formula was consistent with the value predicted by Tivatrainer© with a high preci- sion within the error of 1.4%. Convergence time until the stable state was reached varied depending on the condition. However, it took approximately 150 minutes after the beginning of continuous infusion.for the error between values predicted by the simple formula and Tivatrainer© to stabilize within 5 mg.We prepared a simple formula to esti- mate the total residual amount of rocuronium at a steady state. The value predicted by the simple for- mula agreed with the value predicted by Tivatrainer) with a high precision.

Poorly water-soluble compounds have a potential risk of low and variable bioavailability caused by incomplete dissolution. Incorporation of organic acids as pH modifiers is effective method for solubility enhancement of basic compounds and requires no special technique and equipment. The purpose of this study was to evaluate the effect of manufacturing method on the extent of drug solubility enhancement. We successfully prepared the granules and tablets containing ketoconazole (KZ), which is weakly basic, as a model compound and citric acid as a pH modifier using conventional wet and dry granulations. KZ solubility under non-sink condition was enhanced with supersaturation using both wet and dry granulations. High-shear granulation was the most effective method in terms of KZ dissolution enhancement, because both an intimate contact and strong bonding between KZ and incorporated acid were achieved. KZ dissolved amount from the granules prepared by high-shear granulation was about eight times higher than that from the granules without the acid. The granulation involved to suppress a diffusion of acid dissolved, leading to the effectively maintained supersaturation state. The bioavailability of KZ after oral administration to rats was improved by applying high-shear granulation with citric acid independent of gastrointestinal pH. The granules prepared by high-shear granulation showed the bioavailability about 1.7-fold higher than that of the physical mixture in rats with and without neutralization of stomach. As a result, both the dissolution and absorption rates of KZ after oral administration were enhanced using conventional manufacturing technology.

The physiological role of multidrug resistance protein 4 (Mrp4, Abcc4) in the testes is unknown. We found that Mrp4 is expressed primarily in mouse and human Leydig cells; however, there is no current evidence that Mrp4 regulates testosterone production. We investigated its role in Leydig cells, where testosterone production is regulated by cAMP, an intracellular messenger formed when the luteinizing hormone (LH) receptor is activated. Because Mrp4 regulates cAMP, we compared testosterone levels in Mrp4(-/-) and Mrp4(+/+) mice. Young Mrp4(-/-) mice had significantly impaired gametogenesis, reduced testicular testosterone, and disruption of Leydig cell cAMP homeostasis. Both young and adult mice had impaired testosterone production. In Mrp4(-/-) primary Leydig cells treated with LH, intracellular cAMP production was impaired and cAMP-response element-binding protein (CREB) phosphorylation was strongly attenuated. Notably, expression of CREB target genes that regulate testosterone biosynthesis was reduced in Mrp4(-/-) Leydig cells in vivo. Therefore, Mrp4 is required for normal Leydig cell testosterone production. However, adult Mrp4(-/-) mice are fertile, with a normal circulating testosterone concentration. The difference is that in 3-week-old Mrp4(-/-) mice, disruption of gonadal testosterone production up-regulates hepatic Cyp2b10, a known testosterone-metabolizing enzyme. Therefore, defective testicular testosterone production de-regulates hepatic Cyp-mediated testosterone metabolism to disrupt gametogenesis. These findings have important implications for understanding the side effects of therapeutics that disrupt Mrp4 function and are reported to alter androgen production.

The role of ATP-binding cassette transporters in the urinary excretion of diuretics was investigated. Significant ATP-dependent uptake of hydrochlorothiazide (HCT) and furosemide was observed in membrane vesicles that expressed multidrug resistance-associated protein 4 (MRP4) and breast cancer resistance protein (BCRP). Unlike taurocholate uptake, S-methylglutathione had no effect on the ATP-dependent uptake of both compounds by MRP4. The functional importance of MRP4 and BCRP in the urinary excretion of HCT and furosemide was investigated using gene knockout mice. The renal clearance of HCT and furosemide was reduced significantly but not abolished in Mrp4 knockout mice compared with wild-type mice (9.0 +/- 0.9 versus 15 +/- 2 ml/min per kg for HCT and 1.9 +/- 0.3 versus 2.7 +/- 0.1 ml/min per kg for furosemide), and the amount of HCT that was associated with the kidney specimens was greater in Mrp4 knockout mice (21 +/- 3 versus 13 +/- 1 nmol/g kidney). In contrast, Bcrp makes only a negligible contribution because the urinary excretion was unchanged in Bcrp knockout mice. Our results suggest that Mrp4, together with other unknown transporters, accounts for the luminal efflux of HCT and furosemide from proximal tubular epithelial cells.

