Amplification and expression of genes associated with multidrug resistance in mammalian cells.

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.

Transcriptional regulation of the MDR1 gene by histone acetyltransferase and deacetylase is mediated by NF-Y.

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.

Phenotypic expression in Escherichia coli and nucleotide sequence of two Chinese hamster lung cell cDNAs encoding different dihydrofolate reductases.

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.

Stable transfection of the P-glycoprotein promoter reproduces the endogenous overexpression phenotype: the role of MED-1.

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.

Chromosomal organization of amplified genes in multidrug-resistant Chinese hamster cells.

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.

Sequence-dependent enhancement of cytotoxicity produced by ecteinascidin 743 (ET-743) with doxorubicin or paclitaxel in soft tissue sarcoma cells.

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.

Rapid activation of MDR1 gene expression in human metastatic sarcoma after in vivo exposure to doxorubicin.

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.

Differential utilization of multiple transcription start points accompanies the overexpression of the P-glycoprotein-encoding gene in Chinese hamster lung cells.

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 effects of serum depletion and dexamethasone on growth and differentiation of human neuroblastoma cell lines.

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.

Transcriptional regulation of MDR genes.

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.

A conserved downstream element defines a new class of RNA polymerase II promoters.

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.

Transcription of the multidrug resistance gene MDR1: a therapeutic target.

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.

Transcriptional activation of the MDR1 gene by UV irradiation. Role of NF-Y and Sp1.

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.

P-glycoprotein content and mediation of vincristine efflux: correlation with the level of differentiation in luminal epithelium of mouse small intestine.

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.

Ecteinascidin 743, a transcription-targeted chemotherapeutic that inhibits MDR1 activation.

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.

Positive and negative regulation of the gene for transcription factor IIIA in Xenopus laevis oocytes.

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.

Transcriptional repression by p53 through direct binding to a novel DNA element.

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.

Optimization of a versatile in vitro transcription assay for the expression of multiple start site TATA-less promoters.

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.

Differential utilization of poly (A) signals between DHFR alleles in CHL cells.

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.