Etoposide-induced DNA damage in human tumor cells: requirement for cellular activating factors.

Previous studies with the multidrug-resistant human HL60 cell line have shown a 3-4-fold decrease in VP-16 accumulation compared to the sensitive cell line, while the degree of resistance to VP-16 was 300-fold, indicating that other mechanisms of resistance are also operative. Since VP-16 has been shown to interfere with topoisomerase II activity, we have evaluated VP-16-dependent DNA strand break formation in the drug-sensitive and -resistant HL60 cells. Studies reported here show that the drug-resistant HL60 cells are extremely resistant to VP-16-dependent DNA cleavage compared to the sensitive cells. This decrease in DNA cleavage activity in the presence of VP-16 was, in part, related to a 2-3-fold decrease in both the amount and activity of topoisomerase II in the resistant cell line compared to the sensitive cells. Nuclei from the resistant cell line were markedly more resistant to VP-16-dependent DNA cleavage than the WT cell nuclei. Interestingly, WT nuclei were found to be relatively more resistant to VP-16-induced DNA cleavage than the intact WT cells. Addition of WT cytosolic proteins to WT nuclei, however, significantly stimulated VP-16-dependent DNA cleavage and slightly increased DNA cleavage in resistant cell nuclei. In contrast, cytosolic proteins from the resistant cells had no effect on DNA cleavage in nuclei isolated from either cell line. These observations indicate that a decrease in the amount and activity of topoisomerase II in resistant HL60 cells translates into a decrease in VP-16-dependent DNA breakage and contributes to the resistance to VP-16. Furthermore, the cytosolic fraction from WT cells contains some factor, not present in the resistant cells, which is necessary for the maximal drug-induced DNA cleavage.     

Characterization of an unusual mutant of human melanoma cells resistant to anticancer drugs that inhibit topoisomerase II

The topoisomerase II inhibitor, VP-16 (etoposide), is an important component in many chemotherapeutic regimens. To cahracterize resistance to this drug, the human melanoma cell line, FEM-X, was selected in multiple steps with VP-16. To prevent the development of typical multidrug resistance, an inhibitor of P-glycoprotein, the tiapamil analog, RO-11–2933, was added to the selections. The resultant clone FVP3 is 56-fold resistant to VP-16 and cross-resistant to doxorubicin (Adriamycin) (9-fold) and VM-26 (27-fold). These cells are also two- to fourfold resistant to m-AMSA, daunorubicin, and mitoxantrone. FVP3 is not resistant to the P-glycoprotein substrate vinblastine, does not express the MDR1 gene at detectable levels, and does not show reduced ³H-VP-16 accumulation. Unlike other cell lines that exhibit resistance to inhibitors of topoisomerase II, FVP3 has the same level of topoisomerase II expression and activity as FEM-X. Using live cells treated with VP-16, band depeletion assays and KCI/SDS precipitation assays show that topoisomerase II from FVP3 is much less susceptible to drug-induced cleavable complex formation than is that from FEM-X. This difference in sensitivity to VP-16 is also detected using lysates from disrupted cells, but not with isolated nuclei devoid of cytoplasmic and membrane components. In addijtion, the topoisomerase li present in nuclear edtracts from FVP3 is not resistant to the effects of VP-16 as measured by: (1)inhibition of strand passing activity during decatenation of kinetoplast DNA, (2) drug-induced linearization of plasmid DNA, and (3) immunodepletion by VP-16. These results suggest that some component of the cytoplasm or cellular membranes, or a factor depleted from nuclei during their isolation, is responsible for the resistance to VP-16 in FVP3. © 1993 Wiley-Liss, Inc.     

