Activation of PKR/eIF2α signaling cascade is associated with dihydrotestosterone-induced cell cycle arrest and apoptosis in human liver cells

Androgen receptor (AR) signaling plays an important role in the development and progression of several liver diseases, including hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD). Dihydrotestosterone (DHT) is the active metabolite of the major circulating androgen, testosterone. In this study, we investigated the effect of DHT on human liver cells. We found that DHT not only induces cell cycle arrest but also initiates apoptosis in androgen-sensitive liver cells, such as SMMC-7721 and L02. Importantly, DHT/AR induces the activation of RNA-dependent protein kinase (PKR)/eukaryotic initiation factor-2 alpha (eIF2α) cascades in androgen-sensitive liver cells. PKR/eIF2α activation-induced growth arrest and DNA damage-inducible gene 153 (GADD153) and heat shock protein 27 (Hsp27) expression contribute to cell cycle arrest in response to DHT. It is notable that DHT administration results in androgen-sensitive liver cells apoptosis, at least in part, through PKR/eIF2α/GADD153 cascades. These results suggest that the androgen/AR pathway plays a pivotal role in liver cell growth and apoptosis regulating, whose deregulation might be involved in the pathogenesis of liver diseases.     

Proliferation of a highly androgen-sensitive ductus deferens cell line (DDT1MF-2) is regulated by glucocorticoids and modulated by growth on collagen

Proliferation of the hamster ductus deferens cloned tumor cell line (DDT1MF-2) in monolayer culture is markedly stimulated by androgens in a dose dependent fashion. Furthermore, growth on collagen confers upon these cells a greater dependence on this class of hormones, such that testosterone (10 nM) induces a 15-fold elevation in cell number compared to controls. Addition of either dexamethasone (10 nM) or triamcinolone acetonide (TA; 10 nM) dramatically blocks this stimulation by reversibly arresting the cells in the G1 phase of the cell cycle as assessed by flow cell cytometry. Associated with the decreased growth rate is a change from a rounded to a more flattened morphology that may also implicate cell shape in the regulation of proliferation. These steroid effects presumably are mediated through specific receptor proteins for which dihydrotestosterone (DHT) and TA bind with equilibrium dissociation constants (Kd) of 0.3 and 1.0 nM, respectively. Moreover, not only do androgens increase growth rate but treatment with 1 nM [3H]DHT also results in an elevation in androgen receptor concentration from 1.6 to 3.6 f mol/micrograms DNA in 7 h. Simultaneous treatment with 10 nM TA, however, reduces this increase by 53%. Inasmuch as neither progesterone nor estradiol-17 beta display similar inhibitory activity, this effect also seems to be glucocorticoid specific. These observations may be important in elucidating the mechanism of androgen action and should provide some insight into the role of glucocorticoids in regulating the growth of androgen dependent tissues.     

Delphinidin,a dietary anthocyanidin in pigmented fruits and vegetables: A new weapon to blunt prostate cancer growth

In a recent publication, we have shown that delphinidin, an anthocyanidin induces apoptosis and cell cycle arrest in highly metastatic human prostate cancer (PCa) PC3 cells. Extending these studies, we provide additional evidence that delphinidin induces apoptosis and cell cycle arrest in androgen refractory human PCa 22Rn1 cells and that these effects are concomitant with inhibition of NF-kB. We observed that delphinidin treatment to 22Rn1 cells resulted in a dose-dependent (i) G2/M phase cell cycle arrest, (ii) induction of apoptosis (iii) and inhibition of NF-kB signaling. The induction of apoptosis by delphinidin was mediated via activation of caspases since a general caspase inhibitor Z-VAD-FMK significantly reversed this effect. Delphinidin treatment to cells resulted in a dose-dependent decrease in (i) phosphorylation of IKKgamma (NEMO), (ii) phosphorylation of NF-kB inhibitory protein, (iii) phosphorylation of NF-kB/p65 at Ser536 and NF-kB/p50 at Ser529, (iv) NF-kB/p65 nuclear translocation, and (v) NF-kB DNA binding activity. Taken together, our data show that delphinidin induces apoptosis of both androgen independent and androgen refractory human PCa cells via activation of caspases and in addition, this effect might be due to inhibition of NF-kB signaling. We suggest that delphinidin could be developed as a novel agent against PCa.     

CCAAT/enhancer-binding protein delta regulates mammary epithelial cell G0 growth arrest and apoptosis.

