Identification and characterization of a deletion mutant of DNA topoisomerase I mRNA in a camptothecin-resistant subline of human colon carcinoma.

In previous studies, we established two camptothecin (CPT)-resistant sublines, HT-29 / CPT and St-4 / CPT, from the human colon cancer cell line HT-29 and the human stomach cancer cell line St-4, respectively. Cellular contents of DNA topoisomerase I (topo I) in the resistant cells were eight-fold less than those in the corresponding parental lines. In this study, we have shown expression of two species of the TOP1 mRNA in HT-29 / CPT. The longer mRNA (4.0 kb) is the wild-type TOP1 mRNA, and the shorter mRNA (3.3 kb) proved to have a deletion of 672 bp (nucleotides 58 - 729 or 59 - 730) that caused the in-frame deletion of amino acids 20 - 243 of human topo I. The deleted region is identical to exons 3 - 9 of the TOP1 gene. The expression level of the 3.3-kb mRNA was similar to that of the wild-type mRNA in HT-29 / CPT. St-4 / CPT expressed only the wild-type TOP1 mRNA in lesser amounts than did St-4. Mouse NIH3T3 cells transfected with the wild-type TOP1 cDNA showed higher sensitivity to CPT than the parental cells, whereas those transfected with the deleted TOP1 cDNA showed levels similar to those of the parental cells. Expression of the exogenous TOP1 mRNA was confirmed; however, expression of the truncated topo I was not detected in cells transfected with the deleted TOP1 cDNA. These results suggest that the expression of the deleted TOP1 mRNA led to the low expression of CPT-sensitive topo I in the resistant cells.

Hypoxia up-regulates telomerase activity via mitogen-activated protein kinase signaling in human solid tumor cells.

Solid tumor cells are often exposed to hypoxia in vivo, which has been suggested to promote genetic instability in those cells. Telomere elongation by telomerase is implicated in chromosome stabilization in immortal cells. Here we found that hypoxia enhanced telomerase activity in the solid tumor A2780 and HT-29 cells but not in the leukemia U937 cells. The telomerase activation correlated with activation of mitogen-activated protein kinase (MAPK) and c-fos expression. The MEK1 inhibitor PD98059 repressed telomerase activation in the hypoxic cells. Consistently, a dominant negative MEK1 inhibited telomerase activation by hypoxia. Finally, we found a good correlation between telomerase activation and resistance to apoptotic cell death under hypoxic conditions. These findings indicate that hypoxia up-regulates telomerase activity via MAPK cascade signaling especially in solid tumor cells and suggest that solid tumor cells might enhance the telomerase activity as a stress response against genotoxicity induced by hypoxia.

In vitro antitumor activity of TAS-103, a novel quinoline derivative that targets topoisomerases I and II.

TAS-103 is a novel anticancer agent targeting both topoisomerase (Topo) I and Topo II, that stabilizes cleavable complexes of Topo-DNA at the cellular level. In this study, the in vitro antitumor effects of TAS-103 were compared with those of other known Topo I and Topo II inhibitors. TAS-103 inhibited DNA synthesis more strongly than RNA and protein synthesis, and induced an increase of cell population in the S-G2/M phase. The cytotoxicity of TAS-103 was strongest against S-phase cells, but its cell cycle phase specificity was not clear, and depended on drug concentration and exposure time. The cytotoxicity of TAS-103 (IC50: 0.0030-0.23 microM) against various tumor cell lines was much stronger than that of VP-16 and comparable to that of SN-38. The cytotoxicity of TAS-103 seemed to be more related to the amount of protein-DNA complexes than to the accumulation of TAS-103 in the cells. P-Glycoprotein (P-gp)-mediated MDR, CDDP-resistant and 5-FU-resistant cell lines did not show cross-resistance to TAS-103. Although PC-7/CPT cells bearing a Topo I gene mutation showed cross-resistance to TAS-103, the sensitivity of P388/CPT, HT-29/CPT and St-4/CPT cells, showing decreased Topo I expression, was not changed. KB/VM4 and HT-29/Etp cells, showing decreased Topo II expression, were slightly cross-resistant to TAS-103. These results suggest that TAS-103 may act as an inhibitor of both Topo I and Topo II at the cellular level. This property may be responsible for its strong antitumor effect and broad-spectrum, growth-inhibitory effect on drug-resistant cell lines.

