Nucleosomal Ladder Research Articles

Smad7 mediates transforming growth factor-β–induced apoptosis in mesangial cells

In addition to inhibiting cell growth, transforming growth factor-beta (TGF-beta) has recently been reported to induce apoptosis in various cell lines. Smad proteins are the downstream effectors of TGF-beta signaling. Among them, Smad7 exerts negative feedback control over the action of TGF-beta. However, we do not know how the Smad proteins contribute to TGF-beta-induced apoptosis in mesangial cells. To investigate the function of Smad proteins, we examined the effect of Smad overexpression using adenoviral vector in mesangial cells. Primary cultured rat mesangial cells were transfected with Smad7-promoter-luciferase-plasmid by electroporation. Smad7 promoter activity was investigated by luciferase assay. The apoptotic phenomena elicited by TGF-beta and Smad7 overexpression were investigated using adenoviral vector (AdCMV-Smad7). Apoptosis was detected by the cell death detection ELISA assay, CPP32/caspase-3 assay, and nucleosomal DNA laddering. TGF-beta significantly increased the protein expression and the promoter activity of Smad7 in rat mesangial cells. Overexpression of Smad7 induced DNA fragmentation and significant increases in cell death ELISA and CPP32/caspase-3 assay. On the other hand, overexpression of Smad2 and Smad3 did not elicit any significant increases in CPP32/caspase-3 activity. Furthermore, the antisense oligonucleotide to Smad7 prevented the TGF-beta-induced apoptosis. Overexpression of Smad7 did not affect nuclear factor-kappaB activity in mesangial cells. These data indicate that TGF-beta-induced apoptosis in mesangial cells is mediated through the activation of caspase-3 by Smad7, but not by Smad2 or Smad3. Our results provide new clarification on the function of Smad7 in TGF-beta signaling in mesangial cells.

Attenuation of photodynamically induced apoptosis by an RGD containing peptide.

Research efforts have focused on the improvement of already established photodynamic therapy (PDT) protocols. The use of adjunct therapies is one such route. The integrin class of receptors mediates extracellular matrix signals through a complex maze of intertwining cellular pathways. The Arg-Gly-Asp (RGD) motif is known to bind to several of the 25 known integrin receptor types. Soluble RGD peptides under most circumstances induce apoptosis in a number of cell lines In this study, the effect of an RGD-containing peptide on the photodynamic action of aluminium disulfophthalocyanine (A1PcS(2adj)) was investigated. Adenocarcinoma lung cancer cells (A549) and murine mammary cancer cells (EMT-6) were treated with A1PcS(2adj) in the presence of soluble RGD. At elevated RGD concentrations (10 mM) apoptosis was induced by the peptide alone. It was shown that at lower concentrations, RGD abrogated the apoptotic effect of PDT in both cell lines, as assessed by an MTT cytotoxicity assay, nucleosomal DNA laddering and the formation of apoptotic bodies. RGD protection against apoptosis was more pronounced in the A549 receptor positive cell line which exhibits over 70% cell survival when using 100 microM RGD peptide under LD90 conditions. Different parameters were investigated to clearly establish that the attenuation of cell killing was not solely due to quenching of the excited species by the peptide. Indeed, the phenomenon is not photophysical but biological.

P53 Activates the Mitochondrial Death Pathway and Apoptosis of Ventricular Myocytes Independent of de novo Gene Transcription

The tumor suppressor p53 is known to regulate gene transcription and apoptosis in mammalian cells. In the present study we ascertain whether these events are mutually dependent and obligatorily linked for induction of apoptosis of ventricular myocytes. Adenovirus mediated gene delivery of wild p53 (p53WT) or a mutant form of p53 (p53MT) defective for gene transcription to ventricular myocytes was confirmed by Western blot analysis. A significant increase in the p53 dependent genes Bax and MDM2 was observed with p53WT but not p53MT. Nuclear DNA visualized by agarose gel electrophoresis revealed nucleosomal DNA laddering in the presence of either p53 protein. Apoptosis was substantiated by Hoechst 33258 nuclear staining. Perturbations to mitochondria consistent with the mitochondrial death pathway, including loss of mitochondrial transmembrane potential Δæmand cytochrome c release were observed with p53WT and p53MT. An increase in caspase 3-like activity was noted with either p53WT or p53MT protein that was suppressed by the caspase 3 inhibitor Ac-DEVD-CHO. To our knowledge the experiments described here provide the first indication that p53 activates the mitochondrial death pathway and provokes apoptosis of ventricular myocytes independent of DNA binding and de novo gene activation.