CCAAT/enhancer-binding proteins (C/EBPs) are a highly conserved family of DNA-binding proteins that regulate cell-specific growth, differentiation, and apoptosis. Here, we show that induction of C/EBPdelta gene expression during G0 growth arrest is a general property of mammary-derived cell lines. C/EBPdelta is not induced during G0 growth arrest in 3T3 or IEC18 cells. C/EBPdelta induction is G0-specific in mouse mammary epithelial cells; C/EBPdelta gene expression is not induced by growth arrest in the G1, S, or G2 phase of the cell cycle. C/EBPdelta antisense-expressing cells (AS1 cells) maintain elevated cyclin D1 and phosphorylated retinoblastoma protein levels and exhibit delayed G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. Conversely, C/EBPdelta-overexpressing cells exhibited a rapid decline in cyclin D1 and phosphorylated retinoblastoma protein levels, a rapid increase in the cyclin-dependent kinase inhibitor p27, and accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. When C/EBPdelta levels were rescued in AS1 cells by transfection with a C/EBPdelta "sense" construct, normal G0 growth arrest and apoptosis were restored. These results demonstrate that C/EBPdelta plays a key role in the regulation of G0 growth arrest and apoptosis in mammary epithelial cells.     

The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage

Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR) or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs) and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs), Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.     

Differential androgen sensitivity is associated with clonal heterogeneity in steroid metabolism, ornithine decarboxylase regulation and IL-1alpha action in mouse mammary tumor cells

Upon androgen deprivation, Shionogi (SC-115) mouse mammary tumors undergo phenotypic changes enabling their escape from growth dependence on androgens. Even within androgen-responsive cell populations, marked clonal heterogeneity is observed in the trophic effects of androgens. The present study compares several parameters of androgen action between three SC-115 cell clonal subpopulations exhibiting high (clone 107), low (clone S1A2) and no trophic response (clone 415) to androgens. These parameters pertain to (1) kinetics of androgen binding, (2) metabolism of 5alpha-dihydrotestosterone (DHT), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-diol), (3) ornithine decarboxylase (ODC) activity and (4) interleukin-1alpha (IL-1alpha) action on cell proliferation. Only marginal differences in the affinity and abundance of androgen-specific binding sites were detected between the three clones. While clone S1A2 degraded DHT to 3alpha-diol at a much faster rate than the highly androgen-sensitive 107 cells and androgen-insensitive 415 cells, differences in the rates of intracrine conversion of 3alpha-diol and 3beta-diol to DHT did not correlate with the ability of these steroids to stimulate cell proliferation. Induction of ODC activity at the onset of exponential growth was strongly DHT-dependent in 107 cells, whereas this dependence was markedly attenuated in androgen-hyposensitive cells. Unexpectedly, DHT strongly repressed the marked ODC induction resulting from fresh medium addition in 415 cells which show no growth response to androgens. Low IL-1alpha concentrations were mitogenic in all three SC-115 clones. Whereas the mitogenic action of IL-1alpha was completely androgen-dependent in 107 cells, this dependence was relieved in S1A2 cells, which responded to DHT and IL-1alpha in an additive fashion. Thus, clonal heterogeneity in the pattern of steroid metabolism within Shionogi tumors cannot solely account for loss of androgen dependence, which may rather correlate with the constitutive activation of transduction pathways controlling the expression of growth-associated genes (e.g. ODC) by serum growth factors, including IL-1alpha.     

Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis

Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment. The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM. Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis. While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction. Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells. Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest. In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation. Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively. However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells. In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases. Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate.     

Activation of the RhoB Signaling Pathway by Thyroid Hormone Receptor β in Thyroid Cancer Cells

Thyroid hormone receptor (TR) mediates the crucial effects of the thyroid hormone (T3) on cellular growth, development, and differentiation. Decreased expression or inactivating somatic mutations of TRs have been found in human cancers of the liver, breast, lung, and thyroid. The mechanisms of TR-associated carcinogenesis are still not clear. To establish the function of TRβ in thyroid cancer cell proliferation, we constructed a recombinant adenovirus vector, AdTRβ, which expresses human TRβ1 cDNA. Thyroid cancer cell lines in which TRβ protein levels were significantly decreased as compared to intact thyroid tissues were infected with AdTRβ and the function of TRβ on cell proliferation and migration was analyzed. Ligand-bound TRβ induced HDAC1 and HDAC3 dissociation from, and histone acetylation associated with the RhoB promoter and enhanced the expression of RhoB mRNA and protein. In AdTRβ-infected cells, T3 and farnesyl transferase inhibitor (FTI)-treatment induced the distribution of RhoB on the cell membrane and enhanced the abundance of active GTP-bound RhoB. This RhoB protein led to p21-associated cell-cycle arrest in the G0/G1 phase, following inhibition of cell proliferation and invasion. Conversely, lowering cellular RhoB by small interfering RNA knockdown in AdTRβ-infected cells led to downregulation of p21 and inhibited cell-cycle arrest. The growth of BHP18-21v tumor xenografts in vivo was significantly inhibited by AdTRβ injection with FTIs-treatment, as compared to control virus-injected tumors. This novel signaling pathway triggered by ligand-bound TRβ provides insight into possible mechanisms of proliferation and invasion of thyroid cancer and may provide new therapeutic targets for thyroid cancers.     

