Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

Anticancer activities of cinnamic acid derivatives include induction of apoptosis by irreversible DNA damage leading to cell death. The present work aimed to compare the cytotoxic and genotoxic potential of cinnamic acid in human melanoma cell line (HT-144) and human melanocyte cell line derived from blue nevus (NGM). Viability assay showed that the IC50 for HT-144 cells was 2.4 mM, while NGM cells were more resistant to the treatment. The growth inhibition was probably associated with DNA damage leading to DNA synthesis inhibition, as shown by BrdU incorporation assay, induction of nuclear aberrations and then apoptosis. The frequency of cell death caused by cinnamic acid was higher in HT-144 cells. Activated-caspase 3 staining showed apoptosis after 24 hours of treatment with cinnamic acid 3.2 mM in HT-144 cells, but not in NGM. We observed microtubules disorganization after cinnamic acid exposure, but this event and cell death seem to be independent according to M30 and tubulin labeling. The frequency of micronucleated HT-144 cells was higher after treatment with cinnamic acid (0.4 and 3.2 mM) when compared to the controls. Cinnamic acid 3.2 mM also increased the frequency of micronucleated NGM cells indicating genotoxic activity of the compound, but the effects were milder. Binucleation and multinucleation counting showed similar results. We conclude that cinnamic acid has effective antiproliferative activity against melanoma cells. However, the increased frequency of micronucleation in NGM cells warrants the possibility of genotoxicity and needs further investigation.

Modulation of NF-kappaB activation in Theileria annulata-infected cloned cell lines is associated with detection of parasite-dependent IKK signalosomes and disruption of the actin cytoskeleton

Summary Apicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF-kappaB in a parasite-dependent manner. Activation of NF-kappaB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF-kappaB. Further characterization of these lines demonstrated that elevated levels of activated NF-kappaB correlated with increased resistance to cell death and detection of parasite-associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host- and parasite-dependent NF-kappaB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite-dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite-encoded surface ligand.

BCL6 suppression of BCL2 via Miz1 and its disruption in diffuse large B cell lymphoma

The BCL6 proto-oncogene encodes a transcriptional repressor that is required for germinal center (GC) formation and whose deregulation by genomic lesions is implicated in the pathogenesis of GC-derived diffuse large B cell lymphoma (DLBCL) and, less frequently, follicular lymphoma (FL). The biological function of BCL6 is only partially understood because no more than a few genes have been functionally characterized as direct targets of BCL6 transrepression activity. Here we report that the anti-apoptotic proto-oncogene BCL2 is a direct target of BCL6 in GC B cells. BCL6 binds to the BCL2 promoter region by interacting with the transcriptional activator Miz1 and suppresses Miz1-induced activation of BCL2 expression. BCL6-mediated suppression of BCL2 is lost in FL and DLBCL, where the 2 proteins are pathologically coexpressed, because of BCL2 chromosomal translocations and other mechanisms, including Miz1 deregulation and somatic mutations in the BCL2 promoter region. These results identify an important function for BCL6 in facilitating apoptosis of GC B cells via suppression of BCL2, and suggest that blocking this pathway is critical for lymphomagenesis.

Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

Persistent and compartmentalised disruption of dendritic cell subpopulations in the lung following influenza A virus infection.

Immunological homeostasis in the respiratory tract is thought to require balanced interactions between networks of dendritic cell (DC) subsets in lung microenvironments in order to regulate tolerance or immunity to inhaled antigens and pathogens. Influenza A virus (IAV) poses a serious threat of long-term disruption to this balance through its potent pro-inflammatory activities. In this study, we have used a BALB/c mouse model of A/PR8/34 H1N1 Influenza Type A Virus infection to examine the effects of IAV on respiratory tissue DC subsets during the recovery phase following clearance of the virus. In adult mice, we found differences in the kinetics and activation states of DC residing in the airway mucosa (AMDC) compared to those in the parenchymal lung (PLDC) compartments. A significant depletion in the percentage of AMDC was observed at day 4 post-infection that was associated with a change in steady-state CD11b+ and CD11b- AMDC subset frequencies and significantly elevated CD40 and CD80 expression and that returned to baseline by day 14 post-infection. In contrast, percentages and total numbers of PLDC were significantly elevated at day 14 and remained so until day 21 post-infection. Accompanying this was a change in CD11b+and CD11b- PLDC subset frequencies and significant increase in CD40 and CD80 expression at these time points. Furthermore, mice infected with IAV at 4 weeks of age showed a significant increase in total numbers of PLDC, and increased CD40 expression on both AMDC and PLDC, when analysed as adults 35 days later. These data suggest that the rate of recovery of DC populations following IAV infection differs in the mucosal and parenchymal compartments of the lung and that DC populations can remain disrupted and activated for a prolonged period following viral clearance, into adulthood if infection occurred early in life.

