Characterization of DNA Topoisomerase-1 in Spodoptera exigua for Toxicity Evaluation of Camptothecin and Hydoxy-Camptothecin

Camptothecin (CPT), a plant alkaloid originally isolated from the native Chinese tree, Camptotheca acuminate, exerts the toxic effect by targeting eukaryotic DNA topoisomerase 1 (DNA Topo1). Besides as potent anti-cancer agents, CPT and its derivatives are now being explored as potential pesticides for insect control. In this study, we assessed their toxicity to an insect homolog, the Topo1 protein from beet armyworms (Spodoptera exigua Hübner), a worldwide pest of many important crops. The S. exigua Topo1 gene contains an ORF of 2790 base pairs that is predicted to encode a polypeptide of 930 amino acids. The deduced polypeptide exhibits polymorphism at residue sites V420, L530, A653 and T729 (numbered according to human Topo1) among insect species, which are predicted to confer sensitivity to CPT. The DNA relaxation activity of this protein was subsequently examined using a truncated form that contained the residues 337–930 and was expressed in bacteria BL21 cells. The purified protein retained the ability to relax double-stranded DNA and was susceptible to CPT and its derivative hydroxy-camptothecin (HCPT) in a dose-dependent manner. The same inhibitory effect was also found on the native Topo1 extracted from IOZCAS-Spex-II cells, a cell line established from beet armyworms. Additionally, CPT and HCPT treatment reduced the steady accumulation of Topo1 protein despite the increased mRNA expression in response to the treatment. Our studies provide information of the S. exigua Topo1 gene and its amino acid polymorphism in insects and uncover some clues about potential mechanisms of CPT toxicity against insect pests. These results also are useful for development of more effective Topo1-targeted CPT insecticides in the future.     

Animal model of Mycoplasma fermentans respiratory infection

Background

Mycoplasma fermentans has been associated with respiratory, genitourinary tract infections and rheumatoid diseases but its role as pathogen is controversial. The purpose of this study was to probe that Mycoplasma fermentans is able to produce respiratory tract infection and migrate to several organs on an experimental infection model in hamsters. One hundred and twenty six hamsters were divided in six groups (A-F) of 21 hamsters each. Animals of groups A, B, C were intratracheally injected with one of the mycoplasma strains: Mycoplasma fermentans P 140 (wild strain), Mycoplasma fermentans PG 18 (type strain) or Mycoplasma pneumoniae Eaton strain. Groups D, E, F were the negative, media, and sham controls. Fragments of trachea, lungs, kidney, heart, brain and spleen were cultured and used for the histopathological study. U frequency test was used to compare recovery of mycoplasmas from organs.

Results

Mycoplasmas were detected by culture and PCR. The three mycoplasma strains induced an interstitial pneumonia; they also migrated to several organs and persisted there for at least 50 days. Mycoplasma fermentans P 140 induced a more severe damage in lungs than Mycoplasma fermentans PG 18. Mycoplasma pneumoniae produced severe damage in lungs and renal damage.

Conclusions

Mycoplasma fermentans induced a respiratory tract infection and persisted in different organs for several weeks in hamsters. This finding may help to explain the ability of Mycoplasma fermentans to induce pneumonia and chronic infectious diseases in humans.     

Effect of Mycoplasmas on Apoptosis of 32D Cells Is Species-Dependent

We previously showed that mycoplasmal infection effectively prevented apoptosis of infected cells, whereas other researchers have indicated that mycoplasmal infection promoted apoptosis. To understand the mechanism underlying this discrepancy, five different species of mycoplasmas were investigated for their effects on apoptosis of interleukin (IL)-3–dependent 32D cells. Results revealed that Mycoplasma fermentans and M. penetrans effectively supported continuous growth of 32D cells after IL-3 withdrawal. M. fermentans was more potent than M. penetrans. This effect was achieved by way of preventing apoptosis and stimulating cell proliferation. On the contrary, M. hominis and M. salivarium accelerated apoptosis of 32D cells. M. genitalium had no significant effect on apoptosis. The RNase protection assay indicated that the proapoptotic and antiapoptotic mycoplasmas altered the expression of major apoptosis regulatory genes differently. The difference in apoptosis regulatory gene expression induced by different species of mycoplasmas might be accountable for their effects on host cell apoptosis.     