Apoptosis-Inducing costunolide and a novel acyclic monoterpene from the stem bark of Magnolia sieboldii.

In a course of obtaining more amount of bioactive costunolide and successive phytochemical isolation from Magnolia sieboldii (Magnoliaceae), a novel acyclic monoterpene 1 named deoxygeraniol [2,6(E)-dimethyl-2,6-octadiene] was isolated along with beta-sitosterol 3-O-linoleate (2), trilinolein (3) and high amount of costunolide (4) in the pure state. The structure of compound 1 was determined on the basis of spectroscopic data. Costunolide was found to induce apoptotic cell death in a dose-dependent manner by nucleosomal DNA ladder and flow cytometric analysis. Immunoblot analysis showed that the level of the anti-apoptotic protein, Bcl-2, was decreased, whereas the cleavage of poly-(ADP-ribose) polymerase was activated. Furthermore, the N-acetyl-L-cysteine antioxidant effectively prevented costunolide-induced cytotoxicity. These results suggest that costunolide-induced cell death is mediated by reactive oxygen species

Nucleolytic Cleavage of the Mixed Lineage Leukemia Breakpoint Cluster Region during Apoptosis

VP-16 (etoposide) has recently been shown to induce topoisomerase II (TOP2)-mediated DNA cleavage within the mixed lineage leukemia (MLL) breakpoint cluster region (bcr), suggesting a role of TOP2 in MLL gene rearrangement. In our current studies, we have compared the induction of DNA cleavage within the MLL bcr in different cell lines after treatment with various anticancer drugs. All anticancer drugs tested including VP-16 (a TOP2-directed drug), camptothecin (a topoisomerase I-directed drug), 5-fluorouracil and methotrexate (antimetabolites), and vinblastine (a microtubule inhibitor) induced the same site-specific cleavage within the MLL bcr. This cleavage was shown to be nuclease-mediated but not TOP2-mediated by the following observations: 1) drug-induced cleavage within the MLL bcr was not protein-linked; 2) unlike TOP2-mediated cleavage, drug-induced DNA cleavage within the MLL bcr was kinetically slow and coincided with the formation of the apoptotic nucleosomal DNA ladder; 3) drug-induced cleavage within the MLL bcr was unaffected in cells with reduced nuclear TOP2; and 4) drug-induced cleavage within the MLL bcr was abolished by the caspase inhibitor, Z-Asp(OCH(3))-Glu(OCH(3))-Val-Asp(OCH(3))-FMK. The possibility that an apoptotic nuclease may be involved in cleavage of the MLL bcr and MLL gene translocation is discussed.

Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells

The novel serine-threonine kinase Akt is a critical enzyme in cell survival. We investigated the roles of the Akt pathway and apoptotic signals in (1) Madin-Darby canine kidney (MDCK) cells in a hyperosmotic condition in vitro and (2) in the inner medulla of dehydrated rat in vivo. The in vivo experiments were performed in 24- and 48-hour water-restricted rats. Hyperosmolality-stimulated Akt phosphorylation was examined in MDCK cells. Phosphatidylinositol 3-kinase (PI3-K) inhibitors, the dominant-negative mutant of PI3-K, the dominant-negative mutant of Akt, and the dominant-active form of Akt were used to examine the roles of the PI3-K/Akt pathways in renal tubular cell apoptosis. The amount of phosphorylated Akt protein was increased in the inner medulla of dehydrated rats. Hyperosmolality induced by the addition of NaCl, urea, and raffinose phosphorylated Akt in MDCK cells in an osmolality-dependent manner. PI3-K inhibitors and the dominant-negative mutant of PI3-K inhibited the hyperosmolality-induced phosphorylation of Akt. Raising the media osmolality from a normal level to 500 or 600 mOsm/kg H2O final osmolality elicited apoptotic changes such as nucleosomal laddering of DNA and an increment of caspase-3 activity and increased activity in the cell death enzyme-linked immunosorbent assay. Dominant-active Akt prevented the mild hyperosmolality-induced apoptosis, while inhibition of the PI3-K/Akt pathways promoted apoptosis. The Akt pathway is activated by hyperosmolality in vitro and in vivo, and activation of Akt prevents the mild hyperosmolality-induced apoptotic changes in MDCK cells. PI3-K/Akt pathways are involved in a hypertonic condition that confers the balance between cell survival and apoptosis.

Isolation and characterization of an endonuclease synthesized by barley (Hordeum vulgare L.) uninucleate microspores

Few biochemical and molecular details are available on microspore growth and development. In this work, a nuclease was partially purified from diffusates of barley (Hordeum vulgare L.) microspores by using concanavalin-A as ligand. The chromatographic preparation contained a 34-kDa protein with nucleolytic activity; the enzyme (called BMN: barley microspore nuclease) was very stable at pH > 8.0 and temperatures below 50 degrees C. Activity was highest at pH 5.6 and increased almost exponentially with temperature until a breakpoint between activity and stability was reached at 70 degrees C. Although BMN was able to cleave RNA, the enzyme showed a remarkable preference for DNA, especially in the single-stranded form. The best homopolymeric substrates were poly(dA) and poly(A), whereas poly(dC), poly(G) and poly(I) were almost completely uncleaved. When incubated with intact nuclei, BMN caused a nucleosomal DNA ladder of approximately 200 bp. On the basis of DNA laddering, substrate specificity, Mg2+ -dependence and best performance at apoplastic pH, BMN can be referred to as a putative apoptotic nuclease involved in pollen development.

Ligands for peroxisome proliferator-activated receptor gamma inhibit growth and induce apoptosis of human papillary thyroid carcinoma cells.

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma) induce apoptosis and exert antiproliferative effects on several carcinoma cell lines. The present study investigates the expression of PPARgamma and the possibility that agonists for PPARgamma also inhibit the growth of human thyroid carcinoma cells. We examined this hypothesis using six cell lines, designated BHP thyroid carcinoma cells, which originated from patients with papillary thyroid carcinoma. RT-PCR analysis revealed that the thyroid carcinoma cell lines BHP2-7, 7-13, 10-3, and 18-21 express PPARgamma. More PPARgamma was expressed in carcinoma than in adjacent normal thyroid tissue in three of six samples of human papillary carcinoma of the thyroid. PPARgamma-positive thyroid carcinoma cells were treated with agonists of PPARgamma, troglitazone, BRL 49653, and 15-deoxy-12,14-prostaglandin J2. Troglitazone (10 micromol/L), BRL 49653 (10 micromol/L), and 15-deoxy-12,14-prostaglandin J2 (1 microg/mL) decreased [(3)H]thymidine incorporation and reduced cell number, respectively, in BHP carcinoma cell lines that expressed PPARgamma. Under low serum conditions, ligands for PPARgamma induced condensation of the nucleus and fragmentation of chromatin into nucleosome ladders. These findings indicate that the death of thyroid carcinoma cells is a form of apoptosis. To investigate the molecular mechanism of the apoptosis, we assessed expression of the apoptosis-regulatory genes bcl-2, bax, and c-myc. Troglitazone significantly increased the expression of c-myc messenger RNA but had no effect on the expression of bcl-2 and bax in thyroid carcinoma cells. These results suggest that, at least in part, the induction of apoptosis in human papillary thyroid carcinoma cells may be due to an increase of c-myc. Troglitazone (500 mg/kg.day) significantly inhibited tumor growth and prevented distant metastasis of BHP18-21 tumors in nude mice in vivo. Taken together, these results suggest that PPARgamma agonist inhibit cell growth of some types of human thyroid cancer.

