Autonomic nervous function during haemodialysis assessed by spectral analysis of heart-rate variability

To investigate the effect of neuronal differentiation on the capacity of antisense oligonucleotides (AS-ODNs) to suppress the production of acetylcholinesterase (AChE) in rat pheochromocytoma cells, we tested seven 3'-phosphorothioated AS-ODNs targeted to ACHEmRNA and two control ODNs. Three different administration protocols were used: oligonucleotides were added at 1 microM for 24 hours to nondifferentiated PC12 cells, together with nerve growth factor (NGF) or 24 hours following NGF-induced cholinergic differentiation. The content of free thiol groups in lysed cells was measured to evaluate cell number, therefore, survival, and the rate of acetylthiocholine hydrolysis was the measure of AChE activity. Among nondifferentiated cells, over 95% survived treatment with 8 of 9 of the ODNs. Moreover, two AS-ODN suppressed AChE activity in non-differentiated PC12 cells by 16%-20% as compared with 10% suppression by control ODNs (P < or = 0.01). When added concurrently with NGF, one other AS-ODN suppressed AChE activity significantly better (28%) than the control ODNs (16%). Moreover, when added following NGF treatment, which induced a significant increase in AChE activity, four different AS-ODNs but not the control ODNs suppressed 20%-35% of the enhanced AChE activity (p < or = 0.01). Reduced levels of AChE mRNA but no difference in actin mRNA levels were observed by following the kinetics of RT-PCR amplification in differentiated PC12 cells treated with these four AS-ODNs, as compared with control cells. Our findings demonstrate a differentiation-related increase in the susceptibility of PC12 cells to inhibition by specific AS-ODNs, suggesting the use of this model system to select AS-ODNs for suppression of AChE levels in the treatment of neurodegenerative diseases associated with cholinergic malfunction.     

Biliary lipid composition in idiopathic bile acid malabsorption

Human colon cancer frequently develops liver metastasis. Matrilysin (MMP-7), the smallest member of the matrix metalloproteinase (MMP) family, is commonly produced by human colon carcinoma cells and has been suggested to be involved in the progression and metastasis of this type of cancer. In the present study, we tested the effect of a matrilysin-specific antisense phosphorothioate oligonucleotide on liver metastasis of the human colon carcinoma cell line WiDr in nude mice. In culture, the antisense oligonucleotide moderately inhibited the secretion of matrilysin by WiDr cells. Injection of WiDr cells into the spleen of nude mice produced many metastatic tumor nodules in the liver. When the antisense oligonucleotide was injected daily into the mice for 11 days, the formation of the metastatic tumor nodules was strongly inhibited in a dose-dependent manner. An inhibition of liver metastasis of over 70% was obtained at a dose of 120 micrograms of the oligonucleotide per mouse. The antisense oligonucleotide did not inhibit tumor growth in spleen and in liver. A scrambled control oligonucleotide had no effect on liver metastasis of WiDr cells. Our results demonstrate an important role of matrilysin in liver metastasis of human colon cancer and the therapeutic potential of matrilysin antisense oligonucleotides for the prevention of metastasis.     

Comparison of the MOS short form-12 (SF12) health status questionnaire with the SF36 in patients with rheumatoid arthritis

Cultured HL-60, HeLa S3 and WiDr cells grown in male BALB/c nu/nu mice were studied by conventional and field-inversion DNA gel electrophoresis (FIGE), as well as by means of cytomorphological approaches, including TdT-mediated dUTP nick end labeling (TUNEL) assay. Chemosensitivity tests revealed HL-60 to be sensitive to vindesine (VDS), and HeLa S3 and WiDr to mitomycin C (MMC). Although VDS-treated HL-60 exhibited condensation of chromatin and a DNA ladder, MMC-exposed HL-60 cells showed apoptotic figures without typical DNA ladders. With MMC-treated WiDr cells, neither DNA ladders nor apoptotic figures were observed. Cells characterized by chromatin condensation were TUNEL-positive in both treated and untreated cases with the exception of the MMC-treated WiDr case, in which many TUNEL-positive cells were observed without cytomorphological changes. On FIGE, DNA fragments of approximately 50, 300 and 400 kbp were detected in groups treated with both effective and ineffective drugs, as well as in untreated controls. Furthermore, change of the time parameters in FIGE resulted in different sizes (550 and 850 kbp) of DNA fragments. These findings indicate that i) cell death is not always detectable in terms of apoptotic figures or DNA oligonucleosomal fragmentation, ii) only the TUNEL assay is a reliable tool to detect DNA damage and, iii) FIGE does not provide accurate size profiles of macromolecular DNA fragments.     