Interleukin-1 alpha induced cyclooxygenase-2 expression in bone-derived endothelial cells.

Histological studies have suggested that vascular endothelial cells in bone are members of a complex network that regulates bone development and remodeling by producing soluble factors or by mediating cell-cell adhesion. To clarify the role of bone-derived endothelial cell lines (BDECs) in bone remodeling, we established several clones of BDECs from the femurs of BALB/c mice after transformation with the SV40 virus. Then we examined the response of these clones to interleukin-1alpha (IL-1alpha). IL-1alpha is known to induce bone resorption in part by increasing the expression of cyclooxygenase-2 (COX-2) that is associated with the production of PGE2 in osteoblast-lineage cells. Treating the primary and established BDECs with IL-1alpha induced COX-2 mRNA expression. A transcriptional activation assay revealed that the treatment with IL-1alpha increased COX-2 promoter activity in a dose-dependent manner, and IL-1alpha promoted COX-2 protein expression in BDECs. Treatment with IL-1alpha promoted PGE2 production from BDECs in a dose-dependent manner. These results indicate that IL-1alpha stimulates PGE2 synthesis largely by inducing BDECs to express COX-2. Because PGE2 stimulates bone resorption, these vascular endothelial cells, as well as osteoblast cells, play important roles in bone remodeling.

Basic fibroblast growth factor induces cyclooxygenase-2 expression in endothelial cells derived from bone.

Although histological studies have suggested that endothelial cells in bone (BDECs) are associated with some osteolytic bone diseases, it is still unclear how BDECs contribute to bone remodeling. Here we examined the response of BDECs to basic fibroblast growth factor (bFGF, FGF-2) using primary and cloned murine BDECs isolated from the femurs of BALB/c mice. Treatment of primary and cloned BDECs with bFGF induced cyclooxygenase-2 (COX-2) mRNA and protein expression. Furthermore, bFGF promotes the production of prostaglandin E2 (PGE2), which is known to be a potent stimulator of bone resorption and to induce osteoclast formation. Because the secretion of PGE2 was suppressed by COX-2 specific inhibitor NS-398 and by COX-2 antisense oligodeoxynucleotides, bFGF promotes the synthesis of PGE2 in a COX-2-dependent manner. Therefore, endothelial cells in bone are associated with bone remodeling by controlling COX-2 expression and consequently PGE2 production.

Sequence context profoundly influences the mutagenic potency of trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide-purine nucleoside adducts in site-specific mutation studies.

The postoligomerization method was used to prepare oligonucleotide 16-mers that contained dAdo or dGuo adducts, derived from trans opening of each enantiomer of the two diastereomeric benzo[a]pyrene 7,8-diol 9,10-epoxides, in two sequence contexts. These 16 oligonucleotides, along with the four corresponding oligonucleotides containing unsubstituted purines, were ligated into single-stranded DNA from bacteriophage M13mp7L2 and transfected into Escherichia coli SMH77. The mutagenic effects of replication past these adducts were then evaluated. The various adduct isomers induced point mutations at different frequencies and with different distributions of mutation types, as was anticipated. However, sequence context had the most substantial effects on mutation frequency. A high frequency of deletions of a single guanine was found in a context where the dGuo adduct was at the 3'-end of a run of five guanines, whereas no single base deletion was found in the other context studied, 5'-CGA-3'. Mutation frequencies in constructs containing dAdo adducts were much higher in a 5'-TAG-3' context (37-58%, depending on the individual isomer) than in a 5'-GAT-3' context (5-20%), and for a given adduct, mutation frequency was up to 10-fold higher in the former sequence than in the latter. These findings indicate that sequence context effects need more thorough evaluation if the goal of understanding the mechanism through which DNA adducts lead to mutation is to be achieved.

Production of interleukin-11 in bone-derived endothelial cells and its role in the formation of osteolytic bone metastasis.

