Mitochondria are considered as the power‐generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction‐based methods, and Whole Genome Sequencing, we detect extracellular full‐length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell‐free mitochondria using fluorescence‐activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell‐cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases.

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.

Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C␣ (PKC␣) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC␣ V3 hinge domain. This motif is also required for a direct association between PKC␣ and ␤1 integrin. Efficient binding of ␤1 integrin to PKC␣ requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of ␤1 integrin was shown to block both PKC␣-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC␣ and ␤1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.An early critical step within the metastatic cascade involves cell polarization and directed cell movement. The mechanisms controlling these events, however, are poorly understood. Cell polarization signals can be triggered by the binding of cells to extracellular matrix (ECM) proteins, cytokines or chemoattractants, or ligands that are expressed on the surface of target tissues or vessels. Sensing of these extracellular signals results in a redistribution and trafficking of membrane receptors (1, 19) and/or intracellular signaling proteins, as well as the localized formation of protein complexes which can be stable or transient.Members of the integrin receptor family are vital in mediating cell matrix adhesion and have been implicated in providing the persistence and directionality for cell motility, as in wound healing (7). Specifically, a number of studies have demonstrated that the ␤-subunit cytoplasmic domains are involved in the control of cell migration. Thus, the tyrosine residues of the two NPXY motifs in ␤1 are important for directed cell migration through integrin substrate-coated filters in response to growth factor chemotactic gradients (22,23). In fact, when expressed as chimeric receptors connected to heterologous extracellular domains, such as the interleukin-2 receptor (IL-2R) tac subunit, the ␤1 integrin cytoplasmic tail acts as a dominant negative inhibitor of endogenous integrin function (4). Moreover, inhibition of integrin-ECM interactions by GRGDS peptides or inhibitory antibodies was also shown to abolish the persistence or directionality of neural crest cell movement (7).Protein kinase C (PKC) may be seen as part of the molecular machinery that deciphers...

The generation of antisera specific for the priming phosphorylation sites on protein kinase Calpha (PKCalpha) has permitted analysis of the dephosphorylation of these sites in relation to the down-regulation of the protein. It was demonstrated that these priming sites are subject to agonist-induced dephosphorylation, consistent with inactivation of the protein. Further, the process is shown to be blocked by a PKC inhibitor, indicating a requirement for PKC catalytic activity. This was corroborated by showing that a constitutively active fragment of PKCalpha is able to stimulate the dephosphorylation of wild-type PKCalpha in transfected cells. Consistent with a membrane-traffic event, the process controlled by PKC that leads to dephosphorylation is shown to be temperature-sensitive and to correlate with transient accumulation of PKCalpha on cytoplasmic vesicular structures. It was established that the dephosphorylation of priming sites in PKCalpha is not unique and occurs with other conventional PKC isotypes, demonstrating that this is a general desensitization process for this subclass of kinases. The physiological importance of this desensitization is evidenced by the behaviour of PKCbeta1 in U937 cells, where dephosphorylation of the activation loop site is shown to be a function of cell density.

SOX9 inactivation is frequent in colorectal cancer (CRC) due to SOX9 gene mutations and/or to ectopic expression of MiniSOX9, a dominant negative inhibitor of SOX9. In the present study, we report a heterozygous L142P inactivating mutation of SOX9 in the DLD-1 CRC cell line and we demonstrate that the conditional expression of a wild type SOX9 in this cell line inhibits cell growth, clonal capacity and colonosphere formation while decreasing both the activity of the oncogenic Wnt/ß-catenin signaling pathway and the expression of the c-myc oncogene. This activity does not require SOX9 transcriptional function but, rather, involves an interaction of SOX9 with nuclear ß-catenin. Furthermore, we report that SOX9 inhibits tumor development when conditionally expressed in CRC cells injected either subcutaneous or intraperitoneous in BALB/c mice as an abdominal metastasis model. These observations argue in favor of a tumor suppressor activity for SOX9. As an siRNA targeting SOX9 paradoxically also inhibits DLD-1 and HCT116 CRC cell growth, we conclude that there is a critical level of endogenous active SOX9 needed to maintain CRC cell growth.