The effect of brief illumination on intracellular free calcium concentration in cells with 5-aminolevulinic acid-induced protoporphyrin IX synthesis

The effect of illumination on intracellular free calcium concentration, [Ca2+]i, was studied in a cell line (WiDr cells) derived from a primary adenocarcinoma of the rectosigmoid colon. In these cells the biosynthesis of protoporphyrin IX was stimulated by 5-aminolevulinic acid to reach levels of 600-700 pmol of protoporphyrin IX per mg cell protein. A brief (1-min) exposure of the cells to light (70% of light energy at 340-380 nm) resulted in an increase in [Ca2+]i. This increase was not reversible over a period of at least 20 min following illumination. Elevation of [Ca2+]i most probably represented an influx of calcium ions from the medium to the cell, since it was completely abolished in the presence of extracellular EGTA. The increased [Ca2+]i did not reflect general membrane damage, as determined by trypan blue staining as well as measurement of the intercalation of ethidium bromide into cellular DNA, and neither did the sustained elevation of [Ca2+]i lead to any substantial loss of clonogenicity following illumination of protoporphyrin-containing cells. Together these results indicate that an increased [Ca2+]i level is not per se a cause of cell death during photodynamic therapy.     

Serum interleukin 4 and interleukin 10 levels in patients with chronic hepatitis C virus infection

The antiproliferative effect of 5-fluorouracil (5-FU) in colon cancer can be enhanced by interferons (IFN-alpha and IFN-gamma). The mechanisms by which IFNs modulate 5-FU activity are not completely elucidated. IFN-alpha may elevate the levels of the active 5-FU metabolite 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) in the cell, possibly leading to increased inhibition of the target enzyme thymidylate synthase (TS), which might enhance DNA damage. It has been shown that IFN-gamma can prevent 5-FU induced overexpression of TS. We studied IFN modulation in three colon cancer cell lines (SW948, WiDr, human; C26-10, murine) and the sublines WiDr/F and C26-10/F, which were adapted to low folate levels. A 1.5-fold increase in 5-FU sensitivity was observed in C26-10 and C26-10/F (by murine IFN-alpha, beta); in SW948, WiDr and WiDr/F (by human IFN-gamma) and in SW948 and WiDr/ F (by human IFN-alpha). In none of the cell lines did human IFN-alpha, IFN-gamma or murine IFN-alpha, beta increase FdUMP levels after exposure to 5-FU. TS activity, indirectly measured by incorporation of [6-3H]-deoxyuridine into DNA, was inhibited by 5-FU, but the IFNs did not enhance inhibition. DNA damage was measured as a drug-induced decrease of double-stranded (dss) DNA compared to control cells. After 5-FU exposure, dss DNA decreased to 60-75% in WiDr, WiDr/F and SW948 cells. Human IFN-alpha alone caused minimal DNA damage (95% dss DNA), but increased 5-FU-induced effects to 35-50% dss DNA. IFN-gamma did not cause DNA damage and did not enhance 5-FU-mediated DNA damage. Expression of TS protein, analysed by ELISA, was increased after 5-FU exposure of SW948 cells, but this increase was not affected by addition of either IFN-alpha or IFN-gamma. It is concluded that one of the mechanisms involved in modulation of 5-FU activity is the effect of IFN-alpha on 5-FU-mediated DNA damage, but for IFN-gamma no mechanism of action was found.     

Cholinergic, but not the rod pathway-related glycinergic (All), amacrine cells contain calretinin in the rat retina

Double-label immunocytochemistry was carried out on cryostat sections of rat retina to test for the presence of calretinin in cholinergic starburst and the rod pathway-related glycinergic (All) amacrine cells. All cholinergic cells contained calretinin, but calretinin-immunoreactive cells were much more numerous in both the inner nuclear and ganglion cell layers than the cholinergic cells. Glycinergic All amacrine cells have been found to contain calretinin in cat, monkey and rabbit retinas. Since All amacrine cells in rat can be selectively labeled with antibodies against parvalbumin, in a second experiment we attempted to colocalize these proteins. We found that calretinin- and parvalbumin-immunoreactive neurons belonged to distinct amacrine cell populations permitting the conclusion that, in the rat retina, All amacrine cells do not contain calretinin. The results indicate that even those amacrine cells of the mammalian retina that are highly conserved with respect to morphology and transmitter content, may differ with respect to other neurochemical characteristics, such as their calcium-binding proteins.     

Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotides on human melanoma cells in vitro and and in mice

BACKGROUND: Phosphorothioate oligodeoxynucleotides ([S]ODNs) contain a modified internucleoside phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used with some therapeutic success in animal models human leukemia; however, the potential for antisense [S]ODN treatment of solid tumors has only recently been explored. Purpose: We evaluated the effects of antisense [S]ODNs targeted to the c-myc oncogene on the proliferation of human melanoma cells in vitro and on the growth of human melanoma xenografts in CD-1 nude (nu/nu) mice, METHODS: The effects of 15-mer [S]ODNs containing c-myc sense, c-myc antisense, and two different scrambled sequences on the proliferation and viability of cultures of three established human melanoma cell lines (M14, JR8, and PLF2) were determined by measuring cell numbers and use of the trypan blue exclusion test. The induction of apoptosis in these cells following treatment with [S]ODNs was evaluated by fluorescence-activated cell sorter (FACS) analysis. FACS analysis was also used to determine the effects of [S]ODN treatment on the proliferation of primary cultures of a human melanoma explant (NG cells). The expression of c-Myc protein in cultured NG cells after treatment with [S]ODNs was examined by western blot analysis. The antitumor activity and the toxic effects of several [S]ODN treatment regimens were monitored by measuring differences in tumor weight (percent tumor weight inhibition), tumor growth rate (tumor growth inhibition), animal lifespan (percent increase in lifespan), the number of toxic deaths and the median number of long metastases in treated and control mice bearing NG xenografts. c-Myc protein expression in NG tumor cells following [S]ODN treatment was evaluated by FACS analysis, and the extent of apoptosis in these cells was determined by FACS analysis and morphologic examination. RESULTS: Treatment with antisense [S]ODNs, but not the others, inhibited the growth of all tested melanoma cultures in vitro; FACS analysis revealed that growth inhibition was associated with the induction of apoptosis. Antisense [S]ODN treatment also led to reduced celluLar levels of c-Myc protein. In vivo, [S]ODN antitumor activity and toxicity were dose and schedule dependent; however, only antisense [S]ODNs exhibited antitumor activity. Mice bearing NG xenografts treated with antisense [S]ODNs showed a marked inhibition of tumor growth, a reduction in the number of long metastases, and an increase in life span. Reduced levels of c-Myc protein and increased levels of apoptosis were also observed in NG tumor cells following antisense [S]ODN treatment. CONCLUSIONS: treatment of human melanoma cells and solid tumors with antisense [S]ODNs targeted to c-Myc inhibits their growth and is associated with the induction of apoptosis.     

Synchronization of cells in the S phase of the cell cycle by 3'-azido-3'-deoxythymidine: implications for cell cytotoxicity

The mechanism of synergy between 3'-azido-3'-deoxythymidine (AZT) and anticancer agents was investigated with emphasis on cell-cycle events. Exposure of exponentially growing WiDr human colon carcinoma cells to AZT resulted in synchronization of cells in the S phase of the cell cycle. Following treatment with AZT at 50 or 200 microM, 62% +/- 3% or 82% +/- 4% of the cells were in the S phase as compared with 36% +/- 2% in the control. Bromodeoxyuridine uptake studies revealed that the synchronized cells actively synthesized DNA. At concentrations of up to 200 microM, AZT produced a cytostatic rather than cytotoxic effect as indicated by viability and cell growth measurements. At 200 microM, AZT-induced synchronization was significant (P = < 0.001) after 12 h of drug exposure, reached a maximum at 24 h, and reversed to baseline levels by 72 h even in the continued presence of the drug. This indicates that AZT-induced cytostasis is a transient and reversible effect. The cell-cycle events seen with AZT in WiDr cells were also observed in eight of nine human tumor cell lines tested. Isobologram analysis of WiDr cells preexposed to AZT for 24 h and then exposed to either AZT-5-fluorouracil or AZT-methotrexate for a further 72 h revealed synergy between AZT and the anticancer agents, indicating that AZT-induced synchronization may have therapeutic benefits.     

Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line

It has been shown recently that expression of p21 is enhanced by paclitaxel. This cytotoxic compound induces mitotic spindle damage resulting in blockade of the mitotic cell cycle associated or not with apoptotic cell death. In the present study, we showed that, in MCF-7 cells, paclitaxel induced accumulation of p21 in cells with a G2/M DNA content, corresponding to cells either in abnormal mitosis or in an interphase-like state (decondensed chromatin) with multiple nuclei. In MCF-7 cells, the increase in p21 was subsequent to the mitotic arrest and was associated with the exit from abnormal mitosis leading to formation of cells with micronuclei. In this cell line, we noted a relationship between the elevation of p21 expression and the inhibition of p34cdc2 activity. High levels of p21 protein were also found to be associated with inactive p34cdc2/cyclin B protein complex after treatment with paclitaxel. Treatment with p21 antisense oligonucleotide partially blocked induction of p21 expression by paclitaxel and significantly reduced survival of MCF-7 cells exposed to this agent. In NIH-OVCAR-3 cells, which are deficient in basal and paclitaxel-induced p21 expression, paclitaxel led to a prolonged activation of p34cdc2 and a delayed mitotic exit associated with apoptotic cell death. These observations suggest that p21 is not required for the mitotic arrest in response to paclitaxel, but argue in favor of a role for this inhibitor in facilitating the exit from abnormal mitosis. This effectively enhances cell survival after paclitaxel-induced spindle damage.     

Interruption of a transforming growth factor alpha autocrine loop in Caco-2 cells by antisense oligodeoxynucleotides

BACKGROUND & AIMS: We have previously shown that Caco-2 cell proliferation is driven by basolateral membrane epidermal growth factor receptors. The aim of this study was to investigate whether autocrine production of transforming growth factor alpha (TGF-alpha) activates these receptors and stimulates proliferation using antisense oligodeoxynucleotides. METHODS: Caco-2 cells grown on microporous membranes or Jurkat cells were exposed to conventional or 5' cholesterol-modified oligodeoxynucleotides synthesized with random, antisense, or missense base sequences. Indices of proliferation were measured, including [3H]thymidine or [3H]uridine uptake for studies of short-term stimulation and the methylthiotetrazole assay as an index of cell number increase over longer periods. Secretion of TGF-alpha by cells was detected using a soft agar bioassay. RESULTS: Incubation with antisense oligodeoxynucleotides inhibited TGF-alpha secretion compared with controls. Random and missense oligodeoxynucleotides had no effect on proliferation. The TGF-alpha antisense oligodeoxynucleotides markedly inhibited proliferation, an effect that was abolished by adding TGF-alpha to the medium. Oligonucleotides had no effect on Jurkat cells, a lymphocytic cell line lacking epidermal growth factor receptors. Cholesterol-modified oligodeoxynucleotides were more effective and specific than unmodified oligodeoxynucleotides. CONCLUSIONS: Caco-2 cell proliferation is driven by autocrine stimulation of epidermal growth factor receptors by TGF-alpha. This mechanism may be effectively inhibited by antisense oligodeoxynucleotides, particularly those modified by the 5' attachment of cholesterol.     

Inhibition of BCR-ABL expression with antisense oligodeoxynucleotides restores beta1 integrin-mediated adhesion and proliferation inhibition in chronic myelogenous leukemia hematopoietic progenitors

Chronic myelogenous leukemia (CML) is characterized by the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors. This may be related to the unresponsiveness of CML progenitors to beta1 integrin adhesion receptor-mediated inhibition of progenitor proliferation by the marrow microenvironment. In hematopoietic cell lines, the BCR-ABL oncogene product, p210(BCR-ABL), interacts with a variety of cytoskeletal elements important for normal integrin signaling. We studied the role of p210(BCR-ABL) in abnormal integrin function in CML by evaluating the effect of inhibition of BCR-ABL expression with antisense oligodeoxynucleotides (AS-ODNs) on integrin-mediated adhesion and proliferation inhibition of malignant primary progenitors from CML marrow. Preincubation of CML CD34(+)HLA-DR+ (DR+) cells with breakpoint-specific AS-ODNs significantly increased adhesion of CML progenitors to stroma and fibronectin (FN). Pretreatment with breakpoint-specific ODNs also resulted in significant inhibition of CML progenitor proliferation after ligand or antibody-mediated beta1 integrin engagement. Breakpoint-specific ODNs were significantly more effective in restoring CML progenitor adhesion and proliferation inhibition than control ODNs. BCR-ABL mRNA and p210(BCR-ABL) levels in CML CD34(+) cells were significantly reduced after incubation with breakpoint-specific AS-ODN. These studies indicate a role for BCR-ABL in abnormal circulation and defective integrin-dependent microenvironmental regulation of proliferation of CML hematopoietic progenitors.     