The interactions of the cells in the bone microenvironment play important roles in bone remodeling. Osteoblasts are involved in the bone remodeling through the production of soluble factors that regulate proliferation and differentiation of osteoclasts and through cell-cell interactions. Histological studies have suggested that endothelial cells are also associated with some osteolytic bone diseases. However, it is still unclear how endothelial cells contribute to bone resorption. We established bone-derived endothelial cells (BDECs) to study their roles in bone remodeling. The established BDECs promoted bone resorption in a murine neonatal calvaria organ culture system by secreting a soluble bone resorption-inducing factor(s) when stimulated by several inflammatory cytokines. This bone resorption-inducing factor was identified as interleukin-11 (IL-11). IL-11 is known to enhance bone resorption by promoting osteoclastogenesis and by suppressing the activity of osteoblasts. The production of IL-11 in BDECs was also promoted by conditioned medium of human melanoma A375M cells. Because A375M cells formed osteolytic bone metastasis in vivo, BDECs might be involved in pathological osteolysis by producing IL-11. These results suggest that endothelial cells in bone play important roles in the promotion of bone resorption by secreting IL-11 in physiological and pathological conditions.

Cremophor EL reversed multidrug resistance in vitro but not in tumor-bearing mouse models.

Cremophor EL (CreEL), a polyethylene castor oil used as a vehicle for cyclosporin A and taxol, reverses P-glycoprotein-mediated drug resistance. The vehicle in an i.v. dosage form of PSC 833, [3'-keto-Bmt1]-[Val2]-cyclosporin, contains CreEL and has been presumed to have the potentiation of the reversal activity of PSC 833. To examine this possibility, we compared reversal activities of CreEL and PSC 833 against multidrug resistance (MDR) in vitro and in vivo. Both CreEL and PSC 833 inhibited P-glycoprotein-mediated efflux of [3H]vincristine from adriamycin-resistant myelogenous leukemia K562. The sensitization of multidrug-resistant cell lines to anticancer drugs by CreEL and PSC 833 was selective to MDR-related agents, suggesting a specific interference of the P-glycoprotein function by the two MDR modulators. The concentration-dependent activity of the modulators demonstrated that CreEL is at least 100 times less potent than PSC 833. The in vivo reversal effects of CreEL alone and PSC 833 in the vehicle were investigated in multidrug-resistant tumor-bearing mouse models. In vincristine-resistant P388 leukemia-bearing mice, neither i.v. nor i.p. administration of CreEL even at 1440 mg/kg enhanced the antitumor activity of adriamycin. The in vivo negligible activity of CreEL was confirmed in an HCT-15-bearing athymic mouse model. In contrast, PSC 833 significantly enhanced the antitumor activity of adriamycin in the in vivo models. The reversal activity of CreEL restricted to in vitro leads us to conclude that the vehicle containing CreEL did not potentiate the activity of PSC 833 in the tumor-bearing mouse models.

Potentiation of the reversal activity of SDZ PSC833 on multi-drug resistance by an anti-P-glycoprotein monoclonal antibody MRK-16.

SDZ PSC833 (PSC833), an analogue of cyclosporines, is one of the most potent modulators of multi-drug resistance (MDR). We previously reported that MRK-16, an anti-P-glycoprotein MAb, enhanced MDR reversal activity of cyclosporin A (CsA) through inhibition of P-glycoprotein-mediated CsA transport. We have examined here whether MRK-16 can enhance MDR reversal activity of PSC833. We found that MRK-16 potentiated the MDR reversal activity of PSC833, and of CsA, in MDR sublines of human myelocytic leukemia K562 and human ovarian cancer A2780 cells. Like MRK-16 combined with CsA, MRK-16 enhanced the effect of a sub-optimum dose of PSC833 on vincristine accumulation in MDR cells. However, MRK-16 could not increase cellular accumulation of PSC833 in MDR tumor cells, yet it could increase cellular accumulation of CsA. P-glycoprotein could not transport PSC833 but could transport CsA. Our results indicate that MRK-16 potentiates the MDR reversal activity of both PSC833 and CsA, yet also suggest that the molecular mechanism of the potentiation differs between the two substances.

