Effects of paclitaxel on cultured synovial cells from patients with rheumatoid arthritis

BACKGROUND: Proliferation of synovial cells is considered to play a key role in rheumatoid arthritis (RA). Using paclitaxel, a unique antineoplastic agent known to suppress collagen-induced arthritis, we conducted an in vitro study of cell kinetics on cultured synovial cells from patients with RA. METHODS: Alterations of the cell cycle of cultured fibroblast-like synovial cells (FLSs) from patients with RA were studied using flow cytometry and laser scanning cytometry. Apoptosis and accumulation of cyclin concerning effects of paclitaxel were detected. RESULTS: Paclitaxel induced arrest of the cell cycle at G2/M phase and apoptosis in FLSs. The late stage of apoptosis was determined by the positivity of terminal deoxynucleotidyl transferase assay. Morphological observation by combined usage of both annexin V and propidium iodide on FLSs on a slide glass showed early apoptotic changes in detail. FLSs arrested at G2/M phase showed marked accumulation of cyclin B1. The effects of paclitaxel decreased on FLSs, which diminished proliferative activity. CONCLUSIONS: These data indicate that paclitaxel induces cell arrest at G2/M phase followed by apoptosis in human FLSs, which have high proliferative activity, and possible therapeutic effects of paclitaxel on RA.     

Pro-apoptotic effect and cytotoxicity of genistein and genistin in human ovarian cancer SK-OV-3 cells

We investigated the effects of genistein and genistin on proliferation and apoptosis of human ovarian SK-OV-3 cells and explored the mechanism for these effects. SK-OV-3 cells were treated with genistein and genistin at various concentrations (ranging from 1 to 100 muM) either alone or in combination for 24 and 48 h. Cell proliferation was estimated using an MTT assay, and cell cycle arrest was evaluated using FACS. Caspase-3 activity and annexin-based cell cycle analysis were used as measures of apoptosis. In addition, genistein- and genistin-induced cytotoxicity was determined by measuring release of LDH. Genistein treatment for 24 or 48 h substantially inhibited SK-OV-3 cell proliferation in a dose-dependent manner, and genistin treatment for 48 h also inhibited cell proliferation. Genistein caused cell cycle arrest at G2/M phase in dose- and time-dependent manner, and genistin caused cell cycle arrest not only at G2/M phase but also at G1 phase. Genistein markedly induced apoptosis and significantly increased LDH release, whereas genistin did not affect LDH release. Moreover, exposure to both genistein and genistin in combination for 48 h induced apoptosis without increasing LDH release. Genistein and genistin inhibit cell proliferation by disrupting the cell cycle, which is strongly associated with the arrest induction of either G1 or G2/M phase and may induce apoptosis. Based on our findings, we speculate that both genistein and genistin may prove useful as anticancer drugs and that the combination of genistein and genistin may have further anticancer activity.     

Regulation of survivin by retinoic acid and its role in paclitaxel-mediated cytotoxicity in MCF-7 breast cancer cells

The chemotherapeutic drug paclitaxel induces microtubular stabilization and mitotic arrest associated with increased survivin expression. Survivin is a member of the inhibitor of apoptosis (iap) family which is highly expressed in during G2/M phase where it regulates spindle formation during mitosis. It is also constitutively overexpressed in most cancer cells where it may play a role in chemotherapeutic resistance. MCF-7 breast cancer cells stably overexpressing the sense strand of survivin (MCF-7(survivin-S) cells) were more resistant to paclitaxel than cells depleted of survivin (MCF-7(survivin-AS) despite G2/M arrest in both cell lines. However, survivin overexpression did not protect cells relative to control MCF-7(pcDNA3) cells. Paclitaxel-induced cytotoxicity can be enhanced by retinoic acid and here we show that RA strongly reduces survivin expression in MCF-7 cells and prevents paclitaxel-mediated induction of survivin expression. Mitochondrial release of cytochrome c after paclitaxel alone or in combination with RA was weak, however robust Smac release was observed. While RA/paclitaxel-treated MCF-7 (pcDNA3) cultures contained condensed apoptotic nuclei, MCF-7(survivin-S) nuclei were morphologically distinct with hypercondensed disorganized chromatin and released mitochondrial AIF-1. RA also reduced paclitaxel-associated levels of cyclin B1 expression consistent with mitotic exit. MCF-7(survivin-S) cells displayed a 30% increase in >2N/<4N ploidy while there was no change in this compartment in vector control cells following RA/paclitaxel. We propose that RA sensitizes MCF-7 cells to paclitaxel at least in part through survivin downregulation and the promotion of aberrant mitotic progression resulting in apoptosis. In addition we provide biochemical and morphological data which suggest that RA-treated MCF-7(survivin-S) cells can also undergo catastrophic mitosis when exposed to paclitaxel.     

