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ABCG2, a member of the ABC transporter superfamily, is overexpressed in many human tumors and has long been studied for its ability to export a variety of chemotherapeutic agents, thereby conferring a multidrug resistance (MDR) phenotype. However, several studies have shown that ABCG2 can also confer an MDR-independent survival advantage to tumor cells exposed to stress. While investigating the mechanism by which ABCG2 enhances survival in stressful milieus, we have identified a physical and functional interaction between ABCG2 and SLC1A5, a member of the solute transporter superfamily and the primary transporter of glutamine in cancer cells. This interaction was accompanied by increased glutamine uptake, increased glutaminolysis, and rewired cellular metabolism, as evidenced by an increase in key metabolic enzymes and alteration of glutamine-dependent metabolic pathways. Specifically, we observed an increase in glutamine metabolites shuttled to the TCA cycle, and an increase in the synthesis of glutathione, accompanied by a decrease in basal levels of reactive oxygen species and a marked increase in cell survival in the face of oxidative stress. Notably, the knockdown of SLC1A5 or depletion of exogenous glutamine diminished ABCG2-enhanced autophagy flux, further implicating this solute transporter in ABCG2-mediated cell survival. This is, to our knowledge, the first report of a functionally significant physical interaction between members of the two major transporter superfamilies. Moreover, these observations may underlie the protective role of ABCG2 in cancer cells under duress and suggest a novel role for ABCG2 in the regulation of metabolism in normal and diseased states., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

E2F1-3a overexpression due to amplification or to mutation or loss of the retinoblastoma gene, induces genes involved in DNA synthesis and leads to abnormal cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more of these activating E2Fs is a recognized target in cancer therapeutics. In previous studies we identified by phage display, a novel 7-mer peptide (PEP) that bound tightly to an immobilized consensus E2F1 promoter sequence, and when conjugated to penetratin to increase its uptake into cells, was cytotoxic to several malignant cell lines and human prostate and small cell lung cancer xenografts. Based on molecular simulation studies that showed that the D-Arg penetratin peptide (D-Arg PEP) secondary structure is more stable than the L-Arg PEP, the L-Arg in the peptide was substituted with D-Arg. In vitro studies confirmed that it was more stable than the L- form and was more cytotoxic as compared to the L-Arg PEP when tested against the human castrate resistant cell line, DU145 and the human lung cancer H196 cell line. When encapsulated in PEGylated liposomes, the D-Arg-PEP potently inhibited growth of the DU145 xenograft in mice. Our findings validate D- Arg PEP, an inhibitor of E2F1and 3a transcription, as an improved second generation drug candidate for targeted molecular therapy of cancers with elevated levels of activated E2F(s)., Competing Interests: CONFLICTS OF INTEREST No potential conflicts of interest were disclosed.

NAD + kinase (NADK) catalyzes the phosphorylation of nicotinamide adenine dinucleotide (NAD + ) to nicotinamide adenine dinucleotide phosphate (NADP + ) using ATP as the phosphate donor. NADP + is then reduced to NADPH by dehydrogenases, in particular glucose-6-phosphate dehydrogenase and the malic enzymes. NADPH functions as an important cofactor in a variety of metabolic and biosynthetic pathways. The demand for NADPH is particularly high in proliferating cancer cells, where it acts as a cofactor for the synthesis of nucleotides, proteins, and fatty acids. Moreover, NADPH is essential for the neutralization of the dangerously high levels of reactive oxygen species (ROS) generated by increased metabolic activity. Given its key role in metabolism and regulation of ROS, it is not surprising that several recent studies, including in vitro and in vivo assays of tumor growth and querying of patient samples, have identified NADK as a potential therapeutic target for the treatment of cancer. In this review, we will discuss the experimental evidence justifying further exploration of NADK as a clinically relevant drug target and describe our studies with a lead compound, thionicotinamide, an NADK inhibitor prodrug. Clin Cancer Res; 22(21); 5189-95. ©2016 AACR., (©2016 American Association for Cancer Research.)

The ABC drug transporters, including ABCG2, are well known for their ability to efflux a wide spectrum of chemotherapeutic agents, thereby conferring a multidrug-resistant phenotype. However, studies over the past several years suggest that the ABC transporters may play additional role(s) in cell survival in the face of stress inducers that are not ABCG2 substrates (i.e., nutrient deprivation, ionizing radiation, rapamycin). The mechanism by which this occurs is largely unknown. In the present study, using several cancer cell lines and their ABCG2-overexpressing sublines, we show that cells overexpressing ABCG2 were more resistant to these stressors. This resistance was associated with an elevated level of autophagy flux, as measured by a higher rate of SQSTM1/p62 degradation and greater accumulation of LC3-II when compared to parental cells. Knockdown of ABCG2 reduced autophagic activity in resistant cells to a level similar to that observed in parental cells, confirming that the enhanced autophagy was ABCG2-dependent. Moreover, using cell viability, apoptosis, and clonogenic assays, we demonstrated that the ABCG2-expressing cells were more resistant to amino acid starvation and radiation-induced cell death. Importantly, knockdown of the critical autophagy factors ATG5 and ATG7 greatly reduced cell survival, verifying that enhanced autophagy was critical for this effect. Taken together, these data indicate that autophagy induced by various stressors is enhanced/accelerated in the presence of ABCG2, resulting in delayed cell death and enhanced cell survival. This defines a new role for this transporter, one with potential clinical significance.

Chemotherapeutic resistance can occur by P-glycoprotein (P-gp), a 12-transmembrane ATP-dependent drug efflux pump. Glioblastoma (GBM) has poor survival rate and uniformly acquired chemoresistance to its frontline agent, Temozolomide (TMZ). Despite much effort, overcoming TMZ resistance remains a challenge. We reported on autonomous induction of TMZ resistance by increased transcription MDR1, the gene for P-gp. This study investigated how P-gp and TMZ interact to gain resistance. Using an experimental model of Adriamycin-resistant DC3F cells (DC3F/Adx), we showed that increased P-gp caused TMZ resistance. Increasing concentrations of TMZ competed with Calcein for P-gp, resulting in reduced efflux in the DC3F/Adx cells. Three different inhibitors of P-gp reversed the resistance to TMZ in two different GBM cell lines, by increasing active Caspase 3. Molecular modeling predicted the binding sites to be the intracellular region of P-gp and also identified specific amino acids and kinetics of energy for the efflux of TMZ. Taken together, we confirmed P-gp targeting of TMZ, a crucial regulator of TMZ resistance in GBM. This study provides insights on the effectiveness by which TMZ competes with other P-gp substrates, thereby opening the door for combined targeted therapies., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