Serpin protein CrmA suppresses hypoxia-mediated apoptosis of ventricular myocytes.

In this study, we ascertain whether caspase 8 activation and mitochondrial defects underlie apoptosis of ventricular myocytes during hypoxia. As an approach to circumvent the potential shortcomings surrounding the limited permeability and short half-life of the synthetic peptide inhibitors designed to block caspase activation, we constructed a replication-defective adenovirus encoding the serpin caspase inhibitor protein CrmA to ensure efficient and continual inhibition of caspase 8 activity during chronic hypoxia. In contrast to normoxic cells, oxygen deprivation of postnatal ventricular myocytes for 24 hours resulted in a 9-fold increase (P<0.05) in apoptosis as determined by Hoechst 33258 staining and nucleosomal DNA laddering. Moreover, hypoxia provoked a 1.5-fold increase (P<0.01) in caspase 8-like activity. Furthermore, hypoxia provoked perturbations to mitochondria consistent with the mitochondrial death pathway, including permeability transition pore (PT) opening, loss of mitochondrial membrane potential ((m)), and cytochrome c release. Importantly, CrmA suppressed caspase 8 activity, PT pore changes, loss of (m), and apoptosis but had no effect on hypoxia-mediated cytochrome c release. Furthermore, Bongkrekic acid, an inhibitor of PT pore, prevented hypoxia-induced PT pore changes, loss of (m), and apoptosis but had no effect on hypoxia-mediated cytochrome c release. To our knowledge, we provide the first direct evidence for the operation of CrmA as an antiapoptotic factor in ventricular myocytes during prolonged durations of hypoxia. Furthermore, our data suggest that perturbations to mitochondria including PT pore changes and (m) loss are caspase-regulated events that appear to be separable from cytochrome c release.

Cleavage of caspases-1, -3, -6, -8 and -9 substrates by proteases in skeletal muscles from mice undergoing cancer cachexia.

A prominent feature of several type of cancer is cachexia. This syndrome causes a marked loss of lean body mass and muscle wasting, and appears to be mediated by cytokines and tumour products. There are several proteases and proteolytic pathways that could be responsible for the protein breakdown. In the present study, we investigated whether caspases are involved in the proteolytic process of skeletal muscle catabolism observed in a murine model of cancer cachexia (MAC16), in comparison with a related tumour (MAC13), which does not induce cachexia. Using specific peptide substrates, there was an increase of 54% in the proteolytic activity of caspase-1, 84% of caspase-8, 98% of caspase-3 151% to caspase-6 and 177% of caspase-9, in the gastrocnemius muscle of animals bearing the MAC16 tumour (up to 25% weight loss), in relation to muscle from animals bearing the MAC13 tumour (1–5% weight loss). The dual pattern of 89 kDa and 25 kDa fragmentation of poly (ADP-ribose) polymerase (PARP) occurred in the muscle samples from animals bearing the MAC16 tumour and with a high amount of caspase-like activity. Cytochrome c was present in the cytosolic fractions of gastrocnemius muscles from both groups of animals, suggesting that cytochrome c release from mitochondria may be involved in caspase activation. There was no evidence for DNA fragmentation into a nucleosomal ladder typical of apoptosis in the muscles of either group of mice. This data supports a role for caspases in the catabolic events in muscle involved in the cancer cachexia syndrome. © 2001 Cancer Research Campaign http://www.bjcancer.com

