Drastic reduction of topoisomerase II alpha associated with major acquired resistance to topoisomerase II active agents but minor perturbations of cell growth

V511 and V513 cell lines, derived from Chinese hamster V79 cells following alkylating agent mutagenesis and subsequent selection with VP-16, showed resistance to cytotoxicity and DNA strand breaks induced by topoisomerase (topo) II inhibitors and were resistant to VP-16-induced sister chromatid exchanges. They showed no amplification of the multidrug-resistant p-glycoprotein. In a kinetoplast-DNA decatenation assay, V511 and V513 showed 51% and 49% topo II activity relative to parental V79 cells, respectively. By western-blot analysis all three logarithmically growing cell lines showed similar levels of topo II beta (M(r) 180,000), which increased as cells progressed to quiescence. In contrast, immunoreactive levels of topo II alpha (M(r) 170,000) were 6.8% in V511 and 62.4% in V513 relative to V79. V511 showed drastically decreased topo II alpha in both log growth and quiescence. In a second approach, immunoreactive topo II was analyzed in different phases of the cell cycle in logarithmically growing cells fractionated by fluorescence-activated cell sorting. All cell lines demonstrated relatively stable topo II beta throughout the cell cycle. Topo II alpha showed little cell cycle variation in V79 or V513. However, in V511, it was only detectable at low levels in G2/M phase. When cell growth parameters were measured, V511 and V513 showed a 17% increase in cell doubling time relative to V79. These studies indicate that cells with a drastic reduction in topo II alpha (V511) or mutant topo II alpha (V513) but with normal levels of topo II beta show only minor perturbations of cell growth.     

Maternal and fetal plasma concentrations of endothelin, lipidhydroperoxides, glutathione peroxidase and fibronectin in relation to abnormal umbilical artery velocimetry

The topoisomerase II alpha (topo II alpha) enzyme is the target for several chemotherapeutic agents, including etoposide, teniposide, mitoxantrone, and doxorubicin (topo II poisons). The enzyme also is a marker of cell proliferation. Most cases of Hodgkin's disease (HD) are responsive to combination chemotherapy regimes that include topo II poisons such as doxorubicin. Immunoperoxidase methods for detection of the topo II alpha isoenzyme are now available for use in formalin-fixed, paraffin-embedded tissues, which may provide information about the proliferative capacity and possible sensitivity of tumors to drugs that target topo II. We used a specific antibody to analyze subsets of HD for topo II alpha staining patterns. Formalin-fixed blocks from 49 cases of HD, including 20 nodular sclerosis (NS), 14 mixed-cellularity (MC), and 15 lymphocyte-predominant (LP) subtypes, were analyzed by dual staining for topo II in combination with monoclonal antibodies against Reed-Sternberg (RS) cells consisting of CD15 for the NS and MC subtypes and CD20 for LP lymphocytic and histiocytic (L & H) cells. The number of morphologically appropriate cells coexpressing the RS or L & H marker and topo II alpha was quantitated. Positive nuclear staining for topo II alpha in RS or L & H cells was seen in 100% of cases, irrespective of subtype. Coexpression of CD15 and topo II alpha was seen in 58.4% of the RS cells or mononuclear variants in NSHD cases and 68.4% in MCHD cases. No significant difference in the percentage of neoplastic cells expressing topo II alpha was found between NS and MC subtypes. Cases of LPHD showed coexpression of CD20 and topo II alpha in 84.4% of the L & H cells, a significant increase over the level of tumor cell coexpression seen in NSHD and MCHD (P < .001). Only one case was found to have a low (< 25% of tumor cell coexpression) level of topo II alpha expression. Immunohistochemical detection of a high level of topo II alpha expression in HD, irrespective of subtype, suggests a molecular explanation for the excellent response of most HD to standard combination chemotherapy, which can include topo II poisons. The LP subtype has a higher expression of topo II alpha in the neoplastic cell population than do NS or MC subtypes, perhaps indicating increased sensitivity of these tumors to topo II poisons. It may be possible to identify subsets of HD that are more or less sensitive to conventional chemotherapeutic regimes, which would help in the selection of appropriate treatment.     