In order to map the molecular determinants that dictate the subcellular localization of human protein kinase C ␣ (hPKC␣), full-length and deletion mutants of hPKC␣ were tagged with the green fluorescent protein (GFP) and transiently expressed in GH3B6 cells. We found that upon thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate stimulation, hPKC␣-GFP was localized exclusively in regions of cell-cell contacts. Surprisingly, PKC␣ failed to translocate in single cells despite the presence of TRH receptors, as attested by the TRH-induced rise in intracellular calcium concentration in these cells. TRH-stimulated translocation of hPKC␣-GFP from the cytoplasm to cell-cell contacts was a biphasic process: a fast (measured in seconds) and transient phase, followed by a slower (approximately 1 hour) and long lasting phase. The latter and the translocation induced by phorbol 12-myristate 13-acetate absolutely required the N-terminal V1 region. In contrast to the full-length hPKC␣, the N-terminal regulatory domain alone or associated with the V3 hinge region was spontaneously and uniformly localized at the plasma membrane of single and apposed cells. However, treatment with the calcium chelator BAPTA/AM induced a differential cytoplasmic/nuclear redistribution of the regulatory domain, depending on its association with V3, which suggests the existence of a mechanism controlling the cytoplasmic sequestration of inactive hPKC␣ and involving the V3 region. By using other deletion mutants, we were able to map the sequence required for this sequestration to the C2؉V3 regions. This work points to the existence of a complex interplay between membrane targeting and cytoplasmic sequestration in the control of the spatiotemporal localization of hPKC␣. Protein kinase C (PKC)1 is a term coined to designate a family of isoforms that play key roles in the processes of proliferation/apoptosis, differentiation, or hormone release and the function of which is regulated at multiple levels: transcription, phosphorylation, and subcellular targeting.Subcellular targeting of PKC, and particularly that of the conventional PKC ␣, ␤, and ␥ isoforms, is linked to enzyme activation. Indeed, inactive PKC is mostly cytoplasmic, whereas activated PKC translocates to various membrane compartments, such as the plasma membrane. Physiological activation of the conventional PKC is associated with an increase in diacylglycerol (DAG) and intracellular Ca 2ϩ concentrations (1), which results from the activation of a seven transmembrane receptor coupled to phospholipase C␥ via a heterotrimeric G protein (2). The increase in Ca 2ϩ is thought to be essential for translocation, although it can be bypassed by the phorbol ester phorbol 12-myristate 13-acetate (PMA) (3). The increase in DAG concentration at the plasma membrane allows additional conformational changes to achieve PKC activation at its targeting site. When inactive, PKC is in a "closed" conformation due to the interaction of the pseudosubstrate sequence with the catalytic site (4, 5). ...

Increased protein kinase C (PKC) activity in malignant breast tissue and in most aggressive breast cancer cell lines has suggested a possible role of PKC in breast carcinogenesis and tumor progression. We have investigated here the involvement of PKC in the in vitro invasiveness and motility of several breast cancer cell lines. Modulation of PKC activity by treatment with a phorbol ester (TPA), drastically increased the invasiveness of 2 estrogen receptor‐positive (ER+) lines (MCF7 and ZR 75.1), whereas it markedly decreased the invasiveness of 2 ER− cell lines (MDA‐MB‐231 and MDA‐MB‐435). A PKC inhibitor (H7) reversed the TPA effects in MCF7 cells, whereas it mimicked TPA action in MDA‐MB‐231 cells. All of these effects of TPA also were observed to a similar extent for cell chemotaxis, and they were not dependent on protein neo‐synthesis. In parallel, short TPA treatment induced cell spreading and microtubule organization in MCF7 cells and inverse morphological changes in MDA‐MB‐231 cells. In ER+ cells, constitutive PKC activity and PKCα expression were very low as compared to ER− cells, and this correlated with the invasive potential of the cells. The opposed effects of TPA in ER+ and ER− cells could be due to the abnormal TPA regulation of PKCα observed in ER− cells. Int. J. Cancer 75:750–756, 1998.© 1998 Wiley‐Liss, Inc.

Since the mid 1970s, cancer has been described as a process of Darwinian evolution, with somatic cellular selection and evolution being the fundamental processes leading to malignancy and its many manifestations (neoangiogenesis, evasion of the immune system, metastasis, and resistance to therapies). Historically, little attention has been placed on applications of evolutionary biology to understanding and controlling neoplastic progression and to prevent therapeutic failures. This is now beginning to change, and there is a growing international interest in the interface between cancer and evolutionary biology. The objective of this introduction is first to describe the basic ideas and concepts linking evolutionary biology to cancer. We then present four major fronts where the evolutionary perspective is most developed, namely laboratory and clinical models, mathematical models, databases, and techniques and assays. Finally, we discuss several of the most promising challenges and future prospects in this interdisciplinary research direction in the war against cancer.