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We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.
The Forkhead family of transcription factors participates in the induction of death-related genes. In NMuMG and 4T1 mammary epithelial cells, transforming growth factor beta (TGF beta) induced phosphorylation and cytoplasmic retention of the Forkhead factor FKHRL1, while reducing FHKRL1-dependent transcriptional activity. TGF beta-induced FKHRL1 phosphorylation and nuclear exclusion were inhibited by LY294002, an inhibitor of phosphatidylinositol-3 kinase. A triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated (TM-FKHRL1), did not translocate to the cytoplasm in response to TGF beta. In HaCaT keratinocytes, expression of dominant-negative Akt prevented TGF beta-induced 1) reduction of Forkhead-dependent transcription, 2) FKHRL1 phosphorylation, and 3) nuclear exclusion of FKRHL1. Forced expression of either wild-type (WT) or TM-FKHRL1, but not a FKHRL1 mutant with deletion of the transactivation domain, resulted in NMuMG mammary cell apoptosis. Evidence of nuclear fragmentation colocalized to cells with expression of WT- or TM-FKHRL1. The apoptotic effect of WT-FKHRL1 but not TM-FKHRL1 was prevented by exogenous TGF beta. Serum starvation-induced apoptosis was also inhibited by TGF beta in NMuMG and HaCaT cells. Finally, dominant-negative Akt abrogated the antiapoptotic effect of TGF beta. Taken together, these data suggest that TGF beta may play a role in epithelial cell survival via Akt-dependent regulation of FKHRL1.
Alterations in endothelial cell (EC) signaling could serve as a marker of effective antiangiogenic therapy. We determined the effect of an antiangiogenic tyrosine kinase inhibitor, SU6668, on tumor EC signaling in liver metastases in mice. In vitro immunofluorescence verified that pretreatment of ECs with SU6668 before exposure to VEGF decreased in vitro phosphorylation of Erk and Akt. Using double-fluorescence immunohistochemistry, phosphorylated Erk and Akt were constitutively expressed in ECs in liver metastases in untreated mice, but SU6668 blocked activation of these signaling intermediates. Determining the activation status of the Erk and Akt signaling pathways in tumor ECs may serve as a surrogate marker for the effectiveness of antiangiogenic regimens.
Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.
It is well established that ErbB1 and ErbB2 can cooperate in mammary epithelial cell transformation. Therefore, to understand how ErbB1/ErbB2 signaling contributes to this process, we used the ErbB kinase inhibitor AG1478in ErbB2-dependent BT-474 and SKBR-3 human breast cancer cells. These cells overexpress ErbB2 and also display moderate levels of ErbB1. Treatment with AG1478 resulted in rapid ErbB2 dephosphorylation, reversible G(1) arrest, and interruption of constitutive mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Consequently, both MAPK-dependent transcription of cyclin D1 and phosphorylation of the cyclin-dependent kinase (Cdk) inhibitor p27 were inhibited. The inhibition of PI3K/Akt resulted in increased activity of glycogen synthase kinase-3beta, which phosphorylated cyclin D1, potentially reducing its steady-state levels. The loss of cyclin D1 reduced the amount of cyclin D1/Cdk4 complexes that can sequester p27 in the cytosol. This plus the reduced phosphorylation of p27 by MAPK enhanced the stability of p27 that associated with nuclear Cdk2 at high stoichiometry and inhibited its kinase activity. Antisense p27 oligonucleotides decreased p27 levels and abrogated the G(1) arrest induced by AG1478. Similarly, infection with an adenovirus encoding inducible cyclin D1 also counteracted the antiproliferative effect of AG1478. These data imply that: (a) modulation of both p27 and cyclin D1 are required for the growth arrest that results from blockade of the ErbB2 kinase; and (b) ErbB2 overexpressing cells use both MAPK and PI3K/Akt to modulate p27 and cyclin D1 and, hence, subvert the G(1)-to-S transition.
An inverse correlation between p27(Kip1) expression and proliferation has been recently established in tissues derived from human lymphomas. The nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)/phospholipase C-gamma (PLCgamma) complex also appears to play an important role in cell proliferation and malignant transformation of anaplastic large cell lymphoma (ALCL). In this study, we report that SUDHL-1 and KARPAS 299 ALCL-derived cell lines present different sensitivity to the antiproliferative effect of recombinant adenovirus-mediated p27(Kip1) expression or to serum-starvation in culture media. The results indicate that exogenous p27(Kip1) may interact with the NPM-ALK/PLCgamma pathway in SUDHL-1 but not in KARPAS 299 cells. This interaction correlates with changes in cell cycle and cell morphology observed mainly in SUDHL-1 cells. The percentage of SUDHL-1 cells in S phase declines, whereas it is almost unchanged in KARPAS 299 cells as compared to the controls after 96 h of infection with the recombinant adenovirus. Furthermore KARPAS 299 cells are resistant to serum-starvation due to deficient p27(Kip1)-upregulation and G1 arrest, whereas SUDHL-1 cells respond with increased G1 phase and p27(Kip1)-upregulation after 48 h of serum-starvation. Both cell lines express appropriate variation of levels of cyclins E and A, and Rb-phosphorylation as expected by growing them in culture media with different FBS content. Although both cell lines express cyclin D2, SUDHL-1 cells only present high level of cyclin D3. Moreover SUDHL-1 cells express high level of PTEN and the PKB/Akt pathway is constitutively activated in both cell lines. Lastly SUDHL-1 cells show higher levels of phosphotyrosine-containing proteins that is correlated with a higher NPM-ALK-associated autophosphorylation activity compared to KARPAS 299 cells. Our study clearly identifies some of the biochemical differences that may explain the difference in sensitivity to antiproliferative stimuli shown by two cell lines derived from the same type of lymphoma.
Hemopexin protects cells lacking hemopexin receptors by tightly binding heme abrogating its deleterious effects and preventing nonspecific heme uptake, whereas cells with hemopexin receptors undergo a series of cellular events upon encountering heme-hemopexin. The biochemical responses to heme-hemopexin depend on its extracellular concentration and range from stimulation of cell growth at low levels to cell survival at otherwise toxic levels of heme. High (2-10 microM) but not low (0.01-1 microM) concentrations of heme-hemopexin increase, albeit transiently, the protein carbonyl content of mouse hepatoma (Hepa) cells. This is due to events associated with heme transport since cobalt-protoporphyrin IX-hemopexin, which binds to the receptor and activates signaling pathways without tetrapyrrole transport, does not increase carbonyl content. The N-terminal c-Jun kinase (JNK) is rapidly activated by 2-10 microM heme-hemopexin, yet the increased intracellular heme levels are neither toxic nor apoptotic. After 24 h exposure to 10 microM heme-hemopexin, Hepa cells become refractory to the growth stimulation seen with 0.1-0.75 microM heme-hemopexin but HO-1 remains responsive to induction by heme-hemopexin. Since free heme does not induce JNK, the signaling events, like phosphorylation of c-Jun via activation of JNK as well as the nuclear translocation of NFkappaB, G2/M arrest, and increased expression of p53 and of the cell cycle inhibitor p21(WAF1/CIP1/SDI1) generated by heme-hemopexin appear to be of paramount importance in cellular protection by heme-hemopexin.