Acyclic nucleotide phosphonates (adefovir, cidofovir, and tenofovir) are eliminated predominantly into the urine, and renal failure is their dose-limiting toxicity, particularly for adefovir and cidofovir. In this study, we examined the involvement of multidrug resistance-associated protein (MRP)4 (ABCC4) in their luminal efflux in the kidney. ATP-dependent uptake of adefovir and tenofovir but not cidofovir was observed only in the membrane vesicles expressing MRP4. The ATP-dependent uptake of adefovir and tenofovir by MRP4 was not saturated at 1 mM. The ATP-dependent uptake of adefovir by membrane vesicles expressing MRP4 was osmotic-sensitive. No ATP-dependent uptake of either agent was observed in the membrane vesicles expressing human MRP2 or breast cancer resistance protein. These nucleotide analogs were given to mice by constant intravenous infusion, and the plasma, urine, and tissue concentrations were determined. The kidney accumulation of adefovir and tenofovir was significantly greater in Mrp4 knockout mice (130 versus 66 and 191 versus 87 pmol/g tissue, respectively); thus, the renal luminal efflux clearance was estimated to be 37 and 46%, respectively, of the control. There was no difference in the fraction of mono- and diphosphorylated forms of adefovir in the kidney between wild-type and Mrp4 knockout mice. In mice, cidofovir was also eliminated via the urine by tubular secretion as well as glomerular filtration. There was no change in the kinetic parameters of cidofovir in Mrp4 knockout mice. Our results suggest that MRP4 is involved in the luminal efflux of both adefovir and tenofovir, but it makes only a limited contribution to the urinary excretion of cidofovir.

Summary Maspin is a member of the serpin (serine protease inhibitor) superfamily, and some experimental studies revealed a potential tumor suppressor activity of maspin. To reveal clinical significance of maspin status in non-small cell lung cancer (NSCLC), we quantitatively evaluated maspin gene expression in lung primary tumors cut from a total of 55 resected NSCLC patients. Maspin expression in squamous cell carcinoma (Sq) was significantly higher than that in adenocarcinoma (Ad, p =0.011). Five-year overall survival rates of maspin-high and maspin-low patients were 67.7 and 41.4%, respectively, demonstrating a significant favorable prognosis of maspin-high patients (log-rank, p =0.042). A multivariate analysis confirmed that high maspin expression was an independent and significant factor to predict a favorable overall survival ( p =0.031). These results suggested that maspin expression was significantly increased in Sq than in Ad, and that increased maspin expression was a significant factor to predict a favorable prognosis in resected NSCLC.

Mrp4 is a member of the multidrug resistance–associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive upregulation in response to cholestatic injury or bile acid feeding. However, the relative importance of Mrp4 in a protective adaptive response to cholestatic injury is not known. To address this issue, common bile duct ligation (CBDL) was performed in wild-type and Mrp4−/− mice and animals followed for 7 days. Histological analysis and serum aminotransferase levels revealed more severe liver injury in the absence of Mrp4 expression. Western analyses revealed that Mrp4, but not Mrp3, was significantly increased after CBDL in wild-type mice. Serum bile acid levels were significantly lower in Mrp4−/− mice than in wild-type CBDL mice, whereas serum bilirubin levels were the same, suggesting that Mrp4 was required to effectively extrude bile acids from the cholestatic liver. Mrp3 and Ostα-Ostβ were upregulated in Mrp4−/− mice but were unable to compensate for the loss of Mrp4. High-performance liquid chromatography analysis on liver extracts revealed that taurine tetrahydroxy bile acid/beta-muricholic acid ratios were increased twofold in Mrp4−/− mice. In conclusion, hepatic Mrp4 plays a unique and essential protective role in the adaptive response to obstructive cholestatic liver injury. (HEPATOLOGY 2006;43:1013–1021.)

症例は13歳男性. 小児科で急性骨髄性白血病 (AML) の診断の際, 前縦隔から右胸腔全体を占拠する巨大な縦隔腫瘍を指摘されていた. AMLに対し化学療法, 骨髄移植を行った後, 縦隔腫瘍の摘出術を施行した. 右肺は腫瘍に強く圧迫されており, 含気がなく完全に虚脱していた. 腫瘍摘出後の膨張も不十分なままであり, 術直後より再膨張に伴う右肺水腫を考慮し, 早期からステロイド, 利尿剤を投与した. PEEP補助などの処置を行なったが, 術後2日目には対側肺水腫の併発が疑われ, 呼吸不全のため一時的に人工呼吸管理を必要とした. 再膨張性肺水腫は一般的に予後良好であるが, 本例のような対側肺水腫を併発する重症例も稀であるが報告されている. 長期にわたり肺虚脱があった場合, および化学療法後など, その発症が懸念される時は早期から人工呼吸管理を躊躇しない積極的な治療を行なう必要がある.

The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.

