Melanoma cell lines are susceptible to histone deacetylase inhibitor TSA provoked cell cycle arrest and apoptosis

Melanoma is the most aggressive of skin cancers because of its high resistance to currently available therapy. Although melanoma cells often retain wild-type p53 tumour suppressor protein and express it at high levels, the p53 mediated apoptosis pathway is suppressed. Histone deacetylase (HDAC) inhibitors are a promising group of compounds inducing differentiation, growth arrest and apoptosis in tumour cells in preclinical studies. We have studied the cellular effects of trichostatin A (TSA), a HDAC inhibitor, in a panel of melanoma cell lines and its mechanism of action in relation to p53. TSA stabilized wild-type p53, but p53 protein accumulation was overridden by simultaneous downregulation of p53 mRNA leading to a decrease in p53 protein. While growth arrest was induced in all cell lines studied and apoptosis in most (6/7), these cellular effects were independent of the p53 status of the cells. Inhibiting p53 function by a dominant negative p53 (p53(175His)) confirmed that the HDAC inhibitor induced apoptosis was independent of wild-type p53, even though TSA slightly activated p53 in a reporter assay. The results indicate that while the action of TSA is independent of p53, the activation of the apoptosis pathway by the HDAC inhibitors may provide therapeutic approaches for melanoma treatment.     

Removal of N-glycans from cell surface proteins induces apoptosis by reducing intracellular glutathione levels in the rhabdomyosarcoma cell line S4MH

Expression of determined Asn-bound glycans (N-glycans) in cell surface glycoproteins regulates different processes in tumour cell biology. Specific patterns of N-glycosylation are displayed by highly metastatic cells and it has been shown that inhibition of N-glycan processing restrains cell proliferation and induces cell death via apoptosis. However, the mechanisms by which different N-glycosylation states may regulate cell viability and growth are not understood. Since malignant cells express high levels of intracellular glutathione (GSH) and a reduction of intracellular GSH induces cell death via apoptosis, we investigated whether GSH was involved in the induction of apoptosis by removal of cell surface N-glycans. We found that removal of N-glycans from cell surface proteins by treating the rhabdomyosarcoma cell line S4MH with tunicamycin or N-glycosidase resulted in a reduction in intracellular GSH content and cell death via apoptosis. Moreover, GSH depletion caused by the specific inhibitor of GSH synthesis BSO induced apoptosis in S4MH cells. This data indicates that adequate N-glycosylation of cell surface glycoproteins is required for maintenance of intracellular GSH levels that are necessary for cell survival and proliferation.     

Lgl/aPKC and Crb regulate the Salvador/Warts/Hippo pathway

A key goal of developmental biology is to understand the mechanisms that coordinate organ growth. It has long been recognized that the genes that control apico-basal cell polarity also regulate tissue growth. How loss of cell polarity contributes to tissue overgrowth has been the subject of much speculation. Do loss-of-function mutations in cell polarity regulators result in secondary effects that globally deregulate cell proliferation, or do these genes specifically control growth pathways? Three recent papers have shown that the apico-basal polarity determinants Lgl/aPKC and Crb regulate tissue growth independently of their roles in cell polarity and coordinately regulate cell proliferation and cell death via the Salvador/Warts/Hippo (SWH) pathway. Lgl/aPKC are required for the correct localization of Hippo (Hpo)/Ras associated factor (RASSF), while Crb regulates the levels and localization of Expanded (Ex), indicating that cell polarity determinants modify SWH pathway activity by distinct mechanisms. Here, we review the key data that support these conclusions, highlight remaining questions and speculate on the underlying mechanisms by which the cell polarity complexes interact with the SWH pathway. Understanding the interactions between cell polarity regulators and the SWH pathway will improve our knowledge of how epithelial organization and tissue growth are coordinated during development and perturbed in disease states such as cancer.Key words: Drosophila, tumor suppressor gene, cell polarity, Hippo pathway, Crb, Lgl, aPKC     

