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Biomedical subjects

D P Suttle

Publications and source records attributed to D P Suttle.

At least 19 recordsLinked to original sources
PubMed2000

Catalytic inhibition of DNA topoisomerase II by N-benzyladriamycin (AD 288).

N-Benzyladriamycin (AD 288) is a highly lipophilic, semi-synthetic congener of doxorubicin (DOX). Unlike DOX, which stimulates double-stranded DNA scission by stabilizing topoisomerase II/DNA cleavable complexes, AD 288 is a catalytic inhibitor of topoisomerase II, capable of preventing topoisomerase II activity on DNA. The concentration of AD 288 required to inhibit the topoisomerase II-catalyzed decatenation of linked networks of kinetoplast DNA was comparable to that for DOX. However, AD 288 did not stabilize cleavable complex formation or stimulate topoisomerase II-mediated DNA cleavage. In addition, AD 288 inhibited the formation of cleavable complexes by etoposide in a concentration-dependent manner. Human CCRF-CEM cells and murine J774. 2 cells exhibiting resistance against DOX, teniposide, or 3'-hydroxy-3'-deaminodoxorubicin through reduced topoisomerase II activity remained sensitive to AD 288. These studies suggest that AD 288 inhibits topoisomerase II activity by preventing the initial non-covalent binding of topoisomerase II to DNA. Since AD 288 is a potent DNA intercalator, catalytic inhibition is achieved by prohibiting access of the enzyme to DNA binding sites. These results also demonstrate that specific substitutions on the aminosugar of DOX can alter the mechanism of topoisomerase II inhibition.

Antineoplastic Agents, Phytogenic↗
PubMed1999

Ras stimulates DNA topoisomerase II alpha through MEK: a link between oncogenic signaling and a therapeutic target.

Topoisomerase II alpha (topo II alpha) is a major target of antitumor treatments. In an effort to determine why this protein might be a better target in tumor cells than in normal cells, we attempted to determine if the altered proliferative signaling in a tumor cell might effect the levels of expression of the topo II alpha gene. In support of this idea, it was found that topo II alpha was elevated following microinjection of oncogenic Ras protein. Oncogenic ras was further shown to stimulate the topo II alpha promoter. Stimulation by ras was independent of the normal cell cycle regulation of this promoter. Transactivation of topo II alpha by ras required both the MEK/ERK pathway, and the stress-associated protein kinase (SAPK) signaling pathway. As a direct confirmation that both ERK and SAPK were involved in topo II alpha regulation, a constitutively active MEKK that stimulates these two kinases simultaneously was shown to strongly induce topo II alpha promoter activity. Activation of either pathway alone, on the other hand, only slightly stimulated the topo II alpha promoter. Deletion analyses showed that elements near both the 5' and 3' ends of the promoter were responsible for the ras stimulation. Site-directed mutagenesis further demonstrated that an Ets-like binding site near the 5' end (-480 to -475) was one of the responsive elements. Taken together, these studies demonstrate the direct role of Ras signaling in stimulation of topo II alpha expression, and thereby establish a link between the action of a common tumor mutation and the target of multiple anti-tumor reagents.

3T3 Cells↗
PubMed1997

Inhibition of DNA topoisomerase II alpha gene expression by the p53 tumor suppressor.