Combined Fourier transform infrared and Raman spectroscopic approach for identification of multidrug resistance phenotype in cancer cell lines

Cancer cells escape cytotoxic effects of anticancer drugs by a process known as multidrug resistance (MDR). Identification of cell status by less time-consuming methods can be extremely useful in patient management and treatment. This study aims at evaluating the potentials of vibrational spectroscopic methods to perform cell typing and to differentiate between sensitive and resistant human cancer cell lines, in particular those that exhibit the MDR phenotype. Micro-Raman and Fourier transform infrared (FTIR) spectra have been acquired from the sensitive promyelocytic HL60 leukemia cell line and two of its subclones resistant to doxorubicin (HL60/DOX) and daunorubicin (HL60/DNR), and from the sensitive MCF7 breast cancer cell line and its MDR counterpart resistant to verapamil (MCF7/VP). Principal components analysis (PCA) was employed for spectral comparison and classification. Our data show that cell typing was feasible with both methods, giving two distinct clusters for HL60- and MCF7-sensitive cells. In addition, phenotyping of HL60 cells, i.e., discriminating between the sensitive and MDR phenotypes, was attempted by both methods. FTIR could not only delineate between the sensitive and resistant HL60 cells, but also gave two distinct clusters for the resistant cells, which required a two-step procedure with Raman spectra. In the case of MCF7 cell lines, both the sensitive and resistant phenotypes could be differentiated very efficiently by PCA analysis of their FTIR and Raman point spectra. These results indicate the prospective applicability of FTIR and micro-Raman approaches in the differentiation of cell types as well as characterization of the cell status, such as the MDR phenotype exhibited in resistant leukemia cell lines like HL60 and MCF7.     

Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II

HL-60/AMSA is a human leukemia cell line that is 100 times more resistant to the cytotoxic actions of the antineoplastic, topoisomerase II-reactive DNA intercalating acridine derivative amsacrine (m-AMSA) than is its parent HL-60 line. HL-60/AMSA cells are minimally resistant to etoposide, a topoisomerase II-reactive drug that does not intercalate. Previously we showed that HL-60 topoisomerase II activity in cells, nuclei, or nuclear extracts was sensitive to m-AMSA and etoposide, while HL-60/AMSA topoisomerase II was resistant to m-AMSA but sensitive to etoposide. Now we show that purified topoisomerase II from the two cell lines exhibits the same drug sensitivity or resistance as that in the nuclear extracts although the magnitude of the m-AMSA resistance of HL-60/AMSA topoisomerase II in vitro is not as great as the resistance of the intact HL-60/AMSA cells. In addition HL-60/AMSA cells are cross-resistant to topoisomerase II-reactive intercalators from the anthracycline and ellipticine families and the pattern of sensitivity or resistance to the cytotoxic actions of the various topoisomerase II-reactive drugs is paralleled by topoisomerase II-reactive drug-induced DNA cleavage and protein cross-link production in cells and the production of drug-induced, topoisomerase II-mediated DNA cleavage and protein cross-linking in isolated biochemical systems. In addition to its lowered sensitivity to intercalators, HL-60/AMSA differed from HL-60 in 1) the susceptibility of its topoisomerase II to stimulation of DNA topoisomerase II complex formation by ATP, 2) the catalytic activity of its topoisomerase II in an ionic environment chosen to reproduce the environment found within the living cell, and 3) the observed restriction enzyme pattern on a Southern blot probed with a cDNA for human topoisomerase II. These data indicate that an m-AMSA-resistant form of topoisomerase II contributes to the resistance of HL-60/AMSA to m-AMSA and to other topoisomerase II-reactive DNA intercalating agents. The drug resistance is associated with additional biochemical and molecular alterations that may be important determinants of cellular sensitivity or resistance to topoisomerase II-reactive drugs.     

Identification of molecular targets for selective elimination of TRAIL‐resistant leukemia cells. From spots to in vitro assays using TOP15 charts

The resistance of malignant cells to chemotherapy calls for the development of novel anti‐cancer drugs. TNF‐related apoptosis‐inducing ligand (TRAIL) is a pro‐apoptotic cytokine, which selectively induces apoptosis in malignant cells. We derived two TRAIL‐resistant HL‐60 subclones, HL‐60/P1 and HL‐60/P2, from a TRAIL‐sensitive HL‐60 acute promyelocytic leukemia cell line. To identify therapeutically exploitable “weaknesses” of the TRAIL‐resistant leukemia cells that could be used as molecular targets for their elimination, we performed proteomic (2‐DE) analysis and compared both TRAIL‐resistant subclones with the original TRAIL‐sensitive HL‐60 cells. We identified over 40 differentially expressed proteins. To significantly narrow the lists of candidate proteins, we excluded proteins that are known to be often differentially expressed, regardless of experiment type and tissue (the so‐called “TOP15” proteins). Decreased expression of DNA replication and maintenance proteins MCM7 and RPA32 in HL‐60/P1 cells, and the marked down‐regulation of enzyme adenosine deaminase in HL‐60/P2 cells, suggests increased sensitivity of these cells to DNA‐interfering drugs, and adenosine and its homologues, respectively. In a series of in vitro assays, we confirmed the increased toxicity of etoposide and cisplatin to TRAIL resistant HL‐60/P1 cells, and adenosine and vidarabine to HL‐60/P2, compared with TRAIL‐sensitive HL‐60 cells.     

Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.

The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.     

Purification and characterization of an altered topoisomerase II from a drug-resistant Chinese hamster ovary cell line

The cytotoxicity and DNA damage induced by the epipodophyllotoxins and several intercalating agents appear to be mediated by DNA topoisomerase II. We have purified topoisomerase II to homogeneity both from an epipodophyllotoxin-resistant Chinese hamster ovary cell line, VpmR-5, and from the wild-type parental cell line. Immunoblots demonstrate similar topoisomerase II content in these two cell lines. The purified enzymes are dissimilar in that DNA cleavage by VpmR-5 topoisomerase II is not stimulated by VP-16 or m-AMSA. Furthermore, the VpmR-5 enzyme is unstable at 37 degrees C. Thus, the drug resistance of VpmR-5 cells appears to result from the presence of an altered topoisomerase II in these cells. Purified topoisomerase II from VPMR-5 and wild-type cells has the same monomeric molecular mass as well as equivalent catalytic activity with respect to decatenation of kinetoplast DNA. Etoposide (VP-16) inhibits the activity of both enzymes. Noncovalent DNA-enzyme complex formation, assayed by nitrocellulose filter binding, is also similar, as is protection from salt dissociation of this complex by ATP and VP-16. The data suggest a model in which the drug-resistant cell line, VpmR-5, has religation activity which is less affected by drug than that of the wild-type cells. Drug effect on DNA religation and catalytic activity are dissociated mechanistically. In addition, under certain circumstances, the "cleavable complex" observed following denaturation of a drug-stabilized DNA-enzyme complex may not adequately reflect the nature of the antecedent lesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-2 induces the activities of DNA topoisomerase I and DNA topoisomerase II in HuT 78 cells

The induction by interleukin-2 of DNA topoisomerase I and DNA topoisomerase II activities in the human T cell line HuT 78 was investigated. HuT 78 cells were treated with 1000 U of interleukin-2/ml, and extracts of the HuT 78 nuclei were prepared over a 24 h period. The extracts were assayed quantitatively for the activities of DNA topoisomerase I and DNA topoisomerase II. Three concomitant, transient increases of 3- to 11-fold in the specific activities of both DNA topoisomerase I and DNA topoisomerase II were observed following treatment with IL-2 at 0.5, 4, and 10 h after treatment with interleukin-2. The specific activities of both enzymes returned to base-line values after each of these transient increases. These results reveal that the activities of DNA topoisomerase I and DNA topoisomerase II are highly regulated in HuT 78 cells upon treatment with IL-2.     

Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform.

Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance in HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. We discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant, as measured by the decatenation of Crithidia fasciculata kinetoplast DNA, was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Total cellular topoisomerase II activity in HL-60/MX2 cells was only 50% lower than that in HL-60 cells, however, because the "cytosolic fraction" of the HL-60/MX2 nuclear preparation contained high levels of decatenating activity. Antisera to calf thymus topoisomerase II defined a distinctive immunoreactive pattern of topoisomerase II proteins in crude nuclear extracts from the HL-60/MX2 cells. Both alpha (170 kDa) and beta (180 kDa) forms of topoisomerase II were detected in the HL-60 cell extracts, but only the alpha form was detected in extracts from HL-60/MX2 cells. This finding was associated with the appearance of a new 160-kDa immunoreactive species in nuclear extracts from HL-60/MX2 but not HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-resistance of an amsacrine-resistant human leukemia line to topoisomerase II reactive DNA intercalating agents. Evidence for two topoisomerase II directed drug actions.

