High Molecular Weight Fragments Research Articles

A Role of the Ca2+/Mg2+-dependent Endonuclease in Apoptosis and Its Inhibition by Poly(ADP-ribose) Polymerase

Apoptosis is characterized by various cell morphological and biochemical features, one of which is the internucleosomal degradation of genomic DNA. The role of the human chromatin-bound Ca(2+)- and Mg(2+)-dependent endonuclease (CME) DNAS1L3 and its inhibition by poly(ADP-ribosyl)ation in the DNA degradation that accompanies apoptosis was investigated. The nuclear localization of this endonuclease is the unique feature that distinguishes it from other suggested apoptotic nucleases. Purified recombinant DNAS1L3 was shown to cleave nuclear DNA into both high molecular weight and oligonucleosomal fragments in vitro. Furthermore, exposure of mouse skin fibroblasts expressing DNAS1L3 to inducers of apoptosis resulted in oligonucleosomal DNA fragmentation, an effect not observed in cells not expressing this CME, as well as in a decrease in cell viability greater than that apparent in the control cells. Recombinant DNAS1L3 was modified by recombinant human poly(ADP-ribose) polymerase (PARP) in vitro, resulting in a loss of nuclease activity. The DNAS1L3 protein also underwent poly(ADP-ribosyl)ation in transfected mouse skin fibroblasts in response to inducers of apoptosis. The cleavage and inactivation of PARP by a caspase-3-like enzyme late in apoptosis were associated with a decrease in the extent of DNAS1L3 poly(ADP-ribosyl)ation, which likely releases DNAS1L3 from inhibition and allows it to catalyze the degradation of genomic DNA.

Regression Rate Behavior of Hybrid Rocket Solid Fuels

An experimental investigation of the regression-rate characteristics of hydroxyl-terminated polybutadiene (HTPB) solid fuel burning with oxygen was conducted using a windowed, slab-geometry hybrid rocket motor. A real-time, x-ray radiography system was used to obtain instantaneous solid-fuel regression rate data at many axial locations. Fuel temperature measurements were made using an array of 25- πm e ne-wire embedded thermocouples. The regression rates displayed a strong dependence on axial location near the motor head-end. At lower mass e ux levels, thermal radiation was found to signie cantly ine uence the regression rates. The regression rates werealso affected by theadditionofactivated aluminum powder.A 20%by weightaddition of activated aluminum to HTPB increased the fuel mass e ux by 70% over that of pure HTPB. Correlations were developed to relate the regression rate to operating conditions and port geometry for both pure HTPB and for HTPB loaded with certain fractions of activated aluminum. Thermocouple measurements indicated that the fuel surface temperatures for pureHTPBwerebetween930 and1190 K.TheHTPBactivationenergywasestimatedat11.5 kcal/mole,suggesting that the overall regression process is governed by physical desorption of high-molecular weight fragments from the fuel surface.

DNA damage and DNA damage-inducible protein Gadd45 following ischemia in the P7 neonatal rat

Cerebral ischemia in adult rodents leads to the production of several types of lesions in the genomic DNA, followed by the activation of the damage-response indicator Gadd45. Our purpose was to investigate the structural changes that occur in chromatin DNA and repair processes after ischemic injury in neonatal brain. Neonatal ischemia was induced by the permanent left MCA occlusion in association with 1 h occlusion of the left common carotid artery in 7-day-old Wistar pups. Oligonucleosome fragments that are recognized as the characteristic DNA ladder was observed in a delayed fashion. Double-strand breaks result in high molecular weight fragments of 50- and 300-kbp as demonstrated by pulsed-field gel electrophoresis, and visualized by the TUNEL assay at 24 h of recovery. In contrast, DNA single-strand breaks, shown by the use of DNA polymerase I-mediated biotin-dATP nick translation were not so abundant. Gadd45 immunoreactivity was sequentially increased in vulnerable neurons in the infarct (4 to 24 h) and in sublethally injured neurons in the penumbra (24–48 h). Taken together, these findings suggest that Gadd45 responds to DNA damage following neonatal ischemia. Furthermore, repairing processes seem to be more active in the penumbra and therefore Gadd45 could have also a protective role in cerebral ischemia.