Cellular factors may contribute to the decreased efficacy of chemotherapy in HIV infection. Indeed, prolonged treatment with nucleoside analogues, such as azidothymidine (AZT), 2′,3′-deoxycytidine or 9-(2-phosphonylmethoxyethyl)adenine, induces cellular resistance. We have developed a human T lymphoblastoid cell line (CEM3TC) that is selectively resistant to the antiproliferative effect of 2′,3′-dideoxy-3′-thiacytidine (3TC) because the CEM3TC cells were equally sensitive to AZT, as well as the antimitotic agent, vinblastine. The anti-retroviral activity of 3TC against HIV-1 was also severely impaired in the CEM3TC cells. Despite similar deoxycytidine kinase activity and unchanged uptake of nucleosides such as AZT and 2′-deoxycytidine, CEM3TC had profoundly impaired 3TC accumulation. Further studies indicated that CEM3TC retained much less 3TC. However, despite a small overexpression of multidrug resistance protein (MRP) 4, additional studies with cells specifically engineered to overexpress MRP4 demonstrated there was no impact on either 3TC accumulation or efflux. Finally, an increased expression of the MRP5 homologue, ATP-binding cassette C11 (ABCC11) was observed in the CEM3TC cells. We speculate that the decreased 3TC accumulation in the CEM3TC might be due to the upregulation of ABCC11.

The energy dependent transport of drugs contributes to cellular resistance and is undoubtedly a prime suspect in chemotherapeutic failure of a variety of disease processes. Early studies focused on a single gene, the multidrug resistance gene, MDR1, as a main contributor to chemotherapeutic failure. However, the multifaceted nature of cellular resistance lead to the discovery of the MRP gene. This pivotal finding and the concurrent rapid development of gene databases lead to the expansion of the MRP gene family. The purpose of this review is to discuss two of the recently described MRP family members that were orphans until their role in drug resistance was discovered. This review will provide an overview of the current state of our understanding of MRP4 and 5.

Background & Aims: The molecular basis of cell motility is highly complex and is controlled by a number of molecular systems, whereas angiogenesis is an important biological component of tumor progression. The aims of this study were to investigate the possible involvement of proteins at the cell surface in controlling cell motility and angiogenesis, and to identify the cell surface molecules involved in gastrointestinal tumors. Methods: We addressed these issues using functional monoclonal antibodies, which inhibit cell motility, endothelial cell migration, and tube formation. Furthermore, we investigated the relationship between this antigen and colon cancer, and showed the prognostic significance in human colon cancer. Results: We established a murine monoclonal antibody MH8-11, which inhibits cell motility and in vitro angiogenesis. This epitope was a 165-kilodalton protein, and the sequencing analysis revealed that it was almost identical to aminopeptidase N (APN)/cluster of differentiation (CD) 13. APN/CD13 expression was associated with tumor status ( P = 0.025). The disease-free and overall survival rate for patients with positive APN/CD13 expression tumors was significantly lower than that for patients with negative APN/CD13 expression tumors ( P = 0.014, 0.033, respectively). Among 47 node-positive patients, the survival rate of patients with negative APN/CD13 expression was better than that of those with positive APN/CD13 expression. Conclusions: Our data suggest that APN/CD13 is involved in cell motility and angiogenesis, and APN/CD13 expression may be a useful indicator of a poor prognosis for node-positive patients with colon cancer. GASTROENTEROLOGY 2002;122:376-386

Fas is a death receptor that belongs to the tumor necrosis factor receptor family and is expressed in various cell types, in particular, in lymphoid cells. A loss-of-function mutation in the Fas gene (lpr mutation) causes lymphadenopathy and splenomegaly, and accelerates autoimmune diseases in some strains of mice such as MRL. In this report, Fas cDNA driven by murine lck distal promoter was used to establish transgenic MRL-lpr mouse lines. The transgenic mice expressed functional Fas in mature T cells and B cells. The lymphadenopathy and splenomegaly caused by accumulation of abnormal T cells in the lpr mice were rescued in the transgenic mice. The number of B cells in the periphery as well as the serum IgG level were significantly reduced, and the autoimmune symptoms and mortality were ameliorated. These results indicate that both mature B cells and T cells must undergo Fas-mediated apoptosis to prevent the development of autoimmune diseases.

BACKGROUND Previously, the authors reported the role of the vascular endothelial growth factor (VEGF) as an angiogenic factor in 40 patients with pancreatic carcinoma. In this study, they investigated the mechanism underlying the regulation of VEGF gene expression and evaluated VEGF expression and K-ras gene status in 48 patients with pancreatic carcinoma. METHODS The authors used quantitative reverse transcriptase-polymerase chain reaction analysis and direct sequencing techniques for a retrospective study of VEGF gene expression and K-ras gene status in tumor tissue samples from 48 patients with pancreatic carcinoma. Immunohistochemistry also was used to investigate VEGF protein expression. RESULTS Thirty-one tumors (64.6%) were evaluated with high VEGF expression, and 17 tumors (35.4%) were evaluated with low VEGF expression. Of the 48 primary pancreatic tumors studied, 33 tumors (68.8%) contained mutations of the K-ras gene. There was a significant correlation between VEGF expression and K-ras status. Twenty-five of 33 tumors (75.8%) with mutant K-ras genes showed high VEGF expression, whereas only 6 of 15 tumors with the wild type K-ras (40.0%) showed high VEGF expression (P = 0.038). The mean (± standard error) VEGF conservation rate for the 33 tumors with mutant K-ras was 1.839 ± 1.241, and that for the 15 tumors with wild type K-ras was 1.057 ± 0.983 (P = 0.037). Furthermore, the median survival for patients with mutant K-ras was shorter than for those with wild type K-ras (10.6 months vs. 27.6 months, respectively; P = 0.026), whereas the median survival for patients with high VEGF expression was shorter compared with that for patients with low VEGF expression (9.5 months vs. 26.4 months, respectively; P = 0.002). Cox regression model analysis indicated that only the VEGF status was a significant factor for prognosis (P = 0.024). Other variables, i.e., K-ras status, histopathologic tumor grade, tumor status, lymph node status, metastatic status, gender, and age at surgery, were not significant. CONCLUSIONS The results of this study suggest that K-ras oncogene mutation may be associated with VEGF expression and that patients with pancreatic carcinoma who have high VEGF expression are associated with a poor prognosis. Cancer 2001;92:488–99. © 2001 American Cancer Society.