HL-60/AMSA is a human leukemia cell line that is 50-100-fold more resistant than its drug-sensitive HL-60 parent line to the cytotoxic actions of the DNA intercalator amsacrine (m-AMSA). HL-60/AMSA topoisomerase II is also resistant to the inhibitory actions of m-AMSA. HL-60/AMSA cells and topoisomerase II are cross-resistant to anthracycline and ellipticine intercalators but relatively sensitive to the nonintercalating topoisomerase II reactive epipodophyllotoxin etoposide. We now demonstrate that HL-60/AMSA and its topoisomerase II are cross-resistant to the DNA intercalators mitoxantrone and amonafide, thus strongly indicating that HL-60/AMSA and its topoisomerase II are resistant to topoisomerase II reactive intercalators but not to nonintercalators. At high concentrations, mitoxantrone and amonafide were also found to inhibit their own, m-AMSA's, and etoposide's abilities to stabilize topoisomerase II-DNA complexes. This appears to be due to the ability of these concentrations of mitoxantrone and amonafide to inhibit topoisomerase II mediated DNA strand passage at a point in the topoisomerization cycle prior to the acquisition of the enzyme-DNA configuration that yields DNA cleavage and topoisomerase II-DNA cross-links. In addition, amonafide can inhibit the cytotoxic actions of m-AMSA and etoposide. Taken together, these results suggest that the cytotoxicity of m-AMSA and etoposide is initiated primarily by the stabilization of the topoisomerase II-DNA complex. Other topoisomerase II reactive drugs may inhibit the enzyme at other steps in the topoisomerization cycle, particularly at elevated concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pivotal role of a DEVD-sensitive step in etoposide-induced and Fas-mediated apoptotic pathways.

We investigated the role of proteases in the pathway that leads from specific DNA damage induced by etoposide (VP-16), a topoisomerase II inhibitor, to apoptotic DNA fragmentation in the U937 human leukemic cell line. In a reconstituted cell-free system, Triton-soluble extracts from VP-16-treated cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This effect was inhibited by the tetrapeptide Ac-DEVD-CHO, a competitive inhibitor of the interleukin-1 beta-converting enzyme (ICE)-related protease CPP32, but was not influenced by Ac-YVAD-CHO and Ac-YVAD-CMK, two specific inhibitors of ICE. The three tetrapeptides inhibited Fas-mediated apoptotic DNA fragmentation in the cell-free system. Internucleosomal DNA fragmentation, triggered by either VP-16 or an anti-Fas antibody, was associated with proteolytic cleavage of the poly(ADP-ribose)polymerase (PARP), a decrease in the level of 32 kDa CPP32 proenzyme and the appearance of the CPP32 p17 active subunit. Conversely, the expression of Ich-1L, another ICE-like protease, remained stable in apoptotic U937 cells. Several cysteine and serine protease inhibitors prevented apoptotic DNA fragmentation by acting either upstream or downstream of the DEVD-sensitive protease(s) activation and PARP cleavage. We conclude that a DEVD-sensitive step, which could involve CPP32, plays a central role in the proteolytic pathway that mediates apoptotic DNA fragmentation in VP-16-treated leukemic cells at the crossing with Fas-mediated pathway.     

Oleanolic Acid Induces Apoptosis in Human Leukemia Cells through Caspase Activation and Poly（ADP-ribose） Polymerase Cleavage

It has been shown that Fructus Ligustri Lucidi （FLL）, a promising traditional Chinese medicine, can inhibit the growth of tumors. However, the effective component and molecular mechanism of FLL act to inhibit tumor proliferation are unclear. In this study, we demonstrated that oleanolic acid （OA）, a principal chemical component of FLL, inhibited the proliferation of human leukemia HL60 cells in culture. MTT assay showed that treatment of HL60 cells with FLL crude extracts or OA dramatically blocked the growth of target tumor cell in a time- and dose-dependent manner. Morphological changes of the nuclei and DNA fragmentation showed that apoptotic cell death occurred in the HL60 cells after treating with FLL extracts （20 mg/ml） or OA （3.65×10^-2 mg/ml）. Furthermore, flow cytometry assay showed that treatment of HL60 cells with FLL or OA caused an increased accumulation of G1 and sub-G1 subpopulations. Western blot analysis showed that caspase-9 and caspase-3 were activated, accompanied by the cleavage of poly （ADP-ribose） polymerase （PARP） in the target cells during FLL- or OA-induced apoptosis, These results suggest that OA acts as the effective component of FLL by exerting its cytotoxicity towards target tumor cells through activation of caspases and cleavage of PARP.     