Overexpression of human DNA topoisomerase II alpha by fusion to enhanced green fluorescent protein

DNA topoisomerase (topo) II alpha is a major target for many anticancer agents. However, progress towards understanding how these agents interact with this enzyme in human cells and how resistance to these agents arises is greatly impeded by difficulties in expressing this gene. Here, we report on achieving a high level of expression of a full-length human topo II alpha gene in human cells. We started with the topo II alpha cDNA driven by a strong cytomegalovirus promoter and transiently transfected HeLa cells. Although topo II alpha mRNA was consistently detected in transfected cells, no exogenous topo II alpha protein was detected. By contrast, when the same cDNA was fused to an enhanced green fluorescent protein (EGFP), we detected a high level of expression at both mRNA and protein levels. The exogenous topo II alpha was localized to cell nuclei as expected, indicating that the fusion protein is properly folded. Furthermore, overexpression of the EGFP-topo II alpha fusion protein increased the sensitivity of the transfected cells to teniposide, suggesting that it functions as the endogenous counterpart. Thus, in addition to being used as a gene tag, the GFP fusion approach may be generally applicable for expressing genes, such as topo II alpha, that are difficult to express by conventional methods.     

Antisense-mediated repression of DNA topoisomerase II expression leads to an impairment of HIV-1 replicative cycle

Previously, we have observed a strong restriction of the Moloney murine leukemia virus (MoMLV) replicative cycle in a cell line displaying resistance to topoisomerase II (topo II)-interactive drugs. Resistance towards these antitumoral inhibitors was associated with decreased expression and activity of topo II, suggesting that such a decrease may be responsible for MoMLV restriction. To more specifically assess the role of topo II during the retroviral cycle, we have used the antisense strategy to obtain a selective decrease of cellular topo II expression. The RNA antisense was isolated from a retroviral library expressing random fragments of human topo II (alpha form). This system allowed us to investigate the HIV-1 replicative cycle in two related human CEM cell lines expressing different levels of topo II. Expression of the enzyme is decreased four- to sixfold following formation of a sense-antisense RNA hybrid. Repression of the topo II enzyme results in an impairment of the HIV-1 replicative cycle. Using the polymerase chain reaction, we showed that the number of integration events was decreased in cells repressing the enzyme, although viral DNA synthesis and circularization were equivalent to those in the parent cells.     

Facilitation of apoptosis by cyclosporins A and H, but not FK506 in mouse bronchial eosinophils

This study was undertaken to clarify whether or not binding to cyclophilin is a prerequisite for cyclosporin A-induced modulation of apoptotic cell death in eosinophils. Eosinophils were isolated from bronchoalveolar lavage fluid of mice challenged with inhaled allergen after sensitization. Apoptosis was determined by analysing the DNA content. At 72 h, 99% of the cells had died without addition of cytokines, whereas 55-60% of the cells survived in the presence of 10 U/ml recombinant murine interleukin 5 or recombinant murine granulocyte macrophage-colony stimulation factor (GM-CSF). Apoptotic cell death at 72 h in the presence of 10 U/ml interleukin 5 was increased by addition of cyclosporin H, an analogue of cyclosporin A without cyclophilin binding activity, in a concentration-dependent (0.3 to 3 microM) manner. The increase in apoptosis elicited by cyclosporin A and cyclosporin H took place also in the presence of 10 U/ml GM-CSF but to a lesser extent. There was a significant augmentation of apoptosis in eosinophils cultured in the presence of each cytokine for 72 h or longer. Tacrolimus (FK506) failed to augment apoptotic cell death. Thus, it is unlikely that binding of cyclosporin A to cyclophilin accounts for the increased apoptosis induced by cyclosporin A and its analogue in eosinophils. The increase in apoptosis induced by cyclosporin A, but not FK506, in activated eosinophils from the airways may be attributed to the anti-asthmatic effects of cyclosporin A.     

