The expression of "tissue" transglutaminase in two human cancer cell lines is related with the programmed cell death (apoptosis).

The expression of "tissue" transglutaminase (tTG) in two human tumor cell lines (the cervix adenocarcinoma line HeLa-TV and the neuroblastoma cells SK-N-BE-2) was found to be in correlation with the rate of physiological cell death (apoptosis) in culture. We investigated the effect of retinoic acid (RA) and alpha-difluoromethylornithine (DFMO) in order to elucidate the relationship between tTG expression and apoptosis. RA led to a 6-fold increase of tTG activity in HeLa-TV cells and to a 12-fold increase in SK-N-BE(2) cells, which was paralleled in both cell lines by a proportional increase in the number of apoptotic bodies recovered from the cultures. On the contrary, DFMO determined a dramatic reduction of tTG expression and of the apoptotic index. Immunohistochemical analysis using an anti-tTG antibody showed that the enzyme was accumulated in both cell lines within typical apoptotic bodies. Immunocytochemistry and cell cloning of SK-N-BE(2) line demonstrated that tTG was absent in cells showing neurite outgrowth, indicating that the enzyme expression is not associated with neural differentiation, even though both phenomena are elicited by retinoic acid. On the whole, these data indicate that also in tumors tTG activation takes place in cells undergoing apoptosis. The enzyme is activated in apoptotic cells to form cross-linked protein envelopes which are insoluble in detergents and chaotropic agents. The number of insoluble protein envelopes as well as the N,N-bis(gamma-glutamyl)polyamine cross-links is related with both tTG expression and apoptotic index, strongly suggesting the participation of the enzyme in the apoptotic program.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of "tissue" transglutaminase increases cell survival by preventing apoptosis

Treatment of the human promonocytic cell line U937 with all-trans-retinoic acid (RA) commits these cells to apoptosis, which can be triggered by simply increasing intracellular calcium levels by the ionophore A23187. RA treatment of U937 cells is characterized by a decrease in Bcl-2 and marked induction of "tissue" transglutaminase (tTG) gene expression. In this study, we show that the inhibition of tTG expression in U937 cells undergoing apoptosis prevents their death. In fact, U937 cell-derived clones transfected with the human tTG gene in the antisense orientation showed a pronounced decrease in apoptosis induced by several stimuli. These findings demonstrate that the Ca(2+)-dependent irreversible cross-linking of intracellular proteins catalyzed by tTG represents an important biochemical event in the gene-regulated cell death in monoblasts. In addition, our data indicate that the apoptotic program in promonocytic cells is strictly regulated by RA and that a key role is played by the free intracellular calcium concentration.     

4-Hydroxynonenal modulation of p53 family gene expression in the SK-N-BE neuroblastoma cell line

4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.     

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

'Tissue' transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK-N-BE (2)) in which the tTG-expression was driven by a constitutive promoter. Furthermore, a tet-off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared 'sensitized' to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial-mediated apoptosis by staurosporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a 'sensitizer' towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.     

Synergistic effect of retinoic acid and dehydroepiandrosterone on differentiation of human neuroblastoma cells

Retinoic acid (RA) affects many cell types by either promoting their survival or inducing their differentiation. Dehydroepiandrosterone (DHEA), a precursor for both androgenic and estrogenic steroids and abundantly produced by brain, is known as an inhibitor of cell proliferation. Differentiation of a human neuroblastoma cell line (SK-N-BE) was evaluated measuring growth rate, motility, neurite extension and GAP-43 expression. We report that DHEA enhances the differentiating effect of RA on neuroblastoma cells via a signalling that is not RA receptor-mediated. Instead, we show a differential expression of matrix metalloproteinases: RA enhances the activity of MMP-2, whereas MMP-9 expression is up-regulated by DHEA. The concerted modulation of these proteinases may support the neurite outgrowth observed after co-treatment with the two drugs.     