Previously we showed that MRP-1/CD9 might prevent tumor metastasis by suppression of cell motility and invasion of tissue barriers. The present study explored the possibility of preventing metastasis of mouse melanoma BL6 by expression of MRP-1/CD9 through gene transfer. A replication-deficient adenovirus vector was used for the in vivo transfer of MRP-1/CD9 cDNA. Intratumor injection of an adenovirus vector (rAd-MRP-1/CD9) expressing MRP-1/CD9 resulted in a 73.7% reduction in the number of pulmonary metastases of mice and the median survival time of mice treated with rAd-MRP-1/CD9 was significantly longer than those treated with the rAd-beta-gal vector (103.2 approximately plus;8.5 days vs 71.2 approximately plus;5.2 days, P0.001 respectively). These results support the expression of MRP-1/CD9 through gene transfer as a therapeutic strategy for preventing metastases and prolonging survival, and support the feasibility of gene transfer in a clinically relevant setting.

The DMP1 transcription factor induces the ARF tumor suppressor gene in mouse fibroblasts, leading to cell cycle arrest in a p53-dependent manner. We disrupted sequences encoding the DNA-binding domain of DMP1 in mouse embryonic stem cells and derived animals lacking the functional protein. DMP1-null animals are small at birth, and males develop more slowly than their wild-type littermates. Some adult animals exhibit seizures and/or obstuctive uropathy, each of unknown cause. The growth of explantedDMP1-null mouse embryo fibroblasts (MEFs) is progressively retarded as cells are passaged in culture on defined transfer protocols; but, unlike the behavior of normal cells, p19ARF, Mdm2, and p53 levels remain relatively low and DMP1-null MEFs do not senesce. Whereas the establishment of cell lines from MEFs is usually always accompanied by either p53 or ARF loss of function, continuously passaged DMP1-null cells readily give rise to established 3T3 and 3T9 cell lines that retain wild-type ARFand functional p53 genes. Early-passage DMP1-null cells, like MEFs from either ARF-null or p53-null mice, can be morphologically transformed by oncogenic Ha-Ras (Val-12) alone. Splenic lymphocytes harvested from both DMP1-null andARF-null mice exhibit enhanced proliferative responses in long-term cultures when stimulated to divide with antibody to CD3 and interleukin-2. Although only 1 of 40 DMP1-null animals spontaneously developed a tumor in the first year of life, neonatal treatment with dimethylbenzanthracene or ionizing radiation induced tumors of various histologic types that were not observed in similarly treated DMP1+/+ animals. Karyotypic analyses of MEFs and lymphomas from DMP1-null animals revealed pseudodiploid chromosome numbers, consistent with the retention of wild-type p53. Together, these data suggest that ARF function is compromised, but not eliminated, in animals lacking functional DMP1.

Caspase-activated DNase (CAD) degrades chromosomal DNA during apoptosis, whereas ICAD (inhibitor of CAD) inhibits the CAD's DNase by binding to it. Here, we describe the assignment of murine CAD and ICAD genes to the distal part of murine chromosome 4. Molecular cloning and structural analysis indicated that CAD and ICAD genes are comprised of 7 and 6 exons, respectively. Two different ICAD mRNAs coding for two forms of ICAD proteins (ICAD-S and ICAD-L) were found to be produced by alternative splicing of intron 5. The CAD and ICAD mRNAs were detected ubiquitously in various murine tissues. Analyses of the promoter activity with a series of deletion mutants of their 5' flanking regions indicated that a 190-bp 5' flanking region of the CAD gene was sufficient to promote the transcription. Whereas, a 120-bp flanking region of ICAD gene was required to promote its transcription. These regions do not show similarity between CAD and ICAD genes, suggesting that expression of CAD and ICAD genes is regulated by different mechanisms.