Cervical Stroma Apoptosis in Pregnancy

In Brief Objective To determine whether the percentage of apoptotic nuclei is different in cervical stroma of pregnant laboring women compared with nonpregnant women and pregnant nonlaboring women. Methods We took cervical stromal biopsies during cesarean delivery at the level of the lower uterine segment from ten women in active labor and 13 women before labor. In addition, we took biopsies of cervical stroma at the level of the internal cervical os from hysterectomy specimens in ten reproductive-aged women. Cryosections were then analyzed using terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining. Tissue specimens were analyzed with ligation-mediated polymerase chain reaction to visualize nucleosomal ladders characteristic of apoptosis. To detect a 10% difference in the percentage of apoptotic cells per subject between study groups assuming a power of 0.90, an alpha of .05 in approximately ten subjects per group was needed. Results The median percentage of apoptotic nuclei was 0.7 (interquartile range 0.4, 1.4) for the nonpregnant group, 7.5 (interquartile range 6.6, 11.2) for the pregnant nonlaboring group, and 11.6 (interquartile range 8.3, 16.7) for the pregnant laboring group (P < .001). The percentage of apoptotic nuclei differed significantly across the three study groups. Using ligation-mediated polymerase chain reaction, nucleosomal ladders were seen in the specimens from pregnant women but not in the specimens from nonpregnant women, confirming the increase in stromal apoptosis seen with pregnancy. Conclusion Apoptosis of cervical stromal cells may play a role in the remodeling of the cervix during pregnancy and contribute to cervical changes during labor. Cervical stroma cell apoptosis is increased in pregnancy and in labor compared with nonpregnant women.

DNA fragmentation in cereal roots indicative of programmed root cortical cell death.

In cereals, a progressively increasing root cortical cell death (RCD) occurs from the root tip and upwards when measured with vital staining methods. In this study, nuclear DNA fragmentation was studied in seminal root segments of wheat and barley in order to investigate if the cell death resembled apoptosis. The fraction of cells with TUNEL-positive nuclei increased gradually with increasing root age in both the cortex and the stele. Southern analysis showed a typical ladder pattern, indicating nucleosomal fragmentation already in 2-day-old root segments, and this became more pronounced in older root segments. DNA fragmentation appeared to be more extensive in wheat than in barley roots. These results confirm earlier studies, where RCD has been found to be earlier initiated and to proceed at a faster rate in wheat. The characteristic DNA fragmentation found in the roots indicates programmed cell death with mechanistic similarities to apoptosis. Ultrastructural examination of nuclei in cortex cells with transmission electron microscopy revealed an increased chromatin condensation in older roots, particularly in wheat. In addition, we found nucleosomal DNA ladders in young leaf tissue from wheat but not from barley.

Cervical stroma apoptosis in pregnancy

Objective: To determine whether the percentage of apoptotic nuclei is different in cervical stroma of pregnant laboring women compared with nonpregnant women and pregnant nonlaboring women. Methods: We took cervical stromal biopsies during cesarean delivery at the level of the lower uterine segment from ten women in active labor and 13 women before labor. In addition, we took biopsies of cervical stroma at the level of the internal cervical os from hysterectomy specimens in ten reproductive-aged women. Cryosections were then analyzed using terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining. Tissue specimens were analyzed with ligation-mediated polymerase chain reaction to visualize nucleosomal ladders characteristic of apoptosis. To detect a 10% difference in the percentage of apoptotic cells per subject between study groups assuming a power of 0.90, an alpha of .05 in approximately ten subjects per group was needed. Results: The median percentage of apoptotic nuclei was 0.7 (interquartile range 0.4, 1.4) for the nonpregnant group, 7.5 (interquartile range 6.6, 11.2) for the pregnant nonlaboring group, and 11.6 (interquartile range 8.3, 16.7) for the pregnant laboring group ( P < .001). The percentage of apoptotic nuclei differed significantly across the three study groups. Using ligation-mediated polymerase chain reaction, nucleosomal ladders were seen in the specimens from pregnant women but not in the specimens from nonpregnant women, confirming the increase in stromal apoptosis seen with pregnancy. Conclusion: Apoptosis of cervical stromal cells may play a role in the remodeling of the cervix during pregnancy and contribute to cervical changes during labor.

Mitogen-induced p53 downregulation precedes vascular smooth muscle cell migration from healthy tunica media and proliferation.