Antitumor activity of a novel podophyllotoxin derivative (TOP-53) against lung cancer and lung metastatic cancer.

We synthesized a potent new antitumor podophyllotoxin derivative (4beta-aminoalkyl-4'-O-demethyl-4-desoxypodophyllotoxin; TOP-53) in our search for a drug that has strong activity against lung cancer and lung metastatic cancer. TOP-53 exhibited twice the inhibitory activity of etoposide (VP-16) against topoisomerase II and induced DNA strand breaks but showed no inhibitory activity against tubulin polymerization. The in vitro cytotoxic activity of TOP-53 assessed as IC50 was 0.016-0.37 microg/ml and 0.26-8.9 microg/ml against marine tumor and human non-small cell lung cancer (NSCLC) cell lines, respectively. TOP-53 exerted significant efficacy equivalent to that of VP-16 on s.c.-implanted murine solid tumors (Colon 26, B16-BL6, and Lewis lung carcinoma) at doses 3-5 times lower than that of VP-16. In human tumor xenografts using NSCLC, TOP-53 was active for four of five tumors, whereas VP-16 was active for two of five tumors. Potent inhibitory activity of TOP-53 was also found against a lung tumor (Lewis lung carcinoma) and four lung metastatic tumors (NL-22 and NL-17 colon cancer, UV2237M fibrosarcoma, and K1735M2 melanoma). TOP-53 appeared to be more active against four of them than VP-16. Thus, TOP-53 is not only active against s.c.-implanted lung cancers but also strongly active against lung localized tumor and metastatic tumors in the lungs. The high selectivity of TOP-53 was attributed to its high distribution into the lung and its persistence. TOP-53 is expected to be highly effective against lung cancer including NSCLC and various lung metastatic tumors in the clinical field.

Modulation of multidrug resistance by SDZ PSC 833 in leukemic and solid-tumor-bearing mouse models.

P-Glycoprotein inhibitors, including the nonimmunosuppressive cyclosporin D analog SDZ PSC 833 (PSC 833), have been developed to circumvent multidrug resistance. In the present study, the potential of PSC 833 in reversing multidrug resistance was evaluated in various systemic treatment models with leukemic and solid-tumor-bearing mice. Having a relatively wide therapeutic window of daily p.o. doses from 12.5 to 75 mg/kg, PSC 833 significantly improved the antileukemic activity of the anticancer drugs adriamycin (ADM), vincristine (VCR) and etoposide (VP-16) given i.p. or i.v. against i.p.-inoculated vincristine-resistant P388 tumor (P388/VCR). PSC 833 in combination with i.p.-injected anticancer drugs in optimal schedule and dosage induced apparent cures in some leukemic mice, whereas no cures were obtained with the cyclosporin A/anticancer drug combinations. PSC 833 combined with i.v.-injected anticancer drugs was highly active, but not curative, against P388/VCR and parental P388 tumors (maximum T/C>175%) PSC 833 in combination with intravenous treatment with ADM showed prominent anti-solid-tumor activity against s.c.-inoculated colon adenocarcinoma 26 and human colorectal adenocarcinoma HCT-15. Against colon adenocarcinoma 26, the PSC 833/ADM combinations induced cure in two or three of six mice. PSC 833/ADM combinations significantly inhibited the growth of the tumor with maximum percent inhibitions of 83 and 73% in the early and advanced stages of the HCT-15 tumor models, respectively. The present study demonstrated that PSC 833 is highly active in potentiating the antitumor activity of systemically administered ADM, VCR and VP-16 against four murine and human tumors with a relatively wide therapeutic window of daily p.o. dose range of 12.5-100 mg/kg.

