Pinus kesiya Royle ex Gordon induces apoptotic cell death in hepatocellular carcinoma HepG2 cell via intrinsic pathway by PARP and Topoisomerase I suppression.

Pinus kesiya Royle ex Gordon (PK), widely found in Southeast Asia, has been traditionally used for the treatment of several illnesses. Our previous studies showed that PK was highly cytotoxicity against liver cancer cells. The detailed mechanism of anticancer action of 50% hydro-ethanolic extract of PK's twig was, therefore, investigated in hepatocellular carcinoma HepG2 cells. Cytotoxicity of PK was determined by using NR assay, followed by determination of the mode of cell death by flow cytometry. The apoptosis-inducing effect was determined based on caspases activity, mitochondria membrane potential change, and expression of proteins related to apoptosis by western blot. The biomolecular alteration in the PK-treated HepG2 cells was investigated by FTIR microspectroscopy. Inhibition of topoisomerase I enzyme was determined by using DNA relaxation assay. Results showed that PK displayed high selective cytotoxicity and induced apoptosis against HepG2. FTIR microspectroscopy indicated that PK altered major biomolecules in HepG2 different from melphalan (a positive control), indicating a different mechanism of anticancer action. PK induced apoptotic cell death through the intrinsic pathway by increasing caspases 9 and 3/7 activity, increasing Bax, and decreasing Bcl-2 expression leading to mitochondrial membrane potential changes. PK also inhibited Top I and PARP activity that triggered an intrinsic apoptotic pathway. The phytochemical test presented terpenoids (i.e., α-pinene confirmed by GC-MS), alkaloids, steroids, xanthone, reducing sugar, and saponin. α-Pinene exhibited low cytotoxicity against HepG2, therefore, several terpene derivatives may work synergistically for inducing apoptosis. Our data demonstrated that PK has the potential for further study with chemotherapeutic purposes.

The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants.

DNA replication inhibitors are utilized extensively in studies of molecular biology and as chemotherapy agents in clinical settings. The inhibition of DNA replication often triggers double-stranded DNA breaks (DSBs) at stalled DNA replication sites, resulting in cytotoxicity. In East Asia, some traditional medicines are administered as anticancer drugs, although the mechanisms underlying their pharmacological effects are not entirely understood. In this study, we screened Japanese herbal medicines and identified two benzylisoquinoline alkaloids (BIAs), berberine and coptisine. These alkaloids mildly induced DSBs, and this effect was dependent on the function of topoisomerase I (Topo I) and MUS81-EME1 structure-specific endonuclease. Biochemical analysis revealed that the action of BIAs involves inhibiting the catalytic activity of Topo I rather than inducing the accumulation of the Topo I-DNA complex, which is different from the action of camptothecin (CPT). Furthermore, the results showed that BIAs can act as inhibitors of Topo I, even against CPT-resistant mutants, and that the action of these BIAs was independent of CPT. These results suggest that using a combination of BIAs and CPT might increase their efficiency in eliminating cancer cells.

Natural Products as Anti-Cancerous Therapeutic Molecules Targeted towards Topoisomerases.

Topoisomerases are reported to resolve the topological problems of DNA during several cellular processes, such as DNA replication, transcription, recombination, and chromatin remodeling. Two types of topoisomerases (Topo I and II) accomplish their designated tasks by introducing single- or double-strand breaks within the duplex DNA molecules, and thus maintain the proper structural conditions of DNA to release the topological torsions, which is generated by unwinding of DNA to access coded information, in the course of replication, transcription, and other processes. Both the topoisomerases have been looked at as crucial targets against various types of cancers such as lung, melanoma, breast, and prostate cancers. Conceptually, targeting topoisomerases will disrupt both DNA replication and transcription, thereby leading to inhibition of cell division and consequently stopping the growth of actively dividing cancerous cells. Since the discovery of camptothecin (an alkaloid) as an inhibitor of Topo I in 1958, a number of derivatives of camptothecin were developed as potent inhibitors of Topo I. Two such derivatives of camptothecin, namely, topotecan and irinotecan, have been commonly used as US Food and Drug Administration (FDA) approved drugs against Topo I. Similarly, the first Topo II inhibitor, namely, etoposide, an analogue of podophyllotoxin, was developed in 1966 and got FDA approval as an anti-cancer drug in 1983. Subsequently, several other inhibitors of Topo II, such as doxorubicin, mitoxantrone, and teniposide, were developed. These drugs have been reported to cause accumulation of cytotoxic non-reversible DNA double-strand breaks (cleavable complex). Thus, the present review describes the anticancer potential of plant-derived secondary metabolites belonging to alkaloids, flavonoids and terpenoids directed against topoisomerases. Furthermore, in view of the recent advances made in the field of computer-aided drug design, the present review also discusses the use of computational approaches such as ADMET, molecular docking, molecular dynamics simulation and QSAR to assess and predict the safety, efficacy, potency and identification of these potent anti-cancerous therapeutic molecules.