Thromboxane A2 acts as tonic immunoregulator by preferential disruption of low-avidity CD4+ T cell-dendritic cell interactions

Interactions between dendritic cells (DCs) and T cells control the decision between activation and tolerance induction. Thromboxane A2 (TXA2) and its receptor TP have been suggested to regulate adaptive immune responses through control of T cell-DC interactions. Here, we show that this control is achieved by selectively reducing expansion of low-avidity CD4(+) T cells. During inflammation, weak tetramer-binding TP-deficient CD4(+) T cells were preferentially expanded compared with TP-proficient CD4(+) T cells. Using intravital imaging of cellular interactions in reactive peripheral lymph nodes (PLNs), we found that TXA2 led to disruption of low- but not high-avidity interactions between DCs and CD4(+) T cells. Lack of TP correlated with higher expression of activation markers on stimulated CD4(+) T cells and with augmented accumulation of follicular helper T cells (TFH), which correlated with increased low-avidity IgG responses. In sum, our data suggest that tonic suppression of weak CD4(+) T cell-DC interactions by TXA2-TP signaling improves the overall quality of adaptive immune responses.

Gene disruption of p27Kip1 allows cell proliferation in the postnatal and adult organ of Corti

Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024–1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27Kip1 is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27Kip1-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27Kip1-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ.

Disruption of epithelial gamma delta T cell repertoires by mutation of the Syk tyrosine kinase.

Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial gamma delta T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial gamma delta T cells are similar to B cells, but distinct from splenic gamma delta T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V gamma/V delta gene rearrangements with specific epithelia are also disrupted by Syk deficiency.

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion.

Vinculin, a major constituent of focal adhesions and zonula adherens junctions, is thought to be involved in linking the microfilaments to areas of cell-substrate and cell-cell contacts. To test the role of vinculin in cell adhesion and motility, we used homologous recombination to generate F9 embryonal carcinoma and embryonic stem cell clones homozygous for a disrupted vinculin gene. When compared to wild-type cells, vinculin-mutant cells displayed a rounder morphology and a reduced ability to adhere and spread on plastic or fibronectin. Decreased adhesion of the mutant cells was associated with a reduction in lamellipodial extensions, as observed by time-lapse video microscopy. The locomotive activities of control F9 and the vinculin-null cells were compared in two assays. Loss of vinculin resulted in a 2.4-fold increase in cell motility. These results demonstrate an important role for vinculin in determining cell shape, adhesion, surface protrusive activity, and cell locomotion.

Antitumor activity of 3-ingenyl angelate: Plasma membrane and mitochondrial disruption and necrotic cell death

Options for skin cancer treatment currently include surgery, radiotherapy, topical chemotherapy, cryosurgery, curettage, and electrodes-sication. Although effective, surgery is costly and unsuitable for certain patients. Radiotherapy can leave a poor cosmetic effect, and current chemotherapy is limited by low cure rates and extended treatment schedules. Here, we describe the preclinical activity of a novel topical chemotherapeutic agent for the treatment of skin cancer, 3-ingenyl angelate (PEP005), a hydrophobic diterpene ester isolated from the plant Euphorbia peplus. Three daily topical applications of 42 nmol (18 mug) of PEP005 cured a series of s.c. mouse tumors (B16 melanoma, LK2 UV-induced squamous cell carcinoma, and Lewis lung carcinoma; it = >14 tumors/group) and human tumors (DO4 melanoma, HeLa cervical carcinoma, and PC3 and DU145 prostate carcinoma; it = >4 tumors/group) previously established (5-10 mm(3)) on C57BL/6 or Fox1(nu) mice. The treatment produced a mild, short-term erythema and eschar formation but, ultimately, resulted in excellent skin cosmesis. The LD90 for PEP005 for a panel of tumor cell lines was 180-220 muM. Electron microscopy showed that treatment with PEP005 both ill vitro (230 tot) and ill vivo (42 nmol) rapidly caused swelling of mitochondria and cell death by primary necrosis. Cr-51 release, uptake of propidium iodide, and staining with the mitochondria dye JC1, revealed that PEP005 (230 muM) treatment of tumor cells ill vitro resulted in a rapid plasma membrane perturbation and loss of mitochondrial membrane potential. PEP005 thus emerges as a new topical anti-skin cancer agent that has a novel mode of action involving plasma membrane and mitochondrial disruption and primary necrosis, ultimately resulting in an excellent cosmetic outcome.