Nuclear magnetic resonance solution structure of an undecanucleotide duplex with a complementary thymidine base opposite a 10R adduct derived from trans addition of a deoxyadenosine N6-amino group to (-)-(7R,8S,9R,10S)-7,8-dihydroxy- 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The solution structure of a modified undecamer duplex containing (-)-(7R,8S,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene covalently bonded through trans ring opening at C10 of the epoxide by the N6-amino group of deoxyadenosine (dA) was studied. This diol epoxide 1 diastereomer has the benzylic 7-hydroxyl group and the epoxide oxygen cis. The modified nucleotide residue has R chirality at C10 of the hydrocarbon (10R adduct). The undecamer duplex d(C1G2G3T4C5A*6C7G8A9G10G11).d(C12C13T14C15G16T17G18A19C2 0C21G22) has a complementary T opposite the modified dA (dA*6 is the modified dA). Exchangeable and nonexchangeable proton assignments were made using 2D TOCSY, NOESY, and water/NOESY NMR spectroscopy. The hybrid complete relaxation matrix program MORASS was used to generate NOESY distance constraints for iterative refinement using distance-restrained molecular dynamics calculations. The refined structure showed the hydrocarbon intercalated from the major groove between dA*6-T17 and dC5-dG18 base pairs. The modified dA*6 was in the normal anti configuration and showed Watson-Crick base pairing to T17 opposite. The chemical shifts of the hydrocarbon protons and the unusual shifts of sugar protons were accounted for by the intercalated orientation of the hydrocarbon.

Comparative study on reversal efficacy of SDZ PSC 833, cyclosporin A and verapamil on multidrug resistance in vitro and in vivo.

A non-immunosuppressive cyclosporin, SDZ PSC 833 (PSC833), shows a reversal effect on multidrug resistance (MDR) by functional modulation of MDR1 gene product, P-glycoprotein. The objective of the present study was to compare the reversal efficacy of three multidrug resistance modulators, PSC833, cyclosporin A (CsA) and verapamil (Vp). PSC833 has approximately 3-10-fold greater potency than CsA and Vp with respect to the restoring effect on reduced accumulation of doxorubicin (ADM) and vincristine (VCR) in ADM-resistant K562 myelogenous leukemia cells (K562/ADM) in vitro and also on the sensitivity of K562/ADM to ADM and VCR in in vitro growth inhibition. The in vivo efficacy of a combination of modifiers (PSC833 and CsA: 50 mg/kg, Vp 100 mg/kg administered p.o. 4 h before the administration of anticancer drugs) with anticancer drugs (ADM 2.5 mg/kg i.p., Q4D days 1, 5 and 9, VCR 0.05 mg/kg i.p., QD days 1-5) was tested in ADM-resistant P388-bearing mice. PSC833 significantly enhanced the increase in life span by more than 80%, whereas CsA and Vp enhanced by less than 50%. This reversal potency, which exceeded that of CsA and Vp, was confirmed by therapeutic experiments using colon adenocarcinoma 26-bearing mice. These results demonstrated that PSC833 has significant potency to reverse MDR in vitro and in vivo, suggesting that PSC833 is a good candidate for reversing multidrug resistance in clinical situations.

Induction of resistance to 1-beta-D-arabinofuranosylcytosine in human H9 cell line by simian immunodeficiency virus.

We examined drug sensitivity of human T cell acute lymphoblastic leukemia H9 cells chronically infected with simian immunodeficiency virus (SIVmac) and found that the retrovirus-infected H9 cells showed 8.2-fold resistance to 1-beta-D-arabinofuranosylcytosine (Ara-C). In the infected cells, Ara-CTP levels decreased to 20% of that found in uninfected H9 cells after 3 h incubation at Ara-C concentration of 1 microM, and 8.1-fold increase of cytidine deaminase activity was observed in the infected H9 cells. A competitive inhibitor of cytidine deaminase, 3, 4, 5, 6-tetrahydrouridine (THU), at 100 microM reversed Ara-C resistance in the infected cells. These results indicate that inducing increased cytidine deaminase activity by SIVmac infection conferred Ara-C resistance to H9 cells. An understanding of these cellular differences in drug sensitivity may aid in the development of therapeutic strategies against retrovirus-infected cells.

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by daphnodorins.