Proliferation of a highly androgen-sensitive ductus deferens cell line (DDT1MF-2) is regulated by glucocorticoids and modulated by growth on collagen

Summary Proliferation of the hamster ductus deferens cloned tumor cell line (DDT₁MF-2) in monolayer culture is markedly stimulated by androgens in a dose dependent fashion. Furthermore, growth on collagen confers upon these cells a greater dependence on this class of hormones, such that testosterone (10 nM) induces a 15-fold elevation in cell number compared to controls. Addition of either dexamethasone (10 nM) or triamcinolone acetonide (TA; 10 nM) dramatically blocks this stimulation by reversibly arresting the cells in the G1 phase of the cell cycle as assessed by flow cell cytometry. Associated with the decreased growth rate is a change from a rounded to a more flattened morphology that may also implicate cell shape in the regulation of proliferation. These steroid effects presumably are mediated through specific receptor proteins for which dihydrotestosterone (DHT) and TA bind with equilibrium dissociation constants (Kd) of 0.3 and 1.0 nM, respectively. Moreover, not only do androgens increase growth rate but treatment with 1 nM [³H]DHT also results in an elevation in androgen receptor concentration from 1.6 to 3.6 f mol/μg DNA in 7 h. Simultaneous treatment with 10 nM TA, however, reduces this increase by 53%. Inasmuch as neither progesterone nor estradiol-17β display similar inhibitory activity, this effect also seems to be glucocorticoid specific. These observations may be important in elucidating the mechanism of androgen action and should provide some insight into the role of glucocorticoids in regulating the growth of androgen dependent tissues. This research was supported by Grant R01 CA 36264 from the National Institutes of Health, Bethesda, MD.     

Farnesylated RhoB inhibits radiation-induced mitotic cell death and controls radiation-induced centrosome overduplication

Our previous results demonstrated that expressing the GTPase ras homolog gene family, member B (RhoB) in radiosensitive NIH3T3 cells increases their survival following 2 Gy irradiation (SF2). We have first demonstrated here that RhoB expression inhibits radiation-induced mitotic cell death. RhoB is present in both a farnesylated and a geranylgeranylated form in vivo. By expressing RhoB mutants encoding for farnesylated (RhoB-F cells), geranylgeranylated or the CAAX deleted form of RhoB, we have then shown that only RhoB-F expression was able to increase the SF2 value by reducing the sensitivity of these cells to radiation-induced mitotic cell death. Moreover, RhoB-F cells showed an increased G2 arrest and an inhibition of centrosome overduplication following irradiation mediated by the Rho-kinase, strongly suggesting that RhoB-F may control centrosome overduplication during the G2 arrest after irradiation. Overall, our results for the first time clearly implicate farnesylated RhoB as a crucial protein in mediating cellular resistance to radiation-induced nonapoptotic cell death.     

Interleukin-1beta can mediate growth arrest and differentiation via the leukemia inhibitory factor/JAK/STAT pathway in medullary thyroid carcinoma cells

Interleukin-1beta (IL-1beta) is a pleiotropic cytokine that can induce several cellular signal transduction pathways. Here, we show that IL-1beta can induce cell cycle arrest and differentiation in the human medullary thyroid carcinoma (MTC) cell line, TT. IL-1beta induces cell cycle arrest accompanied by morphological changes and expression of the neuroendocrine marker calcitonin. These changes are blocked by the MEK1/2 specific inhibitor U0126, indicating that MEK1/2 is essential for IL-1beta signaling in TT cells. IL-1beta induces expression of leukemia inhibitory factor (LIF) and activation of STAT3 via the MEK/ERK pathway. This activation of STAT3 could be abrogated by treatment with anti-LIF neutralizing antibody or anti-gp130 blocking antibody, indicating that induction of LIF expression is sufficient and essential for STAT3 activation by IL-1beta. In addition to activation of the LIF/JAK/STAT pathway, IL-1beta also induced an MEK/ERK-mediated intracellular cell-autonomous signaling pathway that is independently sufficient for growth arrest and differentiation. Thus, IL-1beta activates the MEK/ERK pathway to induce growth arrest and differentiation in MTC cells via dual independent signaling mechanisms, the cell-extrinsic LIF/JAK/STAT pathway, and the cell-intrinsic autonomous signaling pathway.     