Enhanced sensitivity of G1 arrested human cancer cells suggests a novel therapeutic strategy using a combination of simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.     

Human immunodeficiency virus type 1 Vpr induces cell cycle arrest at the G(1) phase and apoptosis via disruption of mitochondrial function in rodent cells

Vpr of human immunodeficiency virus type 1 causes cell cycle arrest at the G(2)/M phase and induces apoptosis after G(2)/M arrest in primate cells. We have reported previously that Vpr also induces apoptosis independently of G(2)/M arrest in human HeLa cells. By contrast, Vpr does not induce G(2)/M arrest in rodent cells, but it retards cell growth. To clarify the relationship between cell cycle arrest and apoptosis, we expressed Vpr endogenously in rodent cells and investigated cell cycle profiles and apoptosis. We show here that Vpr induces cell cycle arrest at the G(1) phase and apoptosis in rodent cells. Vpr increased the activity of caspase-3 and caspase-9, but not of caspase-8. Moreover, Vpr-induced apoptosis could be inhibited by inhibitors of caspase-3 and caspase-9, but not by inhibitor of caspase-8. We also showed that Vpr induces the release of cytochrome c from mitochondria into the cytosol and disrupts the mitochondrial transmembrane potential. Finally, we showed that apoptosis occurred in HeLa cells through an identical pathway. These results suggest that disruption of mitochondrial functions by Vpr induces apoptosis via cell cycle arrest at G(1), but that apoptosis is independent of G(2)/M arrest. Furthermore, it appears that Vpr acts species-specifically with respect to induction of cell cycle arrest but not of apoptosis.     

Differential Taxol-dependent arrest of transformed and nontransformed cells in the G1 phase of the cell cycle, and specific-related mortality of transformed cells

Taxol (paclitaxel) induces a microtubule hyperassembled state, and effectively blocks cells in mitosis. Here we report that Taxol also induces a stable late-G1 block in nontransformed REF-52 and WI-38 mammalian fibroblast cells, but not in T antigen-transformed cells of the same parental lineage. G1 arrest is characterized by partially dephosphorylated pRb, and inactive cdk2 kinase. Nontransformed cells recover normally from Taxol arrest. In contrast, T antigen transformed cells continue inappropriately past both G1 and G2-M in the presence of Taxol, and undergo a rapid death upon release. These results demonstrate a microtubule sensitive step in G1 regulation of nontransformed fibroblast cells. Also, Taxol selectively induces death of transformed cells, possibly because they slip the Taxol-dependent G1 arrest, as well as G2/M arrest, which are both specific to nontransformed cells.     

Enhanced Sensitivity of G1 Arrested Human Cancer Cells Suggests a Novel Therapeutic Strategy Using a Combination of Simvastatin and TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells over normal cells. To study the relationship between cell cycle progression and TRAIL-induced apoptosis, SW480 colon cancer and H460 lung cancer cell lines were examined for their sensitivity to TRAIL after arrest in different cell cycle phases. Cells were synchronized in G0/G1, S, and G2/M phase by serum starvation, aphidicolin, or nocodazole treatment, respectively. We found that arrest of cells in G0/G1 phase confers significantly higher susceptibility to TRAIL-induced apoptosis as compared to cells in late G1, S, or G2/M phase. To determine if cell cycle phase could be harnessed for therapeutic gain in the presence of TRAIL, we used the HMG-CoA reductase inhibitor, Simvastatin and lovastatin, to enrich a cancer cell population in G0/G1. Both simvastatin and lovastatin significantly augmented TRAIL-induced apoptosis in tumor cells, but not in normal keratinocytes. The results indicate that TRAIL, in combination with a HMG-CoA reductase inhibitor, may have therapeutic potential in the treatment of human cancer.