Light‐induced Damage in the Retina: Differential Effects of Dimethylthiourea on Photoreceptor Survival, Apoptosis and DNA Oxidation

In the rat, photoreceptor cell death from exposure to intense visible light can be prevented by prior treatment with antioxidants. In this study we subjected albino rats raised in dim cyclic light and rats made more susceptible to light damage by rearing in darkness to exposures of green light that led to similar losses of photoreceptor cells. Rhodopsin and photoreceptor DNA, indicators of the number of surviving photoreceptor cells, were determined at various times over a period of 14 days after light exposure. Fragmentation of DNA was determined over a similar time course by neutral and alkaline agarose gel electrophoresis. Apoptosis in retinal DNA was measured by quantitating the appearance of 180 base pair (bp) nucleosomal fragments. Oxidation of DNA was measured by electrochemical detection of the nucleoside 8-hydroxydeoxyguanosine (8-OHdG) after separation by high-performance chromatography. For albino rats reared in dim cyclic light, 24 h of intense light exposure resulted in the loss of 50% rhodopsin and photoreceptor cell DNA. In dark-reared rats, the losses were 40%, respectively, after only 3 h of intense light treatment. In both cases pretreatment with the antioxidant dimethylthiourea (DMTU) prevented rhodopsin and photoreceptor cell DNA loss. The kinetics of the light-induced apoptosis depended markedly on the rearing environment of the rats. The DNA ladders appeared within 12 h of the onset of intense light in the rats reared in dim cyclic light. In these rats the 180 bp fragment was at two-thirds of its maximum intensity immediately after 24 h of light exposure and reached the maximum 12 h later. Dimethylthiourea partially inhibited ladder formation in rats reared in dim cyclic light and delayed the time of appearance of the 180 bp maximum by 6 h. By contrast, in rats reared in darkness the 180 bp fragment was undetected immediately after 3 h of light exposure and reached its maximum 2 days later. Pretreatment with DMTU completely eliminated DNA ladders in these rats. Alkaline gel electrophoresis revealed a pattern of single-strand DNA breaks, with relatively high molecular weight fragments, 6 h after light exposure of dark-reared rats. Single-strand DNA breaks in cyclic light rats corresponded with the onset of apoptotic ladders, but peak values preceded by 12 h the peak of DNA ladder formation. The quantity of 8-OHdG in retinal DNA remained close to control values in all samples with the exception of a peak of twice the control value 18 h after light exposure in the dark-reared rats and a value 60% higher 16 days after exposure in cyclic light animals. Dimethylthiourea had no effect on the amount of oxidized purine in any of the samples. The differences between dark-reared rats and rats reared in dim cyclic light in the kinetics of DNA fragmentation and in their response to treatment with DMTU is consistent with previous observations of fundamental differences in retinal cell physiology in these animals. In dim light-reared rats, the pathway to apoptosis may be qualitatively different from the pathway to net photoreceptor loss in rats reared in darkness. The lack of effect of DMTU on 8-OHdG formation suggests that the oxidation of DNA bases is not a causal factor in light-mediated photoreceptor cell death.

Prevention of depurination during elution facilitates the reamplification of DNA from differential display gels.

DNA fragments that show a pattern of differential expression on differential display gels must be eluted from the gel matrix and reamplified to enable further analysis. Elution is usually achieved by heating excised gel slices in a small volume of either water or TE. Here we show that this elution step can adversely affect the ability of the eluted DNA to act as a template for PCR reamplification, probably via the process of depurination. Simply switching to an elution solvent designed to minimise depurination (PCR buffer) facilitates the elution of intact DNA fragments. This improvement is likely to be most beneficial when eluting higher molecular weight fragments (e.g. those >500 bp), in situations where the amount of DNA in an excised gel slice is limited or when contaminating differential display products co-migrate with the differentially expressed product.