Three flavans, daphnodorins A, B and C isolated from Dahpne odora THUNB. were tested for their abilities to inhibit human immunodeficiency virus type 1 (HIV-1(IIIB)) replication in MT-4 cells. The effective concentrations (EC50) of daphnodorins A, B and C against HIV-1-induced cytolysis were 0.26 +/- 0.08, 1.8 +/- 0.6 and 3.6 +/- 0.5 micrograms/ml, respectively. Also these three compounds showed inhibitory effects of p24 antigen in human peripheral blood lymphocytes. As compared with 2',3'-dideoxycytidine 5'-triphosphate (DDC-TP), daphnodorin A and daphnodorin C had relatively weak inhibitory effects on the reverse transcriptase of HIV-1, while daphnodorin B did not show any inhibitory effect at concentrations up to 1000 micrograms/ml. These three compounds showed marked inhibitory effects on syncytium formation between HIV-1(IIIB)-infected and uninfected MOLT-4 (clone 8) cells at 3-30 micrograms/ml without inducing cytotoxicity. The concentrations of the compounds blocking syncytium formation were consistent with the effective concentrations (EC50) against HIV-induced cytolysis of MT-4 cells. These results, differing from reverse transcriptase inhibitors, suggest that the daphnodorins exert their anti-HIV-1 activity through inhibition of early events of viral replication including adsorption of the virions to the cells or the subsequent entry.

Transport of cyclosporin A across the brain capillary endothelial cell monolayer by P-glycoprotein.

P-glycoprotein, a multidrug transporter protein, exists in the brain capillary endothelium. To study the function of P-glycoprotein in brain capillary endothelium as a barrier against cyclosporin A, we examined the interaction of cyclosporin A with P-glycoprotein expressed in cultured brain capillary endothelial cells (MBEC4). P-glycoprotein of MBEC4 specifically bound [125I]iodoaryl azidoprazosin, and the binding was inhibited by cyclosporin A and vincristine. Intracellular accumulation of cyclosporin A in MBEC4 was about one-third the amount accumulated in mouse aortic endothelial cells (MAEC3), a cell line that did not express P-glycoprotein. The reduced accumulation of cyclosporin A in MBEC4 was increased by verapamil, a competitive inhibitor of transport function of P-glycoprotein. Cyclosporin A was preferentially transported from basal to apical side when the cell monolayer of MBEC4 was formed; however this transendothelial transport was not observed across cell monolayer of MAEC3. Verapamil inhibited the transendothelial transport of cyclosporin A across the MBEC4 monolayer. Thus P-glycoprotein in brain capillary endothelium could transport cyclosporin A across the endothelium from the basal to the apical side. These observations suggest that P-glycoprotein is involved in the complex function of the blood-brain barrier as a secretory detoxifying transporter of cyclosporin A.

Inhibitory effects of tumor invasion-inhibiting factor 2 and its conjugate on disseminating tumor cells.

Tumor invasion-inhibiting factor 2 (IIF-2) is a polypeptide of 21 amino acids which binds to the surface of tumor cells and inhibits experimental invasion in vitro. An albumin conjugate of IIF-2 was used to examine its potential as an antimetastatic compound. The conjugate inhibits in vivo lung metastasis of various highly metastatic tumor cells, including murine melanoma, colon adenocarcinoma, squamous cell carcinoma, forestomach carcinoma, and human fibrosarcoma. In addition to the anti-lung metastasis activity of this compound, it also showed the inhibitory effects on liver and spleen metastasis of murine T-lymphoma cells. A single administration of the conjugate with melanoma cells resulted in prolonged survival times, and their lung colonization was also inhibited when the conjugate was administrated i.v. at times ranging from 6 h before to 1 h after tumor cell inoculation. Similarly, i.p. administration 1 h prior to melanoma cell injection suppressed lung colonization. Pharmacokinetic analysis revealed that the conjugate was more stable than IIF-2 peptide alone. Approximately 10% of the conjugate remained circulating 2 h postinjection and persisted 20 h without degradation, compared with rapid clearing of the unconjugated IIF-2 peptide within 5 min. Furthermore, spontaneous lung metastasis of murine melanoma and colon adenocarcinoma cell was inhibited by successive i.p. administration of the conjugate before the removal of the primary site, with no effect on primary tumor growth. The conjugate significantly reduced tumor cell arrest in the lung and both the IIF-2 peptide and its conjugate demonstrated potent inhibition of basal as well as cytokine-induced-stimulated tumor cell motility. These results suggest that one mode of IIF-2 action may be inhibition of the extravasation of metastasizing cells which have arrested in a target organ, and that the IIF-2-albumin conjugate may be a potent antimetastatic substance with utility in the prevention of artificial seeding of tumor cells during surgical removal of the primary lesions as well as inhibiting metastasis from established metastases.