Key Words

TRAIL, Synchronization, Simvastatin, Cancer Therapy, Lovastatin, Cell Cycle, Apoptosis     

BAD detects coincidence of G2/M phase and growth factor deprivation to regulate apoptosis

BAD, a member of the Bcl-2 protein family, promotes mitochondria-dependent apoptosis. Here, we report that BAD dissociates from 14-3-3zeta at each G2/M phase of proliferating lymphoid cells. The cell cycle-dependent dissociation of BAD was associated with phosphorylation at Ser-128, whereas mutant S128A-BAD, in which Ser-128 was converted to alanine, remained associated with 14-3-3zeta throughout the cell cycle. Although the cell cycle-dependent dissociation of BAD per se did not induce apoptosis, growth factor deprivation induced prompt apoptosis at the G2/M phase but not at the G1 phase. In cells expressing S128A-BAD, growth factor deprivation-induced apoptosis was markedly delayed and was accompanied by a delayed dephosphorylation of growth factor-dependent regulatory serine residues. These results indicate that BAD induces apoptosis upon detecting the coincidence of G2/M phase and growth factor deprivation.     

Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis.

Hereditary tyrosinemia type I is the most severe metabolic disease of the tyrosine catabolic pathway mainly affecting the liver. It is caused by deficiency of fumarylacetoacetate hydrolase, which prevents degradation of the toxic metabolite fumarylacetoacetate (FAA). We report here that FAA induces common effects (i.e., cell cycle arrest and apoptosis) in both human (HepG2) and rodent (Chinese hamster V79) cells, effects that seem to be temporally related. Both the antiproliferative and apoptosis-inducing activities of FAA are dose dependent and enhanced by glutathione (GSH) depletion with L-buthionine-(S,R)-sulfoximine (BSO). Short treatment (2 h) with 35 microM FAA/+BSO or 100 microM FAA/-BSO induced a transient cell cycle arrest at the G2/M transition (20% and 37%, respectively) 24 h post-treatment. In cells treated with 100 microM FAA/-BSO, an inactivation, followed by a rapid over-induction of cyclin B-dependent kinase occurred, which peaked 24 h post-treatment. Maximum levels of caspase-1 and caspase-3 activation were detected at 3 h and 32 h, respectively, whereas release of mitochondrial cytochrome c was maximal at 24-32 h post-treatment. The G2/M peak declined 24 h later, concomitantly with the appearance of a sub-G1, apoptotic population showing typical nucleosomal-sized DNA fragmentation and reduced mitochondrial transmembrane potential (Deltapsi(m)). These events were prevented by the general caspase inhibitor z-VAD-fmk, whereas G2/M arrest and subsequent apoptosis were abolished by GSH-monoethylester or N-acetylcysteine. Other tyrosine metabolites, maleylacetoacetate and succinylacetone, had no antiproliferative effects and induced only very low levels of apoptosis. These results suggest a modulator role of GSH in FAA-induced cell cycle disturbance and apoptosis where activation of cyclin B-dependent kinase and caspase-1 are early events preceding mitochondrial cytochrome c release, caspase-3 activation, and Deltapsi(m) loss. -Jorquera, R., Tanguay, R. M. Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis.     

Chemosensitivity of human prostate cancer cells PC3 and LNCaP to genistein isoflavone and beta-lapachone

A wide spectrum of anti-cancer activity of genistein and beta-lapachone in various tumors has been reported in single treatments. In this study the combined effects of genistein and beta-lapachone on the chemosensitivity of LNCaP and PC3 human prostate cancer cells was determined in vitro, using 3-[4,5-dimethylthiazol-2-yl]-2-,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) to study treatment-induced growth inhibition and cytotoxicity and, annexin V-fluoresceine (FI) and terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-propidium iodide (PI) assays to determine potential treatment-induced apoptosis and/or necrosis. The results showed: i) that both PC3 and LNCaP are sensitive to single and combination treatments regardless of hormone sensitivity status, ii) that treatment induced dual death pathways (apoptosis and necrosis) in both cell types, iii) that growth inhibition in both cell types correlated positively with cell death via apoptosis at lower drug concentrations and necrosis at higher concentrations, iv) that combination of genistein and beta-lapachone had synergistic inhibitory effects on growth and proliferation in both cell types. The synergistic inhibitory effect was correlated positively with treatment-induced cell death via apoptosis and necrosis. The overall results indicate that combination treatments with beta-lapachone and genistein are more potent in killing both PC3 and LNCaP cancer cells than treatment with either genistein or beta-lapachone alone. beta-lapachone acts at the G1 and S phase checkpoints in the cell cycle, while genistein induces cell cycle arrest at the G2-M stage. The current results are therefore in agreement with the hypothesis that drug combinations that target cell cycles at different critical checkpoints would be more effective in causing cell death. This result provides a rationale for in vivo studies to determine whether beta-lapachone-genistein combination will provide effective chemotherapy for prostate cancer, regardless of the tumor sensitivity to hormone.     