We have previously reported that the methylxanthine caffeine increases expression of the splicing factor SRSF2, the levels of which are normally controlled by a negative autoregulatory loop. In the present study we have investigated the mechanisms by which methylxanthines induce this aberrant overexpression. RT-PCR analyses suggested little impact of caffeine on SRSF2 total mRNA levels. Instead, caffeine induced changes in the levels of SRSF2 3' UTR splice variants. Although some of these variants were substrates for nonsense-medicated decay (NMD), and could potentially have been stabilized by caffeine-mediated inhibition of NMD, down-regulation of NMD by a genetic approach was not sufficient to reproduce the phenotype. Furthermore, cell-based assays demonstrated that some of the caffeine-induced variants were intrinsically more efficiently translated than others; the addition of caffeine increased the translational efficiency of most SRSF2 transcripts. MicroRNA array analyses revealed a significant caffeine-mediated decrease in the expression of two SRSF2-targeting miRs, both of which were shown to repress translation of specific SRSF2 splice variants. These data support a complex model whereby caffeine down-regulates SRSF2-targeting microRNAs, leading to an increase in SRSF2 translation, which in turn induces SRSF2 splicing. SRSF2 splice variants are then stabilized by caffeine-mediated NMD inhibition, breaking the normal negative feedback loop and allowing the aberrant increase in SRSF2 protein levels. These findings highlight the complexity of SRSF2 gene regulation, and suggest ways in which SRSF2 expression may be dysregulated in disease., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Glioblastoma multiforme (GBM) commonly resists the frontline chemotherapy treatment temozolomide. The multidrug resistance gene (MDR1) and its protein, P-glycoprotein (P-gp), are associated with chemoresistance. This study investigated the mechanisms underlying MDR1-mediated resistance by GBM to temozolomide. P-gp trafficking was studied by flow cytometry and Western blot analysis. MDR1 expression was analyzed by real-time PCR and reporter gene assays. AP-1 interaction with MDR1 was studied by chromatin immunoprecipitation assay. EGF production was analyzed by ELISA, EGFR signaling was determined by Western blot analysis, and in vivo response to erlotinib and/or temozolomide was studied in nude mice. During the early phase of temozolomide treatment, intracellular P-gp was trafficked to the cell membrane, followed by conformational change into active P-gp. At the later phase, gene transcription of MDR1 was induced by temozolomide-mediated production of EGF. EGF activated ERK1/2-JNK-AP-1 cofactors (c-jun and c-fos). An inhibitor of EGFR kinase (erlotinib) given to nude mice with GBM prevented temozolomide-induced resistance. The results identified an essential role for activated EGFR in the resistance of GBM to temozolomide. Temozolomide resistance occurred through a biphasic response; first, by a conformational change in P-gp into the active form and, second, by releasing EGF, which caused autocrine stimulation of GBM cells to induce MDR1. Pharmacologic inhibition of EGFR kinase blunted the ability of GBM cells to resist temozolomide. These findings may explain reports on the common occurrence of mutant EGFR (EGFRvIII) and EGFR expansion in the resistance of GBM cells., (©2014 American Association for Cancer Research.)

E2F-1, a key transcription factor necessary for cell growth, DNA repair and differentiation, is an attractive target for development of useful anticancer drugs in tumors that are E2F "oncogene addicted". A peptide, isolated from phage clones, based on its binding to an E2F-1 consensus sequence, was cytotoxic against a wide range of cancer cell lines. The peptide was coupled to penetratin (PEP) and tested against prostate cancer cell lines, and a fresh sample from a patient with metastatic cancer. As the PEP was found to be relatively unstable in serum, it was encapsulated in PEGylated liposomes for in vivo studies. The peptide was cytotoxic against prostate cell lines and a fresh sample from a patient with metastatic prostate cancer. Treatment of mice bearing the human Du-145 human prostate tumor with the PEP encapsulated in PEGylated liposomes (PL-PEP) caused tumor regression without significant toxicity. The liposome encapsulated PEP has promise as an antitumor agent, alone or in combination with inhibitors of DNA synthesis.

E2F-1, a key transcription factor necessary for cell growth, DNA repair, and differentiation, is an attractive target for development of anticancer drugs in tumors that are E2F "oncogene addicted". We identified a peptide isolated from phage clones that bound tightly to the E2F-1 promoter consensus sequence. The peptide was coupled to penetratin to enhance cellular uptake. Modeling of the penetratin-peptide (PEP) binding to the DNA E2F-1 promoter demonstrated favorable interactions that also involved the participation of most of the penetratin sequence. The penetratin-peptide (PEP) demonstrated potent in vitro cytotoxic effects against a range of cancer cell lines, particularly against Burkitt lymphoma cells and small cell lung cancer (SCLC) cells. Further studies in the H-69 SCLC cell line showed that the PEP inhibited transcription of E2F-1 and also several important E2F-regulated enzymes involved in DNA synthesis, namely, thymidylate synthase, thymidine kinase, and ribonucleotide reductase. As the PEP was found to be relatively unstable in serum, it was encapsulated in PEGylated liposomes for in vivo studies. Treatment of mice bearing the human small cell lung carcinoma H-69 with the PEP encapsulated in PEGylated liposomes (PL-PEP) caused tumor regression without significant toxicity. The liposome encapsulated PEP has promise as an antitumor agent, alone or in combination with inhibitors of DNA synthesis.

Dihydrofolate reductase (DHFR) is an essential enzyme involved in de novo purine and thymidine biosynthesis. For several decades, selective inhibition of DHFR has proven to be a potent therapeutic approach in the treatment of various cancers including acute lymphoblastic leukemia, non-Hodgkin's lymphoma, osteogenic sarcoma, carcinoma of the breast, and head and neck cancer. Therapeutic success with DHFR inhibitor methotrexate (MTX) has been compromised in the clinic, which limits the success of MTX treatment by both acquired and intrinsic resistance mechanisms. We report that benzamide riboside (BR), via anabolism to benzamide adenine dinucleotide (BAD) known to potently inhibit inosine monophosphate dehydrogenase (IMPDH), also inhibits cell growth through a mechanism involving downregulation of DHFR protein. Evidence to support this second site of action of BR includes the finding that CCRF-CEM/R human T-cell lymphoblasic leukemia cells, resistant to MTX as a consequence of gene amplification and overexpression of DHFR, are more resistant to BR than are parental cells. Studies of the mechanism by which BR lowers DHFR showed that BR, through its metabolite BAD, reduced NADP and NADPH cellular levels by inhibiting nicotinamide adenine dinucleotide kinase (NADK). As consequence of the lack of NADPH, DHFR was shown to be destabilized. We suggest that, inhibition of NADK is a new approach to downregulate DHFR and to inhibit cell growth.