MDM2 protein overexpression promotes proliferation and survival of multiple myeloma cells

The murine double minute 2 (MDM2) protein facilitates G1 to S phase transition by activation of E2F-1 and can enhance cell survival by suppressing wild-type p53 (wtp53) function. In this study, we examined MDM2 expression and function in multiple myeloma (MM) cells. MDM2 is strongly and constitutively expressed in MM cell lines (ARH-77, RPMI 8226, and OCI-My5) and in the cells of plasma cell leukemia (PCL) patients, but is not expressed in normal bone marrow mononuclear cells (BM MNCs). Treatment of MM cells with MDM2 antisense, but not sense, nonsense, or scrambled, oligodeoxyribonucleotides (ODNs) decreased DNA synthesis and cell viability; it also induced G1 growth arrest, as evidenced by propidium iodide (PI) staining and induction of retinoblastoma protein (pRB) to E2F-1 binding. Moreover, inhibition of MDM2 using antisense ODNs also triggered MM cell apoptosis as evidenced by acridine orange-ethidium bromide staining. We next studied the association of MDM2 with wtp53 and/or mutant p53 (mtp53), E2F-1, CDK4, and p21. MDM2 constitutively binds to E2F-1 in all MM cells, to both wtp53 and mtp53, and to p21 in tumor cells lacking p53. These data suggest that MDM2 may enhance cell-cycle progression in MM cells both by activating E2F-1 and by downregulating cell-cycle inhibitory proteins (wtp53 and p21). Overexpression of MDM2 may therefore contribute to both growth and survival of MM cells, suggesting the potential utility of treatment strategies targeting MDM2 in MM.     

Okadaic acid-induced apoptosis in neuronal cells: evidence for an abortive mitotic attempt

There is increasing evidence that apoptosis in postmitotic neurons is associated with a frustrated attempt to reenter the mitotic cycle. Okadaic acid, a specific protein phosphatase inhibitor, is currently used in models of Alzheimer's research to increase the degree of phosphorylation of various proteins, such as the microtubule-associated protein tau. Okadaic acid induces programmed cell death in the human neuroblastoma cell lines TR14 and NT2-N, as evidenced by fragmentation of DNA and attenuation of this process by protein synthesis inhibitors. In differentiated TR14 cells, okadaic acid increases the fraction of cells in the S phase, induces the appearance of cyclin B1 and cyclin D1 markers of the cell cycle, and triggers a time-dependent increase in DNA fragmentation after release of a thymidine block. Fully differentiated NT2-N cells are forced to enter the mitotic cycle as shown by DNA staining. Chromatin condensation and chromosome formation are initiated, but the cells fail to complete their mitotic cycle. These data suggest that okadaic acid forces differentiated neuronal cells into the mitotic cycle. This pattern of cyclin up-regulation and cell cycle shift is compared with apoptosis induced by neurotrophic factor deprivation in differentiated rat pheochromocytoma PC12 cells.     

Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the Bcl-2 coding sequence

BACKGROUND: The emergence of resistance to chemotherapy remains a major problem in the treatment of patients with small-cell lung cancer. Elevated expression of Bcl-2, a protein that inhibits programmed cell death or apoptosis, has been associated with radiation and drug resistance and has been observed in the majority of small-cell lung cancer specimens and cell lines. PURPOSE: To test the hypothesis that Bcl-2 expression levels are critical for inhibiting apoptosis in small-cell lung cancer cells, we used an antisense strategy to reduce Bcl-2 expression in these cells in an attempt to restore the natural occurrence of apoptosis. METHODS: Thirteen antisense oligodeoxynucleotides (ODNs) targeting various regions of the bcl-2 messenger RNA and a control scrambled-sequence ODN were tested to identify the most effective sequence(s) for reducing Bcl-2 protein levels. Northern and western blot analyses were used to examine basal bcl-2 messenger RNA and protein levels, respectively, in four human small-cell lung cancer cell lines (SW2, NCI-H69, NCI-H82, and NCI-N417). SW2 cells were treated with the antisense ODNs in the presence of cationic lipids (to facilitate uptake), and cytotoxic effects were measured by use of a cell viability assay. Flow cytometric analysis of DNA fragmentation and cell morphology was also performed. The cytotoxic effect of the most potent antisense ODN was also tested on the three other cell lines. RESULTS: The viability of SW2 cells was effectively reduced by ODNs that targeted the translation initiation and termination sites of the bcl-2 messenger RNA, but ODN 2009 that targeted the coding region was the most cytotoxic. Treatment of SW2 cells with 0.15 microM ODN 2009 for 96 hours reduced their viability by 91% (95% confidence interval [CI] = 88%-94%) and caused a dose-dependent reduction in Bcl-2 levels that became detectable 24 hours after treatment and persisted up to 96 hours; analysis of cellular morphology demonstrated that viability was reduced through apoptosis. Moreover, ODN 2009 at 0.15 microM was cytotoxic to NCI-H69, NCI-H82, and NCI-N417 cells, resulting in decreases in cell viability of 82% (95% CI = 78%-86%), 100%, and 100%, respectively, after 96 hours of treatment. The cytotoxic effects were inversely correlated with the basal Bcl-2 levels in the cell lines (r = -9964). A control scrambled-sequence oligodeoxynucleotide had no statistically significant effect on the cell lines (P values ranging from .38 to .89). CONCLUSION: We have identified a novel antisense ODN sequence (ODN 2009) that effectively reduces the viability of small-cell lung cancer cells by reducing Bcl-2 levels and facilitating apoptosis.     

Regulation of serotonin2A receptor expression by an antisense oligodeoxynucleotide

The regulation of 5-HT2A receptor expression by an antisense oligodeoxynucleotide, complementary to the coding region of rat 5-HT2A receptor mRNA, was examined in a cortically derived cell line and in rat brain. Treatment of A1A1 variant cells, which express the 5-HT2A receptor coupled to the stimulation of phosphatidylinositol (PI) hydrolysis, with antisense oligodeoxynucleotide decreased the maximal stimulation of PI hydrolysis by the partial agonist quipazine and the number of 5-HT2A receptor sites as measured by the binding of 2-[125I]-iodolysergic acid diethylamide. Treatment of cells with random, sense, or mismatch oligodeoxynucleotide did not alter the stimulation of PI hydrolysis by quipazine or 5-HT2A receptor number. Intracerebroventricular infusion of antisense, but not mismatch, oligodeoxynucleotide for 8 days resulted in a significant increase in cortical 5-HT2A receptor density and an increase in headshake behavior induced by the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. The density of cortical 5-HT2A receptors was not altered by administration of antisense oligodeoxynucleotide for 1, 2, or 4 days. We hypothesize that in brain this antisense oligodeoxynucleotide relieved some form of translational suppression, resulting in an increase in 5-HT2A receptor expression.     

Different effects of inorganic and dimethylated arsenic compounds on cell morphology, cytoskeletal organization, and DNA synthesis in cultured Chinese hamster V79 cells

Changes in cytoskeletal organization of cultured V79 cells exposed to arsenite and dimethylarsinic acid (DMAA), a methylated derivative of inorganic arsenics, and related changes, such as mitotic arrest and induction of multinucleated cells, were investigated in comparison with their effects on DNA synthesis. DMAA caused mitotic arrest and induction of multinucleated cells with a delay of 12 h relative to the mitotic arrest. By contrast, arsenite at equitoxic concentrations to DMAA was less effective than DMAA in causing mitotic arrest and in inducing multinucleated cells. Post-mitotic incubation of cells arrested in metaphase by 6 h incubation with 10 mM DMAA showed that the incidence of multinucleated cells increased conversely with a rapid decrease in metaphase cells. This suggests that metaphase-arrested cells can escape from metaphase, resulting in the appearance of multinucleated cells. The mitotic arrest caused by DMAA was accompanied by disruption of the microtubule network. By contrast, both arsenite and DMAA did not cause disorganization of actin stress fibers even when incubated at concentrations that caused a marked retardation of cell growth. Cells exposed to arsenite for 6 h showed marked inhibition of DNA synthesis, whereas inhibition by DMAA was not observed. When incubation was prolonged by 18 h, the arsenite-induced inhibition of DNA synthesis was mitigated. By contrast, inhibition of DNA synthesis by DMAA occurred in parallel with an increase in the population of mitotic cells. These results suggest that DMAA caused growth retardation and morphological changes via disruption of the microtubule network, and that arsenite-induced retardation of cell growth and inhibition of DNA synthesis were not attributable to the cytoskeletal changes.