Distinct mechanisms underlay DNA disintegration during apoptosis induced by genotoxic and nongenotoxic agents in neuroblastoma cells

Exposure of mouse NB-2a neuroblastoma cells to genotoxic (etoposide or cytosine arabinoside) or nongenotoxic challenges (serum deprivation or okadaic acid) resulted in progressive cell death with biochemical and morphological characteristics typical of apoptosis. Apoptotic cell death induced by nongenotoxic agents was associated with the disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal-DNA fragments, while the formation of HMW-DNA fragments, but not oligonucleosomal-DNA ladder accompanied apoptosis induced by genotoxic agents. Combination of genotoxic and nongenotoxic insults, i.e. incubation of etoposide-treated cells in the serum-free medium, resulted in an additive effect on the profile of DNA disintegration, which involved both HMW fragmentation pattern as in etoposide alone treated cells and the oligonucleosomal-DNA ladder observed with serum-deprived cells. On the other hand, incubation of serum-deprived cells in the presence of Zn 2+-ions led to the abrogation of internucleosomal DNA fragmentation but accumulation of HMW-DNA fragments. Differences in the pattern of DNA fragmentation were reproducible in a cell free apoptotic system after treatment of isolated normal nuclei with cytosolic extracts prepared from the cells treated with genotoxic or nogenotoxic apoptotic inducers. Cell free experiments also revealed that activities responsible for the formation of HMW- and oligonucleosomal-DNA fragments are separable in cytosolic extract prepared from the serum-deprived cells. Finally, DNA fragmentation induced by nongenotoxic apoptotic inducers was effectively prevented by cycloheximide and suramin, while both cycloheximide and suramin had only a slight inhibitory effect on DNA fragmentation induced by genotoxic agents. The results presented suggest that distinct pathways underlay disintegration of nuclear DNA during apoptosis induced by genotoxic and nongenotoxic inducers, and that the formation of HMW- and oligonucleosomal-DNA fragments proceeds via separate mechanisms in NB-2a neuroblastoma cells.

Hyperphosphorylation of replication protein A middle subunit (RPA32) in apoptosis.

Replication protein A (RPA) is a trimeric single-stranded DNA (ssDNA)-binding complex of eukaryotic cells that plays an important role in DNA metabolism by stabilising single-stranded regions of DNA. The functionally important binding activity towards ssDNA is mainly localised on the large subunit, RPA70, whereas the middle subunit, RPA32, appears to have a regulatory function. It has been shown previously that RPA32 is phosphorylated both during the S-phase of a normal cell cycle and in response to DNA damage. In this study we demonstrate that phosphorylation of RPA32 is rapidly induced during apoptotic cell death of Jurkat T-lymphocytes, resulting in a hyperphosphorylated form with reduced electrophoretic mobility. In contrast, the large subunit of RPA is neither modified nor cleaved during apoptosis. Phosphorylation of RPA32 begins in parallel to the degradation of DNA to high molecular weight fragments, and slowly continues until late apoptosis. Experiments with specific kinase inhibitors indicate that RPA32 hyperphosphorylation requires the activities of DNA-dependent protein kinase and of a cyclin-dependent protein kinase. Interestingly, the hyperphosphorylated, but not the less phosphorylated forms of RPA32, sediments independently from the trimeric complex in sucrose gradients under high ionic strength, and is not bound to the complex in immunoprecipitation assays.

Delayed oligodendrocyte degeneration induced by brief exposure to hydrogen peroxide.

An in vitro model system of cultured oligodendrocytes was used to determine the susceptibility of these cells to oxidative stress induced by 15 min exposure to millimolar concentrations of hydrogen peroxide (H2O2). Following the exposure, the cells were incubated in normal growth medium, and analyzed at different time points. Although no cell loss was observed during the exposure period, there was a progressive depletion of adherent cells during the postexposure period as seen from either the number of recoverable nuclei, or from total RNA content of the cultures. Both the rate and the extent of cell deletion was directly dependent on H2O2 concentration. Cell death was preceded by structural alterations in the nuclear envelope resulting in "fragile" nuclei which disintegrated during isolation. Northern blot analysis showed that the expression of myelin-specific genes was rapidly downregulated in H2O2-treated cells. On the other hand, the expression of antiapoptotic gene, bcl-2 featured massive but transient upregulation. Oligodendrocyte degeneration also featured genomic DNA degradation into high molecular weight fragments, which are likely to represent cleaved chromosomal loops. The results demonstrate vulnerability of oligodendrocytes to oxidative stress that induces rapid degeneration and ultimately leads to delayed cell death. This feature is highly relevant to oligodendrocyte damage and depletion following ischemic, traumatic, or inflammatory insults to the central nervous system (CNS).