ABCG2 is an ATP-binding-cassette (ABC) transporter that confers multidrug resistance (MDR) to tumor cells by extruding a broad variety of chemotherapeutic agents, ultimately leading to failure of cancer therapy. Thus, the down-regulation of ABCG2 expression and/or function has been proposed as part of a regimen to improve cancer therapeutic efficacy. In this study, we found that a group of xanthines including caffeine, theophylline, and dyphylline can dramatically decrease ABCG2 protein in cells that have either moderate (BeWo, a placental choriocarcinoma cell line) or high (MCF-7/MX100, a breast cancer drug-resistant cell subline) levels of ABCG2 expression. This down-regulation is time-dependent, dose-dependent, and reversible. Using lysosomal inhibitors, we found that xanthines decreased ABCG2 by inducing its rapid internalization and lysosome-mediated degradation. As a consequence, caffeine treatment significantly increased the retention of an established ABCG2 substrate in MCF-7/MX100 cells but not in parental MCF-7 cells and sensitized the MDR cells to the chemotherapeutic agent mitoxantrone (MX); combination treatment with MX and caffeine decreased the IC(50) of MX ~10-fold and induced a greater degree of apoptotic cell death than MX treatment alone. Taken together, our results describe a novel function for this large class of therapeutically relevant compounds and suggest that a subset of xanthines could be developed as combination therapy to improve the efficacy of anticancer drugs that are ABCG2 substrates.

The multidrug resistance 1 gene (MDR1) encodes P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) transporter family that confers tumor drug resistance by actively effluxing a number of antitumor agents. We had previously shown that MDR1 transcription is regulated by epigenetic events such as histone acetylation, and had identified the histone acetylase P/CAF and the transcription factor NF-Y as the factors mediating the enzymatic and DNA-anchoring functions, respectively, at the MDR1 promoter. It has also been shown that MDR1 activation is accompanied by increased methylation on lysine 4 of histone H3 (H3K4). In this study, we further investigated histone methylation in MDR1 regulation and function. We show that the mixed lineage leukemia 1 (MLL1) protein, a histone methyltransferase specific for H3K4, is required for MDR1 promoter methylation, as knockdown of MLL1 resulted in a decrease in MDR1 expression. The regulation of MDR1 by MLL1 has functional consequences in that downregulation of MLL1 led to increased retention of the Pgp-specific substrate DIOC(2)(3), as well as increased cellular sensitivity to several Pgp substrates. Regulation of MDR1 by MLL1 was dependent on the CCAAT box within the proximal MDR1 promoter, similar to what we had shown for MDR1 promoter acetylation, and also requires NF-Y. Finally, overexpression of the most prevalent MLL fusion protein, MLL-AF4, led to increased MDR1 expression. This is the first identification of a histone methyltransferase and its leukemogenic rearrangement that regulates expression of an ABC drug transporter, suggesting a new target for circumvention of tumor multidrug resistance., (©2010 AACR.)

We have observed that rodent cell lines (mouse, hamster) contain approximately 10 times the levels of dihydrofolate reductase as human cell lines, yet the sensitivity to methotrexate (ED(50)), the folate antagonist that targets this enzyme, is similar. Our previous studies showed that dihydrofolate reductase protein levels increased after methotrexate exposure, and we proposed that this increase was due to the relief of feedback inhibition of translation as a consequence of methotrexate binding to dihydrofolate reductase. In the current report, we show that unlike what was observed in human cells, dihydrofolate reductase (DHFR) levels do not increase in hamster cells after methotrexate exposure. We provide evidence to show that although there are differences in the putative mRNA structure between hamster and human mRNA in the dihydrofolate reductase binding region previously identified, "hamsterization" of this region in human dihydrofolate reductase mRNA did not change the level of the enzyme or its induction by methotrexate. Further experiments showed that human dihydrofolate reductase is a promiscuous enzyme and that it is the difference between the hamster and human dihydrofolate reductase protein, rather than the DHFR mRNA, that determines the response to methotrexate exposure. We also present evidence to suggest that the translational up-regulation of dihydrofolate reductase by methotrexate in tumor cells is an adaptive mechanism that decreases sensitivity to this drug.

Alternative splicing of pre-mRNA contributes significantly to human proteomic complexity, playing a key role in development, gene expression and, when aberrant, human disease onset. Many of the factors involved in alternative splicing have been identified, but little is known about their regulation. Here we report that caffeine regulates alternative splicing of a subset of cancer-associated genes, including the tumor suppressor KLF6. This regulation is at the level of splice site selection, occurs rapidly and reversibly, and is concentration dependent. We have recapitulated caffeine-induced alternative splicing of KLF6 using a cell-based minigene assay and identified a "caffeine response element" within the KLF6 intronic sequence. Significantly, a chimeric minigene splicing assay demonstrated that this caffeine response element is functional in a heterologous context; similar elements exist within close proximity to caffeine-regulated exons of other genes in the subset. Furthermore, the SR splicing factor, SC35, was shown to be required for induction of the alternatively spliced KLF6 transcript. Importantly, SC35 is markedly induced by caffeine, and overexpression of SC35 is sufficient to mimic the effect of caffeine on KLF6 alternative splicing. Taken together, our data implicate SC35 as a key player in caffeine-mediated splicing regulation. This novel effect of caffeine provides a valuable tool for dissecting the regulation of alternative splicing of a large gene subset and may have implications with respect to splice variants associated with disease states.