DNA topoisomerase II (topo II) is an essential nuclear enzyme involved in major cellular functions such as DNA replication, transcription, recombination, and mitosis. While an elevated level of topo II alpha is associated with cell proliferation, wild-type (wt) p53 inhibits the expression of various growth-stimulatory genes. To determine if p53 downregulates topo II alpha gene expression, a murine cell line, (10)1val, that expresses a temperature-sensitive p53 was utilized. The (10)1val cells had significantly lower levels of topo II alpha mRNA and protein following incubation for 24 h at 32 degrees C (p53 with wt conformation) than at 39 degrees C (p53 with mutant conformation). The effect of p53 on the human topo II alpha gene promoter was determined by using luciferase reporter plasmids containing varying lengths of the topo II alpha promoter transiently cotransfected into p53-deficient (10)1 cells together with wt or mutant p53 expression plasmids. Transcription from the full-length (bp -557 to +90) topo II alpha promoter was decreased 15-fold by wt p53 in a concentration-dependent manner, whereas mutant p53 exerted much weaker inhibition. Consecutive deletion of the five inverted CCAAT elements (ICEs) from the topo II alpha promoter reduced both the basal promoter activity and wt p53-induced suppression. Transcription of the minimal promoter (-32 to +90), which contains no ICE, was slightly stimulated by wt or mutant p53 expression. When point mutations were introduced into the most proximal ICE (-68), the inhibitory effect of wt p53 was alleviated and stimulation of topo II alpha expression resulted. Our study suggests that wt p53 functions as a transcriptional repressor of topo II alpha gene expression, possibly through a functional interaction with specific ICEs. Inactivation of wt p53 may reduce normal regulatory suppression of topo II alpha and contribute to abortive cell cycle checkpoints, accelerated cell proliferation, and alterations in genomic stability associated with neoplasia.

Animals↗
PubMed1995

UMP synthase activity expressed in deficient hamster cells by separate transferase and decarboxylase proteins or by linker-deleted bifunctional protein.

Segments of the human UMP synthase cDNA coding for the orotate phosphoribosyl transferase (OPRT) and orotidylate decarboxylase (ODC) domains of the bifunctional protein UMP synthase were produced by polymerase chain reaction techniques and cloned into a eukaryotic expression vector. The separate OPRT and ODC vectors, along with a selectable marker, were cotransfected into UMP synthase-deficient hamster cells (Urd-C) that require exogeneous uridine for growth. Transfected Urd-C cells surviving selection in media without added uridine were isolated and designated transferase decarboxylase Urd-C (TDU). All of the selected colonies contained DNA corresponding to the OPRT and ODC expression vectors. Two cell lines (TDU3 and TDU5) integrated many more copies of the OPRT and ODC vectors into their genomes compared to the other TDU lines. A 28.6-kDa ODC protein band and a 24.4-kDa OPRT band were detected on western blots with UMP synthase-specific polyclonal antiserum. The OPRT activity of the TDU lines was up to 8.7 times the OPRT activity of control CHL cells, and the ODC activity was up to 12.5 times control levels. Both OPRT and ODC activities in the monofunctional proteins were less heat stable than in the bifunctional UMP synthase protein. The monofunctional OPRT protein was less stable than the ODC protein at 45 degrees C. Growth of transfected cells in 6-azauridine resulted in striking increases in activity and temperature stability for the monofunctional ODC protein. A UMP synthase bifunctional protein was constructed with a deletion of the suspected linker region joining the two catalytic domains. The linker-deleted UMP synthase showed no significant change in either OPRT or ODC activity or temperature stability. The increased stability of the bifunctional protein may be a factor in its evolutionary selection in mammalian cells.

Animals↗
PubMed1993

Isolation of genetic suppressor elements, inducing resistance to topoisomerase II-interactive cytotoxic drugs, from human topoisomerase II cDNA.

Many cytotoxic anticancer drugs act at topoisomerase II (topo II) by stabilizing cleavable complexes with DNA formed by this enzyme. Several cell lines, selected for resistance to topo II-interactive drugs, show decreased expression or activity of topo II, suggesting that such a decrease may be responsible for drug resistance. In the present study, etoposide resistance was used as the selection strategy to isolate genetic suppressor elements (GSEs) from a retroviral library expressing random fragments of human topo II (alpha form) cDNA. Twelve GSEs were isolated, encoding either peptides corresponding to short segments of the topo II alpha molecule (2.4-6.5% of the protein) or 163- to 220-bp-long antisense RNA sequences. Expression of a GSE encoding antisense RNA led to decreased cellular expression of the topo II alpha protein. Both types of GSE induced resistance to several topo II poisons but not to drugs that do not act at topo II. These results provide direct evidence that inhibition of topo II results in resistance to topo II-interactive drugs, indicate structural domains of topo II capable of independent functional interactions, and demonstrate that expression selection of random fragments constitutes an efficient approach to the generation of GSEs in mammalian cells.

Antineoplastic Agents↗
PubMed1993

Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells.