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.     

The cytoplasmic trafficking of DNA topoisomerase IIalpha correlates with etoposide resistance in human myeloma cells

In this study we have investigated the role of topoisomerase (topo) IIalpha trafficking in cellular drug resistance. To accomplish this, it was necessary to separate the influence of cell cycle, drug uptake, topo protein levels, and enzyme trafficking on drug sensitivity. Thus, we developed a cell model (called accelerated plateau) using human myeloma H929 cells that reproducibly translocates topo IIalpha to the cytoplasm. Compared to log-phase cells, the cytoplasmic redistribution of topo IIalpha in plateau-phase cells correlated with a 10-fold resistance to VP-16 and a 40-60% reduction in the number of drug-induced double-strand DNA breaks. In addition, 7-fold more VP-16 was necessary to achieve 50% topo IIalpha band depletion, suggesting that there are fewer drug-induced topo-DNA complexes formed in quiescent cells than in log-phase cells. The total cellular amount of topo IIalpha and topo IIbeta protein in log- and plateau-phase cells was similar as determined by Western blot analysis. There was a 25% reduction in S-phase cell number in plateau cells (determined by bromodeoxyuridine (BrdU) incorporation), while there was no significant difference in the equilibrium concentrations of [(3)H]-VP-16 when log cells were compared with plateau cells. Furthermore, the nuclear/cytoplasmic ratio of topo IIalpha is increased 58-fold in accelerated-plateau H929 cells treated with leptomycin B (LMB) when compared to untreated cells. It appears that the nuclear-cytoplasmic shuttling of topo IIalpha, which decreases the amount of nuclear target enzyme, is a major mechanism of drug resistance to topo II inhibitors in plateau-phase myeloma cells.     

Hyperactivated m-calpain affects acquisition of doxorubicin resistance in breast cancer cells

Background

Doxorubicin is commonly using chemotherapeutic agents for breast cancer. However, doxorubicin has limitations in clinical use because of dose-dependent cardiotoxicity and drug resistance. Despite of previously reported studies about mechanisms of doxorubicin resistance including overexpression of P-gp and abnormal expression and mutation of topoisomerase IIα, resistance to this agent still abundantly occur and is regarded as a major obstacle to successful treatment.

The production of topoisomerase II-mediated DNA cleavage in human leukemia cells predicts their susceptibility to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)

Protein-associated DNA cleavage is produced in mammalian cells treated with active antileukemic DNA intercalating agents such as 4'(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). We have examined the ability of m-AMSA to produce DNA cleavage in 3 human myeloid leukemic cell lines with different sensitivities to the cytotoxic actions of m-AMSA to see if the magnitude of DNA cleavage correlated with the degree of m-AMSA sensitivity. DNA alkaline elution was used to quantify DNA cleavage. The amount of m-AMSA-induced DNA cleavage in the two lines sensitive to m-AMSA was 1-2 orders of magnitude greater than that in an m-AMSA-resistant leukemic line. The m-AMSA resistant line had been developed by prolonged exposure of one of the sensitive lines to m-AMSA. This finding was not secondary to a decreased uptake of m-AMSA in the resistant cell line. m-AMSA treatment of the nuclei isolated from the three lines produced DNA cleavage frequencies comparable to the cleavage frequencies produced by m-AMSA treatment of the whole cells from which the nuclei were isolated. The DNA cleaving ability stimulated by m-AMSA is thought to be mediated by drug-induced effects on topoisomerase II, a nuclear enzyme that mediates alterations in DNA conformation. Alterations in the manner in which this enzyme interacts with antineoplastic agents may explain the emergence of resistant cells following initially successful chemotherapy.     