Deletion of Topoisomerase 1 in excitatory neurons causes genomic instability and early onset neurodegeneration.

Topoisomerase 1 (TOP1) relieves torsional stress in DNA during transcription and facilitates the expression of long (>100 kb) genes, many of which are important for neuronal functions. To evaluate how loss of Top1 affected neurons in vivo, we conditionally deleted (cKO) Top1 in postmitotic excitatory neurons in the mouse cerebral cortex and hippocampus. Top1 cKO neurons develop properly, but then show biased transcriptional downregulation of long genes, signs of DNA damage, neuroinflammation, increased poly(ADP-ribose) polymerase-1 (PARP1) activity, single-cell somatic mutations, and ultimately degeneration. Supplementation of nicotinamide adenine dinucleotide (NAD+) with nicotinamide riboside partially blocked neurodegeneration, and increased the lifespan of Top1 cKO mice by 30%. A reduction of p53 also partially rescued cortical neuron loss. While neurodegeneration was partially rescued, behavioral decline was not prevented. These data indicate that reducing neuronal loss is not sufficient to limit behavioral decline when TOP1 function is disrupted.

Repurposing of ginseng extract as topoisomerase I inhibitor based on the comparative analysis of gene expression patterns.

Repositioning of plant extracts and chemical drugs can accelerate drug development. However, its success rate may depend on what the clue is for the repositioning. Recently, repositioning based on correction of unwarranted gene expression pattern has suggested the possibility of new drug development. Here, we designed a similar method for the repositioning of nutraceutical ginseng (Panax ginseng C.A.Mey.), which is one of the most validated natural therapeutic products for various diseases. We analyzed ginseng-induced gene expression profiles using the connectivity map algorithm, which is a database that connects diseases, chemical drugs, and gene expression. Ginseng was predicted to show the same effects as those of topoisomerase I inhibitors. In a subsequent in vitro assay, ginseng extract unwound coiled or supercoiled DNA, an effect comparable to that of the topoisomerase I inhibitor, camptothecin. Furthermore, ginseng extract induced synthetic lethality with suppression of the Werner syndrome gene. The collected data implicate ginseng as a candidate antitumor agent owing to its topoisomerase I inhibitory activity and further validate the usefulness of differentially expressed gene similarity-based repurposing of other natural products.

Alpha-terpineol affects synthesis and antitumor activity of triterpenoids from Antrodia cinnamomea mycelia in solid-state culture.

To enhance production of Antrodia cinnamomea triterpenoids (ACTs) from mycelia in solid-state culture, α-terpineol was added to the medium as an elicitor at an optimal concentration of 0.05 mL L-1. Multi-stage solvent extraction and HPLC analysis were performed, and the compositions of ACTs-E (from culture with elicitor) and ACTs-NE (from culture without elicitor) were found to be quite different. In assays of in vitro antitumor activity, ACTs-E, in comparison with ACTs-NE, produced stronger viability reduction in several tumor cell lines and stronger apoptosis induction in HeLa in a dose-dependent manner. Several related proteins involved in the mitochondrial pathway of apoptosis (p53, Bax, caspase-3) did not show expression upregulation by ACTs-E, suggesting that apoptosis induction occurred through a p53-independent process. Further analysis revealed that ACTs-E strongly inhibited synthesis of topoisomerase I (TOP1) and tyrosyl-DNA phosphodiesterase I (TDP1), which are involved in DNA repair, at both transcriptional and protein levels. Our findings suggest that ACTs-E have potential for applications in the pharmaceutical, clinical, and functional food industries, as a novel antitumor agent and a dual TOP1/TDP1 inhibitor.

The RTR Complex Partner RMI2 and the DNA Helicase RTEL1 Are Both Independently Involved in Preserving the Stability of 45S rDNA Repeats in Arabidopsis thaliana.