A molecular staging protocol using reliable markers is of importance in predicting the prognosis of patients with non-small cell lung cancer (NSCLC) and for instituting their appropriate post-surgical treatment. We analysed tumor tissues from 187 NSCLC patients. The DNA and mRNA were extracted from frozen specimens, and then polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to investigate mutations of p53 from exons 5-8, and mutations of K-ras at exon 1. To determine MRP-1/CD9 gene and KA11/CD82 gene expression, which have been postulated to be metastasis suppressor genes, we have applied quantitative RT-PCR. A Cox multivariate regression analysis showed that nodal status, MRP-1/CD9 and K-ras status were significant factors for prognosis (P

To evaluate whether angiogenic factors are of clinical relevance to actual human pancreatic cancers, we studied the intratumoral microvessel density (IMD), and PD-ECGF, VEGF protein expression in 40 pancreatic cancers using immunohistochemistry. We also investigated PD-ECGF and VEGF gene expression using reverse transcriptase-PCR (RT-PCR). Of the 40 pancreatic cancers studied, 30 carcinomas (75.0%) were evaluated to be PD-ECGF-positive and 10 carcinomas (25.0%) were determined to be PD-ECGF-negative. In contrast, 27 carcinomas (67.5%) were evaluated to be VEGF-positive, whereas 13 carcinomas (32.5%) were VEGF-negative. VEGF gene expression was moderately associated with an increase in the IMD (r2 = 0.181, P = 0.006), but no significant relationship was found between PD-ECGF gene expression and the IMD (r2 = 0.093, P = 0.059). However, tumours with positive expression for both PD-ECGF and VEGF had a higher IMD (P = 0.027). The results of the immunohistochemistry agreed well with the results of the quantitative RT-PCR. The median survival time of the hypervascular group was significantly shorter than that of the hypovascular group (P < 0.0001). In comparing the survival according to PD-ECGF and VEGF gene expression, the median survival time of the patients with positive PD-ECGF expression was significantly shorter than those with negative PD-ECGF expression (P = 0.040). Furthermore, the median survival time of the patients with positive VEGF expression was significantly shorter than those with negative VEGF expression (P = 0.048). However, the Cox multivariate analysis indicated that the IMD and VEGF expression were independent prognostic factors of the various clinicopathologic variables in pancreatic cancer patients (P = 0.0021 and P = 0.0443, respectively). © 1999 Cancer Research Campaign

Several members of the transmembrane 4 superfamily (TM4SF) have been reported to be related to tumor progression and metastasis. The aims of our study were to clarify the relationship between TM4SF and pancreatic cancer and to determine the prognostic significance of TM4SF in human pancreatic cancer. The mRNA levels for MRP-1/CD9, KAI1/CD82 and ME491/CD63, which belong to the TM4SF gene family, were evaluated in 40 resectable pancreatic adenocarcinomas using reverse transcriptase-PCR. MRP-1/CD9 gene expression was associated with lymph node status, and with pathological status. Moreover, MRP-1/CD9 expression was inversely associated with histo-pathological grading. KAI1/CD82 gene expression was inversely associated with tumor status. ME491/CD63 gene expression, however, was conserved in all pancreatic cancers. The overall survival rate for the 22 patients whose tumors had decreased MRP-1/CD9 gene expression was strikingly lower than that for the 18 patients with MRP-1/CD9-positive tumors. The overall survival rate of the 15 patients who were KAI1/CD82-positive was significantly higher than that of the 25 patients with decreased KAI1/CD82 gene expression. In a multivariate analysis using the Cox proportional hazards model, MRP-1/CD9 and KAI1/CD82 status was found to be the most significant.

Fas is a member of the TNF receptor family. Binding of Fas ligand to Fas induces apoptosis in Fas-bearing cells. Fas is expressed in various cells, including thymocytes, peripheral T cells, and activated B cells. The mouse lpr mutation is a loss of function mutation of Fas. MRL-lpr/lpr mice develop lymphadenopathy and splenomegaly, and produce multiple autoantibodies, which results in autoimmune disease. In this report, we describe the establishment of a line of Fas transgenic MRL-lpr mice in which mouse Fas cDNA was expressed using the T cell-specific murine lck promoter. The transgenic mice expressed functional Fas in thymocytes and peripheral T cells, but not in B cells. The transgenic mice did not accumulate abnormal T cells (Thy-1+ B220+), but still accumulated B cells (Thy-1− B220+); they produced a large quantity of Igs (IgG1 and IgG2a), including anti-DNA Abs, and developed glomerulonephritis. These results suggest that autoreactive or activated B cells must be killed through Fas expressed in the B cells by the Fas ligand expressed in activated T cells.

