Regulation of G(1) cell-cycle progression by oncogenes and tumor suppressor genes.

Progression of resting quiescent G(0) cells into early G(1) and transition across the restriction point are highly regulated processes. Mutation of proto-oncogenes and tumor suppressor genes regulating these transitions are targeted during oncogenesis. Recent work has underscored the importance of the G(0) to early G(1) transition and metabolism to neoplastic cells.     

Morphological changes and nuclear translocation of DLC1 tumor suppressor protein precede apoptosis in human non-small cell lung carcinoma cells

We have previously shown that reactivation of DLC1, a RhoGAP containing tumor suppressor gene, inhibits tumorigenicity of human non-small cell lung carcinoma cells (NSCLC). After transfection of NSCLC cells with wild type (WT) DLC1, changes in cell morphology were observed. To determine whether such changes have functional implications, we generated several DLC1 mutants and examined their effects on cell morphology, proliferation, migration and apoptosis in a DLC1 deficient NSCLC cell line. We show that WT DLC1 caused actin cytoskeleton-based morphological alterations manifested as cytoplasmic extensions and membrane blebbings in most cells. Subsequently, a fraction of cells exhibiting DLC1 protein nuclear translocation (PNT) underwent caspase 3-dependent apoptosis. We also show that the RhoGAP domain is essential for the occurrence of morphological alterations, PNT and apoptosis, and the inhibition of cell migration. DLC1 PNT is dependent on a bipartite nuclear localizing sequence and most likely is regulated by a serine-rich domain at N-terminal part of the DLC1 protein. Also, we found that DLC1 functions in the cytoplasm as an inhibitor of tumor cell proliferation and migration, but in the nucleus as an inducer of apoptosis. Our analyses provide evidence for a possible link between morphological alterations, PNT and proapoptotic and anti-oncogenic activities of DLC1 in lung cancer.     

Cycloalkyl-substituted aryl chloroethylureas inhibiting cell cycle progression in G0/G1 phase and thioredoxin-1 nuclear translocation

1-(2-Chloroethyl)-3-(4-cyclohexylphenyl)urea (cHCEU) has been shown to abrogate the presence of thioredoxin-1 into the nucleus through its selective covalent alkylation. In the present letter we have evaluated the structure-activity relationships of the substituents at positions 3 and 4 of the phenyl ring of cHCEU derivatives on cell cycle progression and thioredoxin-1 nuclear translocation. Active CEU derivatives exhibited GI(50) ranging from 1.9 to 49muM on breast carcinoma MCF-7, skin melanoma M21, and colon carcinoma HT-29 cells. On one hand, compounds 1, 2, 9c, 10c, 13, and 14 arrested the cell cycle in G(2)/M phase while CEUs 3, 4, 5c, 6c, 11c, and 12c blocked the cell division in G(0)/G(1) phase. On the other hand, CEUs 2-4, 5c, 7c, 8c, 11c, and 12c abrogated the translocation of thioredoxin-1 while the other CEU derivatives were inactive in that respect. Our results suggest that CEU substituted on the phenyl ring at position 3 or 4 by lower cycloalkyl or cycloalkoxy groups arrest cell progression in G(0)/G(1) phase through mechanism of action different from their antimicrotubule counterparts, presumably via thioredoxin-1 alkylation and modulation of its activity. The mechanism of action of these new molecules is still undetermined. However, the significant accumulation of cells in G(0)/G(1) phase suggests that these molecules may act similarly to known chemopreventive agents against cancers. In addition, the inhibition of Trx-1 nuclear localization also suggests the abrogation of an important chemoresistance mechanism towards a variety of chemotherapeutic agents.     