The stability of repetitive sequences in complex eukaryotic genomes is safeguarded by factors suppressing homologues recombination. Prominent in this is the role of the RTR complex. In plants, it consists of the RecQ helicase RECQ4A, the topoisomerase TOP3α and RMI1. Like mammals, but not yeast, plants harbor an additional complex partner, RMI2. Here, we demonstrate that, in Arabidopsis thaliana, RMI2 is involved in the repair of aberrant replication intermediates in root meristems as well as in intrastrand crosslink repair. In both instances, RMI2 is involved independently of the DNA helicase RTEL1. Surprisingly, simultaneous loss of RMI2 and RTEL1 leads to loss of male fertility. As both the RTR complex and RTEL1 are involved in suppression of homologous recombination (HR), we tested the efficiency of HR in the double mutant rmi2-2 rtel1-1 and found a synergistic enhancement (80-fold). Searching for natural target sequences we found that RTEL1 is required for stabilizing 45S rDNA repeats. In the double mutant with rmi2-2 the number of 45S rDNA repeats is further decreased sustaining independent roles of both factors in this process. Thus, loss of suppression of HR does not only lead to a destabilization of rDNA repeats but might be especially deleterious for tissues undergoing multiple cell divisions such as the male germline.

Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression levels of HIF-1-associated proteins in human perihilar cholangiocarcinomas, (2) investigate the role of HIF-1 in PDT-treated human perihilar cholangiocarcinoma cells, and (3) determine whether HIF-1 inhibition reduces survival signaling and enhances PDT efficacy. RESULTS: Increased expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was confirmed in human perihilar cholangiocarcinomas. PDT with liposome-delivered zinc phthalocyanine caused HIF-1α stabilization in SK-ChA-1 cells and increased transcription of HIF-1α downstream genes. Acriflavine was taken up by SK-ChA-1 cells and translocated to the nucleus under hypoxic conditions. Importantly, pretreatment of SK-ChA-1 cells with acriflavine enhanced PDT efficacy via inhibition of HIF-1 and topoisomerases I and II. METHODS: The expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was determined by immunohistochemistry in human perihilar cholangiocarcinomas. In addition, the response of human perihilar cholangiocarcinoma (SK-ChA-1) cells to PDT with liposome-delivered zinc phthalocyanine was investigated under both normoxic and hypoxic conditions. Acriflavine, a HIF-1α/HIF-1ß dimerization inhibitor and a potential dual topoisomerase I/II inhibitor, was evaluated for its adjuvant effect on PDT efficacy. CONCLUSIONS: HIF-1, which is activated in human hilar cholangiocarcinomas, contributes to tumor cell survival following PDT in vitro. Combining PDT with acriflavine pretreatment improves PDT efficacy in cultured cells and therefore warrants further preclinical validation for therapy-recalcitrant perihilar cholangiocarcinomas.

An improved assay for the detection of alterations in bacterial DNA supercoiling in vivo.

Due to the increasing prevalence of antibiotic resistance and the yet low output of the genomics-based drug discovery approach novel strategies are urgently needed to detect new antibiotics. One such strategy uses known ubiquitous targets like DNA topoisomerases. However, to detect inhibitors of these enzymes by an in vitro assay time-consuming isolation of enzymes and DNA followed by electrophoretic separation of topoisomers are required. Instead, this study aimed at developing an in vivo assay for the detection of alterations in DNA supercoiling indicative of topoisomerase inhibition by a reporter gene assay. A pair of plasmids was developed which carry the reporter gene luc for firefly luciferase under control of either promoter ptopA (pPHB90) or pgyrA (pPHB91), whose activities are reciprocally affected by alterations of the supercoiling degree. Each plasmid is individually transferred into E. coli cells. The quotient of the luciferase activities determined using cells with either plasmid was taken as relative measure of the global supercoiling degree Qsc (quotient of supercoiling). Using isogenic reference strains with known alterations of the global DNA supercoiling degree due to mutations in either gyrB or topA, the reporter gene system was able to detect both a decrease and an increase of the negative supercoiling degree compared to the isogenic parent strain. Treating cells with known inhibitors of DNA gyrase, like fluoroquinolones, novobiocin as well as simocyclinone D8 from Streptomyces antibioticus which has been identified as an inhibitor of DNA gyrase in vitro, also caused decreases of the Qsc value in vivo. The suitability of this reporter gene system to screen for anti-topoisomerase I and II compounds from various natural sources like plant extracts by sensing alterations of the DNA supercoiling was demonstrated and offers a new application to identify novel compounds active against bacterial topoisomerases I and gyrase.