Effect of Clostridium difficile toxin A on human colonic lamina propria cells: early loss of macrophages followed by T-cell apoptosis

We have previously shown that Clostridium difficile toxin A induces detachment of human colonic epithelial cells from the basement membrane and subsequent cell death by apoptosis. Because these cells require adhesion-dependent signalling from the extracellular matrix for survival, their detachment from the basement membrane by other means also induces apoptosis. The role of toxin A in the induction of apoptosis therefore remains to be determined. In addition, sensitivities to C. difficile toxin A of lamina propria lymphocytes, macrophages, and eosinophils, which lie below the surface epithelium, are not known. In contrast to epithelial cells, these lamina propria cells do not require adhesion-dependent signalling from the extracellular matrix for survival, and this may allow the mechanisms of toxin A-induced cell death to be further investigated. The aim of this study was to investigate the effect of purified C. difficile toxin A on human colonic lamina propria T cells, macrophages, and eosinophils. We show that C. difficile toxin A induces loss of viability in isolated colonic lamina propria cell preparations containing the three different cell types in a dose- and time-dependent fashion. Exposure to high concentrations of the toxin led to loss of macrophages within 72 h. T-lymphocyte and eosinophil cell death was prominent at later time points and occurred by apoptosis. Exposure to toxin A also induced the production of tumor necrosis factor alpha by the isolated colonic lamina propria cells. However, the presence of neutralizing antibodies to this cytokine did not influence C. difficile toxin A-induced T-cell apoptosis. Moreover, purified T cells also underwent apoptosis following exposure to toxin A, implying that apoptosis occurred as a consequence of a direct interaction between T cells and the toxin. Our studies suggest that C. difficile toxin A is capable of suppressing human colonic mucosal immune responses by inducing early loss of macrophages followed by T-cell apoptosis.     

Genomic fluidity is a necessary event preceding the acquisition of tumorigenicity during spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells

We have shown that both DNA topoisomerase (topo) IIalpha and beta are in vivo targets for etoposide using a new assay which directly measures topo IIalpha and beta cleavable complexes in individual cells after treatment with topo II targeting drugs. CCRF-CEM human leukemic cells were exposed to etoposide for 2 hr, then embedded in agarose on microscope slides before cell lysis. DNA from each cell remained trapped in the agarose and covalently bound topo II molecules from drug-stabilized cleavable complexes remained associated with the DNA. The covalently bound topo II was detected in situ by immunofluorescence. Isoform-specific covalent complexes were detected with antisera specific for either the alpha or beta isoform of topo II followed by a fluorescein isothiocyanate-conjugated second antibody. DNA was detected using the fluorescent stain Hoechst 33258. A cooled slow scan charged coupled device camera was used to capture images. A dose-dependent increase in green immunofluorescence was observed when using antisera to either the alpha or beta isoforms of topo II, indicating that both isoforms are targets for etoposide. We have called this the TARDIS method, for trapped in agarose DNA immunostaining. Two key advantages of the TARDIS method are that it is isoform-specific and that it requires small numbers of cells, making it suitable for analysis of samples from patients being treated with topo II-targeting drugs. The isoform specificity will enable us to extend our understanding of the mechanism of interaction between topo II-targeting agents and their target, the two human isoforms.     

The role of activin type I receptors in activin A-induced growth arrest and apoptosis in mouse B-cell hybridoma cells

Activins transduce their signals by binding to activin type I receptors and activin type II receptors, both of which contain a serine/threonine kinase domain. In this study, we established stable transfectants expressing two types of activin receptors, ActRI and ActRIB, to clarify the role of these receptors in activin signalling for growth inhibition in HS-72 mouse B-cell hybridoma cells. Over-expression of ActRI suppressed activin A-induced cell-cycle arrest in the G1 phase caused by inhibition of retinoblastoma protein phosphorylation through induction of p21CIP1/WAF1, a cyclin-dependent kinase inhibitor, and subsequent apoptosis. In contrast, HS-72 clones that over-expressed ActRIB significantly facilitated activin A-induced apoptosis. These results indicate that ActRI and ActRIB are distinct from each other and that the ActRI/ActRIB expression ratio could regulate cell-cycle arrest in the G1 phase and subsequent apoptosis in HS-72 cells induced by activin A.     

Phase I clinical and plasma and cellular pharmacological study of topotecan without and with granulocyte colony-stimulating factor