A novel domain of caveolin‐2 that controls nuclear targeting: regulation of insulin‐specific ERK activation and nuclear translocation by caveolin‐2

Herein, we report that insulin‐activated extracellular signal‐regulated kinase (ERK) is translocated to the nuclear envelope by caveolin‐2 (cav‐2) and associates with lamin A/C in the inner nuclear membrane in response to insulin. We identified that the Ser¹⁵⁴–Val¹⁵⁵–Ser¹⁵⁶ domain on the C‐terminal of cav‐2 is essential for insulin‐induced phosphorylation and nuclear targeting of ERK and cav‐2. In human embryonic kidney 293T cells, ERK was not activated and translocated to the nucleus by insulin in comparison to insulin‐like growth factor‐1 (IGF‐1). However, insulin‐stimulated activation of ERK was induced by exogenous addition of cav‐2. The activated ERK associated and translocated with the cav‐2 to the nucleus. In turn, cav‐2 promoted phospho‐ERK interaction with lamin A/C in the inner nuclear membrane. In contrast, ERK, but not cav‐2, was phosphorylated and translocated to the nucleus by IGF‐1. The nuclear targeted phospho‐ERK failed to localize in the nuclear envelope in response to IGF‐1. Together, our data demonstrate that translocation of phospho‐ERK to the nuclear envelope is mediated by Ser¹⁵⁴–Val¹⁵⁵–Ser¹⁵⁶ domain of cav‐2 and this event is an insulin‐specific action.     

Troglitazone inhibits angiotensin II-induced extracellular signal-regulated kinase 1/2 nuclear translocation and activation in vascular smooth muscle cells.

The thiazolidinedione troglitazone inhibits angiotensin II-induced extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase activity in vascular smooth muscle cells. Activation of extracellular signal-regulated kinase 1/2 by angiotensin II is a multistep process involving both its phosphorylation by mitogen-activated protein kinase extracellular signal-regulated kinase kinase in the cytoplasm and a subsequent translocation to the nucleus. The cytoplasmic activation of extracellular signal-regulated kinase 1/2 in vascular smooth muscle cells proceeds through the protein kinase Czeta --> mitogen-activated protein kinase extracellular signal-regulated kinase kinase --> extracellular signal-regulated kinase pathway. Troglitazone did not affect the angiotensin II-induced activation of protein kinase Czeta or its downstream signaling kinases extracellular signal-regulated kinase 1/2 in the cytosol. In contrast, angiotensin II-induced activation of protein kinase Czeta and extracellular signal-regulated kinase 1/2 in the nucleus were both inhibited by troglitazone. Nuclear translocation of extracellular signal-regulated kinase 1/2 induced by angiotensin II was completely blocked by troglitazone. Protein kinase Czeta, however, did not translocate upon angiotensin II stimulation. Troglitazone, therefore, inhibits both angiotensin II-induced nuclear translocation of extracellular signal-regulated kinase 1/2 and the nuclear activity of its upstream signaling kinase protein kinase Czeta. Since extracellular signal-regulated kinase 1/2 nuclear translocation may be a critical signaling step for multiple growth factors that stimulate vascular smooth muscle cells proliferation and migration, troglitazone may provide a new therapeutical approach for the prevention and treatment of atherosclerosis and restenosis.     

Cyclophilin A is required for CXCR4-mediated nuclear export of heterogeneous nuclear ribonucleoprotein A2, activation and nuclear translocation of ERK1/2, and chemotactic cell migration

The chemokine receptor CXCR4-mediated signaling cascades play an important role in cell proliferation and migration, but the underlying mechanisms by which the receptor signaling is regulated remain incompletely understood. Here, we demonstrate that CXCR4 was co-immunoprecipitated with cyclophilin A (CyPA) from the lysate of HEK293 cells stably expressing CXCR4. Although both the glutathione S-transferase-CXCR4 N- and C-terminal fusion proteins were associated with the purified CyPA, truncation of the C-terminal domain of CXCR4 robustly inhibited the receptor co-immunoprecipitation with CyPA in intact cells, thereby suggesting a critical role of the receptor C terminus in this interaction. Ligand stimulation of CXCR4 induced CyPA phosphorylation and nuclear translocation, both of which were inhibited by truncation of the C-terminal domain of CXCR4. CyPA was associated with transportin 1, and knockdown of transportin 1 by RNA interference (RNAi) blocked CXCL12-induced nuclear translocation of CyPA, thereby suggesting a transportin 1-mediated nuclear import of CyPA. CyPA formed a complex with heterogeneous nuclear ribonucleoprotein (hnRNP) A2, which underwent nuclear export in response to activation of CXCR4. Interestingly, the CXCR4-mediated nuclear export of hnRNP A2 was blocked by RNAi of CyPA. Moreover, CXCR4-evoked activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was attenuated by CyPA RNAi, by overexpression of a PPIase-deficient mutant of CyPA (CyPA-R55A), and by pretreatment of the immunosuppressive drugs, cyclosporine A and sanglifehrin A. Finally, CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 or Jurkat T cells was inhibited by CyPA RNAi or CsA treatment.     