Mechanism study of goldenseal-associated DNA damage.

Goldenseal has been used for the treatment of a wide variety of ailments including gastrointestinal disturbances, urinary tract disorders, and inflammation. The five major alkaloid constituents in goldenseal are berberine, palmatine, hydrastine, hydrastinine, and canadine. When goldenseal was evaluated by the National Toxicology Program (NTP) in the standard 2-year bioassay, goldenseal induced an increase in liver tumors in rats and mice; however, the mechanism of goldenseal-associated liver carcinogenicity remains unknown. In this study, the toxicity of the five goldenseal alkaloid constituents was characterized, and their toxic potencies were compared. As measured by the Comet assay and the expression of γ-H2A.X, berberine, followed by palmatine, appeared to be the most potent DNA damage inducer in human hepatoma HepG2 cells. Berberine and palmatine suppressed the activities of both topoisomerase (Topo) I and II. In berberine-treated cells, DNA damage was shown to be directly associated with the inhibitory effect of Topo II, but not Topo I by silencing gene of Topo I or Topo II. In addition, DNA damage was also observed when cells were treated with commercially available goldenseal extracts and the extent of DNA damage was positively correlated to the berberine content. Our findings suggest that the Topo II inhibitory effect may contribute to berberine- and goldenseal-induced genotoxicity and tumorigenicity.

Inactivation of PbTopo IIIß causes hyper-excision of the Pathogenicity Island HAI2 resulting in reduced virulence of Pectobacterium atrosepticum.

Topoisomerase III enzymes are present only in a limited set of bacteria and their physiological role remains unclear. Here, we show that PbTopo IIIß, a homologue of topoisomerase III encoded on the chromosome of Pectobacterium atrosepticum strain SCRI1043 (Pba SCRI1043), is involved in excision of HAI2, a discrete ~100 kb region, from the Pba SCRI1043 chromosome. HAI2 is a Pathogenicity Island (PAI) that encodes coronafacic acid (Cfa), a major virulence determinant required for infection of potato. PAIs are horizontally acquired genetic elements that in some instances are able to excise from the chromosome of their host cell to form a circular episome prior to transfer to a recipient bacterium. We demonstrate excision of HAI2 from the chromosome, a process that is independent of growth phase and that results in the production of a circular intermediate. Inactivation of PbTopo IIIß causes a 10(3) - to 10(4) -fold increase in excision, leading to reduced fitness in vitro and a decrease in the virulence of Pba SCRI1043 on potato. These results suggest that PbTopo IIIß is required for stable maintenance of HAI2 in the chromosome of Pba SCRI1043 and may control as yet unidentified genes involved in viability and virulence of Pba SCRI1043 on potato.

Correlation of camptothecin-producing ability and phylogenetic relationship in the genus Ophiorrhiza.

Camptothecin (CPT) is an essential precursor of semisynthetic chemotherapeutic agents for cancers throughout the world. In spite of the rapid growth of market demand, CPT raw material is still harvested by extraction from Camptotheca acuminata and Nothapodytes foetida because its total synthesis is not cost-effective. In this study, we examined eight species of the genus Ophiorrhiza (Rubiaceae) from Thailand as novel alternative sources of CPT. CPT and/or 9-methoxy camptothecin (9-MCPT) were detected at different amounts in the leaf and root extracts of five species. We found that the CPT production ability of Ophiorrhiza spp. in Thailand was related mainly to species, not habitat. Chloroplast MATK and nuclear TOPI genes of eight species were investigated and compared with those of other Ophiorrhiza sequences from GenBank in order to classify and study the evolution in this genus. The molecular phylogenetic trees of both separated and combined MATK and TOPI nucleotide sequences revealed a major clade of Ophiorrhiza taxa correlated with production of CPT and its derivatives. Several amino acid markers of CPT- or 9-MCPT-producing Ophiorrhiza plants were also suggested from the alignment of TopI amino acid sequences. Our findings suggest that genetic factors play an important role in determining the CPT- and 9-MCPT-producing properties of Ophiorrhiza plants. Consequently, MATK and TOPI gene sequences could be utilized for the prediction of CPT and 9-MCPT production ability of members of Ophiorrhiza.

[Comparative analysis of mitochondrial and nuclear DNA topoisomerase I from maize].