Endocytic control of epithelial polarity and proliferation in Drosophila

Intracellular protein transport is a key factor in epithelial cell polarity. Here we report that mutations in two core components of the vesicle trafficking machinery - a syntaxin and a Rab protein - cause an expansion of the apical membrane domain of Drosophila melanogaster epithelia; this polarity defect is coupled with overproliferation to form neoplastic tumours. Surprisingly, these proteins are associated with the endocytic, and not the exocytic, pathway. The syntaxin Avalanche (Avl) localizes to early endosomes, and loss of avl results in the cellular accumulation of specific membrane proteins, including the Notch signalling receptor and the polarity determinant Crumbs (Crb). Protein accumulation results from a failure in endosomal entry and progression towards lysosomal degradation; these and other avl phenotypes are also detected in Rab5 null mutant cells. Overexpression of Crb alone is sufficient to induce overproliferation of wild-type imaginal tissue, suggesting that polarity alterations in avl and Rab5 mutants directly contribute to tumour formation. Our findings reveal a critical and specific role for endocytic traffic in the control of both apico-basal polarity and cell proliferation.     

The frizzled extracellular domain is a ligand for Van Gogh/Stbm during nonautonomous planar cell polarity signaling

The Frizzled (Fz) receptor is required cell autonomously in Wnt/beta-catenin and planar cell polarity (PCP) signaling. In addition to these requirements, Fz acts nonautonomously during PCP establishment: wild-type cells surrounding fz(-) patches reorient toward the fz(-) cells. The molecular mechanism(s) of nonautonomous Fz signaling are unknown. Our in vivo studies identify the extracellular domain (ECD) of Fz, in particular its CRD (cysteine rich domain), as critical for nonautonomous Fz-PCP activity. Importantly, we demonstrate biochemical and physical interactions between the FzECD and the transmembrane protein Van Gogh/Strabismus (Vang/Stbm). We show that this function precedes cell-autonomous interactions and visible asymmetric PCP factor localization. Our data suggest that Vang/Stbm can act as a FzECD receptor, allowing cells to sense Fz activity/levels of their neighbors. Thus, direct Fz-Vang/Stbm interactions represent an intriguing mechanism that may account for the global orientation of cells within the plane of their epithelial field.     

Lgl/aPKC and Crb regulate the Salvador/Warts/Hippo pathway

A key goal of developmental biology is to understand the mechanisms that coordinate organ growth. It has long been recognized that the genes that control apico-basal cell polarity also regulate tissue growth. How loss of cell polarity contributes to tissue overgrowth has been the subject of much speculation. Do loss-of-function mutations in cell polarity regulators result in secondary effects that globally deregulate cell proliferation, or do these genes specifically control growth pathways? Three recent papers have shown that the apico-basal polarity determinants Lgl/aPKC and Crb regulate tissue growth independently of their roles in cell polarity and coordinately regulate cell proliferation and cell death via the Salvador/Warts/Hippo (SWH) pathway. Lgl/aPKC are required for the correct localization of Hippo (Hpo)/Ras associated factor (RASSF), whilst Crb regulates the levels and localization of Expanded (Ex), indicating that cell polarity determinants modify SWH pathway activity by distinct mechanisms. Here, we review the key data that support these conclusions, highlight remaining questions and speculate on the underlying mechanisms by which the cell polarity complexes interact with the SWH pathway. Understanding the interactions between cell polarity regulators and the SWH pathway will improve our knowledge of how epithelial organization and tissue growth are coordinated during development and perturbed in disease states such as cancer.     

FcγRIIb and BAFF differentially regulate peritoneal B1 cell survival

B1 cells produce most natural Abs in unimmunized mice and play a key role in the response to thymus-independent Ags and microbial infection. Enlargement of B1 cell number in mice is often associated with autoimmunity. However, the factors that control peripheral B1 cell survival remain poorly characterized. Mice lacking the inhibitory receptor FcγRIIb exhibit a massive expansion in peritoneal B1 cells, implicating this receptor in B1 cell homeostasis. In this study, we show that peritoneal B1 cells express the highest levels of FcγRIIb among B cell subsets and are highly susceptible to FcγRIIb-mediated apoptosis. B1 cells upregulate FcγRIIb in response to innate signals, including CpG, and the B cell homeostatic cytokine BAFF efficiently protects activated B1 cells from FcγRIIb-mediated apoptosis via receptor downregulation. BAFF-transgenic mice manifest an expansion of peritoneal B1 cells that express lower levels of FcγRIIb and exhibit reduced susceptibility to apoptosis. Whereas both peritoneal B1 cells from wild-type and BAFF-transgenic mice immunized with CpG exhibit an increase in FcγRIIb levels, this change is blunted in BAFF-transgenic animals. Our combined results demonstrate that FcγRIIb controls peritoneal B1 cell survival and this program can be modulated by the BAFF signaling axis.     