The EphB2 tumor suppressor induces autophagic cell death via concomitant activation of the ERK1/2 and PI3K pathways

EphB2 is a tyrosine kinase receptor that has been shown to be a tumor suppressor gene in various cancers. However the mechanisms of this function are unknown. We report that EphB2 induces a form of cell death that does not involve the formation of apoptotic bodies or nuclear fragmentation and is instead accompanied by extensive vacuolization. Transmission electron microscopy demonstrates cytoplasmic vacuoles in EphB2-overexpressing cells that resembled autophagosomes. Using an EYFP-LC3 fusion protein and immunoblotting, we detected LC3 aggregation and conversion from form I to form II, both hallmarks of autophagy, in EphB2-transfected cells. Silencing of the autophagy regulating genes ATG5 or ATG7 using shRNAs, strongly prevented EphB2-induced cell death, further confirming its autophagic nature. EphB2 expression results in mitochondrial depolarization and translocation of cytochrome c from the mitochondria to the cytosol. Mapping of signaling pathways revealed novel information about the mechanisms of action of EphB2. We demonstrated that the MAPK pathway is important in the pro-death action of EphB2, through ERK1/2 phosphorylation and inhibition of this pathway using PD98059 counters EphB2-driven cell death. In addition, we found that inhibition of class III PI3K pathway, using the autophagy inhibitor 3MA, but not class I PI3K inhibition using LY294002, also effectively blocks EphB2-induced cell death. Finally, EphB2 expression inactivates Akt, which is a known inhibitor of autophagy. In conclusion, the EphB2 receptor induces an autophagic cell death that is mediated through the ERK1/2 and PI3K/Akt pathways.     

Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by rac1 and phosphatidylinositol-3 kinase activation

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that causes no currently known pathology in humans. Despite the fact that this virus is of increasing interest to molecular medicine as a vector for gene delivery, relatively little is known about the cellular mechanisms controlling infection. In this study, we have examined endocytic and intracellular trafficking of AAV-2 using fluorescent (Cy3)-conjugated viral particles and molecular techniques. Our results demonstrate that internalization of heparan sulfate proteoglycan-bound AAV-2 requires alphaVbeta5 integrin and activation of the small GTP-binding protein Rac1. Following endocytosis, activation of a phosphatidylinositol-3 (PI3) kinase pathway was necessary to initiate intracellular movement of AAV-2 to the nucleus via both microfilaments and microtubules. Inhibition of Rac1 using a dominant N17Rac1 mutant led to a decrease in AAV-2-mediated PI3 kinase activation, indicating that Rac1 may act proximal to PI3 kinase during AAV-2 infection. In summary, our results indicate that alphaVbeta5 integrin-mediated endocytosis of AAV-2 occurs through a Rac1 and PI3 kinase activation cascade, which directs viral movement along the cytoskeletal network to the nucleus.     