We conducted a comparative study of the properties of topoisomerase I isolated from maize nuclei and mitochondria. We found that nuclear and mitochondrial enzymes possess different ability to bind single stranded DNA. Study of the enzyme activity dependence on Mg2+ demonstrated an absolute dependence of the mitochondrial topoisomerase activity. Contrary, nuclear enzyme activity was not absolutely dependent but stimulated by the magnesium cation. Mitochondrial topoisomerase formed covalent bond with the 5'-end of the cleaved DNA what is unique property of prokaryotic topoisomerase I. Nuclear enzyme bound covalently to the 3'-end like all eukaryotic topoisomerases I. The search through databases revealed genes which could encode mitochondrial topoisomerase I in the genomes of higher plants. Using both cDNA sequencing and in silico methods we demonstrated an existence of the ortholog gene in the maize genome. This gene shares significant homology with prokaryotic topoisomerase I genes that may explain differences in the properties of the mitochondrial and nuclear enzyme. Data obtained is of a significant interest both from the point of view of plant organelle evolution and mitochondrial genome expression mechanisms study.

Increased negative superhelical density in vivo enhances the genetic instability of triplet repeat sequences.

The influence of negative superhelical density on the genetic instabilities of long GAA.TTC, CGG.CCG, and CTG.CAG repeat sequences was studied in vivo in topologically constrained plasmids in Escherichia coli. These repeat tracts are involved in the etiologies of Friedreich ataxia, fragile X syndrome, and myotonic dystrophy type 1, respectively. The capacity of these DNA tracts to undergo deletions-expansions was explored with three genetic-biochemical approaches including first, the utilization of topoisomerase I and/or DNA gyrase mutants, second, the specific inhibition of DNA gyrase by novobiocin, and third, the genetic removal of the HU protein, thus lowering the negative supercoil density (-sigma). All three strategies revealed that higher -sigma in vivo enhanced the formation of deleted repeat sequences. The effects were most pronounced for the Friedreich ataxia and the fragile X triplet repeat sequences. Higher levels of -sigma stabilize non-B DNA conformations (i.e. triplexes, sticky DNA, flexible and writhed DNA, slipped structures) at appropriate repeat tracts; also, numerous prior genetic instability investigations invoke a role for these structures in promoting the slippage of the DNA complementary strands. Thus, we propose that the in vivo modulation of the DNA structure, localized to the repeat tracts, is responsible for these behaviors. Presuming that these interrelationships are also found in humans, dynamic alterations in the chromosomal nuclear matrix may modulate the -sigma of certain DNA regions and, thus, stabilize/destabilize certain non-B conformations which regulate the genetic expansions-deletions responsible for the diseases.

Topoisomerase I and ATP activate cDNA synthesis of human immunodeficiency virus type 1.

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated at reverse transcription. Cellular topoisomerase I has been reported to be carried into HIV-1 virions and enhance cDNA synthesis in vitro, suggesting that topoisomerase I expressed in virus producer cells regulates reverse transcription. Here, by employing both indicator cell assay and endogenous reverse transcription (ERT) assay, we show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV-1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I-RNA complex and enhance cDNA synthesis in vitro. These findings suggest that cellular topoisomerase I and ATP play a pivotal role in the synthesis of cDNA of HIV-1.

Regulation of mouse DNA topoisomerase IIIalpha gene expression by YY1 and USF transcription factors.

To investigate the mechanisms responsible for the regulation of DNA topoisomerase IIIalpha (TOP3alpha) gene expression, the promoter region of the mouse gene has been cloned and analyzed. The promoter region is moderately high in GC content and lacks a canonical TATA box, typical for promoters of a number of housekeeping genes. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences demonstrated that the 34-bp region from -137 to -170 upstream of the transcription initiation site contains a positive regulatory element(s) for the efficient expression of mouse TOP3alpha gene. Combined analyses by gel mobility shift and supershift assays revealed that both YY1 and USF transcription factors were capable of binding to the 34-bp region. When YY1 and USF-binding elements were selectively mutated, the luciferase activity of the resulted constructs was greatly reduced, indicating that both YY1 and USF function as transcriptional activators. Interestingly, YY1 and USF-binding elements are conserved in both human and mouse TOP3alpha promoters. This suggests that mammalian TOP3alpha genes may possess a common mechanism of transcription regulation through these elements.

Retroviral expression of a mutant (Gly-533) human DNA topoisomerase I cDNA confers a dominant form of camptothecin resistance.

