Agnès Loubat

Monoterpenes inhibit proliferation of human colon cancer cells by modulating cell cycle-related protein expression

The monoterpene perillyl alcohol (POH) is a naturally occurring anti-cancer compound which is effective against a variety of rodent organ-specific tumor models. To establish the molecular mechanisms of POH and its major metabolite perillic acid (PA) as anti-proliferative agents, their effects on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in HCT 116 human colon cancer cells. POH, and to a lesser extent, PA, exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed that monoterpenes increased expression of cdk inhibitor p21(Waf1/Cip1) and cyclin E, and decreased expression of cyclin D1, cyclin-dependent kinase (cdk) 4 and cdk2. Our results suggest that monoterpenes induce growth arrest of colon cancer cells through the up-regulation of p21(Waf1/Cip1) and the down-expression of cyclin D1 and its partner cdk4.

Microphthalmia-Associated Transcription Factor Controls the DNA Damage Response and a Lineage-Specific Senescence Program in Melanomas

Apoptosis and senescence are cellular failsafe programs that counteract excessive mitogenic signaling observed in cancer cells. Melanoma is known for its notorious resistance to apoptotic processes; therefore, senescence, which remains poorly understood in melanomas, can be viewed as a therapeutic alternative. Microphthalmia-associated transcription factor (MITF), in which its M transcript is specifically expressed in melanocyte cells, plays a critical role in melanoma proliferation, and its specific inhibition is associated with G(0)-G(1) growth arrest. Interestingly, decreased MITF expression has been described in senescent melanocytes, and we have observed an inhibition of MITF expression in melanoma cells exposed to chemotherapeutic drugs that induce their senescence. All these observations thereby question the role of MITF in controlling senescence in melanoma cells. Here, we report that long-term depletion of MITF in melanoma cells triggers a senescence program characterized by typical morphologic and biochemical changes associated with a sustained growth arrest. Further, we show that MITF-silenced cells engage a DNA damage response (DDR) signaling pathway, leading to p53 upregulation, which is critically required for senescence entry. This study uncovers the existence of a lineage-restricted DDR/p53 signaling pathway that is inhibited by MITF to prevent senescence and favor melanoma cell proliferation.

AS602868, a pharmacological inhibitor of IKK2, reveals the apoptotic potential of TNF- α in Jurkat leukemic cells

NF-κB transcription factors promote survival in numerous cell types via induction of antiapoptotic genes. Pharmacological blockade of the IKK2 kinase with AS602868, a specific inhibitor that competes with ATP binding, prevented TNF-α-induced NF-κB activation in Jurkat leukemic T cells. While TNF-α by itself had no effect on Jurkat survival, the addition of AS602868 induced cell death, visualized by DNA fragmentation and sub-G1 analysis. A disruption of the mitochondrial potential followed by activation of caspases 9 and 3 was observed in cells treated by the combination TNF-α+AS602868. Quantitative real-time PCR demonstrated that AS602868 prevented TNF-α induction of the antiapoptotic genes coding for c-IAP-2, Bclx, Bfl-1/A1 and Traf-1. The use of a specific IKK2 inhibitor appears, therefore, as an interesting pharmaceutical strategy to increase the cells sensitivity towards apoptotic effectors.

Focal adhesion kinase pp125FAK interacts with the large conductance calcium-activated hSlo potassium channel in human osteoblasts: potential role in mechanotransduction.

Molecular events of mechanotransduction in osteoblasts are poorly defined. We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock. This could convert mechanical signals in biochemical events, leading to osteoblast activation.

The protective effect of phorbol esters on Fas-mediated apoptosis in T cells. Transcriptional and postranscriptional regulation.

Phorbol esters are tumor promoters that bind and activate both conventional and new Protein kinase C (PKC) isoforms. In various circumstances, PKC-dependent signaling pathways can promote cell survival and protect against cell death. This was first analysed in Jurkat T cells where Phorbol Myristate Acetate (PMA) was found to inhibit Fas-mediated apoptosis as judged by DiOC6(3) staining, caspase activation and DNA fragmentation, indicating that PMA exerts its protective effect upstream or at the mitochondrial level in these cells. PMA activated most of the main kinase pathways in T cells such as PKCs, p42/44MAPK, p38MAPK and p90Rsk but not JNK and Akt. A pharmacological approach allowed us to identify that nPKCs are both necessary and likely sufficient to promote T cell survival. Besides this post-transcriptional regulation, nPKCs may also regulate apoptosis at the transcriptional level. cDNA arrays were used to identify a set of genes whose expression was modulated in death versus survival conditions. Following PMA treatment, expression of Mcl-1 and Bcl-x increased while that of c-Myc was significantly reduced. Moreover, survivin expression decreased upon CH11 or PMA treatment. c-Myc, survivin and Bcl-x modulation seems to be regulated at the transcriptional level while decrease in Mcl-1 protein in CH11-treated cells resulted especially from a caspase-dependent proteolysis. Taken together, our data demonstrate that PMA-mediated inhibition of apoptosis is a complex process that is integrated at both the transcriptional and post-transcriptional level and point out to the potential role of Mcl-1, Bcl-x, c-Myc and survivin in this process.