Drug resistance caused by overexpression of P-glycoprotein (P-gp), the MDR1 (ABCB1) gene product, limits the therapeutic outcome. Expression of MDR1 can be induced by divergent stimuli, and involves a number of transcriptional factors. We found that the expression of CtBP1 (C-terminal-binding protein 1), a transcriptional co-regulator, was increased (approximately 4-fold) in human multidrug resistant (MDR) cancer cell lines, NCI/ADR-RES and A2780/DX, as compared to their sensitive counterparts. Silencing of CtBP1 expression by RNAi decreased the MDR1 mRNA and P-gp. Knockdown of CtBP1 also enhanced the sensitivity of MDR cells to chemotherapeutic drugs that are transported by P-gp and increased intracellular drug accumulation. In a reporter gene assay, co-transfection of MDR1 promoter constructs with a CtBP1 expression vector resulted in a approximately 2-4-fold induction of MDR1 promoter activity. CtBP1 appeared to contribute to the activation of MDR1 transcription through directly interacting with the MDR1 promoter, as evidenced by its physical binding to the promoter region of the MDR1 gene in chromatin immunoprecipitation and electromobility shift assays. Histone modifications at the MDR1 promoter, such as mono-methylation, di-methylation, and acetylation of histone H3, were not found to be affected by silencing of CtBP1 expression. Our results reveal a novel role for CtBP1 as an activator of MDR1 gene transcription, and suggest that CtBP1 might be one of the key transcription factors involved in the induction of MDR1 gene. Therefore, CtBP1 may represent a potentially new target for inhibiting drug resistance mediated by overexpression of the MDR1 gene.

Although the p53 family members share a similar structure and function, it has become clear that they differ with respect to their role in development and tumor progression. Because of the high degree of homology in their DNA binding domains (DBDs), it is not surprising that both p63 and p73 activate the majority of p53 target genes. However, recent studies have revealed some differences in a subset of the target genes affected, and the mechanism underlying this diversity has only recently come under investigation. Our laboratory has demonstrated previously that p53 represses transcription of the P-glycoprotein-encoding MDR1 gene via direct DNA binding through a novel p53 DNA-binding site (the HT site). By transient transfection analyses, we now show that p63 and p73 activate rather than repress MDR1 transcription, and they do so through an upstream promoter element (the alternative p63/p73 element (APE)) independent of the HT site. This activation is dependent on an intact DNA binding domain, because mutations within the p63DBD or p73DBD are sufficient to prevent APE-mediated activation. However, neither p63 nor p73 directly interact with the APE, suggesting an indirect mechanism of activation through this site. Most interestingly, when the p53DBD is replaced by the p63DBD, p53 is converted from a repressor working through the HT site to an activator working through the APE. Taken together, these data indicate that, despite considerable homology, the DBD of the p53 family members have unique properties and can differentially regulate gene targeting and transcriptional output by both DNA binding-dependent and -independent mechanisms.

P-glycoprotein, the founding member of the ATP-binding cassette (ABC) family of drug transporters, was first identified almost three decades ago and shown to confer resistance to multiple chemotherapeutic agents when overexpressed in human tumors. Subsequent years have witnessed a tremendous effort to characterize the function and regulation of P-glycoprotein, initially spurred by the hope that its inhibition was the key to overcoming clinical resistance to multiple anticancer agents. However, the identification of MRP1, another member of the ABC drug transporter family, led to the realization that the multidrug resistance (MDR) phenotype is considerably more complex than initially believed. Indeed, at the present time at least 10 members of the ABC transporter family have been implicated in an MDR phenotype, and it is likely that more will be added to this list as studies progress. With this complexity comes the imperative to improve our understanding of the function of individual transporters, as well as to delineate the mechanisms underlying their expression in normal and tumor cells, particularly those that may be amenable to therapeutic intervention. Several articles within this volume address the structure and function of drug transporters. This review will focus on our current understanding of the regulation of ABC drug transporters at the level of transcription.

Ecteinascidin 743 (ET-743) is a potent antitumor agent from the Caribbean tunicate Ecteinascidia turbinata and is presently in clinical trials for human cancers. The aim of this study was to assess the nature of the interaction between ET-743 and other antineoplastic agents using the combination index method of Chou and Talalay to better understand how ET-743 might be used clinically. We examined the cytotoxic effect of ET-743 combined with six other antineoplastic agents on human breast cancer cell lines, MX-1, MCF7, and P-glycoprotein overexpressing MCF7/DXR to different schedules. Pretreatment with paclitaxel for 24 h before ET-743 was the most effective combination regimen in all three breast cancer cell lines. Furthermore, sequential treatment with paclitaxel followed by ET-743 increased the antitumor effects in nude mice bearing MX-1 mammary carcinoma xenografts without increasing toxicity. These results suggest that the combination of ET-743 and paclitaxel should be assessed in clinical trials for the treatment of breast cancer.

Ecteinascidin-743 (ET-743) is a promising chemotherapeutic agent currently in Phase III clinical trials. Previous studies indicated a novel spectrum of activity for this agent, including transcriptional inhibition. Initially hypothesized to target a single transcription factor (NF-Y), we now show that ET-743 is a more general inhibitor of activated transcription. Induction of the Sp1-regulated p21 gene by Trichostatin A (TSA) was blocked by ET-743 at concentrations that had minimal effect on uninduced (constitutive) expression. Moreover, ET-743 blocked induction of Gal4 fusion proteins by TSA without affecting activation mediated by the fusion proteins in the absence of the inducer. Finally, microarray analysis of SW620 cells treated with TSA and/or ET-743 indicated that activation of TSA-responsive promoters was blocked by ET-743 with little affect on nonresponsive promoters. These results, taken together with previous reports, leads us to suggest a mechanism whereby ET-743 is a novel, potent, and general inhibitor of activated but not uninduced transcription.

Ecteinascidin-743 (ET-743), an anti-tumor agent derived from the marine tunicate, Ecteinascidia turbinata, is active against various solid tumor cell lines, including soft tissue sarcoma, breast, ovarian, non-small-cell lung and prostate cancers and melanoma, and has a broad spectrum of anti-cancer activity in vivo. For reasons as yet unclear, sarcoma cell lines are exquisitely sensitive to ET-743. The drug has a unique mechanism of action that makes it a novel anti-tumor agent. ET-743 is a DNA-binding agent that covalently interacts with the minor groove of the DNA double helix to bend the molecule towards the major groove. Defects in DNA repair pathways have paradoxical effects on the anti-tumor activity of ET-743: loss of mismatch repair does not affect its toxicity; loss of DNA-dependent protein kinase activity enhances its toxicity; defects in transcription-coupled nucleotide excision repair confer resistance to ET-743. As a DNA repair capability appears to be necessary for at least one mechanism of ET-743-mediated cytotoxicity, the drug may interact with the DNA repair machinery to induce lethal strand breaks. One of the most novel aspects of ET-743 is its effect on RNA polymerase II-mediated gene transcription. ET-743 selectively inhibits activation of the multidrug resistance gene, while leaving constitutive gene expression relatively unaffected. Preliminary studies of other genes and transcriptional inducers indicate that ET-743 may be a more general inhibitor of activated, but not basal, transcription.