PURPOSE We investigated the possible association between integrin alpha3 and motility-related protein (MRP-1), cluster of differentiation antigen 9 (CD9) gene expression in non-small-cell lung cancer (NSCLC) and evaluated the prognostic significance of integrin alpha3 expression. PATIENTS AND METHODS We performed a retrospective study of integrin alpha3 and MRP-1/CD9 expression in resected tumor tissues from 151 NSCLC patients using quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. RESULTS The ratio of integrin alpha3/beta-actin expression ranged from 0 to 5.87 (mean was 0.80; median, 0.70). Using the cutoff value of 0.7, there were 78 (52%) integrin alpha3-positive tumors and 73 (48%) tumors with reduced integrin alpha3 expression. The immunohistochemical results agreed well with those of the RT-PCR assays, and 88% had no discrepancy. In case of discrepancy, the results of RT-PCR were used in specimen classification. Integrin alpha3 gene expression was independent from MRP-1/CD9 gene expression. No significant association was found between integrin alpha3 expression and the patients' clinical characteristics. The overall survival rate of patients with integrin alpha3-positive NSCLCs was only slightly better than that of individuals whose tumors had reduced integrin alpha3 expression (55.9% v 47.1%; P = .085). By comparison, the overall survival rate of patients with integrin alpha3-positive adenocarcinomas was strikingly greater than in those whose tumors had reduced gene expression (54.4% v 35.2%; P = .004). Multivariate analysis with the Cox regression model of NSCLC and adenocarcinoma indicated that integrin alpha3 expression correlated better (P = .0188 and P = .0008, respectively) with the overall survival rate than other variables, except lymph node status. CONCLUSION No significant association was found between integrin alpha3 and MRP-1/CD9 gene expression in lung cancer. However, reduced integrin alpha3 expression is a poor prognosis factor in patients with adenocarcinomas.

The mitogen-dependent induction of cyclin D–dependent kinase activity is required for cells to enter the DNA synthetic (S) phase of their division cycle. Immature 32Dcl3 myeloid cells (32D) proliferating in the presence of interleukin-3 (IL-3) normally express cyclins D2 and D3, which assemble into binary holoenzyme complexes with their catalytic subunits, CDK4 and CDK6. When 32D cells are switched to medium containing granulocyte colony-stimulating factor (G-CSF ) instead of IL-3, D-type cyclins are degraded and, in the absence of their associated kinase activity, the cells arrest in the first gap phase (G1 ) of the cell cycle and differentiate to neutrophils. We derived 32D cells in which the expression of p19INK4d, a specific polypeptide inhibitor of CDK4 and CDK6, is regulated by the heavy metal-inducible sheep metallothionein promoter. Induction of p19INK4d in response to zinc prolonged cell survival in the absence of growth factor treatment. When maintained in medium containing both IL-3 and zinc, these cells lost cyclin D–dependent kinase activity, underwent G1 phase arrest, and acquired certain morphologic, antigenic, and functional properties of mononuclear phagocytes. Cells induced to express p19INK4d did not synthesize receptors for macrophage colony-stimulating factor (M-CSF/CSF-1) and reverted to an immature myeloid phenotype when shifted back into medium containing IL-3 alone. These cells exhibited accelerated differentiation to neutrophils in response to G-CSF but also gave rise to macrophage-like cells when maintained in medium containing both G-CSF and zinc. Therefore, the acquisition of macrophage properties in response to zinc treatment neither depended upon IL-3 nor upon G1 phase arrest per se and instead reflects some ability of p19INK4d, and presumably cyclin D–dependent kinases, to affect myeloid differentiation.

Motility-related protein-1 (MRP-1)/CD9 is a trans-membrane glycoprotein closely associated with suppression of cell motility and reduced metastatic potential of some tumor cells. We currently report that, according to the RT-PCR method for MRP-1/CD9 gene expression, patients with low expression of MRP-1/CD9 in non-small-cell lung cancer, especially the adenocarcinoma type, showed short overall survival. Then, to determine accurately the prognostic value of MRP-1/CD9 product levels in lung-adenocarcinoma cells, we immunohistochemically investigated its expression in 132 lung-adenocarcinoma patients undergoing potentially curative surgery. Of these patients, 44 (33%) showed reduced expression of MRP-1/CD9 in cancer cells, and an inverse association was observed between its expression and factors associated with tumor progression, such as nodal involvement (p = 0.029) or stage (p = 0.028). Patients with reduced expression of MRP-1/CD9 showed a significantly worse prognosis in overall survival (p = 0.005) and disease-free survival (DFS; p < 0.0001) than those with stronger expression; and even among patients with stage-I disease, similar results were obtained (overall survival, p = 0.038; DFS, p = 0.012). In a multivariate analysis, immunohistochemical MRP-1/CD9-expression level was an independent prognostic factor for DFS (p = 0.021), but not for overall survival (p = 0.572). Thus, immunohistochemical MRP-1/CD9-expression level solely in lung-adenocarcinoma cells within the tumor tissue appears to be a prognostic factor for DFS, and may be useful for detecting a high-risk sub-group of recurrence during the post-operative clinical course of the disease.