PGE1 protection against apoptosis induced by D-galactosamine is not related to the modulation of intracellular free radical production in primary culture of rat hepatocytes

d -galactosamine ( d -GalN) toxicity is a useful experimental model of liver failure in human. It has been previously observed that PGE 1 treatment reduced necrosis and apoptosis induced by d -GalN in rats. Primary cultured rat hepatocytes were used to evaluate if intracellular oxidative stress was involved during the induction of apoptosis and necrosis by d -GalN (0-40 mM). Also, the present study investigated if PGE 1 (1 μM) was equally potent reducing both types of cell death. The presence of hypodiploid cells, DNA fragmentation and caspase-3 activation were used as a marker of hepatocyte apoptosis. Necrosis was measured by lactate dehydrogenase (LDH) release. Oxidative stress was evaluated by the intracellular production of hydrogen peroxide (H 2 O 2 ), the disturbances on the mitochondrial transmembrane potential (MTP), thiobarbituric-reacting substances (TBARS) release and the GSH/GSSG ratio. Data showed that intermediate range of d -GalN concentrations (2.5-10 mM) induced apoptosis in association with a moderate oxidative stress. High d -GalN concentration (40 mM) induced a reduction of all parameters associated with apoptosis and enhanced all those related to necrosis and intracellular oxidative stress, including a reduction of GSH/GSSG ratio and MTP in comparison with d -GalN (2.5-10 mM)-treated cells. Although PGE 1 reduced apoptosis induced by d -GalN, it was not able to reduce the oxidative stress and cell necrosis induced by the hepatotoxin in spite to its ability to abolish the GSH depletion.     

Cell cycle G2/M arrest and activation of cyclin-dependent kinases associated with low-dose paclitaxel-induced sub-G1 apoptosis

Paclitaxel is a potential anti-cancer agent for several malignancies including ovary, breast, and head and neck cancers. This study investigated the kinetics of paclitaxel-induced cell cycle perturbation in two human nasopharyngeal carcinoma (NPC) cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with higher concentrations (0.1 or 1 μM) of paclitaxel showed obvious G2/M arrest and then converted to a cell population with reduced DNA content, which was detected as a sub-G2 peak in the flow cytometric histographs. If a low concentration (5 nM) of paclitaxel was used instead, transient G2/M arrest was observed in NPC cells, which subsequently converted to a sub-G1 form during the treatment period. Internucleosomal fragmentation and chromatin condensation were detectable in these sub-G1 and sub-G2 cells, suggesting that persistent or transient G2/M arrest is a prerequisite step for apoptosis elicited by varying doses of paclitaxel. The levels of cyclins A, B1, D1, E, CDK 1 (CDC 2), CDK 2 and proliferating cell nuclear antigen (PCNA) were unchanged in NPC cells following treatment with any concentration of paclitaxel; however, apoptosis-related cyclin B1-associated CDC 2 kinase was highly activated by paclitaxel even at concentrations as low as 5 nM, which is consistent with the finding that low-dose paclitaxel is also able to induce apoptosis in NPC cells. Activation of cyclin B1-associated CDC 2 kinase seems to be an important G2/M event required for paclitaxel-induced apoptosis, and this activation of cyclin B1/CDC 2 kinase could be attributed to the increased activity of CDK 7 kinase. This revised version was published online in November 2006 with corrections to the Cover Date.     

PGE 1 Protection against Apoptosis Induced by d -galactosamine is Not Related to the Modulation of Intracellular Free Radical Production in Primary Culture of Rat Hepatocytes