De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus.

The relationship between co-suppression and DNA methylation was explored in transgenic plants showing inducible co-suppression following infection with a cytoplasmically replicating RNA virus. Induction resulted in a loss of transgene mRNA and resistance to further infection, factors typical of post-transcriptional gene silencing. In infected plants, de novo methylation of the transgene appeared to precede the onset of resistance and only occurred in plants where the outcome was co-suppression. The methylation was limited to sequences homologous to the viral RNA and occurred at both symmetric and non-symmetric sites on the DNA. Although methylation is predicted to occur in mitotic cells, the virus was found not to access the meristem. A diffusible sequence-specific signal may account for the epigenetic changes in those tissues.

Distinct mode of apoptosis induced by genotoxic agent etoposide and serum withdrawal in neuroblastoma cells

Here we compared the features of apoptosis induced by DNA-damaging agent, etoposide, and by withdrawal of the growth factors in NB 2a neuroblastoma cells. We showed that serum deprivation and etoposide induced a distinct pattern of regulation of c-Fos, c-Jun and p53 protein levels, as well as the differential changes in DNA-binding activity of AP-1 and NF-κB transcription factors. The late phase of apoptesis induced by serum withdrawal was associated with disintegration of nuclear DNA both into high molecular weight (HMW) and oligonucleosomal DNA fragments, whereas etoposide induced the formation of HMW-DNA fragments without internucleosomal DNA cleavage. Incubation of etoposide-treated cells without serum resulted in an additive effect on the pattern of DNA fragmentation. Differences in DNA fragmentation profiles induced by serum withdrawal and etoposide in NB 2a cells were reproducible in nonproliferating cerebellar granule cells and also in a cell free system assay after treatment of isolated normal nuclei with cytosolic extracts prepared from serum-deprived or etoposide-treated cells. Both HMW and oligonucleosomal DNA fragmentation in serum-deprived cells was inhibited by aurintricarboxylic acid and was completely abrogated by cycloheximide. In contrast, DNA fragmentation in etoposide-treated cells was insensitive to the inhibitory effect of aurintricarboxylic acid, and was not prevented by cycloheximide. Our results indicate that in NB 2a neuroblastoma cells etoposide and serum withdrawal induce a distinct mode of apoptosis which is associated with a distinct pattern of regulation of immediately early response genes in the early phase, and with recruitment of different mechanisms for DNA disintegration in the late phase of apoptosis.

The use of capillary viscometry, reducing end-group analysis, and size exclusion chromatography combined with multi-angle laser light scattering to characterize endo-1,4-β- d-glucanases on carboxymethylcellulose: a comparative evaluation of the three methods