Fas is a 45-kDa membrane protein that transduces an apoptotic signal. The mouse lymphoproliferation (lpr) mutation is a leaky mutation of Fas. In this study, we examined lymphocyte development in Fas-null mice generated by gene targeting. The Fas-/- mice progressively accumulated abnormal T cells (Thy1+, B220+, CD4-, and CD8-) and developed lymphadenopathy and splenomegaly, which were much more accelerated and pronounced than those in lpr mice. In addition, the Fas-null mice showed lymphocytosis, accompanied by lymphocytic infiltration in the lungs and liver. The number of apparently normal B cells also increased, and large amounts of immunoglobulins, including anti-DNA antibodies, were produced. Thymic clonal deletion, assessed by deletion of T cells reactive to mouse endogenous superantigens, was apparently normal in the Fas-/- mice, whereas the peripheral clonal deletion of mature T cells against a bacterial superantigen was impaired. These results suggested that Fas plays a decisive role in peripheral clonal deletion but not in negative selection in the thymus.

We immunohistochemically evaluated lymphoid cell infiltration and HLA-DR antigen expression in gastric tumor tissue obtained from advanced gastric cancer patients I day after the completion of the treatment with mito-mycin C (MMC) 12 mg/m 2 i.v. on day 1 and recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Then the results were compared with those in 11 patients pretreated with MMC alone, 5 treated with IL-2 alone, and 24 untreated patients. Widespread lymphoid infiltration was observed in 17% of untreated tumors, 27% of MMC-pretreated tumors, and 40% of tumors treated with IL-2 alone. However, 71% of carcinomas pretreated with MMC plus IL-2 exhibited widespread infiltration. The frequency of cases with high-grade infiltration of CD4 + cells was significantly higher in either group of patients treated with MMC alone or MMC plus IL-2. Because the CD8 + cell infiltration was not significantly altered, the ratio of CD4 + to CD8 + cells estimated as being >1 was more frequently noted in patients given MMC alone or MMC plus IL-2, as compared with untreated control. Furthermore, 86% of tumors pretreated with MMC plus IL-2 exhibited positive HLA-DR antigen expression, whereas 29% of untreated carcinomas did so. MMC or IL-2 alone did not significantly increase HLA-DR expression. These results indicate that the combination of low-dose of IL-2 with MMC enhances the intensity of lymphoid cell infiltration in tumors, with the predominance of CD4 + cells, and HLA-DR antigen expression on tumor cells in patients with advanced gastric carcinoma.

The effects of intravenous cisplatin (CDDP) administration on the generation of lymphokine-activated killer (LAK) activity in peripheral blood mononuclear (PBM) cells were investigated in cancer patients. The ability of PBM to generate LAK activity was significantly augmented 3, 5 and 7 days after a single dose, 50 mg m-2, of CDDP injection when compared to that before injection. NK activity of PBM was not altered. The distribution of lymphocyte subsets exhibited no significant change following CDDP injection, except CD2+ cells. However, the ability of monocytes in PBM to produce TNF-alpha was significantly enhanced 5 days after the drug administration, although IL-1-alpha and IL-1-beta production was not augmented.

リソパ球の分化過程でのアポトーシスを介しての細胞死の誘導は免疫機構の調節において重要な役割を担っている.細胞内にアポトーシスめシグナルを伝達する機能をもつ細胞表面タンパク質のFas抗原の異常が自己免疫疾患の発症機序に関与している可能性が高いことを示した.すなわち,異常なT細胞を蓄積して,自己免疫疾患のSLE様症状を示すlpr (lymphoproliferation)変異マウスではFas抗原遺伝子のイントロンの中にトランスポゾンが挿入されていたり,あるいは点突然変異を起こしているためにFas抗原の機能が失われていた.つまり, Fas抗源はTリンパ球の発生段階において生と死の判別機構に関与しており,このバランスが障害されることが自己免疫疾患の素因になると考えられる.

The purpose of this study was to confirm that the grades of lymph vessel tumor emboli in biopsy specimens obtained before neoadjuvant therapy and in the surgical specimens obtained after neoadjuvant therapy according to the grading system we devised are significant histological outcome predictor for invasive ductal carcinoma (IDC) patients who received neoadjuvant therapy. The subjects of this study were the 318 consecutive IDC patients who had received neoadjuvant therapy in our institution. The lymph vessel tumor embolus grades in the biopsy specimens and in the surgical specimens were significantly associated with the increases in mean number of nodal metastases. Multivariate analyses with well-known prognostic factors and p53 expression in tumor-stromal fibroblasts clearly showed that the lymph vessel tumor embolus grade based on the biopsy specimens and based on the surgical specimens significantly increased the hazard rates for tumor recurrence and tumor-related death in all the IDC patients as a whole, in the IDC patients who did not have nodal metastasis, and in the IDC patients who had nodal metastasis, and the outcome-predictive power of the lymph vessel tumor embolus grades based on the surgical specimens was superior to that of the lymph vessel tumor embolus grades based on the biopsy specimens. The grades in the grading system for lymph vessel tumor emboli were significantly associated with nodal metastasis, and the histological grading system is an excellent system for accurately predicting the outcome of patients with IDC of the breast who have received neoadjuvant therapy.