d -galactosamine ( d -GalN) toxicity is a useful experimental model of liver failure in human. It has been previously observed that PGE 1 treatment reduced necrosis and apoptosis induced by d -GalN in rats. Primary cultured rat hepatocytes were used to evaluate if intracellular oxidative stress was involved during the induction of apoptosis and necrosis by d -GalN (0-40 mM). Also, the present study investigated if PGE 1 (1 &#119 M) was equally potent reducing both types of cell death. The presence of hypodiploid cells, DNA fragmentation and caspase-3 activation were used as a marker of hepatocyte apoptosis. Necrosis was measured by lactate dehydrogenase (LDH) release. Oxidative stress was evaluated by the intracellular production of hydrogen peroxide (H 2 O 2 ), the disturbances on the mitochondrial transmembrane potential (MTP), thiobarbituric-reacting substances (TBARS) release and the GSH/GSSG ratio. Data showed that intermediate range of d -GalN concentrations (2.5-10 mM) induced apoptosis in association with a moderate oxidative stress. High d -GalN concentration (40 mM) induced a reduction of all parameters associated with apoptosis and enhanced all those related to necrosis and intracellular oxidative stress, including a reduction of GSH/GSSG ratio and MTP in comparison with d -GalN (2.5-10 mM)-treated cells. Although PGE 1 reduced apoptosis induced by d -GalN, it was not able to reduce the oxidative stress and cell necrosis induced by the hepatotoxin in spite to its ability to abolish the GSH depletion.     

Arsenic trioxide selectively induces early and extensive apoptosis via the APO2/caspase-8 pathway engaging the mitochondrial pathway in myeloma cells with mutant p53

Arsenic trioxide (ATO) is effective in the treatment of acute promyelocytic leukemia (APL) and induces apoptosis in APL cells and in a great variety of other cancer cells. We have previously shown that ATO induces apoptosis in myeloma cells in two different modes depending on p53 status in the cells. In cells expressing mutated p53, ATO induced, G2/M arrest and activation caspase 8 and 3 and rapid and extensive apoptosis. Myeloma cells expressing w.t. p53, ATO induced G1 arrest and delayed apoptosis with activation of caspase 9 and 3. APO2/TRAIL receptor expression was induced in both cell types and APO2/TRAIL synergized with ATO in the induction of apoptosis. Here we tested the effect of ATO on mitochondrial membrane potential (MMP) in myeloma cells expressing mutated or w.t. p53. In myeloma cells expressing mutated p53, depolarization of MMP occurred early, concomitant with induction of APO2/TRAIL, activation of BID and release of AIF, preceding apoptosis. However, in cells expressing w.t. p53, APO2/TRAIL is not induced, BID is not cleaved and depolarization of MMP occurs concurrently with cytochrome c release and apoptosis. These results explain the greater sensitivity to ATO of cells with mutated p53 and suggest perhaps a general mechanism for ATO-induced apoptosis.     

Arsenic Trioxide Selectively Induces Early and Extensive Apoptosis via the APO2/Caspase-8 Pathway Engaging the Mitochondrial Pathway in Myeloma Cells with Mutant p53

Arsenic trioxide (ATO) is effective in the treatment of acute promyelocytic leukemia (APL) and induces apoptosis in APL cells and in a great variety of other cancer cells. We have previously shown that ATO induces apoptosis in myeloma cells in two different modes depending on p53 status in the cells. In cells expressing mutated p53, ATO induced, G2/M arrest and activation caspase 8 and 3 and rapid and extensive apoptosis. Myeloma cells expressing w.t. p53, ATO induced G1 arrest and delayed apoptosis with activation of caspase 9 and 3. APO2/TRAIL receptor expression was induced in both cell types and APO2/TRAIL synergized with ATO in the induction of apoptosis. Here we tested the effect of ATO on mitochondrial membrane potential (MMP) in myeloma cells expressing mutated or w.t. p53. In myeloma cells expressing mutated p53, depolarization of MMP occurred early, concomitant with induction of APO2/TRAIL, activation of BID and release of AIF, preceding apoptosis. However, in cells expressing w.t. p53, APO2/TRAIL is not induced, BID is not cleaved and depolarization of MMP occurs concurrently with cytochrome c release and apoptosis. These results explain the greater sensitivity to ATO of cells with mutated p53 and suggest perhaps a general mechanism for ATO-induced apoptosis.     