Antibody response to MfGL-II, a phosphocholine-containing major lipid of Mycoplasma fermentans membranes

The choline-containing phosphoglycolipid, MfGL-II, is the major polar lipid of Mycoplasma fermentans PG18. Anti-MfGL-II antisera raised in rabbits using the purified MfGL-II as an immunogen were employed in immunogold electron microscopic and immunofluorescence studies showing that MfGL-II is uniformly distributed and exposed on the cell surface of M. fermentans cells. The specificity of the antibodies was determined by immunostaining of lipid extracts separated by thin layer chromatography. The antibodies recognize lipids specific to M. fermentans but did not cross-react with lipid extracts of M. penetrans, M. capricolum, M. gallisepticum or Acholeplasma laidlawii. As phosphocholine almost completely abolished antibody interaction with MfGL-II in an ELISA assay it is suggested that the anti-MfGL-II repertoire is composed primarily of anti-phosphocholine antibodies. The anti-MfGL-II antisera inhibit the attachment of M. fermentans to Molt-3 lymphocytes suggesting that MfGL-II plays a major role in M. fermentans-host cell interaction.     

Phosphorylation and stabilization of topoisomerase IIα protein by p38γ mitogen-activated protein kinase sensitize breast cancer cells to its poisons

Cancer drugs suppress tumor cell growth by inhibiting specific cellular targets. However, most drugs also activate several cellular nonspecific stress pathways, and the implications of these off-target effects are mostly unknown. Here, we report that p38γ, but not p38α, MAPK is specifically activated by treatment of breast cancer cells with topoisomerase II (Topo II) drugs, whereas paclitaxel (Taxol) does not have this effect. The activated p38γ in turn phosphorylates and stabilizes Topo IIα protein, and this enhances the growth inhibition by Topo II drugs. Moreover, p38γ activity was shown to be necessary and sufficient for Topo IIα expression, the drug-p38γ-Topo IIα axis is only detected in intrinsically sensitive but not resistant cells, and p38γ is co-overexpressed with Topo IIα protein in primary breast cancers. These results reveal a new paradigm in which p38γ actively regulates the drug-Topo IIα signal transduction, and this may be exploited to increase the therapeutic activity of Topo II drugs.     

Identification of Phosphocholine-Containing Glycoglycerolipids Purified from Mycoplasma fermentans-Infected Human Helper T-Cell Culture as Components of M. fermentans

Previously, we have reported the occurrence of novel phosphocholine-containing glycoglycerolipids (GGPLs: GGPL-I and GGPL-III) in human helper T-cell culture (MT-4 cell line) (Matsuda et al, Glycoconjugate J. 10: 340). However, the GGPLs disappeared from the MT-4 cells after treatment with an antimycoplasma agent. This disappearance suggested the involvement of microorganisms in the GGPL expression. In this paper, we show that the novel lipids are components of Mycoplasma fermentans itself. The supernatant fluid of the antimycoplasma agent-untreated MT-4 cell culture produced mycoplasma-like colonies on PPLO agar plates, and PCR and immunological methods revealed the presence of M. fermentans. GGPLs were expressed again in the treated MT-4 cells after infection with the isolated M. fermentans. The isolated M. fermentans had glycoglycerolipids corresponding to GGPL-I and GGPL-III. Thin-layer chromatography-mass spectrometry and immunological analyses showed that these glycoglycerolipids which were derived from the isolated M. fermentans were identical with GGPL-I and GGPL-III previously obtained. This is the first report that shows mycoplasma has phosphocholine-containing glycoglycerolipids.     