Recently, we described a platelet antibody against a putative collagen receptor (P62), which was found in a patient with idiopathic thrombocytopenic purpura (ITP) (Blood 69:1712). We now report a deficiency of the P62 receptor in a young man whose platelets showed defective collagen-induced platelet aggregation. He had a mild bleeding tendency and slight thrombocytopenia. The results of coagulation and fibrinolysis studies were normal. The patient's platelets were partially unresponsive to collagen, although aggregation in response to ADP, thrombin, ristocetin, and calcium ionophore (A23187) was almost normal. Adhesion of his platelets to bovine collagen was markedly reduced. Addition of collagen caused no synthesis of thromboxane (TX)B2 in platelet rich plasma (PRP) from this patient. Furthermore, collagen produced no rise of cytosolic free calcium ([Ca2+]i) in fura2-loaded platelets. In contrast, thrombin caused TXB2 formation and an increase of [Ca2+]i in his platelets. These results suggest defective interaction between the platelets and collagen. The IgG from the ITP-patient induced irreversible aggregation in normal PRP, but caused no aggregation of the young man's platelets. Immunoblot studies showed that normal platelets had antigens with a molecular weight of 62 KDa under reducing conditions and of 57 KDa under nonreducing conditions. In contrast, the young man's platelets had no P62 band, although GPIa/IIa and thrombospondin were normally present. These results indicate that impaired collagen-induced aggregation in the patient's platelets was due to a deficiency of P62 and confirm that P62 may play a crucial role as a collagen receptor in platelet activation.

In this paper, investigate the relation between emotions represented by speech and features included in speech. There are two kinds of approaches in comparing experiment. Firstly, speech data are non-verbal such as white-noise, sine wave and vowel /a:/, so that investigate the relation between emotions and prosodic features only. Secondly, speech data include verbal information such as ‘ohayou’(hello), ‘usotsuki’(Ananias), so that investigate case speech data are words. Result of comparing shows the effect that of verbal information on emotions is strong, in case of words used this experiment. On the other hand, emotions felt by subjects are changed by variation of prosodic features in some words.

A 61-year-old man with Philadelphia chromosome-positive chronic myelogenous leukemia developed megakaryoblastic leukemia. In the blast phase, his blast cells showed undifferentiated megakaryoblastic characteristics with no alpha-granules or demarcation membranes but with detectable platelet peroxidase (PPO) activity and surface glycoprotein (GP) IIb/IIIa. The patient has remained reasonably well for at least 12 months after blastic crisis, and 6-mercaptopurine alone has been effective in controlling leukocytosis and megakaryoblast proliferation. The expression of mRNA for platelet-specific proteins, such as GPIIb and platelet factor 4 (PF4), was studied in the patient's blast cells by the Northern blot analysis. Both GPIIb and PF4 mRNA were detected in the blast cells. Cytoplasmic maturation occurs later than the synthesis of the surface GP during megakaryocyte maturation. Therefore, PF4 mRNA expression should be a marker of mature megakaryoblasts. The PF4 mRNA expression in megakaryoblastic leukemia may indicate that a patient will have long survival and a good response to chemotherapy.

Olmesartan, a novel angiotensin II AT1-receptor antagonist, is excreted into both bile and urine, with minimal metabolism. Because olmesartan is a hydrophilic anionic compound, some transporters could be involved in its hepatic and renal clearance. In this study, we characterized the role of human drug transporters in the pharmacokinetics of olmesartan and determined the contribution of each transporter to the overall clearance of olmesartan. Olmesartan was significantly taken up into human embryonic kidney 293 cells expressing organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, organic anion transporter (OAT) 1, and OAT3. We also observed its saturable uptake into human hepatocytes and kidney slices. Estimated from the relative activity factor method and application of specific inhibitors, the relative contributions of OATP1B1 and OATP1B3 to the uptake of olmesartan in human hepatocytes were almost the same, whereas OAT3 was predominantly involved in its uptake in kidney slices. The vectorial transport of olmesartan was observed in OATP1B1/multidrug resistance-associated protein (MRP) 2 double transfectants, but not in OATP1B1/multidrug resistance (MDR) 1 and OATP1B1/breast cancer resistance protein (BCRP) transfectants. ATP-dependent transport into membrane vesicles expressing human MRP2 and MRP4 was clearly observed, with K(m) values of 14.9 and 26.2 microM, respectively, whereas the urinary excretion of olmesartan in Mrp4-knockout mice was not different from that of control mice. We also investigated the transcellular transport of olmesartan medoxomil, a prodrug of olmesartan. Vectorial basal-to-apical transport was observed in OATP1B1/MRP2, OATP1B1/MDR1 double, and OATP1B1/BCRP double transfectants, suggesting the possible involvement of MRP2, MDR1, and BCRP in the limit of intestinal absorption of olmesartan medoxomil. From these results, we suggest that multiple transporters make a significant contribution to the pharmacokinetics of olmesartan and its prodrug.

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2′-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [125I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy. [Cancer Res 2007;67(14):6965–72]