A MAPK-positive feedback mechanism for BLR1 signaling propels retinoic acid-triggered differentiation and cell cycle arrest

MAPK signaling is required for retinoic acid (RA)-triggered G(0) cell cycle arrest and cell differentiation, but the mechanism is not well defined. In this study, RA is found to cause MAPK activation with sustained association of RAF to MEK or ERK, leading to a MAPK-dependent accumulation of p21(Waf1/Cip1) and binding to CDK2 blocking G(1)/S transition. BLR1, a chemokine receptor, was found to function as a critical component of RA-triggered MAPK signaling. Unlike wild-type parental cells, RA-treated BLR1 knock-out cells failed to show RAF and consequential MEK and ERK phosphorylation, failed to accumulate CDK inhibitors that control G(1)/S transition, and failed to differentiate and arrest in response to RA, whereas ectopically overexpressing BLR1 enhanced MAPK signaling and caused accelerated RA-induced differentiation and arrest. Ectopic overexpression of RAF enhanced BLR1 expression in response to RA, whereas inhibition of RAF or MEK by inhibitors or knockdown of RAF by short interfering RNA diminished RA-induced BLR1 expression and attenuated differentiation and growth arrest. Ectopic expression of the RAF CR3, the catalytically active domain, in the BLR1 knock-out restored RA-induced MAPK activation and the ability to differentiate and arrest, indicating that RAF effects MAPK signaling by BLR1 to propel differentiation/arrest. Taken together, RA induces cell differentiation and growth arrest through activation of a novel MAPK pathway with BLR1 as a critical component in a positive feedback mechanism that may contribute to the prolonged MAPK signaling propelling RA-induced cell cycle arrest and differentiation.     

Retinoic acid increases amount of phosphorylated raf; Ectopic expression of cFMS reveals that retinoic acid-induced differentiation is more strongly dependent on ERK2 signaling than induced go arrest is

Summary Retinoic acid is known to cause the myeloid differentiation and G1/0 cell cycle arrest of HL-60 cells in a process that requires mitogen-activated protein/extracellular signal regulated kinase (MEK)-dependent extracellular signal regulated kinase (ERK)2 activation. It has also been shown that ectopic expression of cFMS, a platelet-derived growth factor (PDGF)-family transmembrane tyrosine kinase receptor, enhances retinoic acid-induced differentiation and G1/0 arrest. The mechanism of how the retinoic acid and cFMS signaling pathways intersect is not known. The present data show that the ectopic expression of cFMS results in the differential loss of sensitivity of retinoic acid-induced differentiation or G1/0 arrest to inhibition of ERK2 activation. PD98059 was used to inhibit MEK and consequently ERK2. In wild-type HL-60 cells, PD98059 blocked retinoic acid-induced differentiation; but in cFMS stable transfectants, PD98059 only attenuated the induced differentiation, with the resulting response resembling that of retinoic acid-treated wild-type HL-60. In wild-type HL-60, PD98059 greatly attenuated the retinoic acid-induced G1/0 arrest allied with retinoblastoma (RB) hypophosphorylation; but in cFMS stable transfectants, PD98059 had no inhibitory effect on RB hypophosphorylation and G1/0 arrest. This differential sensitivity to PD98059 and uncoupling of retinoic acid-induced differentiation and G1/0 arrest in cFMS transfectants is associated with changes in mitogen-activated protein kinase signaling molecules. The cFMS transfectants had more activated ERK2 than did the wild-type cells, which surprisingly was not attributable to enhanced mitogen-activated protein-kinase-kinase-kinase (RAF) phosphorylation. Retinoic acid increased the amount of activated ERK2 and phosphorylated RAF in both cell lines. But PD98059 eliminated detectable ERK2 activation, as well as inhibited RAF phosphorylation, in untreated and retinoic acid-treated wild-type HL-60 and cFMS transfectants, consistent with MEK or ERK feedback-regulation of RAF, in all four cases. Since PD98059 blocks the cFMS-conferred enhancement of the retinoic acid-induced differentiation, but not growth arrest, the data indicate that cFMS-enhanced differentiation acts primarily through MEK and ERK2, but cFMS-enhanced G1/0 arrest allied with RB hypophosphorylation depends on another cFMS signal route, which by itself can effect G1/0 arrest without activated ERK2. Ectopic expression of cFMS and differential sensitivity to ERK2 inhibition thus reveal that retinoic acid-induced HL-60 cell differentiation and G1/0 arrest are differentially dependent on ERK2 and can be uncoupled. A significant unanticipated finding was that retinoic acid caused a MEK-dependent increase in the amount of phosphorylated RAF. This increase may help sustain prolonged ERK2 activation.