Differential regulation of tissue transglutaminase in rat hepatoma cell lines McA-RH7777 and McA-RH8994: Relation to growth rate and cell death

Close correlation between tissue transglutaminase (tTG) induction and growth regulation and/or cell death processes has been suggested in many cell lineages. In this study, the regulation of the tTG levels by various growth and differentiation factors and its relation to growth rate and cell death processes were investigated in two rat hepatoma cell lines, McA-RH7777 and McA-RH8994, using a monoclonal antibody against liver tTG. Transforming growth factor-β1 (TGF-β1) and retinoic acid (RA) each increased tTG to the level of 8- to 32-fold above that of control cultures in both cell lines after 72-h treatment. Dexamethasone (DEX) induced a 16- to 32-fold of tTG in McA-RH8994 cells while it did not change the enzyme level in McA-RH7777 cells. Simultaneous addition of DEX and RA increased the tTG level to more than 50-fold in McA-RH7777 cells as well as McA-RH8994 cells. Other factors, such as TGF-α, hepatocyte growth factor, dimethyl sulfoxide, and protein kinase C activator, did not show significant increases of the tTG levels. Although tTG induction by TGF-β1 or DEX appeared to be correlated with their growth suppressive effects, RA increased the tTG level without suppressing the growth rate of hepatoma cells. TGF-β1 was also shown to induce cell death in both cell lines. Our results demonstrate that RA and DEX are capable of modulating the TGF-β1-induced cell death processes independent of the tTG levels. We present evidence here that tTG induction by itself is not the direct cause of growth suppression and cell death in these hepatoma cells.     

Protective role of tissue transglutaminase in the cell death induced by TNF-alpha in SH-SY5Y neuroblastoma cells

Tissue transglutaminase (tTGase) regulates various biological processes, including extracellular matrix organization, cellular differentiation, and apoptosis. Here we report the protective role of tTGase in the cell death that is induced by the tumor necrosis factor alpha (TNF-alpha) and ceramide, a product of the TNF-alpha signaling pathway, in human neuroblastoma SH-SY5Y cells. Treatment with retinoic acid (RA) induced the differentiation of the neuroblastoma cells with the formation of extended neurites. Immunostaining and Western blot analysis showed the tTGase expression by RA treatment. TNF-alpha or C(2) ceramide, a cell permeable ceramide analog, induced cell death in normal cells, but cell death was largely inhibited by the RA treatment. The inhibition of tTGase by the tTGase inhibitors, monodansylcadaverine and cystamine, eliminated the protective role of RA-treatment in the cell death that is caused by TNF-alpha or C(2)-ceramide. In addition, the co-treatment of TNF-alpha and cycloheximide decreased the protein level of tTGase and cell viability in the RA-treated cells, supporting the role of tTGase in the protection of cell death. DNA fragmentation was also induced by the co-treatment of TNF-alpha and cycloheximide. These results suggest that tTGase expressed by RA treatment plays an important role in the protection of cell death caused by TNF-alpha and ceramide.     

Soluble Integrin Ligands and Growth Factors Independently Rescue Neuroblastoma Cells from Apoptosis under Nonadherent Conditions

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N-BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (cromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment-induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 ± 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 ± 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca²⁺levels nor onde novoprotein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since ([1]) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to β₁and α₁subunits and ([2]) the antibody-induced clustering of α₁, α₃, α_v, β₁, and β₃integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 μg/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. Thede novoprotein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-β₁antibody was associated with the increased expression ofbcl-2.In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension-induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.     

Increased expression of LGI1 gene triggers growth inhibition and apoptosis of neuroblastoma cells

The LGI1 gene has been implicated in the malignant progression of glioblastoma and it has also been genetically linked to a form of partial epilepsy (ADLTE). In this study, we investigated the relevance of LGI1 expression for neuroblastoma cells. The analysis of two cell lines (SH-SY5Y and SK-N-BE) revealed unpredictably low levels of LGI1 and stable cell transfection with LGI1 cDNA yielded moderate increases of LGI1 expression. Neuroblastoma cell clones exhibited impaired cell growth and survival ability in relation to LGI1 levels. The process of growth inhibition could be discerned under experimental conditions of low cell density, since conditions of elevated cell density, which enhance the requirement for survival stimuli, resulted in massive cellular death. At high cell density, spontaneous apoptosis of LGI1 cells was clearly shown by the release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria and by phosphatydil serine exposure and nuclear fragmentation. Activation of apoptotic effectors caspase-3/7 also occurred, however, the broad caspase inhibitor Z-VAD-FMK substantially failed to block cell death. Thus the possibility that LGI1-triggered apoptosis may involve initiator caspases linked to activation of death receptors, appears unlikely. The decreased ratio of Bcl-2 to Bax suggests that apoptosis is initiated by the intrinsic mitochondrial pathway through the release of caspase-dependent and -independent apoptogenic molecules. This study provides the first evidence that LGI1 controls neuronal cell survival, suggesting its role in the development of the nervous system in relation to the pathogenesis of neuroblastoma and ADLTE.     

Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.     

The fine structure of continuous human neuroblastoma lines SK-N-SH,SK-N-BE(2), and SK-N-MC

Summary Cell cultures of the continuous human neuroblastoma lines SK-N-SH, SK-N-BE(2), and SK-N-MC at exponential and stationary growth phase have been examined by electron microscopy. At the level of fine structure these cells did not show typical neuronal differentiation such as extensive granular endoplasmic reticulum or neurites with microtubules and neurofilaments. Instead they were characterized by abundant free ribosomes, moderate Golgi complexes, and usually scant granular endoplasmic reticulum, features similar to the fine structure of early normal embryonic autonomic neurons. However, in several respects appearance of differentiated features of the neuroblastoma cells did not follow the pattern observed for normal neurons, suggesting noncoordinate, expression of neuronal phenotypic properties. First, an occasional neuroblastoma cell had as extensive granular endoplasmic reticulum as would be found at later stages in normal developing neurons. Second, the cellular processes of these neuroblastoma cells did not have the fine structure of developing or mature axons in vivo. Third, few dense core vesicles were found in SK-N-SH and SK-N-BE(2), though these organelles are numerous in early normal adrenergic neurons and the adrenergic character of these two lines is apparent from other studies that have demonstrated expression of neurotransmitter synthesizing enzymes (SK-N-MC is cholinergic). The fine structural characterization of these continuous human neuroblastoma cell lines will allow this parameter to be utilized with other approaches in future experimental studies. This work was supported by PSC-BHE Research Award 11612 from the City University of New York and in part by the National Cancer Institute Core Grant CA-08748 to the Sloan-Kettering Institute. E. N. B. was the recipient of a predoctoral fellowship under USPHS Training Grant GM02050.     

Cordycepin induces apoptosis in human neuroblastoma SK-N-BE(2)-C and melanoma SK-MEL-2 cells

In this study, the effect of cordycepin (3'-deoxyadenosine), a major component of Cordyceps militaris, an ingredient of traditional Chinese medicine was investigated for the first time on apoptotsis in human neuroblastoma SK-N-BE(2)-C and melanoma SK-MEL-2 cells. Cordycepin significantly inhibited the proliferation of human neuroblastoma SK-N-BE(2)-C and human melanoma SK-MEL-2 cells with IC50 values of 120 microM and 80 microM, respectively. Cordycepin treatment at 120 microM and 80 microM, respectively, induced apoptosis in both cells and caused the increase of cell accumulation in a time-dependent manner at the apoptotic sub-G1 phase, as evidenced by the flow cytometry (FCM) and annexin V-fluorescein isothiocyanate (FITC) analyses. Western blot analysis revealed the induction of active caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage by cordycepin treatment. These results suggest that cordycepin is a potential candidate for cancer therapy of neuroblastoma and melanoma.     

An autocrine loop involving ret and glial cell-derived neurotrophic factor mediates retinoic acid-induced neuroblastoma cell differentiation

In several neuroblastoma cell lines, retinoic acid (RA)-induced differentiation is coupled to increased expression of functional neurotrophic factor receptors, including Trk family receptors and the glial cell-derived neurotrophic factor receptor, Ret. In several cases, increased expression is dependent on signaling through TrkB. Unlike TrkA and TrkB, Ret has never been implicated as a prognostic marker for neuroblastomas. SK-N-BE(2) cells do not express any of Trk family receptors; therefore, they are a choice system to study the specific role of Ret in RA-induced differentiation. Using a 2'-fluoro-RNA aptamer and a truncated Ret protein as specific inhibitors of Ret, we show that RA-induced differentiation is mediated by a positive autocrine loop that sustains Ret downstream signaling and depends on glial cell-derived neurotrophic factor expression and release. This report shows that in SK-N-BE(2) cells, stimulation of Ret is a major upstream mechanism needed to mediate RA-induced differentiation. These results provide important insights on the molecular mechanism of RA action, which might be relevant for the development of biologically based therapeutic strategies.     