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase IIα associated with nuclear matrices from wild-type and drug-resistant Chinese hamster ovary cell lines

Topo IIα is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo IIα associated with the nuclear matrix in drug-resistant SMR₁₆ and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo IIα in individual nuclear matrices. There are significant variations in topo IIα amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix–associated topo IIα than the resistant cell line matrices. Nuclear matrix–associated topo IIα from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo IIα in the same matrix; in fact they were inversely related. In situ whole-mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures. J. Cell. Biochem. 67:112–130, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of VP-16-induced DNA damage in isolated nuclei from L1210 cells

Based on the observation that VP-16-induced DNA damage can be demonstrated in isolated nuclei but not in purified DNA, and that this effect is temperature-dependent, it is postulated that the mechanism of action of VP-16 involves an essential intranuclear event, perhaps enzyme-mediated, which is a prerequisite for the cleavage of DNA. Using alkaline elution to assay single-strand breaks in isolated L1210 nuclei, we have further characterized conditions influencing this putative intranuclear reaction. We have found drug activity to be dependent on magnesium and pH and to be stimulated by low concentrations of ATP (0.05–1 mM), an effect which was not observed with a nonhydrolyzable analog of ATP. Heat-labile activity in a nuclear non-histone protein extract was critical to VP-16-mediated DNA damage. This new evidence lends further credence to the hypothesis that activity of an intranuclear enzyme, possessing characteristics consistent with a type II DNA topoisomerase, is a prerequisite for the cleavage of DNA by VP-16.     

DNA Fragmentation Induced by Protease Activation in p53-Null Human Leukemia HL60 Cells Undergoing Apoptosis Following Treatment with the Topoisomerase I Inhibitor Camptothecin: Cell-Free System Studies

We studied the role of proteases in apoptosis using a cell-free system prepared from a human leukemia cell line. HL60 cells are p53 null and extremely sensitive to a variety of apoptotic stimuli including DNA damage induced by the topoisomerase I inhibitor, camptothecin. We measured DNA fragmentation induced in isolated nuclei by cytosolic extracts using a filter elution assay. Cytosol from camptothecin-treated HL60 cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This fragmentation was suppressed by serine protease inhibitors. Serine proteases (trypsin, endoproteinase Glu-C, chymotrypsin A, and proteinase K) and papain by themselves induced DNA fragmentation in naive nuclei. This effect was enhanced in the presence of cytosol from untreated cells. Cysteine protease inhibitors (E-64, leupeptin, Ac-YVAD-CHO [ICE inhibitor]) did not affect camptothecin-induced DNA fragmentation. The apopain/Yama inhibitor, Ac-DEVD-CHO, and the proteasome inhibitor, MG-132, were also inactive both in the cell-free system and in whole cells. Interleukin-1β converting enzyme (ICE) or human immunodeficiency virus protease failed to induce DNA fragmentation in naive nuclei. Together, these results suggest that DNA damage activates serine protease(s) which in turn activate(s) nuclear endonuclease(s) during apoptosis in HL60 cells.     

Differential effects of Ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

Ａｐｏｐｔｏｓｉｓｏｒｐｒｏｇｒａｍｍｅｄｃｅｌｌｄｅａｔｈ(ＰＣＤ)ｉｓａｐｒｏｃｅｓｓｏｆｃｅｌｌｄｅｌｅｔｉｏｎｗｈｉｃｈｏｃｃｕｒｓｉｎｒｅｓｐｏｎｓｅｔｏａｎｕｍｂｅｒｏｆｃｙｔｏｔｏｘｉｃａｎｄｐｈｙｓｉｏｌｏｇｉｃａｌｌｙｒｅｌｅｖａｎｔｓｔｉｍｕｌｉ.Ｔｈｉｓｐｒｏｃｅｓｓｉｓｃｈａｒａｃｔｅｒｉｚｅｄｂｙｓｅｖｅｒａｌｅａｒｌｙｍｏｒｐｈｏｌｏｇｉｃａｌｔｅｒａｔｉｏｎｓｉｎｃｌｕｄｉｎｇｐｌａｓｍａａｎｄｎｕｃｌｅａｒｍｅｍｂｒａｎｅｂｌｅｂｂｉｎｇ.Ｅｎｄｏｇｅｎｏｕｓｅｎｄｏ…