Trophic support promotes survival of bcl-x-deficient telencephalic cells in vitro

Survival of immature neurons is regulated by Bcl-xL, as targeted disruption of bcl-x significantly increases cell death in vivo and in vitro. Death of cultured bcl-x-deficient and wild-type telencephalic cells can be prevented by fetal calf serum or chemically-defined medium (ITS), suggesting trophic factors in these media potentiate survival through a pathway independent of Bcl-xL. Addition of trophic factors to basal medium revealed that insulin and insulin-like growth factors (IGFs), but not other trophic factors, reduced apoptosis of wild-type and bcl-x-deficient telencephalic cells. Antibodies raised against IGF-I receptors and wortmannin both attenuated the effects of IGF-I, indicating survival was mediated by IGF-I receptors and phosphatidylinositol 3'-kinase signaling, whereas effects of ITS were only partially reduced by these agents. The survival promoting effects of ITS were reduced in cells lacking both bcl-x and bcl-2, indicating Bcl-2 plays a supportive role to Bcl-xL in maintaining telencephalic cell survival. Furthermore, the ratio of expression of the pro-apoptotic bax gene to the anti-apoptotic bcl-2 gene was reduced in bcl-x-deficient cultures grown in ITS, suggesting that the interaction between these bcl-2 family members may, in part, regulate a Bcl-xL independent survival pathway. Finally, the pro-apoptotic bad gene does not appear to play a role in these interactions as targeted disruption of bad did not alter apoptosis in telencephalic cultures.     

Evidence that phosphatidylinositol 3-kinase- and mitogen-activated protein kinase kinase-4/c-Jun NH2-terminal kinase-dependent Pathways cooperate to maintain lung cancer cell survival

Cancer cells in which the PTEN lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than PTEN gene loss. The role of PI3K in the survival of cancer cells that express wild-type PTEN has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type PTEN, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the PTEN lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85alpha regulatory subunit of PI3K (Deltap85) induced apoptosis. Whereas PTEN and Delta85 both inhibited activation of AKT/protein kinase B, only Deltap85 inhibited c-Jun NH2-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant Rac-1 (Val12), an upstream activator of JNK, abrogated Deltap85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (MKK)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of MKK4-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.     

Varicella-Zoster Virus ORF12 Protein Activates the Phosphatidylinositol 3-Kinase/Akt Pathway To Regulate Cell Cycle Progression

Varicella-zoster virus (VZV) activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and alters cell cycle progression, but the viral protein(s) responsible for these activities is unknown. We previously reported that the VZV open reading frame 12 (ORF12) protein triggers phosphorylation of ERK. Here, we demonstrate that the VZV ORF12 protein also activates the PI3K/Akt pathway to regulate cell cycle progression. Transfection of cells with a plasmid expressing the ORF12 protein induced phosphorylation of Akt, which was dependent on PI3K. Infection of cells with wild-type VZV triggered phosphorylation of Akt, while infection with an ORF12 deletion mutant induced less phosphorylated Akt. The activation of Akt by ORF12 protein was associated with its binding to the p85 subunit of PI3K. Infection of cells with wild-type VZV resulted in increased levels of cyclin B1, cyclin D3, and phosphorylated glycogen synthase kinase 3β (GSK-3β), while infection with the ORF12 deletion mutant induced lower levels of these proteins. Wild-type VZV infection reduced the G₁ phase cell population and increased the M phase cell population, while infection with the ORF12 deletion mutant had a reduced effect on the G₁ and M phase populations. Inhibition of Akt activity with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 reduced the G₁ and M phase differences observed in cells infected with wild-type and ORF12 mutant viruses. In conclusion, we have found that the VZV ORF12 protein activates the PI3K/Akt pathway to regulate cell cycle progression. Since VZV replicates in both dividing (e.g., keratinocytes) and nondividing (neurons) cells, the ability of the VZV ORF12 protein to regulate the cell cycle is likely important for VZV replication in various cell types in the body.     