Antitumor activity of idarubicin, a derivative of daunorubicin, against drug sensitive and resistant P388 leukemia.

Idarubicin (IDA) possessed high antitumor activity against mouse P388 leukemia. When P388 leukemia bearing mice were administered an optimal dose of IDA intravenously, 200-250% of T/C and some cured mice were obtained. IDA showed a partial but significant effect against daunorubicin (DNR)-resistant P388 leukemia, whereas DNR, its parent drug, did not. Although IDA failed to show activity against doxorubicin (DX)- and aclarubicin (ACR)-resistant P388 leukemia in this study, IDA might have chemotherapeutic effects in patients in relapse and refractory to other anthracyclines. This assumption is supported by the results on the growth inhibition study in vitro. Among the metabolites, only idarubicinol showed cytotoxicity against P388 leukemia and idarubicinol showed 38% of the activity of IDA, indicating that the active components in animals could be IDA and idarubicinol. The combination of IDA and Ara-C induced some additive effect in P388 leukemia bearing mice as compared to the results with single drug usage.

Functional involvement of P-glycoprotein in blood-brain barrier.

P-glycoprotein, an active efflux pump of antitumor agents in multidrug-resistant tumor cells, exists in various normal tissues, including brain capillaries. To study the physiological function of P-glycoprotein expressed in brain capillary endothelium, we established nine mouse brain capillary endothelial cell (MBEC) lines and examined the transport of antitumor agents across the monolayer of MBEC epithelia. In the MBECs, the activities of alkaline phosphatase and gamma-glutamyl transpeptidase, specific markers for brain capillary endothelial cells, were about three times higher than those in other cells including human umbilical vein endothelial cells. By immunoblot analysis, P-glycoprotein was detected in all of the nine MBEC clones. The P-glycoprotein expressed in MBECs specifically bound [125I]iodoaryl azidoprazosin as that in multidrug-resistant cells, and efflux of vincristine was observed in the MBECs. When MBECs were grown on a porous filter membrane, they formed a monolayer of epithelium. By immunoelectron microscopic analysis, P-glycoprotein in MBEC epithelia was shown to be localized to the apical surface of the cells. Moreover, the unidirectional transepithelial transport of vincristine from basal side to apical side was demonstrated in vitro. These observations indicate that P-glycoprotein in brain capillary endothelium prevents vincristine from entering the central nervous system and thus may be one of the functional components of the blood-brain barrier.

Human immunodeficiency virus type 1 induces 1-beta-D-arabinofuranosylcytosine resistance in human H9 cell line.

We have found that chronically HIV-1(IIIB)-infected H9 cells showed 21-fold resistance to 1-beta-D-arabinofuranosylcytosine (ARA-C) compared with uninfected H9 cells. In the infected H9 cells, a 37% increase of dCTP pool and a 34% increase of dATP were observed, and no alteration of dTTP and dGTP was observed, compared with the uninfected H9 cells. A marked decrease of ARA-CTP generation was observed in the infected H9 cells after 3-h incubation with 0.1-10 microM ARA-C. The level of deoxycytidine kinase activity with ARA-C as substrate was similar in both the infected and the uninfected cells; however, a 37-fold increase of cytidine deaminase activity was observed in the infected H9 cells. These results indicate that the induction of cytidine deaminase activity by HIV-1(IIIB) infection conferred ARA-C resistance to H9 cells. This conclusion was supported by the observation that a marked reversal of ARA-C resistance in the infected H9 cells occurred after treatment with the inhibitor of cytidine deaminase, 3,4,5,6-tetrahydrouridine. The understanding of these cellular alterations in drug sensitivity may facilitate the development of effective therapeutic strategies against HIV-1-infected cells.