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.

Adenosine Triphosphate↗
PubMed1993

Altered DNA topoisomerase II in multidrug resistance.

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II alpha gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Expression of catalytic domains of human UMP synthase in uridine auxotrophic bacteria.

Orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC), which catalyze the last two steps in de novo UMP biosynthesis, are two distinct monofunctional proteins in bacteria and lower eukaryotes. In mammals, OPRT and ODC activities are contained in a single bifunctional protein labeled UMP synthase. The human UMP synthase cDNA was separated into the predicted OPRT and ODC domains using polymerase chain reaction techniques and the domains inserted into pUC19 expression vectors. Following transformation into OPRT- and ODC-deficient E. coli, the strains were able to grow on minimal media without uridine. The ODC-transformed bacteria expressed up to 24 times the level of activity found in a wild-type E. coli line. The OPRT-transformed E. coli contained only 4-9% of wild-type activity. Western blot analysis with antiserum to human UMP synthase demonstrates that OPRT and ODC domains are being produced in the deficient cells by the respective vectors. The level of the domain protein approximates the level of enzyme activity. The complementation of the OPRT and ODC activities in the transformed deficient E. coli strains demonstrates that human UMP synthase can be separated into active monofunctional domains that will function in the bacterial cell environment.

Base Sequence↗
PubMed1993

Gene amplification and chromosome rearrangements: a study of a single cell lineage selected for amplification and deamplification of the UMP synthase gene.

The UMP synthase gene is stably amplified in Chinese hamster lung cells selected for resistance to pyrazofurin (PF) and 6-azauridine (6AUR), inhibitors of the decarboxylase activity of the bifunctional UMP synthase enzyme. The amplified DNA is located intrachromosomally as expanded chromosomal regions (ECRs). Growth of these cells in 5-fluorouracil enables rapid selection of cells that have undergone deamplification and consequently lost resistance to PF + 6AUR. Detailed cytogenetic analyses and fluorescence in situ hybridization on three consecutive amplification-deamplification cycles in descendants of the same cloned cell showed a unique position and structure for the ECR. In the first cycle of amplification, the ECR forms a homogeneously staining region on a small marker chromosome (M3). In the second cycle of amplification, a chromosomal break was noted at the site of the endogenous UMP synthase gene on another derivative chromosome, M2, with amplification resulting in an abnormally banded region on a third marker (M3). The third cycle of amplification produced a cell line with an ECR on the distal portion of M2. This ECR was unstable, showing variations in size as well as translocations and other chromosome rearrangements. Our data, taken in its entirety, suggest a relationship between amplification as an ECR and chromosome rearrangements in Chinese hamster cells.

Amides↗
PubMed1991

Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide.

Nuclear extracts from teniposide (VM-26)-resistant sublines of the human leukemic cell line CCRF-CEM have decreased levels of DNA topoisomerase II catalytic activity and decreased capacity to form drug-stabilized covalent protein-DNA complexes. The ATP concentration required for equivalent activity in a DNA-unknotting assay is 2- to 8-fold higher in nuclear extracts from drug-resistant cell lines as compared with the parental line. When adenosine 5'-[beta,gamma-imido]triphosphate is substituted for ATP in complex-formation assays, no significant change is seen with drug-sensitive cells, but a 50-65% reduction is seen with VM-26-resistant cells. Collectively, these results indicate that an alteration in ATP binding may be involved in the resistance phenotype. Therefore, we identified regions of the topoisomerase II sequence that conform to previously identified nucleotide-binding sites. Starting with cDNA as the template we determined the sequence of the topoisomerase II mRNA surrounding these sites by sequencing DNA fragments produced by the polymerase chain reaction. In the region corresponding to the consensus B ATP-binding sequence described by Walker et al. [Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. (1982) EMBO J. 1, 945-951], the cDNA from the two VM-26-resistant sublines contained an altered sequence having a G----A base change. This base substitution results in the replacement of the conserved arginine at position 449 with a glutamine. Hybridization with allele-specific oligonucleotides confirmed the presence of both the normal and the altered sequence in the resistant cell lines, whereas only the normal sequence was found in the sensitive CEM cells. A chemical mismatch cleavage procedure for the detection of mispaired bases in DNA duplexes identified no other alterations in the 5' third of the mRNA coding sequence, which contains the complete ATP-binding domain of topoisomerase II. The presence of mRNA encoding topoisomerase II with Gln449 correlates both with the presence of a topoisomerase II protein whose interaction with ATP is altered and with increased resistance to the cytotoxicity of VM-26.