Induction of tissue transglutaminase expression by propionate and n-butyrate in colon cancer cell lines

Short-chain fatty acids (SCFAs) have been demonstrated to induce differentiation and/or apoptosis in colon cancer cells. A close correlation between tissue transglutaminase (tTG) expression and differentiation and/or apoptosis has been suggested in many cell lineages. However, the effects of SCFAs on tTG expression in colon cancer cells have not yet been reported. In this report, the relationship between cytosolic tTG levels and differentiation state was investigated in six human colon cancer cell lines. Effects of four kinds of SCFAs (acetate, propionate, n-butyrate, and isobutyrate) on the expression of tTG then were investigated in association with their effects on apoptosis induction. High expression of tTG protein and mRNA were found in SW480 and WiDr cell lines, which exhibited well differentiated phenotypes. tTG expression was hardly detectable in the less differentiated cell lines COLO201, COLO320DM, and CW-2. However, n-butyrate and propionate significantly increased cytosolic tTG levels at concentrations above 0.5 mM in these less differentiated colon cancer cells. n-Butyrate and propionate induced growth suppression and apoptosis in these cell lines at concentrations that can induce tTG expression. Acetate and isobutyrate did not induce tTG expression or growth suppression at concentrations up to 8 mM. In conclusion, tTG induction by propionate and n-butyrate was suggested to be closely linked to their differentiation- and apoptosis-inducing effects in colon cancer cells. These findings may explain the mechanisms by which dietary fiber show preventive effects against colon carcinogenesis.     

Targeting Neuroblastoma Stem Cells with Retinoic Acid and Proteasome Inhibitor

Background

Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients.

Methodology/Principal Findings

To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA) combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2) neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132.

Conclusions

Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.     

Induction of apoptosis by all-trans retinoic acid in the human myeloma cell line RPMI 8226 and negative regulation of some of its typical morphological features by dexamethasone.

We investigated the effects of all-trans retinoic acid (RA) and dexamethasone (Dex) on the in vitro growth of the human myeloma cell line RPMI 8226. RA inhibited RPMI 8226 cell growth by both antiproliferative effect and induction of apoptosis. Typical morphological and biochemical characteristics of apoptosis including chromatin condensation, apoptotic bodies formation and internucleosomal DNA cleavage were detected after 4 days of treatment with 1 microM RA. In situ TUNEL assay demonstrated that DNA cleavage preceded chromatin condensation. The expression of tissue transglutaminase (tTG), an enzyme proposed to play a role in apoptosis was induced with RA, as shown by both enzymatic assay and in situ immunofluorescence detection. Dex, when used alone, had no effect on cell growth and apoptosis. When combined to RA, Dex did not interfere with the RA-dependent inhibition of cell proliferation, but unexpectedly inhibited both quantitatively and qualitatively several morphological and biochemical features of the apoptosis induced by RA. Dex did not affect RA-induced DNA breaks formation but impeded the progression of chromatin condensation and the formation of apoptotic bodies. Interestingly, Dex also inhibited the RA-dependent induction of tTG. RU486, a glucocorticoid antagonist, counteracted all Dex effects. Taken together these data demonstrate that key cytoplasmic and nuclear events occurring during apoptosis are differentially regulated by RA and Dex in myeloma cell line RPMI 8226.     

HeLa细胞中与组织型转谷氨酰胺酶相互作用蛋白质的筛选

组织型转谷氨酰胺酶 (tissuetransglutaminase ,tTG ,TGII)是转谷氨酰胺酶家族成员之一 ,多数细胞凋亡过程中均有tTG表达水平的升高。为研究tTG在细胞凋亡过程中发挥作用的机制 ,利用Gal 4酵母双杂交系统筛选了HeLa细胞中与tTG相互作用的蛋白质 ,获得了 17个阳性酵母克隆。序列测定显示其中 1个克隆所含cDNA序列编码TIA 1相关蛋白 (TIA 1 relatedprotein ,TIAR )C端 12 9个氨基酸残基序列 ,GST下拉 (pull down)实验也证实tTG与TIAR能相互作用 ,而且这种相互作用需要Ca2 参与作用。这些结果提示tTG可能通过其Ca2 依赖的转谷氨酰胺活性对TIAR进行修饰从而影响TIAR的功能 ,可能在细胞凋亡中发挥着一定的作用。