Kaposi's sarcoma-associated herpesvirus ori-Lyt-dependent DNA replication: involvement of host cellular factors

Herpesvirus lytic DNA replication requires both the cis-acting element, the origin, and trans-acting factors, including virally encoded origin-binding protein, DNA replication enzymes, and auxiliary factors. Two lytic DNA replication origins (ori-Lyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified, and two virally encoded proteins, namely, RTA and K8, have been shown to bind to the origins. In this study, we sought to identify cellular factors that associate with ori-Lyt by using DNA affinity purification and mass spectrometry. This approach led to identification of several cellular proteins that bind to KSHV ori-Lyt. They include topoisomerases (Topo) I and II, MSH2/6, RecQL, poly(ADP-ribose) polymerase I (PARP-1), DNA-PK, Ku86/70 autoantigens, and scaffold attachment factor A (SAF-A). RecQL appears to associate with prereplication complexes and be recruited to ori-Lyt through RTA and K8. Topoisomerases, MSH2, PARP-1, DNA-PK, and Ku86 were not detected in prereplication complexes but were present in replication initiation complexes on ori-Lyt. All these cellular proteins accumulate in viral replication compartments in the nucleus, indicating that these proteins may have a role in viral replication. Topo I and II appear to be essential for viral DNA replication as inhibition of their activities with specific inhibitors (camptothecin and ellipticine) blocked ori-Lyt-dependent DNA replication. Furthermore, inhibition of PARP-1 with chemical inhibitors (3-aminobenzamide and niacinamide) resulted in decreased ori-Lyt-dependent DNA replication, whereas hydroxyurea, which raises PARP-1 activity, caused an increase in the DNA replication, suggesting a positive role for PARP-1 in KSHV lytic DNA replication.     

PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1^−/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP inhibitor or PARP-1–specific small interfering RNAs show similar results. The expression of human PARP-1 restores fork slowing in PARP-1^−/− DT40 cells. PARP-1 affects SSB repair, homologous recombination (HR), and nonhomologous end joining; therefore, we analyzed the effect of CPT on DT40 clones deficient in these pathways. We find that fork slowing is correlated with the proficiency of HR-mediated repair. Our data support the presence of a novel checkpoint pathway in which the initiation of HR but not DNA damage delays the fork progression.     

Delocalization of nucleolar poly(ADP-ribose) polymerase-1 to the nucleoplasm and its novel link to cellular sensitivity to DNA damage

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme activated by binding to DNA breaks, which causes PARP-1 automodification. PARP-1 activation is required for regulating various cellular processes, including DNA repair and cell death induction. PARP-1 involved in these regulations is localized in the nucleoplasm, but approximately 40% of PARP-1 can be found in the nucleolus. Previously, we have reported that nucleolar PARP-1 is delocalized to the nucleoplasm in cells exposed to DNA-damaging agents. However, the functional roles of this delocalization in cellular response to DNA damage is not well understood, since this approach simultaneously induces the delocalization of PARP-1 and its automodification. We therefore devised an approach for separating these processes. Unmodified PARP-1 was first delocalized from the nucleolus using camptothecin. Then, PARP-1 was activated by exposure of cells to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). In contrast to treatment with MNNG alone, delocalization of PARP-1 by CPT, prior to its activation by MNNG, induced extensive automodification of PARP-1. DNA repair activity and consumption of intracellular NAD⁺ were not affected by this activation. On the other hand, activation led to an increased formation of apoptotic cells, and this effect was suppressed by inhibition of PARP-1 activity. These results suggest that delocalization of PARP-1 from the nucleolus to the nucleoplasm sensitizes cells to DNA damage-induced apoptosis. As it has been suggested that the nucleolus has a role in stress sensing, nucleolar PARP-1 could participate in a process involved in nucleolus-mediated stress sensing.     