Alleles↗
PubMed1989

A reversible selection system for UMP synthase gene amplification and deamplification.

The bifunctional enzyme UMP synthase provides a unique reversible selection system whereby cells that have amplified the UMP synthase gene can be isolated from a wild-type population and cells that have deleted the extra genes can be selected from a population with amplified copies of the gene. UMP synthase catalyzes the conversion of orotic acid to orotidine 5'-monophosphate (OMP) and then OMP to UMP. In the amplification step, Chinese hamster lung cells are selected for resistance to pyrazofurin and 6-azauridine, two inhibitors of the orotidine 5'-decarboxylase activity that converts OMP to UMP. The resistant cells have increased levels of both activities of UMP synthase as a result of a stable amplification of the UMP synthase gene. The deamplification step depends on 5-fluorouracil (5FU), which is converted to its monophosphate form by the orotate phosphoribosyltransferase activity of UMP synthase. Thus cells with increases in this activity are more sensitive to 5FU cytotoxicity, permitting single-step selection of revertants that have lost their amplified UMP synthase genes. These 5FU-selected cells are similar to the parental cell line in their level of UMP synthase activity and number of UMP synthase gene copies. Reselection in increasing concentrations of pyrazofurin and 6-azauridine allows one to isolate cells that have reamplified the UMP synthase gene. The ability to cycle cells of a single lineage through states of amplification and deamplification will facilitate study of the gene amplification process and the factors that influence the composition and stability of amplified regions.

Amides↗
PubMed1989

Cytogenetic analysis of amplification and deamplification of UMP synthase genes in Chinese hamster cells.

Chinese hamster lung cells selected for resistance to pyrazofurin and 6-azauridine contain amplified UMP synthase genes. With selection in 5-fluorouracil, cells that have lost the amplified gene copies can be isolated. Reselection of deamplified cells in pyrazofurin and 6-azauridine results in reamplification of the UMP synthase genes. We have used chromosomal banding and in situ hybridization techniques to characterize this cyclic process of amplification and deamplification. Homogeneously staining regions (HSRs) were observed in cells containing amplified copies of the UMP synthase gene but not in cells in which the amplified UMP synthase genes had been lost. After reselection in pyrazofurin and 6-azauridine, abnormally banded regions (ABRs) were observed. Both HSRs and ABRs were located at a single site on the distal regions of a small acrocentric autosome, and both were shown to contain the amplified genes. The majority of 5-fluorouracil-selected cells showed residual marker acrocentric chromosomes of various sizes, suggesting excision of portions of the HSR or ABR as the mechanism of deamplification. The acrocentrics carrying the amplified genes resulted from rearrangements involving chromosome 4, site of the endogenous gene. This reversible selection system provides a unique model for investigating gene amplification and deamplification in association with chromosomal rearrangements and the relationship of G-banding to underlying DNA structure.

Animals↗
PubMed1989

Localization of the gene for uridine monophosphate synthase to human chromosome region 3q13 by in situ hybridization.

The bifunctional enzyme uridine monophosphate (UMP) synthase catalyzes the last two steps in de novo pyrimidine biosynthesis. A genetic deficiency in the activity of this enzyme causes the inherited human disease orotic aciduria. We used a human cDNA probe to localize the gene for UMP synthase to human chromosome region 3q13 by the technique of in situ hybridization.

Autoradiography↗
PubMed1988

Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26.