Previous work from our laboratory has allowed for the subdivision of RNA polymerase II TATA-less promoters into two classes: those that initiate at a single start site (SSS) and those that initiate at multiple start sites (MSS). MSS promoters are defined by the lack of a TATA box and the presence of a transcription initiation window and a downstream MED-1 element (GCTCCC/G) [Ince, T. A., and Scotto, K. W. (1995) J. Biol. Chem. 270, 30249-30252]. Further insight into the mechanisms regulating TATA-less MSS promoters has been hampered by the lack of an in vitro transcription assay in which multiple start sites can be reproduced. In the present study, we describe the development of a versatile in vitro transcription system optimized for the expression of MSS promoters, termed the multiple promoter comparison (MPC) assay. By alteration of assay parameters including template length, cation and nucleotide concentrations, and RNA isolation method, the accurate and robust transcription of two MSS promoters, pgp1 (hamster P-glycoprotein class I homologue) and HPRT (human hypoxanthine phosphoribosyltransferase), was accomplished. Moreover, both TATA-containing and TATA-less single start site promoters were also transcribed in the MPC assay, making this the first general in vitro transcription system for the simultaneous analysis of all three classes of RNA polymerase II genes.

Ecteinascidin 743 (ET-743) is a potent antitumor agent from the Caribbean tunicate Ecteinascidin turbinata and is presently in clinical trials for human cancers. To better understand how ET-743 might be used clinically, the present study used SRB assays to examine the cytotoxicity resulting from combining ET-743 with three other antineoplastic agents: doxorubicin (DXR), trimetrexate, and paclitaxel in different administration schedules in two soft tissue sarcoma cell lines, HT-1080 and HS-18, in vitro. Concurrent exposure of ET-743 with DXR resulted in synergistic interactions in both cell lines. Addition of ET-743 for 24 h before DXR was the most effective cytotoxic regimen against both cell lines. Morphological study by fluorescence microscopy revealed that combination treatment of both cells with ET-743 and DXR induced apoptosis. Exposure to paclitaxel before ET-743 was also an effective regimen. These results encourage studies of the combination of ET-743 and DXR in the treatment of soft tissue sarcoma, because each of these agents have activity in this disease.

The tumor suppressor protein p53 has been well documented as a transcriptional activator involved in the regulation of a number of critical genes involved in the cell cycle, response to DNA damage, and apoptosis. Activation by p53 requires the interaction of the protein with a consensus binding site consisting of two half-sites, each comprising two copies of the sequence PuPuPuC(A/T) arranged head-to-head and separated by 0-13 base pairs. In addition to activation, p53 has been shown to be a potent repressor of transcription. However, the basis for p53-mediated repression is not well understood and has been proposed to occur indirectly through interactions with other promoter-bound transcription factors. In the present study, we show that p53 can repress transcription directly by binding to a novel head-to-tail (HT) site within the MDR1 promoter. A mutation that disrupted p53 binding to the MDR1 HT site blocked p53-mediated repression of the MDR1 promoter in transfection assays. Replacement of the HT site with a head-to-head (HH) site converted the activity of p53 from repression to activation, indicating that simple recruitment of p53 to the promoter is not sufficient for repression and that the orientation of the binding element determines the fate of p53-regulated promoters.

Two decades ago, the overexpression of P-glycoprotein (Pgp) was first demonstrated to mediate the energy-dependent efflux of a variety of chemotherapeutic agents from tumor cells, resulting in the development of multidrug resistance (MDR). Not surprisingly, this discovery triggered an ongoing search for agents that would inhibit Pgp function, with the hope that by doing so the MDR phenotype could be reversed. As our understanding of Pgp function and pharmacokinetics has increased, this quest has become more urgent, as well as more complex.

Ecteinascidin 743 (ET-743), a highly promising marine-based antitumor agent presently in phase II clinical trials, has been shown to interfere with the binding of minor-groove-interacting transcription factors, particularly NF-Y, with their cognate promoter elements in vitro. We have shown that NF-Y is a central mediator of activation of transcription of the human P glycoprotein gene (MDR1) by a variety of inducers and that NF-Y functions by recruiting the histone acetyltransferase PCAF to the MDR1 promoter. In the present study, we tested whether ET-743 could block activation of the MDR1 promoter by agents that mediate their effect through the NF-Y/PCAF complex. We report that physiologically relevant concentrations of ET-743 abrogate transcriptional activation of both the endogenous MDR1 gene and MDR1 reporter constructs by the histone deacetylase inhibitors as well as by UV light, with minimal effect on constitutive MDR1 transcription. Notably, this inhibition does not alter the promoter-associated histone hyperacetylation induced by histone deacetylase inhibitors, suggesting an in vivo molecular target downstream of NF-Y/PCAF binding. ET-743 is therefore the prototype for a distinct class of transcription-targeted chemotherapeutic agents and may be an efficacious adjuvant to the treatment of multidrug-resistant tumors.

Background/purpose: Neuroblastoma is the most solid common extracranial malignancy in childhood. Despite multimodality treatment, high-risk disease continues to carry a poor prognosis. Glucocorticoids have been shown previously to induce differentiation in murine neuroblastoma cell lines, but no such effect has been documented in human neuroblastoma cells. Glucocorticoids are known to be active in the differentiation process of the neural crest. These studies describe the effects of dexamethasone on 6 human neuroblastoma cell lines., Methods: Dexamethasone was added to cultured neuroblastoma cell lines (LA1-5S, LA1-15N, BE[2]S, BE[2]N, SH-EP-1, SH-SY5Y) maintained in media supplemented with either normal serum or charcoal-depleted serum. Proliferation assays were performed, and flow cytometry was used to assess alterations in cell cycle. Cells were closely monitored for morphological changes with serial phase-contrast microscopy. Immunohistochemistry (3F8, NF-1, TRK-A) of cultured cells was used to evaluate differentiation. Glucocorticoid receptor levels was assessed using immunoblotting., Results: Dexamethasone decreased the rate of cellular proliferation in both standard and charcoal-depleted conditions. Flow cytometry showed a G1 accumulation. Increased expression of the differentiation-associated antigens was found in cells cultured in charcoal-depleted media, and a further augmentation was seen with the addition of dexamethasone. In standard media, dexamethasone had no detectable effect on the expression of these antigens. Glucocorticoid receptor expression was found to be comparable in all cell lines., Conclusions: Human neuroblastoma cells are sensitive to the differentiating effects of dexamethasone in an environment of charcoal-depleted serum. This phenomenon may be caused by the existence of growth and mitogenic factors in serum that are inhibiting differentiation.