Topotecan, a semisynthetic water-soluble analogue of camptothecin, inhibits human topoisomerase I (topo I). We performed a Phase I clinical and plasma pharmacological study of topotecan administered by 24-h continuous infusion without and with granulocyte colony-stimulating factor (G-CSF). We also measured topo I-DNA complexes in peripheral blood mononuclear cells (PBMCs) in an attempt to correlate formation of topo I-DNA complexes in patients treated with topotecan with toxicity and/or response. One hundred four courses of topotecan at doses of 2.5-15.0 mg/m2 were administered to 44 patients with solid tumors. The maximum tolerated dose without G-CSF was 10.0 mg/m2; granulocytopenia was the dose-limiting toxic effect. The maximum tolerated dose could not be increased with G-CSF because of severe thrombocytopenia. Plasma pharmacology was obtained in 11 patients treated at 12.5 mg/m2 and 15.0 mg/m2. The topotecan lactone end-infusion plasma levels correlated strongly with the area under the curve. Lactone elimination was biexponential with a mean t1/2alpha of 28 min and a t1/2beta of 3.8 h at 12.5 mg/m2. Topo I-DNA complexes were measured before and after treatment in PBMCs from seven patients. Pretopotecan topo I-DNA complexes were available on two additional patients treated at 15 mg/m2. The mean increase in topo I-DNA complexes at the end of the topotecan infusion was 1.25 times the pretreatment value. There was a statistically significant relationship (P = 0.02) between lack of disease progression and the level of topo I-DNA complexes measured in PBMCs before therapy. For Phase II studies of minimally treated adults with solid tumors, the recommended topotecan starting dose administered by 24-h continuous infusion is 10 mg/m2 without G-CSF.     

Smad7 is an activin-inducible inhibitor of activin-induced growth arrest and apoptosis in mouse B cells

Members of the transforming growth factor-beta (TGF-beta) family, which includes the activins, relay signals from serine/threonine kinase receptors in membrane to nucleus via intracellular Sma- and Mad-related (Smad) proteins. Inhibitory Smad proteins were found to prevent the interaction between the serine/threonine kinase receptors and pathway-restricted Smad proteins. Smad7 was identified as a TGF-beta-inducible antagonist of TGF-beta signaling, and it may participate in a negative feedback loop to control TGF-beta signaling. Here we demonstrate that the mRNA expression of Smad7 is induced by activin A in mouse B cell hybridoma HS-72 cells, which undergo growth arrest and apoptosis upon exposure to activin A. The ectopic expression of mouse Smad7 in HS-72 cells suppressed the activin A-induced cell cycle arrest in the G1 phase by abolishing the activin A-induced expression of p21(CIP1/WAF1) and hypophosphorylation of retinoblastoma protein. Furthermore, Smad7 expression suppressed activin A-induced apoptosis in HS-72 cells. Thus, our data indicate that Smad7 is an activin A-inducible antagonist of activin A-induced growth arrest and apoptosis of B lineage cells.     

Effects of drugs affecting endogenous amines or cyclic nucleotides on ethanol withdrawal head twitches in mice

1 Twenty-four hours after ethanol withdrawal, dependent mice exhibited frequent head twitching. Naive mice exhibited similar twitching 15 min after treatment with 5-hydroxytryptophan (5-HTP) or 6 h after alpha-methyl-p-tyrosine (AMPT). Ethanol lessened the incidence of head twitches induced by any of these treatments. 5-HTP and AMPT each increased the incidence of head twitches induced by withdrawal of ethanol from dependent mice. 2 Drugs that affect the amount or activity of endogenous amines or cyclic nucleotides modified the incidence of head twitches. Nearly all drugs acted in the same direction on twitching elicited by any of these three treatments. 3 The incidence was lessened by: (a) methysergide, methergoline, MA 1420, p-chlorophenylalanine and p-chloroamphetamine; (b) dopamine, noradrenaline, L-DOPA, amphetamine and apomorphine; (c) hyoscine and nicotine; and (d) adenosine triphosphate, dibutyryl cyclic adenosine-3',5'-monophosphate (db cyclic AMP) and prostaglandins E1 and E2. 4 The incidence was increased by: (a) acetylcholine, carbachol and physostigmine; and (b) guanosine triphosphate, dibutyryl cyclic guanosine monophosphate (db cyclic GMP), theophylline and 3-isobutyl-1-methyl-xanthine. 5 These findings suggest that head twitching induced by these three treatments arises from a common biochemical mechanism, which may ultimately be a change in favour of cyclic GMP of the balance between this nucleotide and cyclic AMP within appropriate neurones. This imbalance appears to be elicited or increased by 5-hydroxytryptamine and acetylcholine and to be decreased by dopamine, noradrenaline and E prostaglandins. 6 Neither actinomycin D nor cycloheximide, given during the induction of ethanol dependence, altered the incidence of head twitches after ethanol withdrawal.     