Three methods were used to characterize seven purified endo-1,4-β- d-glucanases derived from different microbial sources ( Trichoderma reesei, Thermomonospora fusca, and Acidothermus cellulolyticus) on carboxymethylcellulose (CMC). The methods included capillary viscometry, reducing end-group analysis, and high performance size exclusion chromatography combined with multi-angle laser light scattering (HPSEC-MALLS). The investigation was performed with the objective of comparative evaluation of the different methods for characterizing endoglucanases, particularly in relation to their endo- and exomode of action. The measurement of the initial rate of reducing end-group formation using disodium 2,2′-bicinchoninate (BCA) was found to be the most accurate method for determination of endoglucanase activity. The BCA method was highly sensitive, simple to perform, and directly gave the number of bonds broken, thus allowing for expression of endoglucanase activity in international units (μmol of β-1,4-glucosidic bonds hydrolyzed in 1 min during the initial period of hydrolysis). The viscometric method was simple to perform and highly sensitive for the internal bonds cleaved, but did not account for the hydrolysis of CMC near the chain end, and thus only allowed for expression of endoglucanase activity in arbitrary viscometric units. The HPSEC-MALLS technique provided the number-average molecular weight ( M n) of CMC, thus allowing the quantification of the number of the bonds broken during degradation of CMC; however, reproducibility of the method was low, especially for the high-molecular weight fragments of CMC at the beginning of hydrolysis. As hydrolysis proceeded to the more advanced stages, the HPSEC-MALLS method gave an overestimated (compared to the reducing end-group analysis) values for M n, probably due to insufficient sensitivity of the light-scattering detector for the low-molecular weight products of CMC degradation. The combined use of the three methods allowed characterization of endoglucanases according to their selectivity for hydrolysis of internal bonds within a CMC molecule which was expressed as the ratio of the initial rate of viscosity decrease to the initial rate of glucosidic bonds broken. This ratio was found to be unique for each endoglucanase, and, therefore, no universal equation could be established for all endoglucanases for conversion of arbitrary viscometric units to international units of activity.

Disassembly of genome of higher eukaryotes: pulsed-field gel electrophoretic study of initial stages of chromatin and DNA degradation in rat liver and thymus nuclei by VM-26 and selected proteases

Nuclei were treated with VM-26, topoisomerase II inhibitor, or exogenous proteases. Initial stages of multi-step chromatin degradation were analyzed by pulsed-field gel (PFG) electrophoresis. VM-26 induced the total genome breakdown into discrete chromatin fragments containing 0.3 Mbp DNA. Treatment of nuclei with recombinant cathepsin B and chymotrypsin also revealed 0.3-Mbp DNA fragments which were mediated by endonucleolysis. Longer incubation of nuclei with chymotrypsin exhibited the appearance of 0.05-Mbp DNA fragments and their oligomers. Proteases might trigger the detachment of chromatin from nuclear matrix and contribute to the release or activation of the endonucleolytic activity leading to the initial degradation of the DNA into high-molecular weight fragments. The fragments observed correspond in size to the suggested chromatin higher-order structures. The presented data imply that the initial stages of chromatin degradation represents in reality the genome disassembly into two basic units of genome topology—0.3-Mbp DNA chromatin loop-domains which are presumably the bits of biological information, and fundamental 0.05-Mbp DNA loopsize units which might generate the functional loops of different sizes.

Higher spontaneous apoptotic index in small cell compared with non-small cell lung carcinoma cell lines; lack of correlation with Bcl-2/Bax

Spontaneous apoptosis was assessed in ten small-cell (SCLC) and five non-small cell (NSCLC) lung carcinoma cell lines by the TUNEL assay and chromatin cleavage. TUNEL staining showed significantly higher apoptotic index (AI) in SCLC (2–20%) compared with NSCLC lines (0.2–1%) in untreated exponentially growing cells. Six out of ten SCLC and none of the NSCLC showed DNA fragmentation when analysed by agarose gel electrophoresis. Field inversion pulse gel electrophoresis was used in a subset of cell lines and showed the presence of high molecular weight fragments in untreated SCLC lines U-1285 and U-1906 cells, but not in the NSCLC line U-1810. Important molecular determinants of apoptosis were studied by Western blot. Bcl-2 was detected at highest level in SCLC. There was no correlation between the ratio Bcl-2/Bax and AI in all tested cell lines. Neither p53 nor c-Myc protein status correlated to AI. Pro-caspase-3 was expressed in all cell lines without correlation to AI and no difference between the SCLC and NSCLC groups was found. In conclusion, this study shows a high degree of spontaneous apoptosis in SCLC lines compared to NSCLC lines unrelated to Bcl-2/Bax ratio.