The MDR1 promoter is subject to control by various internal and external stimuli. We have previously shown that the CCAAT box-binding protein, NF-Y, mediates MDR1 activation by the histone deacetylase inhibitors, trichostatin A and sodium butyrate, through the recruitment of the co-activator, P/CAF. We have now extended our investigation to the activation of MDR1 by genotoxic stress. We show that activation of the MDR1 promoter by UV irradiation is also dependent on the CCAAT box (-82 to -73) as well as on a proximal GC element (-56 to -42). Gel shift and supershift analyses with nuclear extracts prepared from human KB-3-1 cells identified NF-Y as the transcription factor interacting with the CCAAT box, while Sp1 was the predominant factor binding to the GC element. Mutations that abrogated binding of either of these factors reduced or abolished activation by ultraviolet irradiation; moreover, co-expression of a dominant-negative NF-Y protein (NF-YA29) reduced UV-activated transcription. Interestingly, YB-1, a transcription factor that also recognizes the CCAAT motif and had been reported to mediate induction of the MDR1 promoter by ultraviolet light, was incapable of interacting with the double-stranded MDR1 CCAAT box oligonucleotide in nuclear extracts, although it did interact with a single-stranded oligonucleotide. Furthermore, a mutation that abolished activation of MDR1 by UV-irradiation had no effect on YB-1 binding and co-transfection of a YB-1 expression plasmid had a repressive effect on UV-inducible transcription. Taken together, these results indicate a role for both NF-Y and Sp1 in the transcriptional activation of the MDR1 gene by genotoxic stress, and indicate that YB-1, if involved, is not sufficient to mediate this activation.

Overexpression of P-glycoprotein (Pgp), a multidrug transporter encoded by the MDR1 gene, is associated with chemoresistance in some human solid tumor malignancies. To date, analyses of MDR1 levels in solid tumors have examined constitutive increases in expression at relapse. In the present study, we have evaluated the acute induction of MDR1 gene expression in a solid human tumor as a function of time in response to in vivo exposure to chemotherapy. Five patients with unresectable sarcoma pulmonary metastases underwent isolated single lung perfusion with doxorubicin. Relative MDR1 gene expression was measured in metastatic tumor nodules and normal lung specimens after initiation of chemoperfusion. In four of five patients, a 3-15-fold (median, 6.8) increase in MDR1 RNA levels was detected in tumors at 50 min after administration of doxorubicin. In contrast, normal lung samples had very low levels of MDR1 RNA prior to perfusion, and no acute increases were observed after therapy. These findings demonstrate, for the first time, that MDR1 gene expression can be rapidly activated in human tumors after transient in vivo exposure to cytotoxic chemotherapy.

The emergence of resistance in a tumor population is most often associated with a disregulation of gene expression, usually at the level of transcription. A major goal in the field of cancer chemotherapy is to define the mechanisms underlying transcriptional regulation of drug resistance genes in an effort to identify targets for therapeutic intervention. Recently, considerable progress has been made in identifying the molecular mechanisms involved in the transcriptional regulation of the P-glycoprotein (Pgp) gene. When overexpressed in tumor cells, Pgp confers resistance to a variety of chemotherapeutic agents; this resistance has been termed MDR (multidrug resistance). Moreover, Pgp is a normal component of a variety of highly differentiated cell types and, as such, is regulated by both internal and external environmental stimuli. In this review, we will discuss the current knowledge regarding the DNA elements and protein factors involved in both constitutive and inducible regulation of Pgp transcription in normal and tumor cells.

Recent studies have shown that the histone-modifying enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in transcriptional activation and repression, respectively. However, little is known about the endogenous genes that are regulated by these enzymes or how specificity is achieved. In the present report, we demonstrate that HAT and HDAC activities modulate transcription of the P-glycoprotein-encoding gene, MDR1. Incubation of human colon carcinoma SW620 cells in 100-ng/ml trichostatin A (TSA), a specific HDAC inhibitor, increased the steady-state level of MDR1 mRNA 20-fold. Furthermore, TSA treatment of cells transfected with a wild-type MDR1 promoter/luciferase construct resulted in a 10- to 15-fold induction of promoter activity. Deletion and point mutation analysis determined that an inverted CCAAT box was essential for this activation. Consistent with this observation, overexpression of p300/CREB binding protein-associated factor (P/CAF), a transcriptional coactivator with intrinsic HAT activity, activated the wild-type MDR1 promoter but not a promoter containing a mutation in the CCAAT box; deletion of the P/CAF HAT domain abolished activation. Gel shift and supershift analyses identified NF-Y as the CCAAT-box binding protein in these cells, and cotransfection of a dominant negative NF-Y expression vector decreased the activation of the MDR1 promoter by TSA. Moreover, NF-YA and P/CAF were shown to interact in vitro. This is the first report of a natural promoter that is modulated by HAT and HDAC activities in which the transcription factor mediating this regulation has been identified.

Previous studies suggest that dihydrofolate reductase (DHFR) regulates its own translation. Moreover, intracellular levels of DHFR protein increase following exposure to the antifolate methotrexate (MTX), suggesting that MTX may release the translational inhibition mediated by DHFR [Chu et al. (1993) Biochemistry 32,4756-4760; Ercikan et al. (1993) Adv. Exp. Med. Biol. 338, 537-540]. To further investigate the role of DHFR in translational autoregulation, we have considered the possibility that DHFR directly contacts its cognate mRNA. Binding studies using a series of truncated DHFR mRNAs as probes localized the DHFR/RNA interaction to a 100-base-pair region containing two putative stem-loop structures; initial studies indicated that both of these loop structures are involved in protein binding. Moreover, the binding of MTX to DHFR prevents interaction of the protein with its cognate mRNA, thereby relieving translational autoregulation.