The tumor suppressor protein p53 plays an important role in the cell-cycle G(1) and G(2) checkpoints. In response to DNA damage, p53 can induce the transcription of p21, which inhibits the activation of various G(1) cyclin/cyclin-dependent kinase complexes. It is not known whether p53 plays a role in the initial migration of vascular smooth muscle cells from the arterial tunica media (mVSMCs). In this study, we have investigated whether mVSMC migration from healthy tunica media of young pigs and proliferation are regulated by p53. After 6 hours of incubation in mitogen-rich medium, explanted porcine tunica media tissue showed complete downregulation of p53 protein and p53 mRNA. The blockage of gene activity was not due to DNA methylation at the 5' control region of the gene. The mVSMC outgrowth did not show p53 expression. Mitogen-depletion of cultured p53(-)/mVSMCs did not restore p53 expression. Incubation of explanted porcine tunica media tissue in mitogen-deprived medium increased p53 protein content and blocked mVSMC outgrowth from the explant. As in p53-deficient rodent cells, mVSMCs incubated with colcemid overrode the spindle-dependent checkpoint, giving polyploidy and chromosomal pairing. UV-induced DNA damage in mVSMCs incubated with mitogen-free medium induced p53 expression and apoptotic cell death showing DNA nucleosomal laddering. However, UV-irradiated mVSMCs incubated in mitogen-rich medium did not express p53 and did not show cell death. In conclusion, our results demonstrate that early mVSMC migration from the tunica media requires mitogen-induced suppression of p53 that is highly expressed in contractile mVSMCs residing in the healthy vessel wall.

DNA Laddering during Hypersensitive Cell Death in Cultured Rice Cells Induced by an Incompatible Strain of Pseudomonas avenae.

Clear 180-bp nucleosomal DNA laddering in cultured rice cells was induced only by an incompatible strain of Pseudomonas avenae. This supports the current view that the hypersensitive cell death of a plant is programmed cell death. The flagellin-deficient mutant of the incompatible strain did not induce DNA laddering, suggesting that DNA laddering was mediated by incompatible flagellin perception.

Transition between two forms of heterochromatin at plant subtelomeres.

The manner of packing of the terminal DNA loci into nucleosomes and higher order structures may strongly influence their functional interactions. Besides the structural flexibility of telomeric DNA sequences, conserved features of their chromatin including short nucleosome phasing (157 bp) and nucleosome sliding have been described previously. To gain a complementary knowledge of subtelomeres, we have analysed the chromatin structure of two subtelomeric tandem repeats from the plant Silene latifolia: X43.1 and 15Ssp. X43.1 shows two distinct nucleosome periodicities--157 and 188 bp. Preferred positions of its two nucleosomes have been mapped at both low and high resolution and the experimental results correspond to computer-predicted positions. 15Ssp is a newly-discovered sequence showing a telomere-associated position by PCR and a subtelomeric location by pulsed-field gel electrophoresis and fluorescence in situ hybridisation. Its 159 bp sequence unit shows a tandem arrangement and the presence of micrococcal nuclease-hypersensitive sites when either naked DNA or chromatin is digested. Use of a chemical nuclease results in a regular nucleosome ladder of 157 bp periodicity. Moreover, 15Ssp mononucleosomes show instability and absence of specific positioning, features typical for telomeric chromatin.

Involvement of mitochondrial permeability transition and caspase-9 activation in dimethyl sulfoxide-induced apoptosis of EL-4 lymphoma cells