Lemon albedo cell walls contain distinct populations of pectic hairy regions

Alcohol insoluble solids from lemon albedo were extracted to obtain a chelating agent soluble pectin fraction (ChSS), a diluted sodium hydroxide soluble pectin fraction (ASS) which was separated into a water soluble and water insoluble part (ASSws and ASSwi), and an insoluble residue (Residue). ChSS, ASSws, ASSwi and Residue represented 64.3%, 10.4%, 0.4% and 8.1% of the galacturonic acid residues present in the lemon albedo, respectively. The pectin fractions and the residue were characterised by their sugar composition, degrees of methylation and acetylation, and molecular weight distribution. After extensive digestion with polygalacturonase and pectinesterase, the remaining high molecular weight fragments, representing the so-called pectic hairy regions (HR), were isolated and further characterised. After cold alkaline treatment (de-esterification), HR shown to be accessible to a further degradation by rhamnogalacturonan hydrolase (RGase) and the digests were analysed by high-performance size-exclusion chromatography (HPSEC) and high-performance anion-exchange chromatography (HPAEC). The various HR fractions differed in their degradation pattern: typically 2–4% of all glycosidic linkages were hydrolysed. The amount of RGase oligomers formed from the various fractions decreased in the order these were extracted while the degree of galactose substitution of the oligomers increased. Indications were found for the presence of small quantities of xylogalacturonan fragments within the pectic hairy regions of lemon albedo. On the basis of these results the structure of lemon pectin is compared with the structure of pectins extracted from other sources.

Electrophoretic DNA analysis for the detection of apoptosis.

There are many techniques available for the detection of apoptotic cells; some are based on morphological changes, others on biochemical events. However, electrophoretic detection of the systematic cleavage of DNA into oligonucleosomal multimers of 180-200 bp remains the "hallmark" of apoptosis. Conventional constant field agarose gel electrophoresis of DNA from apoptotic cells can be used to resolve the multimers into the characteristic DNA ladders indicative of apoptotic cell death. More recently, it has become clear that the generation of the lower molecular weight oligonucleosomal DNA is preceded by the generation of higher molecular weight fragments. In some cell types, DNA cleavage proceeds no further than the formation of 300 and/or 50 kbp cleavage products. DNA fragmentation of this size can only be resolved using a form of pulsed-field gel electrophoresis. Basic "starter" protocols for conventional and pulsed field electrophoresis for the detection of apoptotic cell DNA are described in this article.

The extracellular polysaccharide of Pichia (Hansenula) holstii NRRL Y-2448: the phosphorylated side chains

The exopolysaccharide produced by Pichia (Hansenula) holstii NRRL Y-2448 is composed of a phosphomannan core to which oligosaccharide diester phosphate side chains are appended. The oligosaccharides of the side chains were released as oligosaccharide phosphates and neutral oligosaccharides by mild hydrolysis with aqueous acetic acid and aqueous hydrogen fluoride, respectively. The liberated oligosaccharide phosphates were studied by NMR spectroscopy and by electrospray and fast atom bombardment mass spectrometry. The structures of the neutral oligosaccharides were determined by 1D and 2D NMR spectroscopic experiments. Further insight into the length of the side chains was obtained from a matrix assisted laser desorption ionisation–time of flight mass spectrometric study of high and low molecular weight fragments obtained from partial acid hydrolysis of the native polysaccharide.

Low level of polymorphism detected by SSR probes in bread wheat

In‐gel hybridization patterns were studied in a set of nine diverse bread wheat (Triticum aestivum L. em. Thell) genotypes using 23 simple sequence repeat (SSR) probes in combination with 14 different restriction enzymes. Multilocus fingerprints due to SSR probes, shown earlier to be characteristic of a majority of plant genomes, were not obtained and only a very low level of polymorphism was detected when using as many as 142 probe‐enzyme combinations. The hybridization of a prominent solitary high molecular weight fragment (> 23 kb) with a number of SSR probes suggested the presence of these SSRs (microsatellites) within the long stretches of repeated DNA sequences. This indicates that the genome of bread wheat differs from that of other plants in the organization and distribution of SSRs and that SSR probes detect very little polymorphism.