Evidence that the stimulus properties of apomorphine are mediated by both D1 and D2 receptor activation

Male and female rats were trained to discriminate between the stimulus properties of apomorphine (0.16 mg/kg i.p.) and saline in a two-lever, food-motivated operant procedure. Apomorphine, at doses different than the training dose, produced a similar dose-dependent relationship in both sexes. Consistent with the hypothesis that the behavioral effects of apomorphine are mediated by D2 activation, the apomorphine interoceptive cue generalized to bromocriptine, a drug considered to be a preferential D2 agonist. In addition, the dose-response curve after 5-15 mg/kg bromocriptine administration was parallel to that of apomorphine. Consistent with the biochemical evidence that apomorphine's effects are mediated, to a lesser extent, by D1 activation, the apomorphine cue partially generalized to the selective D1 agonist SKF 38393. Furthermore, the apomorphine cue was not blocked by the selective D1 antagonist SCH 23390. Somewhat surprising was the partial generalization of the apomorphine cue to SCH 23390. However, this is not the first time that the administration of SCH 23390 has resulted in unexpected behavioral responses. Other novel findings include the lack of sex differences in acquisition training to the apomorphine cue and in the generalization tests to the selective agonists. The behavioral results are consistent with previous biochemical evidence that the effects of apomorphine are mediated by both D1 and D2 activation and is further behavioral support that apomorphine's effects are not the result of D2 activation alone, as previously hypothesized.     

Mutation analysis of large tumor suppressor genes LATS1 and LATS2 supports a tumor suppressor role in human cancer

In recent years, human cancer genome projects provide unprecedented opportunities for the discovery of cancer genes and signaling pathways that contribute to tumor development. While numerous gene mutations can be identified from each cancer genome, what these mutations mean for cancer is a challenging question to address, especially for those from less understood putative new cancer genes. As a powerful approach, in silico bioinformatics analysis could efficiently sort out mutations that are predicted to damage gene function. Such an analysis of human large tumor suppressor genes, LATS1 and LATS2, has been carried out and the results support a role of hLATS1//2 as negative growth regulators and tumor suppressors.     

Immune responses induced by oligogalacturonides are differentially affected by AvrPto and loss of BAK1/BKK1 and PEPR1/PEPR2

Plants possess an innate immune system capable of restricting invasion by most potential pathogens. At the cell surface, the recognition of microbe‐associated molecular patterns (MAMPs) and/or damage‐associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) represents the first event for the prompt mounting of an effective immune response. Pathogens have evolved effectors that block MAMP‐triggered immunity. The Pseudomonas syringae effector AvrPto abolishes immunity triggered by the peptide MAMPs flg22 and elf18, derived from the bacterial flagellin and elongation factor Tu, respectively, by inhibiting the kinase function of the corresponding receptors FLS2 and EFR, as well as their co‐receptors BAK1 and BKK1. Oligogalacturonides (OGs), a well‐known class of DAMPs, are oligomers of α‐1,4‐linked galacturonosyl residues, released on partial degradation of the plant cell wall homogalacturonan. We show here that AvrPto affects only a subset of the OG‐triggered immune responses and that, among these responses, only a subset is affected by the concomitant loss of BAK1 and BKK1. However, the antagonistic effect on auxin‐related responses is not affected by either AvrPto or the loss of BAK1/BKK1. These observations reveal an unprecedented complexity among the MAMP/DAMP response cascades. We also show that the signalling system mediated by Peps, another class of DAMPs, and their receptors PEPRs, contributes to OG‐activated immunity. We hypothesize that OGs are sensed through multiple and partially redundant perception/transduction complexes, some targeted by AvrPto, but not necessarily comprising BAK1 and BKK1.     

The tumor suppressor Rb and its related Rbl2 genes are regulated by Utx histone demethylase

Utx is a candidate tumor suppressor gene that encodes histone H3 lysine 27 (H3K27) demethylase. In this study, we found that ectopic expression of Utx enhanced the expression of retinoblastoma tumor suppressor gene Rb and its related gene Rbl2. This activation was dependent on the demethylase activity of Utx, and was suggested to contribute to the decreased cell proliferation induced by Utx. A chromatin immunoprecipitation assay showed that over-expressed Utx was associated with the promoter regions of Rb and Rbl2 resulting in the removal of repressive H3K27 tri-methylation and the increase in active H3K4 tri-methylation. Furthermore, siRNA-mediated knockdown of Utx revealed the recruitment of endogenous Utx protein on the promoters of Rb and Rbl2 genes. These results indicate that Rb and Rbl2 are downstream target genes of Utx and may play important roles in Utx-mediated cell growth control.