Cellular resistance to multiple chemotherapeutic agents is most often due to the overexpression of P-glycoprotein (Pgp). The mechanisms(s) underlying Pgp overexpression had not been determined, due, in part, to a failure to reproduce the overexpression in transient transfection assays. We now report that stable transfection of a Pgp (pgp1) promoter/luciferase construct in the drug-sensitive Chinese hamster cell line DC-3F and its drug-resistant sublines reproduced the overexpression phenotype, with up to 18-fold higher activity observed in the resistant cell lines compared with DC-3F. Moreover, mutation of a pgp1 promoter element, multiple start site element downstream (MED-1), decreased transcription in drug-resistant cells without affecting activity in drug-sensitive cells. This is the first report of a Pgp promoter element differentially regulated in drug-resistant cells. Moreover, these data suggest that the regulation of Pgp transcription is modulated by chromatin structure, and that stable transfection may be more suitable for identifying promoter elements important for overexpression in drug-resistant cells.

Although many TATA-less promoters transcribed by RNA polymerase II initiate transcription at multiple sites, the regulation of multiple start site utilization is not understood. Beginning with the prediction that multiple start site promoters may share regulatory features and using the P-glycoprotein promoter (which can utilize either a single or multiple transcription start site(s)) as a model, several promoters with analogous transcription windows were grouped and searched for the presence of a common DNA element. A downstream protein-binding sequence, MED-1 (Multiple start site Element Downstream), was found in the majority of promoters analyzed. Mutation of this element within the P-glycoprotein promoter reduced transcription by selectively decreasing utilization of downstream start sites. We propose that a new class of RNA polymerase II promoters, those that can utilize a distinctive window of multiple start sites, is defined by the presence of a downstream MED-1 element.

Sorcin is a 22-kDa calcium-binding protein initially identified in multidrug-resistant cells; however, its patterns of expression and function in normal tissues are unknown. Here we demonstrate that sorcin is widely distributed in rodent tissues, including the heart, where it was localized by immunoelectron microscopy to the sarcoplasmic reticulum. A > 500-kDa protein band immunoprecipitated from cardiac myocytes by sorcin antiserum was indistinguishable in size on gels from the 565-kDa ryanodine receptor/calcium release channel recognized by ryanodine receptor-specific antibody. Association of sorcin with a ryanodine receptor complex was confirmed by complementary co-immunoprecipitations of sorcin with the receptor antibody. Forced expression of sorcin in ryanodine receptor-negative Chinese hamster lung fibroblasts resulted in accumulation of the predicted 22-kDa protein as well as the unexpected appearance of ryanodine receptor protein. In contrast to the parental host fibroblasts, sorcin transfectants displayed a rapid and transient rise in intracellular calcium in response to caffeine, suggesting organization of the accumulated ryanodine receptor protein into functional calcium release channels. These data demonstrate an interaction between sorcin and the ryanodine receptor and suggest a role for sorcin in modulation of calcium release channel activity, perhaps by stabilizing the channel protein.

The overproduction of P-glycoprotein (Pgp) has been associated with the development and maintenance of the multidrug resistant (MDR) phenotype, although the regulatory events responsible have not yet been elucidated. We have analyzed the overexpression of the TATA-less hamster class-I Pgp-encoding gene (Pgp1) in several MDR Chinese hamster cell lines. The MDR lung cell line DC-3F/VCRd5L, as well as the MDR ovary cell line CHRC5, express a level of Pgp1 RNA commensurate with the increase in Pgp1 dosage; in contrast, the actinomycin D (ActD)-selected sublines of DC-3F overexpress Pgp1 mRNA without a concomitant increase in Pgp1 gene-copy number. Analysis of Pgp1 transcription start point (tsp) utilization revealed that drug-sensitive DC-3F cells, as well as DC-3F/VCRd5L and CHRC5 cells, utilize one major tsp; in contrast, the ActD-resistant sublines 'switch' to a more complex pattern, using four additional Pgp1 tsp 32, 42, 52, and 67 bp downstream from the major parental tsp (+1). This observation of a difference in the regulation of transcription of Pgp in MDR vs. drug-sensitive cells suggests that the 'switch' in tsp selection may be involved in the increased expression of Pgp1 mRNA. Interestingly, despite the existence of several hundred MDR cell lines, very few have been analyzed with respect to tsp selection; it is therefore possible that alternate tsp selection is a relatively common yet heretofore unobserved component of the MDR phenotype. Moreover, these cells provide an excellent system in which to evaluate the sequence elements and protein factors that govern the selection of tsp in TATA-less promoters.

The Chinese hamster cell line, DC-3F, is heterozygous at the DHFR locus, and each allele can be distinguished on the basis of a unique DNA restriction pattern, protein isoelectric profile and in the abundancy of the DHFR mRNAs it expresses. Although each allele produces four transcripts, 1000, 1650 and 2150 nucleotides [corrected] in length, the relative distribution of these RNAs differs for each; the 2150 nt mRNA represents the major (60%) species generated from one allele, while the 1000 nt mRNA is the major species generated from the other. The allele that predominantly expresses the 2150 nt transcript is preferentially overexpressed when DC-3F cells are subjected to selection in methotrexate. We have analyzed the 3' ends of both DHFR alleles and have found that the three major mRNAs arise by readthrough of multiple polyadenylation signals. A four base deletion in one allele changes the consensus polyadenylation signal AAUAAA to AAUAAU, resulting in the utilization of a cryptic polyadenylation signal lying 21 bp upstream. Surprisingly, this mutation in the third polyadenylation signal appears to affect not only the utilization of this signal, but also the efficiency with which the first signal, located 1171 bp upstream from the third site, is utilized.

Luminal epithelium from mouse small intestine was fractionated into four distinct components ranging from the most differentiated (absorptive epithelial cells, fraction I) to the least differentiated (proliferative crypt cells, fraction IV). Immunoblot analysis of these fractions with monoclonal antibodies to P-glycoprotein, C219, and HYB-241, indicated that the amount of P-glycoprotein increased in cells as they progressed in level of differentiation from crypt to the villar surface of the lumen of the small intestine. P-glycoprotein in these normal intestinal cells functioned as a transporter of vincristine, one of a group of cancer chemotherapeutic agents to which cells can develop multidrug resistance. Transport ability was shown by efflux studies with vincristine as substrate. Efflux increased with differentiation and correlated with the increase in the amount of P-glycoprotein. Fraction I cells effluxed vincristine about seven times more rapidly than fraction IV cells. Efflux of vincristine was almost entirely inhibited from fraction I cells after treatment with antibody HYB-241, which recognizes an external epitope of P-glycoprotein. This finding would appear to demonstrate that P-glycoprotein was directly responsible for at least the majority of efflux of vincristine in these cells. The observed Mr of P-glycoprotein in fraction I cells was 140,000 when cells were solubilized with sodium dodecyl sulfate at room temperature before electrophoresis and immunoblot analysis. When solubilized fraction I protein was heated with 2-mercaptoethanol before electrophoresis, P-glycoprotein was seen by immunochemical procedures as an Mr 50,000-55,000 protein. No Mr 170,000 or 180,000 forms of P-glycoprotein (forms that are found in multidrug-resistant cell lines and are stable to heat or sulhydryl agents) were detected in any fraction of these epithelial cells under the conditions used. Functional P-glycoprotein was, therefore, associated with differentiation of luminal epithelium in small intestine, and the P-glycoprotein produced by these cells may be a type not previously described.