Disruption of BRCA I function results in telomere lengthening and increased anaphase bridge formation in immortalized cell lines

BRCA1 is a tumor suppressor that functions in controlling cell growth and maintaining genomic stability. BRCA1 has also been implicated in telomere maintenance through its ability to regulate the transcription of hTERT, the catalytic subunit of telomerase, resulting in telomere shortening, and to colocalize with the telomere-binding protein TRF1. The high incidence of nonreciprocal translocations in tumors arising from BRCA1 mutation carriers and Brca1-null mice also raises the possibility that BRCA1 plays a role in telomere protection. To date, however, the consequences for telomere status of disrupting BRCA1 have not been reported. To examine the role of BRCA1 in telomere regulation, we have expressed a dominant-negative mutant of BRCA1 (trBRCA1), known to disrupt multiple functions of BRCA1, in telomerase-positive mammary epithelial cells (SVCT) and telomerase-negative ALT cells (GM847). In SVCT cells, expression of trBRCA1 resulted in an increased incidence of anaphase bridges and in an increase in telomere length, but no change in telomerase activity. In GM847 cells, trBRCA1 also increased anaphase bridge formation but did not induce any change in telomere length. BRCA1 colocalized with TRF2 in telomerase-positive cells and with a small subset of ALT-associated PML bodies (APBs) in ALT cells. Together, these results raise the possibility that BRCA1 could play a role in telomere protection and suggest a potential mechanism for one of the phenotypes of BRCA1 deficient cells. (c) 2005 Wiley-Liss, Inc.

On uniquely determining cell-wall material properties with the compression experiment

A finite element model (FEM) of the cell-compression experiment has been developed in dimensionless form to extract the fundamental cell-wall-material properties (i.e. the constitutive equation and its parameters) from experiment force-displacement data. The FEM simulates the compression of a thin-walled, liquid-filled sphere between two flat surfaces. The cell-wall was taken to be permeable and the FEM therefore accounts for volume loss during compression. Previous models assume an impermeable wall and hence a conserved cell volume during compression. A parametric study was conducted for structural parameters representative of yeast. It was shown that the common approach of assuming reasonable values for unmeasured parameters (e.g. cell-wall thickness, initial radial stretch) can give rise to nonunique solutions for both the form and constants in the cell-wall constitutive relationship. Similarly, measurement errors can also lead to an incorrectly defined cell-wall constitutive relationship. Unique determination of the fundamental wall properties by cell compression requires accurate and precise measurement of a minimum set of parameters (initial cell radius, initial cell-wall thickness, and the volume loss during compression). In the absence of such measurements the derived constitutive relationship may be in considerable error, and should be evaluated against its ability to predict the outcome of other mechanical experiments. (C) 1998 Elsevier Science Ltd. All rights reserved.

Nuclear localization of RelB is associated with effective antigen-presenting cell function

Dendritic cells (DC) are potent APCs that enter resting tissues as precursors and, after Ag exposure, differentiate and migrate to draining lymph nodes. The phenotype of RelB knockout mice implicates this member of the NF kappa B/Rel family in DC differentiation. To further elucidate the role of RelB in DC differentiation, mRNA, intracellular protein expression, and DNA binding activity of RelB were examined in immature and differentiated human DC, as well as other PB mononuclear cell populations. RelB protein and mRNA were detected constitutively in lymphocytes and in activated monocytes, differentiated DC, and monocyte-derived DC. Immunohistochemical staining demonstrated RelB within the differentiated lymph node interdigitating DC and follicular DC, but not undifferentiated DC in normal skin. Active nuclear RelB was detected by supershift assay only in differentiated DC derived from either PB precursors or monocytes and in activated B cells. These RelB(+) APC were potent stimulators of the MLR. The data indicate that RelB expression is regulated both transcriptionally and post-translationally in myeloid cells. Within the nucleus, RelB may specifically transactivate genes that are critical for APC function.

Transient disruption of liver gap junctional intercellular communication and induction of apoptosis after administration of 1,4-bis[2-(3,5 dichloropyridyloxy)]benzene in mice

Gap junctional intercellular communication (GJIC) and connexin expression (Cx26 and Cx32) in mouse liver were studied after administration of 4-bis[2-(3,5 dichloropyridyloxy)]benzene (TCPOBOP), a phenobarbital-like enzyme inducer. Female C57BI/6 mice were administered TCPOBOP (5.8 mg/kg BW) and euthanized 0, 24, 48 and 72 hours later. Liver samples were snap frozen, or fixed in formalin, or submitted to GJIC analysis. The proliferating cell nuclear antigen (PCNA) immunohistochemistry and the Western blotting for Cx26 and Cx32 were performed. After 48 and 72 h of drug administration the liver-to-body weight ratio was increased 70% and 117% (p < 0.0001), respectively. There were temporal-dependent alterations in liver histopathology and a significant increase in cell proliferation was noted after 48h and sustained after 72h, though to a lesser extent (p < 0.0001). In addition. TCPOBOP administration induced apoptosis, which appeared to be time-dependent showing statistical significance only after 72h (p < 0.0001). Interestingly, a transient disruption by nearly 50% of GJIC capacity was detected after 48 h of drug ingestion, which recovered after 72 h (p = 0.003). These GJIC changes were due to altered levels of Cx26 and Cx32 in the livers of TCPOBOP-treated mice. We concluded that a single administration of TCPOBOP transiently disrupted the levels of GJIC due to decreased expression of connexins and increased apoptotic cell death in mouse liver. (C) 2009 Elsevier GmbH. All rights reserved.