dMyc transforms cells into super-competitors

Overexpression of myc protooncogenes has been implicated in the genesis of many human tumors. Myc proteins seem to regulate diverse biological processes, but their role in tumorigenesis remains enigmatic. Here we use Drosophila imaginal discs to mimic situations in which cells with unequal levels of Myc protein are apposed and show that this invariably elicits a win/lose situation reminiscent of cell competition; cells with lower levels of dMyc are eliminated by apoptosis whereas cells with higher levels of dMyc over-proliferate. We find that this competitive behavior correlates with, and can be corrected by, the activation of the BMP/Dpp survival signaling pathway. Hence the heritable increase in dMyc levels causes cells to behave as "super-competitors" and reveals a novel mode of clonal expansion that causes, but also relies on, the killing of surrounding cells.     

Regulation of apoptosis in osteoclasts and osteoblastic cells

In postnatal life, the skeleton undergoes continuous remodeling in which osteoclasts resorb aged or damaged bone, leaving space for osteoblasts to make new bone. The balance of proliferation, differentiation, and apoptosis of bone cells determines the size of osteoclast or osteoblast populations at any given time. Bone cells constantly receive signals from adjacent cells, hormones, and bone matrix that regulate their proliferation, activity, and survival. Thus, the amount of bone and its microarchitecture before and after the menopause or following therapeutic intervention with drugs, such as sex hormones, glucocorticoids, parathyroid hormone, and bisphosphonates, is determined in part by effects of these on survival of osteoclasts, osteoblasts, and osteocytes. Understanding the mechanisms and regulation of bone cell apoptosis will enhance our knowledge of bone cell function and help us to develop better therapeutics for the management of osteoporosis and other bone diseases.     

Micro-environmental factors in the survival of human B-lymphoma cells

Burkitt lymphoma (BL) cells retain a high inherent propensity to undergo apoptosis indicating that net growth of the tumour population in vivo is likely to be influenced profoundly by its micro-environment. Here we investigate micro-environmental factors that affect BL-cell survival in vitro. We show that survival, and consequently net production, of tumour cells is enhanced by autocrine factors and, to a greater extent, by paracrine factors provided by relevant stromal elements of the tumour (fibroblasts and follicular dendritic cells) and by macrophages. Promotion of BL-cell survival by paracrine elements was mediated by cell/cell contact and by short-range soluble factor(s). IL-4, IL-10 and TNF-alpha promoted, whereas TGF-beta1 inhibited, tumour-cell production. Macrophages engaged in phagocytosis of apoptotic BL cells were less effective than untreated macrophages in supporting net expansion of BL populations. These results suggest that the net production of tumour cells in BL is supported by multiple micro-environmental factors that modulate apoptosis.     

Estimating the selective advantage of mutant p53 tumour cells to repeated rounds of hypoxia

The tumour suppressor gene, p53, plays an important role in tumour development. Under low levels of oxygen (hypoxia), cells expressing wild-type p53 undergo programmed cell death (apoptosis), whereas cells expressing mutations in the p53 gene may survive and express angiogenic growth factors that stimulate tumour vascularization. Given that cells expressing mutations in the p53 gene have been observed in many forms of human tumour, it is important to understand how both wild-type and mutant cells react to hypoxic conditions. In this paper a mathematical model is presented to investigate the effects of alternating periods of hypoxia and normoxia (normal oxygen levels) on a population of wild-type and mutant p53 tumour cells. The model consists of three coupled ordinary differential equations that describe the densities of the two cell types and the oxygen concentration and, as such, may describe the growth of avascular tumours in vitro and/or in vivo. Numerical and analytical techniques are used to determine how changes in the system parameters influence the time at which mutant cells become dominant within the population. A feedback mechanism, which switches off the oxygen supply when the total cell density exceeds a threshold value, is introduced into the model to investigate the impact that vessel collapse (and the associated hypoxia) has on the time at which the mutant cells become dominant within vascular tumours growing in vivo. Using the model we can predict the time it takes for a subpopulation of mutant p53 tumour cells to become the dominant population within either an avascular tumour or a localized region of a vascular tumour. Based on independent experimental results, our model suggests that the mutant population becomes dominant more quickly in vivo than in vitro (12 days vs 17 days).     