Nucleotide sequence analysis of two cDNA clones, one shown to direct the synthesis in Escherichia coli of the pI 6.7 form of the 20,000-molecular-weight class of Chinese hamster lung cell dihydrofolate reductase, and the other shown to direct the synthesis of the pI 6.5 form of the 21,000-molecular-weight class of the enzyme, has revealed the following: (i) the differences in physical and enzymatic properties displayed by these two proteins are due to two variations in their respective amino acid sequences with the conversion of Leu to Phe at position 22 probably responsible for the differential sensitivity of these two enzymes to methotrexate and methasquin; (ii) the multiple mRNAs responsible for the synthesis of each of these proteins differ in size due, at least in part, to a length heterogeneity at their 3' ends; (iii) these two proteins are encoded by different genes; and (iv) the sequence AAATATA appears to be a major polyadenylation signal in one Chinese hamster lung cell dihydrofolate reductase gene and a minor signal in another.

Purification of the dihydrofolate reductases overproduced by 16 independently-derived antifolate-resistant sublines of Chinese hamster lung cells and their drug-sensitive parental cell line, DC-3F, has confirmed our original observation that two molecular weight classes of this enzyme, viz. Mr = 21,000 can be overproduced by drug-resistant cells (Melera, P. W., Wolgemuth, D., Biedler, J. L., and Hession, C. (1980) J. Biol. Chem. 255, 319-322). Of the 16 drug-resistant sublines analyzed, 12 overproduce the 21,000-dalton enzyme, while 4 overproduce the 20,000-dalton enzyme. Both molecular weight classes are found in the drug-sensitive parental line. Two-dimensional polyacrylamide gel electrophoresis of the two molecular weight classes of enzyme has shown that each is composed of two isoelectric forms, and that the relative distributions of the isoelectric forms within a molecular weight class are maintained during overproduction. In vitro translation experiments have demonstrated that the synthesis of these isoelectric forms and their relative distributions are mediated by mRNA. Finally, evidence is presented for the existence of two sets of three dihydrofolate reductase mRNAs in overproducing sublines. Both sets, within a subline, encode the same molecular weight class of dihydrofolate reductase, while each set directs the synthesis of only one of the isoelectric forms within that class. Although the genetic basis for these observations is not yet understood, it is clear that the production of dihydrofolate reductase by Chinese hamster lung cells is complex and probably involves the expression of multiple dihydrofolate reductase genes and/or alleles.

Expression of the positively acting 5S gene-specific transcription factor, TFIIIA, is regulated during development, with highest levels of mRNA and protein occurring during oogenesis. By analysis of TFIIIA promoter mutants microinjected into late stage Xenopus oocytes, we have determined DNA sequences required for the transcription of this gene and we have identified proteins that bind to these regulatory sequences. A negative element lies between positions -306 and -289. Three positive-acting sequences are located between positions -289 and -253, -250 and -173, and -144 and -101. Gel shift analyses of TFIIIA promoter fragments incubated with Xenopus oocyte extracts have identified two DNA-protein complexes. One complex, designated B1, requires sequences within the promoter region extending from -271 to -253 while the second complex, designated B2, involves promoter sequences from -235 to -221. The protein involved in formation of the B1 complex has been found to be related to the human adenovirus major late transcription factor, USF.

Six independently derived multidrug-resistant Chinese hamster cell lines selected with vincristine, daunorubicin, actinomycin D, or colchicine were probed by in situ hybridization techniques with the cloned cDNA, p5L-18, to chromosomally localize known or presumed amplified P-glycoprotein genes. One or two clusters of amplified genes were demonstrable in all of the highly resistant sublines and were localized to homogeneously staining regions and/or abnormally banding regions, gene amplification-associated cytogenetic abnormalities, on eight different chromosomes. Analysis of trypsin-Giemsa banded karyotypes revealed additional, multiple chromosomal rearrangements that were apparently nonspecific. Mapping studies localized the native P-glycoprotein gene(s) to the region q23 to 31 (most probably band 26) on the long arm of chromosome 1 of normal Chinese hamster bone marrow fibroblasts and normal chromosome 1 homologues in resistant cells. Southern blot analysis of restriction endonuclease fragments indicated the amplification of one or both of (at least) two wild-type nonallelic genes in four of the lines and the presence in one line (DC-3F/DMM XX) of a unique 5.0-kilobase BamHI fragment resulting from a recombinational event during amplification. Comparison with the cytogenetic data indicated no correlation between restriction patterns generated by EcoRI, HindIII, PstI, or BamHI and chromosomal location of amplified genes. However, the only sublines in which the homogeneously staining region or abnormally banding region is positioned at 1q26 (at or near the site of the native gene) exhibit either alterations in gene structure (DC-3F/DM XX) or in regulation of gene expression (DC-3F/AD X), suggesting a process more complex than simply amplification of the gene in loco.

In multidrug resistance, which is observed clinically and in tissue culture, cells that are challenged with certain cytotoxic drugs develop resistance not only to the selective agent but also to other, seemingly unrelated, agents. The multidrug-resistant phenotype is associated with DNA sequence amplification and with the overproduction of a number of cytosolic and membrane glycoproteins. The differential amplification and altered expression of at least two related genes, termed multidrug-resistant associated genes has been shown in multidrug-resistant Chinese hamster cells. In multidrug-resistant mouse and human cells, genes homologous to those in Chinese hamster cells are also amplified. The level of expression of these genes varied and did not correlate with their copy number. Furthermore, in Chinese hamster cells, the development of resistance to a single drug and multidrug resistance were closely related, but uncoupled, events. The overexpression of the multidrug-resistant genes was better correlated with the degree of resistance to the selective agent than it was with the extent of multidrug resistance.