Differential Responses of Single Cells and Aggregates of Mycoplasmas to Ultraviolet Irradiation

Except for Mycoplasma fermentans strain PG 18, single-cell suspensions of M. arthritidis, M. fermentans (ATCC 19989), M. hominis type 1, M. orale types 1 and 2, M. pneumoniae, and M. salivarium were inactivated exponentially by ultraviolet (UV) irradiation, in contrast to broth cultures containing clusters of elementary bodies. The susceptibility of the mycoplasmas was unaffected by storage at 2-4 C and at -70 C, by sonication, and by filtration. The rate of inactivation was dependent on the intensity of the radiations but independent of the concentration of the cells. Therefore, single-cell suspensions of these mycoplasmas could be differentiated from aggregates of cells by exponential inactivation of the colony-forming units (CFU). By this criterion, the CFU of M. arthritidis in the exponential phase of growth consisted of single cells, in contrast to the other species in which the CFU contained two or more elementary bodies. Even though the cultures of M. fermentans (PG 18) were grown from single cells, they were not homogeneous in their susceptibility to UV light. Neither were cultures of M. arthritidis and M. orale type 1 grown from single cells which had survived irradiation.     

Synthesis and Biological Evaluation of 7‐Alkenyl Homocamptothecins as Potent Topoisomerase I Inhibitors

Homocamptothecin (hCPT) is a camptothecin (CPT) derivative with a seven‐membered β‐hydroxylactone E ring, which shows higher lactone stability and improves topoisomerase I (Topo I) inhibition activity. In an attempt to improve the antitumor activity of homocamptothecins, a series of 7‐alkenyl‐homocamptothecin derivatives was designed and synthesized based on a semisynthetic route starting from CPT. Most of the synthesized compounds exhibit higher cytotoxic activities on the A‐549 tumor cell line than topotecan (TPT). Some compounds such as 2a and 2o show a broad in vitro antitumor spectrum and exhibit superior Topo I‐inhibition activity.     

Bacillus cereus DNA topoisomerase I and IIIalpha: purification, characterization and complementation of Escherichia coli TopoIII activity

The Bacillus cereus genome possesses three type IA topoisomerase genes. These genes, encoding DNA topoisomerase I and IIIα (bcTopo I, bcTopo IIIα), have been cloned into T7 RNA polymerase-regulated plasmid expression vectors and the enzymes have been overexpressed, purified and characterized. The proteins exhibit similar biochemical activity to their Escherichia coli counterparts, DNA topoisomerase I and III (ecTopo I, ecTopo III). bcTopo I is capable of efficiently relaxing negatively supercoiled DNA in the presence of Mg²⁺ but does not possess an efficient DNA decatenation activity. bcTopo IIIα is an active topoisomerase that is capable of relaxing supercoiled DNA at a broad range of Mg²⁺ concentrations; however, its DNA relaxation activity is not as efficient as that of bcTopo I. In addition, bcTopo III is a potent DNA decatenase that resolves oriC-based plasmid replication intermediates in vitro. Interestingly, bcTopo I and bcTopo IIIα are both able to compensate for the loss of ecTopo III in E.coli cells that lack ecTopo I. In contrast, ecTopo I cannot substitute for ecTopo III under these conditions.     

Mycoplasma fermentans glycolipid-antigen as a pathogen of rheumatoid arthritis

Mycoplasma fermentans has been suspected as one of the causative pathogenic microorganisms of rheumatoid arthritis (RA) however, the pathogenic mechanism is still unclear. We, previously, reported that glycolipid-antigens (GGPL-I and III) are the major antigens of M. fermentans. Monoclonal antibody against the GGPL-III could detect the existence of the GGPL-III antigens in synovial tissues from RA patients. GGPL-III antigens were detected in 38.1% (32/84) of RA patient’s tissues, but not in osteoarthritis (OA) and normal synovial tissues. Immunoelectron microscopy revealed that a part of GGPL-III antigens are located at endoplasmic reticulum. GGPL-III significantly induced TNF-α and IL-6 production from peripheral blood mononulear cells, and also proliferation of synovial fibroblasts. Further study is necessary to prove that M. fermentans is a causative microorganism of RA; however, the new mechanisms of disease pathogenesis provides hope for the development of effective and safe immunotherapeutic strategies based on the lipid-antigen, GGPL-III, in the near future.     