The simultaneous development of resistance to the cytotoxic effects of several classes of natural product anticancer drugs, after exposure to only one of these agents, is referred to as multiple drug resistance (MDR). At least two distinct mechanisms for MDR have been postulated: that associated with P-glycoprotein and that thought to be due to an alteration in DNA topoisomerase II activity (at-MDR). We describe studies with two sublines of human leukemic CCRF-CEM cells approximately 50-fold resistant (CEM/VM-1) and approximately 140-fold resistant (CEM/VM-1-5) to VM-26, a drug known to interfere with DNA topoisomerase II activity. Each of these lines is cross-resistant to other drugs known to affect topoisomerase II but not cross-resistant to vinblastine, an inhibitor of mitotic spindle formation. We found little difference in the amount of immunoreactive DNA topoisomerase II in 1.0 M NaCl nuclear extracts of the two resistant and parental cell lines. However, topoisomerase II in nuclear extracts of the resistant sublines is altered in both catalytic activity (unknotting) of and DNA cleavage by this enzyme. Also, the rate at which catenation occurs is 20-30-fold slower with the CEM/VM-1-5 preparations. The effect of VM-26 on both strand passing and DNA cleavage is inversely related to the degree of primary resistance of each cell line. Our data support the hypothesis that at-MDR is due to an alteration in topoisomerase II or in a factor modulating its activity.

Cell Cycle↗
PubMed1988

Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase.

The last two steps in the de novo biosynthesis of UMP are catalyzed by orotate phosphoribosyltransferase (OPRT; orotidine-5'-phosphate:pyrophosphate phosphoribosyltransferase; EC 2.4.2.10) and orotidine-5'-monophosphate decarboxylase (orotidine-5'-phosphate carboxy-lyase; EC 4.1.1.23). In mammals these two activities are found in a single, bifunctional protein called UMP synthase. A human T-lymphoblastic cell cDNA library constructed in lambda gt10 was screened with a UMP synthase-specific rat cDNA probe. Human UMP synthase cDNAs were isolated and then used to select UMP synthase gene fragments. The complete coding sequence of the mRNA for UMP synthase was determined by analysis of overlapping cDNA and genomic fragments. One of the cDNAs appears to have been synthesized from an incompletely or alternatively processed form of the UMP synthase mRNA. This cDNA lacks a poly(A) tail and has an extended 3'-nontranslated region that hybridizes with larger forms of the UMP synthase mRNA. The UMP synthase protein is composed of 480 amino acids with a molecular weight of 52,199. The two activities of UMP synthase reside in distinct domains encoded by the 3' and 5' halves of the mRNA. The COOH-terminal 258 amino acids of the human UMP synthase protein contain the orotidine-5'-monophosphate decarboxylase catalytic domain. This region is highly homologous to the mouse orotidine-5'-monophosphate decarboxylase sequence. The NH2-terminal 214 amino acids contain the OPRT domain. There is amino acid homology between this protein domain and specific regions of the Escherichia coli OPRT. The human OPRT domain also contains the putative catalytic site common to other human phosphoribosyltransferases.

Amino Acid Sequence↗
PubMed1988

Analysis of UMP synthase gene and mRNA structure in hereditary orotic aciduria fibroblasts.

Hereditary orotic aciduria is an autosomal recessive disease in which there is a severe deficiency in the activity of the de novo pyrimidine pathway enzyme uridine 5'-monophosphate (UMP) synthase. UMP synthase is a bifunctional enzyme containing the two activities orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase, which catalyze the two-step conversion of orotic acid to UMP. Cell lines from three orotic aciduria patients have been characterized for UMP synthase gene and mRNA content. Restriction-enzyme analysis of DNA from the deficient cells revealed no changes in the gene structure compared with normal cell DNA structure. The amount of UMP synthase mRNA was not decreased, nor was there a detectable difference in the size of the UMP synthase mRNA in the deficient cells. Analysis of the mRNA by hybridization with a nearly full-length UMP synthase cDNA followed by S1 nuclease digestion showed no alteration in the mRNA structure. The UMP synthase activity of the deficient cells ranges from 2% to 7% of the normal cell level. The activity can be significantly increased by growing the deficient cells in barbituric acid. Our data indicate that UMP synthase gene transcription in the orotic aciduria cells produces the expected amount of a stable, correctly processed mRNA. The mRNA appears to code for a mutant enzyme that has reduced stability or altered kinetic properties.

Carboxy-Lyases↗