In previous studies, we isolated a mutant DNA topoisomerase I cDNA from a camptothecin (CPT)-resistant human T-lymphoblastic leukemia cell line, CPT-K5, and demonstrated that an amino acid change from Asp to Gly at residue 533 is responsible for the CPT resistance of the enzyme. In the present study, we have constructed a bicistronic retroviral vector, Ha-TM1-IRES-neo, that carries the mutant (Gly-533) TOP1 cDNA (TM1) and a neomycin-resistance gene to examine the effect of mutant DNA topoisomerase I (topo I) expression on CPT resistance of cells. HeLa S3 cells were transduced with Ha-TM1-IRES-neo, and the transduced cells were selected with G418. Two independently isolated populations of the G418-resistant cells and 2 clones showed 1.7- to 1.8-fold higher resistance to CPT than the control cells. Integration and expression of the exogenous TOP1 were confirmed by genomic and RT-PCR analyses. The topo I enzyme (mixture of mutant and wild-type) expressed in the transduced cells showed 3-fold resistance to CPT in cleavable-complex-formation assay and DNA-relaxation assay. Mutant topo I activity in the transduced cells was as much as 10% that of the endogenous enzyme. Our results clearly show that expression of Gly-533 topo I confers a dominant form of CPT resistance in cells expressing wild-type topo I. The mutant TOP1 could be used for the protection of normal bone marrow cells of cancer patients from the severe hematotoxicity of CPT-derivative anti-tumor agents.

Cloning and characterization of the 5'-flanking region for the human topoisomerase III gene.

The human DNA topoisomerase III (hTOP3) gene encodes a topoisomerase homologous to the Escherichia coli DNA topoisomerase I subfamily. To understand the mechanisms responsible for regulating hTOP3 expression, we have cloned the 5'-flanking region of the gene coding for the hTOP3 and analyzed its promoter activity. The presence of a single transcription initiation site was suggested by primer extension analysis. The hTOP3 gene promoter is moderately high in GC content and lacks a canonical TATA box, suggesting that hTOP3 promoter has overall similarity to promoters of a number of housekeeping genes. Examination of the promoter sequence indicated the presence of four Sp-1 consensus binding sequences and a putative initiator element surrounding the transcription initiation site. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the 52-base pair region from -326 to -275 upstream of the transcription initiation site includes a positive cis-acting element(s) for the efficient expression of hTOP3 gene. On the basis of gel mobility shift and supershift assays, we demonstrated that both YY1 and USF1 transcription factors can bind to the 52-base pair region. When HeLa cells were transiently transfected with a mutant construct which had disabled both YY1- and USF1-binding sites, the luciferase activity was greatly reduced, suggesting that these binding elements play a functional role in the basal activation of the hTOP3 promoter. Transfection studies with mutations that selectively impaired YY1 or USF1 binding suggested that both YY1 and USF1 function as activators in the hTOP3 promoter.

Cloning, expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea.

We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg2+. In the presence of Mg2+ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.

Expression of PKC isozyme and MDR-associated genes in primary and relapsed state AML.

For investigation of relative differences in mRNA expression levels and of correlations in the expression of genes possibly involved in multidrug resistance (MDR) of acute myelogenous leukemias (AML), a complementary DNA polymerase chain reaction (cDNA-PCR) analysis was established for the genes encoding MDR1/P-glycoprotein, the multidrug resistance-associated protein (MRP), topoisomerase II alpha, topoisomerase II beta, topoisomerase I, glutathione S-transferase pi, protein kinase C (PKC) isozymes alpha, beta 1, beta 2, epsilon, eta, theta and cyclin A. In a first descriptive study comprising samples of childhood or adult AML we calculated the mean values from primary (n=14) or relapsed (n=23) states of the diseases, respectively. We found in the latter significant increases of MDR1, MRP, gst pi, and PKC theta gene expression. MDR1 and MRP gene expression levels were generally correlated (rs= +0.4128, P<0.02, n=37), as well as topoisomerase II alpha and cyclin A gene expression levels (rs= +0.8727, P<0.0001, n=35). Within the group of relapsed state AML a significant negative correlation between the gene expression levels of MDR1 and topoisomerase II alpha (rs= -0.5500, P<0.01, n=22) was observed. Remarkably, highly significant positive correlations were found for MDR1/PKC eta (rs= +0.5560, P<0.001, n=32), MRP/PKC theta (rs= +0.6573, P<0.0001, n=34) and MRP/PKC eta (rs= +0.5241, P<0.005, n=32).