Characterization of an IS Element in <Emphasis Type="Italic">Mycoplasma orale</Emphasis> That Is Highly Homologous to <Emphasis Type="Italic">M. fermentans</Emphasis> ISLE

In a previous study, using a primer set designed from Mycoplasma fermentans, we amplified a PCR fragment from Mycoplasma orale similar to the 206-bp DNA fragment amplified from M. fermentans insertion-sequence-like element (ISLE). The presence of this similar ISLE fragment has the potential to cause confusion in the PCR diagnosis of M. fermentans and M. orale, which have significantly different clinical scenarios. An ISLE from three different M. orale strains was amplified by using a primer set designed from sequence within the left and right terminal stem and loop (S&L) structures flanking the ISLE of M. fermentans. Sequence analysis showed that the M. orale ISLE is 93% identical to that of M. fermentans at the nucleotide level and codes for two open reading frames also found in the M. fermentans ISLE. This is the first finding that two different mycoplasma species harbor highly homologous IS elements. This finding has great significance in clinical diagnosis and suggests a possibility of horizontal transfer of an IS element between two different mycoplasma species. Received: 17 April 2002 / Accepted: 9 July 2002     

Design,synthesis and biological research of novel N-phenylbenzamide-4-methylamine acridine derivatives as potential topoisomerase I/II and apoptosis-inducing agents

A series of novel N-phenylbenzamide-4-methylamine acridine derivatives were designed and synthesized based initially on the structure of amsacrine (m-AMSA). Molecular docking suggested that the representative compound 9a had affinity for binding DNA topoisomerase (Topo) II, which was comparable with that of m-AMSA, and furthermore that 9a could have preferential interactions with Topo I. After synthesis of 9a and analogues 9b-9f, these were all tested in vitro and the synthesized compounds displayed potent antiproliferative activity against three different cancer cell lines (K562, CCRF-CEM and U937). Among them, compounds 9b, 9c and 9d exhibiting the highest activity with IC₅₀ value ranging from 0.82 to 0.91 μM against CCRF-CEM cells. In addition, 9b and 9d also showed high antiproliferative activity against U937 cells, with IC₅₀ values of 0.33 and 0.23 μM, respectively. The pharmacological mechanistic studies of these compounds were evaluated by Topo I/II inhibition, western blot assay and cell apoptosis detection. In summary, 9b effectively inhibited the activity of Topo I/II and induced DNA damage in CCRF-CEM cells and, moreover, significantly induced cell apoptosis in a concentration-dependent manner. These observations provide new information and guidance for the structural optimization of more novel acridine derivatives.     

Propolis cinnamic acid derivatives induce apoptosis through both extrinsic and intrinsic apoptosis signaling pathways and modulate of miRNA expression

Propolis cinnamic acid derivatives have a number of biological activities including anti-oxidant and anti-cancer ones. In this study, we aimed to elucidate the mechanism of the anti-cancer activity of 3 representative propolis cinnamic acid derivatives, i.e., Artepilin C, Baccharin and Drupanin in human colon cancer cell lines. Our study demonstrated that these compounds had a potent apoptosis-inductive effect even on drug-resistant colon cancer cells. Combination treatment of human colon cancer DLD-1 cells with 2 of these compounds, each at its IC₂₀ concentration, induced apoptosis by stimulating both intrinsic and extrinsic apoptosis signaling pathways. Especially, Baccharin plus Drupanin exhibited a synergistic growth-inhibitory effect by strengthening both intrinsic and extrinsic apoptotic signaling transduction through TRAIL/DR4/5 and/or FasL/Fas death-signaling loops and by increasing the expression level of miR-143, resulting in decreased expression levels of the target gene MAPK/Erk5 and its downstream target c-Myc. These data suggest that the supplemental intake of these compounds found in propolis has enormous significance with respect to cancer prevention.