We observed that dimethyl sulfoxide (DMSO) induced apoptotic changes in the EL-4 murine lymphoma cell line and that effect was dependent on the concentration and time period. Incubating cells over a period of 18 h, 2.5% DMSO was found to induce sub-G1 peak in DNA histograms analyzed by flowcytometer and nucleosomal ladder formation in DNA gel electrophoresis. We also found down-regulation of Bcl-2, collapse of mitochondrial membrane potential (Δ Ψ m) occurred following DMSO treatment, and release of cytochrome c from the mitochondria to cytosol. These observations suggest that DMSO converted its pro-apoptotic signal at the mitochondria. In the involvement of caspases, caspase-9 and -3, but not caspase-8, were found to be activated responding to DMSO treatment. Inhibitory experiments demonstrated that caspase cascade of mitochondrial apoptotic pathway was indispensable for DMSO-induced apoptosis. In the caspase cascade, caspase-9 was an upstream initiator and its primary signal could be transduced and amplified by caspase-3, -6 and -7. Kinetic study of these data showed mitochondrial dysfunction and caspase activation occurred at 12 h and apoptotic change of nuclear DNA at 18 h, providing another support for the transduction of DMSO pro-apoptotic signal via the mitochondrial pathway.

Troglitazone induces apoptosis via the p53 and Gadd45 pathway in vascular smooth muscle cells

Thiazolidinediones, activators of peroxisome proliferator-activated receptor (PPAR)γ, have been reported to induce apoptosis in many types of cells. In the present study, we investigated the effects of thiazolidinediones, troglitazone, and pioglitazone on the cell growth of vascular smooth muscle cells, and identified a specific effect of troglitazone in addition to PPARγ activation. Subconfluent rat culture vascular smooth muscle cells were treated with or without PPARγ activators, troglitazone (1–30 μM), or pioglitazone (1–30 μM) for 72 h. After treatment, cell viability was significantly reduced by troglitazone in concentration of 5–30 μM but not by pioglitazone. Vascular smooth muscle cells appeared to float and shrink 48 h after treatment with 20 μM of troglitazone. In situ DNA labeling showed that the nuclei of these cells were positively stained, and genomic DNA extracted from the cells showed nucleosomal laddering. Messenger RNA expression levels of c-myc, p21, bax, bcl-2, and bcl-x were not changed by the treatment with troglitazone. In contrast, along with the induction of vascular smooth muscle cell apoptosis, both the mRNA and protein expression levels of p53 and Gadd45 markedly increased in response to troglitazone. These results strongly suggest that troglitazone can induce vascular smooth muscle cell apoptosis and that this effect is caused primarily by activation of the p53 and Gadd45 pathway but not by PPARγ activation.

Molecular Mechanisms of Cell Cycle Block by Methionine Restriction in Human Prostate Cancer Cells

Previous studies have shown that dietary or pharmacological methionine restriction inhibits growth of human prostate cancer cells in vitro or as xenografts in mice. We undertook the present studies to clarify the molecular mechanisms by which methionine restriction inhibits prostate cancer cell growth. We found that PC-3 and DU 145 cells stopped proliferating within six days in growth medium containing homocysteine in place of methionine. In contrast, proliferation of LNCaP cells was only partially inhibited by the absence of methionine. Using flow cytometry, we found that methionine restriction caused PC-3 cells to arrest in all phases of the cell cycle, but predominantly in the G2/M phase, whereas LNCaP cells accumulated exclusively in the G1 phase. Methionine restriction led to accumulation of the cyclin-dependent kinase inhibitors p21 and p27, as determined by Western blot analysis, and inhibited the enzymatic activities of the cyclin-dependent kinases CDK2 and cdc2, as determined by an in vitro kinase assay. However, methionine restriction had little or no effect on CDK2 or cdc2 protein levels. Methionine restriction also induced PC-3 cells to undergo apoptosis, as indicated by the appearance of a typical nucleosomal ladder on gel electrophoresis of genomic DNA. We conclude that methionine restriction has potential as a novel treatment strategy for prostate cancer.

Hormone activation induces nucleosome positioning in vivo.

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the transition in chromatin structure following hormone activation. This revealed two novel findings: hormone activation led to the establishment of specific translational positioning of nucleosomes despite the lack of significant positioning in the inactive state; and, in the active promoter, a subnucleosomal particle encompassing the glucocorticoid receptor (GR)-binding region was detected. The presence of only a single GR-binding site was sufficient for the structural transition to occur. Both basal promoter elements and ongoing transcription were dispensable. These data reveal a stepwise process in the transcriptional activation by glucocorticoid hormone.