Relationship between amine-carboxyboranes and TNF alpha for the regulation of cell growth in different tumor cell lines

The amine-carboxyboranes were shown to be synergistic with tumor necrosis factor alpha (TNF alpha) in cytotoxicity and inhibition of DNA synthesis in select types of cancer cells depending on the presence of a TNF alpha high affinity receptor on the membrane of the cell. Initially both TNF alpha and the amine-carboxyboranes reduce the influx of calcium but later cause a significant increase intracellularly. This influx is not linked with the amine-carboxyborane activating the calcitonin receptor in the tumor cells. Neither the agents nor TNF alpha directly inhibits DNA topoisomerase II activity but both did cause decreased phosphorylation of the enzyme by protein kinase C (PKC). The two agents caused synergistic inhibition. This event correlated with increased DNA protein linked breaks, DNA fragmentation and cell death. These protein linked breaks are additive with etoposide's effects but the latter agent's mechanism is different than phosphorylation of topoisomerase II. There was no evidence that the DNA fragmentation was caused by a calcium induced endonuclease enzyme in these cancer cells. The low-molecular weight amine-carboxyboranes appear to play an identical function as TNF alpha in its role to cause DNA breaks and fragmentation to cause apoptosis.     

Bcl-2 and adenovirus E1B 19 kDA protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose) polymerase

The E1A oncoproteins of adenovirus type 5 are potent inducers of apoptotic cell death. To manifest growth promoting and transforming properties, therefore, E1A requires the co-expression of a suppressor of apoptosis. During normal viral infection, this function is provided by the E1B 19 kDa protein. However, the cellular suppressor Bcl-2 can substitute for 19K during infection, and both proteins can effectively cooperate with E1A to facilitate transformation of primary cells in culture. How E1A induces apoptosis and at what point(s) on this pathway Bcl-2 and E1B 19K act are not presently known. Here, we demonstrate that E1A-induced apoptosis is accompanied by specific endo-proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), an event that is linked to the Ced-3/ICE apoptotic pathway in other systems. PARP cleavage was also observed in p53-null cells infected with 19K- virus expressing 13S E1A. In addition to PARP cleavage, expression of E1A caused processing of the zymogen form of CPP32, a Ced-3/ICE protease that cleaves PARP and is required for apoptosis in mammalian cells. These events were prevented when E1A was co-expressed with E1B 19K or BCL-2, which places these suppressors of apoptosis either at or upstream of processing of pro-CPP32.     

Cyclic AMP protects against staurosporine and wortmannin-induced apoptosis and opioid-enhanced apoptosis in both embryonic and immortalized (F-11kappa7) neurons

The mechanism by which opiates affect fetal development is unknown, but one potential target is the programmed cell death (apoptosis) pathway of neurons. Apoptosis was induced in both primary neuronal cultures from embryonic day 7 cerebral hemispheres of chick brain (E7CH) and the F-11kappa7 cell line (an immortalized mouse neuroblastoma x dorsal root ganglion hybrid stably transfected to overexpress kappa-opioid receptors) by either staurosporine or the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. Cells pretreated with either the mu-specific opioid agonist morphiceptin (E7CH) or the kappa-specific opioid agonist U69,593 (F-11kappa7) for 24 h showed increased apoptosis in response to staurosporine or wortmannin when compared with non-pretreated cells. The effects of morphiceptin and U69,593 were time- and dose-dependent and antagonist-reversible, suggesting that they were receptor-mediated. Neither morphiceptin nor U69,593 by themselves had any measurable effect on cell viability or DNA fragmentation, and coaddition of opiates at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. Time course studies indicated a maximal opioid effect at a time (16-24 h) when inhibition of adenylate cyclase had been maximal for many hours. Addition of dibutyryl cyclic AMP either before or at the time of opioid addition protected against apoptosis and reduced fragmentation to levels seen for staurosporine plus dibutyryl cyclic AMP alone. The specificity for cyclic AMP was confirmed by showing protection with the specific agonist Sp-adenosine 3',5'-cyclic monophosphothioate and increased killing with the antagonist Rp-adenosine 3',5'-cyclic monophosphothioate. We conclude that the opioid enhancement of apoptosis is based on the inhibition of adenylate cyclase and that the effect is time-dependent.