Phenotypic variations of TRAIL sensitivity in cloned populations of prostate cancer cells

Factors that regulate the induction of apoptosis of tumour cells are potential candidates for therapeutic intervention for the majority of cancers. Studying modifiers of apoptotic responses, such as members of the tumour necrosis factor receptor superfamily, may give clues as to how induction of apoptosis in tumours could be maximized to enhance the benefit of treatment regimes. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is a promising anti‐tumour molecule since its activity is specific for tumour cell populations. TRAIL binds to death receptors, inducing apoptosis in susceptible cells. The mechanisms which determine whether tumour cells are susceptible to TRAIL are unclear, and several mechanisms have been proposed, including expression of osteoprotegerin (OPG), decoy receptors, and factors that affect intracellular signalling of pro‐apoptotic molecules, such as c‐FLIP. Here we show that experiments to modulate the activity of one of these factors, OPG, by over‐expression and also by stable knockdown of OPG expression, alters the TRAIL sensitivity of PC3 prostate cancer cells. However we show that some observed effects, which appear to support the hypothesis that OPG prevents TRAIL‐induced apoptosis of tumour cells, may be due to variation of the TRAIL response of sub‐clones of tumour cells, even within a cloned population. These results highlight potential limitations of experiments designed to test contribution of factors affecting intrinsic apoptosis susceptibility using cloned tumour cell populations. J. Cell. Biochem. 104: 1452–1464, 2008. © 2008 Wiley‐Liss, Inc.     

Apoptosis initiated by dependence receptors: a new paradigm for cell death?

A distinct group of receptors including DCC, UNC5, RET and Ptc1 is known to function in ligand-dependent neuronal growth and differentiation or axon guidance. Acting as "dependence receptors", they may also regulate neuronal cell survival by inducing apoptosis in the absence of cognate ligand. Receptor-initiated apoptosis requires proteolytic (caspase) cleavage and exposure of a pro-apoptotic region in the cytoplasmic domains of the receptors. In contrast, classical apoptosis induced by growth factor or cytokine deprivation involves loss of survival signaling without receptor cleavage. DCC, UNC5, RET and Ptc1 are downregulated or mutated in diverse cancers, and show properties characteristic of tumor suppressors, consistent with their ability to promote neuronal cell death. Dysfunctional dependence receptors have been linked to the loss of specific neurons in certain inherited and neurodegenerative diseases. Dependence receptor-initiated apoptosis represents a novel paradigm for the controlled removal of specific cells during neural development and elimination of malignant cells that have strayed beyond regions of ligand availability.     

Local,cell-nonautonomous feedback regulation of myosin dynamics patterns transitions in cell behavior: a role for tension and geometry?

How robust patterns of tissue dynamics emerge from heterogeneities, stochasticities, and asynchronies in cell behavior is an outstanding question in morphogenesis. A clear understanding of this requires examining the influence of the behavior of single cells on tissue patterning. Here we develop single-cell manipulation strategies to uncover the origin of patterned cell behavior in the amnioserosa during Drosophila dorsal closure. We show that the formation and dissolution of contractile, medial actomyosin networks previously shown to underlie pulsed apical constrictions in the amnioserosa are apparently asynchronous in adjacent cells. We demonstrate for the first time that mechanical stresses and Rho1 GTPase control myosin dynamics qualitatively and quantitatively, in amplitude and direction, both cell autonomously and nonautonomously. We then demonstrate that interfering with myosin-dependent contractility in single cells also influences pulsed constrictions cell nonautonomously. Our results suggest that signals and stresses can feedback regulate the amplitude and spatial propagation of pulsed constrictions through their influence on tension and geometry. We establish the relevance of these findings to native closure by showing that cell delamination represents a locally patterned and collective transition from pulsed to unpulsed constriction that also relies on the nonautonomous feedback control of myosin dynamics.