Analysis of nuclear degradation during lens cell differentiation.

Lens cells demonstrate a terminal differentiation process with loss of their organelles including nuclei. Chromatin disappearance is characterised by the same changes as most apoptotic cells, i.e. condensation of chromatin and cleavage into high molecular weight fragments and oligonucleosomes. The endo-deoxyribonucleases (bicationic (Ca2+, Mg2+), mono-cationic (Ca2+ or Mg2+) and acidic non-cationic dependent nucleases) are present in lens fibre cells. Our results suggest that the acidic non-cationic nuclease (DNase II) plays a major role in chromatin cleavage. This nuclease, known to be lysosomal, is found in lens fibre nuclei and only an antibody directed against DNase II inhibits the acidic DNA cleavage of lens fibre nuclei. In addition, there must be another DNase implicated in the process which is not DNase I but appears to be a Ca2+, Mg2+ dependent molecule. Regulation of these DNase activities may be accomplished by the effect of post-translational modifications, acidic pH, mitochondrial release molecules, growth factors or oncogenes. Finally, fibre cells lose organelles without cytoplasmic elimination. The survival of these differentiated cells might be due to the action of survival factors such as FGF 1.

Role of Protein Tyrosine Phosphorylation in Etoposide-Induced Apoptosis and NF-κB Activation

When a human myeloid cell line, U937, was incubated with etoposide (10 μg/mL), morphologically apoptotic cells first appeared at 3 hr and increased with time to 50% at 6 hr. Pretreatment of U937 cells for 30 min with a potent tyrosine kinase inhibitor, herbimycin A (10 μM), significantly suppressed the appearance of apoptotic morphological changes. Concomitantly, herbimycin A pretreatment prevented both high molecular weight and internucleosomal DNA fragmentation induced by etoposide. Two major bands at 30 and 66 kDa with enhanced tyrosine phosphorylation inhibited by herbimycin A were detectable after 30 min of incubation with etoposide. In addition, herbimycin A prevented etoposide-induced NF-κB activation. The expressions of Bcl-2 and Bax, on the other hand, were not affected by herbimycin A pretreatment. Herbimycin A was also found to inhibit 1-β- d-arabinofuranosylcytosine-induced apoptotic changes and NF-κB activation. These results suggest that activation of tyrosine kinase(s) may play an important role in apoptotic processes induced by a variety of anti-cancer drugs.

Natural resistance of acute myeloid leukemia cell lines to mitoxantrone is associated with lack of apoptosis

The purpose of this study was to characterize mitoxantrone-induced cytotoxicity in KG1a and TF-1, two P-glycoprotein expressing AML cell lines which display early differentiation phenotypes, compared to more mature HL-60 and U937 cells. KG1a and TF-1 cells were found to be 30-40-fold more resistant to mitoxantrone than HL-60 and U937 cells. Uptake and efflux of mitoxantrone were similar for all cell lines. Moreover, a potent P-glycoprotein blocker (PSC833) had no impact on either accumulation or efflux. No differences were found in the appearance and removal of mitoxantrone-induced DNA-protein complexes. These results suggest that resistance of KG1a and TF-1 cells is not related to a decreased interaction between mitoxantrone and topoisomerase II. Further studies showed that the mechanisms of cell death were different for sensitive and resistant cell lines. Thus, mitoxantrone induced rapid apoptotic cell death in sensitive cells as indicated by characteristic morphological changes and both high molecular weight and internucleosomal DNA fragmentation. In contrast, mitoxantrone induced a G2-M block in resistant cells followed by a progressive loss of viability with necrotic features. Neither oligonucleosomal nor large DNA fragments were detected in these cells during a post-treatment period of up to 96 h. Finally, drug-induced activation of the AP-1 transcription factor was higher in resistant cell lines than in sensitive ones whereas activation of NF-kappaB was comparable. Therefore, our study provides evidence that certain AML cells display natural resistance to mitoxantrone which is independent of drug transport and drug-target interactions but appears to be associated with the inability of the drug to induce apoptosis in these cells.