Beta-catenin regulation during the cell cycle: implications in G2/M and apoptosis

Beta-catenin is a multifunctional protein involved in cell-cell adhesion and Wnt signal transduction. Beta-catenin signaling has been proposed to act as inducer of cell proliferation in different tumors. However, in some developmental contexts and cell systems beta-catenin also acts as a positive modulator of apoptosis. To get additional insights into the role of beta-catenin in the regulation of the cell cycle and apoptosis, we have analyzed the levels and subcellular localization of endogenous beta-catenin and its relation with adenomatous polyposis coli (APC) during the cell cycle in S-phase-synchronized epithelial cells. Beta-catenin levels increase in S phase, reaching maximum accumulation at late G2/M and then abruptly decreasing as the cells enter into a new G1 phase. In parallel, an increased cytoplasmic and nuclear localization of beta-catenin and APC is observed during S and G2 phases. In addition, strong colocalization of APC with centrosomes, but not beta-catenin, is detected in M phase. Interestingly, overexpression of a stable form of beta-catenin, or inhibition of endogenous beta-catenin degradation, in epidermal keratinocyte cells induces a G2 cell cycle arrest and leads to apoptosis. These results support a role for beta-catenin in the control of cell cycle and apoptosis at G2/M in normal and transformed epidermal keratinocytes.     

Arsenic Trioxide Arrests Cells Early in Mitosis Leading to Apoptosis

Arsenic trioxide (As2O3) is highly effective in the treatment of acute promyelocytic leukemias that express the promyelocytic leukemia-retinoic acid receptor-a (PML-RARa) fusion protein. However, evidence has accumulated that As2O3 induces apoptosis regardless of PML-RARa status. Here we show that, at clinically relevant concentrations, As2O3 causes S and G2M phase arrest of both PML-RARa-positive and -negative leukemia cell lines, thus inhibiting their growth. Apoptotic cells are generated predominately from the G2M fraction. Using several independent methods, we demonstrate that the cells accumulated in the G2M peak consist primarily of cells arrested in the early stages of mitosis, prophase, prometaphase and metaphase. In mitotic cells, there was significant activation of caspases, PARP cleavage, and morphological changes characteristic of apoptosis. Unlike microtubule-active drugs that arrest cells in metaphase, arsenic trioxide did not affect the architecture of microtubules. Our data suggest that the antileukemic activities of arsenic may be a result of mitotic arrest which culminates in apoptosis.

Key Words:

PML nuclear bodies (NB), Phosphorylated histone H3     

Arsenic trioxide arrests cells early in mitosis leading to apoptosis

Arsenic trioxide (As(2)O(3)) is highly effective in the treatment of acute promyelocytic leukemias that express the promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARalpha) fusion protein. However, evidence has accumulated that As(2)O(3) induces apoptosis regardless of PML-RARalpha status. Here we show that, at clinically relevant concentrations, As(2)O(3) causes S and G(2)M phase arrest of both PML-RARalpha-positive and -negative leukemia cell lines, thus inhibiting their growth. Apoptotic cells are generated predominately from the G(2)M fraction. Using several independent methods, we demonstrate that the cells accumulated in the G(2)M peak consist primarily of cells arrested in the early stages of mitosis, prophase, prometaphase and metaphase. In mitotic cells, there was significant activation of caspases, PARP cleavage, and morphological changes characteristic of apoptosis. Unlike microtubule-active drugs that arrest cells in metaphase, arsenic trioxide did not affect the architecture of microtubules. Our data suggest that the antileukemic activities of arsenic may be a result of mitotic arrest which culminates in apoptosis.     

Roles for basal and stimulated p21(Cip-1/WAF1/MDA6) expression and mitogen-activated protein kinase signaling in radiation-induced cell cycle checkpoint control in carcinoma cells

We investigated the role of the cdk inhibitor protein p21(Cip-1/WAF1/MDA6) (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0-10 min) and secondary (90-240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G(1) that were p21 and MAPK dependent, whereas the elevation of cells present in G(2)/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G(2)/M phase 24-48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G(2)/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G(2)/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G(2)/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G(1)/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G(2)/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.     

MEKK1 is essential for DT40 cell apoptosis in response to microtubule disruption

Vinblastine and other microtubule-damaging agents, such as nocodazole and paclitaxel, cause cell cycle arrest at the G2/M transition and promote apoptosis in eukaryotic cells. The roles of these drugs in disrupting microtubule dynamics and causing cell cycle arrest are well characterized. However, the mechanisms by which these agents promote apoptosis are poorly understood. We disrupted the MEKK1 kinase domain in chicken bursal B-cell line DT40 by homologous recombination and have shown that it is essential for both vinblastine-mediated apoptosis and vinblastine-mediated c-Jun N-terminal protein kinase activation. In addition, our data indicate that vinblastine-mediated apoptosis in DT40 cells requires new protein synthesis but does not require G2/M arrest, suggesting that vinblastine-mediated cell cycle arrest and apoptosis are two independent processes.