Early G1 growth arrest of hybridoma b cells by DMSO involves cyclin D2 inhibition and p21|CIP1| induction

Dimethylsulfoxide (DMSO) was shown to inhibit the proliferation of several B cell lines including Raji, Daudi, and SKW6-CL4 but the mechanisms involved in this growth arrest are still unclear. We show that in 7TD1 mouse hybridoma cells a DMSO-induced reversible G1 arrest involves inactivation of Rb kinases, cyclin D2/CDK4 and cyclin E/CDK2. This occurs by at least three distinct mechanisms. Inhibition of cyclin D2 neosynthesis leads to a dramatic decrease of cyclinD2/CDK4 complexes. This in turn enables the redistribution of p27[KIP1] from cyclin D2/CDK4 to cyclin E/CDK2 complexes. In addition, the simultaneous accumulation of p21[CIP1] entails increasing association with cyclin D3/CDK4 and cyclin E/CDK2. Thus, p21[CIP1] and p27[KIP1], act in concert to inhibit cyclin E/CDK2 activity which, together with CDK4 inactivation, confers a G1-phase arrest.

CAL72: a human osteosarcoma cell line with unique effects on hematopoietic cells.

Abstract: Permanent osteoblastic cell lines are potential tools to study the interactions between osteoblastic and hematopoietic cells in the bone marrow cavity. In a recent work we have shown that the osteosarcoma cell line CAL72 may be more closely related to normal osteoblasts than the osteosarcoma cells previously described. In the present work we continued the characterisation of the CAL72 cell line with regard to its effects on various hematopoietic cells, in coculture experiments. We show here that CAL72 cells, in contrast to MG‐63 or SaOS‐2 osteosarcoma cell lines, do not inhibit hematopoietic colony formation and sustain the limited expansion of hematopoietic progenitors in a similar way to that described for normal osteoblasts. We also demonstrate that CAL72 cells induce the monocytic differentiation of the promyelocytic HL‐60 cell line like MG‐63 and SaOS‐2, but support a better maturation and a longer survival of the differentiated cells than the two other osteosarcoma cell lines. In order to better understand the differential effects observed between CAL72 and MG‐63 or SaOS‐2, we analysed the cytokine and chemokine mRNA expression of these cells using the RNase protection quantitative assay. We show here that the expression profile of CAL72 is clearly different from that of MG‐63 or SaOS‐2 and may explain, at least in part, its specific effects on hematopoietic cells. Taken together these experiments confirm that CAL72 has particular properties and is an interesting tool to study the role of osteoblastic cells in hematopoietic cell growth and differentiation.

Active stromelysin‐3 (MMP‐11) increases MCF‐7 survival in three‐dimensional Matrigel culture via activation of p42/p44 MAP‐kinase

Stromelysin‐3 (ST3) has the characteristic structure of matrix metalloproteinases (MMP), but its substrate specificity and pattern of expression differ markedly from that of other MMP family members. ST3 was originally isolated on the basis of its expression in primary breast cancers and has been shown to be overexpressed in virtually all primary carcinomas, suggesting that ST3 participates in the initial development of epithelial malignancies. Recent data using murine models reported that ST3 expression was able to increase tumor take by suppressing cell apoptosis. Our present goal was to set up an in vitro model in which we could study this new function. For this purpose, we analyzed survival of MCF‐7 transfectants expressing either wild‐type or catalytically inactive ST3 (ST3wt or ST3cat‐) in three‐dimensional (3‐D) culture conditions by inclusion in Matrigel. In such conditions, that mimic the in vivo microenvironment, we found a marked decrease in the percentage of cell death when active ST3 was expressed (ST3wt transfectants vs. ST3cat‐ or vector only transfectants) as assessed by FACS and TUNEL analysis. The addition of batimastat, a broad spectrum MMP inhibitor, reversed the increased cell survival in ST3wt transfectants, confirming that ST3 enzymatic activity was required for this effect. Finally, we analyzed the expression of anti‐ and pro‐apoptotic proteins as well as activation of cell survival pathways and we found that ST3‐mediated cell survival was accompanied by activation of both p42/p44 MAPK and AKT. Our data confirm and extend the anti‐apoptotic function of ST3 and provide a useful model to dissect this new role and identify new physiological substrates.

SigmaR1 regulates membrane electrical activity in response to extracellular matrix stimulation to drive cancer cell invasiveness

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K(+) channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition.

Human Receptors Patched and Smoothened Partially Transduce Hedgehog Signal When Expressed in Drosophila Cells

In humans, dysfunctions of the Hedgehog receptors Patched and Smoothened are responsible for numerous pathologies. However, signaling mechanisms involving these receptors are less well characterized in mammals than in Drosophila. To obtain structure-function relationship information on human Patched and Smoothened, we expressed these human receptors in Drosophila Schneider 2 cells. We show here that, as its Drosophila counterpart, human Patched is able to repress the signaling pathway in the absence of Hedgehog ligand. In response to Hedgehog, human Patched is able to release Drosophila Smoothened inhibition, suggesting that human Patched is expressed in a functional state in Drosophila cells. We also provide experiments showing that human Smo, when expressed in Schneider cells, is able to bind the alkaloid cyclopamine, suggesting that it is expressed in a native conformational state. Furthermore, contrary to Drosophila Smoothened, human Smoothened does not interact with the kinesin Costal 2 and thus is unable to transduce the Hedgehog signal. Moreover, cell surface fluorescent labeling suggest that human Smoothened is enriched at the Schneider 2 plasma membrane in response to Hedgehog. These results suggest that human Smoothened is expressed in a functional state in Drosophila cells, where it undergoes a regulation of its localization comparable with its Drosophila homologue. Thus, we propose that the upstream part of the Hedgehog pathway involving Hedgehog interaction with Patched, regulation of Smoothened by Patched, and Smoothened enrichment at the plasma membrane is highly conserved between Drosophila and humans; in contrast, signaling downstream of Smoothened is different.