Marcello A. Barcinski

Tumor-derived microvesicles modulate the establishment of metastatic melanoma in a phosphatidylserine-dependent manner

Exposure of phosphatidylserine (PS) on cellular membranes and membrane-derived microvesicles stimulates a number of anti-inflammatory responses involved in malignant processes. Herein we show that B16F10 cells, a highly metastatic melanoma cell line, produce large quantities of PS-containing microvesicles in vitro. Tumor microvesicles increased TGF-beta(1) production by cultured macrophages and, in vivo, enhanced the metastatic potential of B16F10 cells in C57BL/6 mice, both effects being reversed by annexin V. Most strikingly, microvesicles induced melanoma metastasis in BALB/c mice, which are normally resistant to this tumor cell line. Altogether, this is the first demonstration that tumor-derived microvesicles favor the establishment of melanoma metastasis in a PS-dependent manner, possibly by down-regulating the hosts inflammatory and/or anti-tumoral immune responses.

Antisense intronic non-coding RNA levels correlate to the degree of tumor differentiation in prostate cancer

A large fraction of transcripts are expressed antisense to introns of known genes in the human genome. Here we show the construction and use of a cDNA microarray platform enriched in intronic transcripts to assess their biological relevance in pathological conditions. To validate the approach, prostate cancer was used as a model, and 27 patient tumor samples with Gleason scores ranging from 5 to 10 were analyzed. We find that a considerably higher fraction (6.6%, [23/346]) of intronic transcripts are significantly correlated (P⩽0.001) to the degree of prostate tumor differentiation (Gleason score) when compared to transcripts from unannotated genomic regions (1%, [6/539]) or from exons of known genes (2%, [27/1369]). Among the top twelve transcripts most correlated to tumor differentiation, six are antisense intronic messages as shown by orientation-specific RT-PCR or Northern blot analysis with strand-specific riboprobe. Orientation-specific real-time RT–PCR with six tumor samples, confirmed the correlation (P=0.024) between the low/high degrees of tumor differentiation and antisense intronic RASSF1 transcript levels. The need to use intron arrays to reveal the transcriptome profile of antisense intronic RNA in cancer has clearly emerged.

Apoptotic mimicry by an obligate intracellular parasite downregulates macrophage microbicidal activity

Programmed cell death by apoptosis of unnecessary or potentially harmful cells is clearly beneficial to multicellular organisms. Proper functioning of such a program demands that the removal of dying cells proceed without an inflammatory reaction. Phosphatidylserine (PS) is one of the ligands displayed by apoptotic cells that participates in their noninflammatory removal when recognized by neighboring phagocytes. PS ligation induces the release of transforming growth factor-beta (TGF-beta), an antiinflammatory cytokine that mediates the suppression of macrophage-mediated inflammation. In Hydra vulgaris, an organism that stands at the base of metazoan evolution, the selective advantage provided by apoptosis lies in the fact that Hydra can survive recycling apoptotic cells by phagocytosis. In unicellular organisms, it has been proposed that altruistic death benefits clonal populations of yeasts and trypanosomatids. Now we show that advantageous features of the apoptotic process can operate without death as the necessary outcome. Leishmania spp are able to evade the killing activity of phagocytes and establish themselves as obligate intracellular parasites. Amastigotes, responsible for disease propagation, similar to apoptotic cells, inhibit macrophage activity by exposing PS. Exposed PS participates in amastigote internalization. Recognition of this moiety by macrophages induces TGF-beta secretion and IL-10 synthesis, inhibits NO production, and increases susceptibility to intracellular leishmanial growth.

Mimicry of Apoptotic Cells by Exposing Phosphatidylserine Participates in the Establishment of Amastigotes of Leishmania (L) amazonensis in Mammalian Hosts

Signaling through exposed phosphatidylserine (PS) is fundamental for the TGFβ1-dependent, noninflammatory phagocytosis of apoptotic cells. This same mechanism operates in the internalization of amastigotes of Leishmania (L) amazonensis (L(L)a) in a process quoted as apoptotic mimicry. Now we show that the host modulates PS exposure by the amastigotes and, as a consequence, BALB/c mice-derived amastigotes expose significantly more PS than those derived from C57BL/6 mice. Due to this difference in the density of surface PS molecules, the former are significantly more infective than the latter, both in vivo, in F1 (BALB/c × C57BL/6) mice, and in vitro, in thioglycollate-derived macrophages from this same mouse strain. PS exposure increases with progression of the lesion and reaches its maximum value in amastigotes obtained at the time point when the lesion in C57BL/6 mice begins to decrease in size and the lesions in BALB/c mice are still growing in size. Synthesis of active TGFβ1, induction of IL-10 message, and inhibition of NO synthesis correlate with the amount of surface PS displayed by viable (propidium iodide-negative) infective amastigote. Furthermore, we also show that, similar to what happens with apoptotic cells, amastigotes of L(L)a are internalized by macropinocytosis. This mechanism of internalization is consistent with the large phagolysosomes characteristic of L(L)a infection. The intensity of macrophage macropinocytic activity is dependent on the amount of surface PS displayed by the infecting amastigote.

Cooperation between Apoptotic and Viable Metacyclics Enhances the Pathogenesis of Leishmaniasis

Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PSPOS) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PSPOS metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNELPOS promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PSPOS and PS-negative (PSNEG) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PSNEG promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PSPOS apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen.

Apoptosis and parasitism: from the parasite to the host immune response.

Apoptosis, a form of programmed cell death (PCD), plays a central role in normal tissue development as well as in the pathogenesis of different diseases. PCD is responsible for the non-inflammatory physiological elimination of potentially harmful or unnecessary cells during embryogenesis, and for the proper functioning of continuous cell renewal systems in adult organisms. Maturation of the immune system and the specific immune response are examples of situations where PCD plays important roles. This review discusses the importance of apoptosis in two fundamental elements of a host-parasite interaction: the parasite (Section 1), and the hosts immune response (Section 2). Section 1 discusses questions raised by the description of apoptosis in unicellular eukaryotes, such as the evolutionary origin of the molecular components of PCD, its role in the emergence and maintenance of parasitism, and the constraints of a multicellular organization for the proper operation of a cell death programme. The proposal is that PCD can occur in any situation where living cells display features of an organized network which operates through interactions within themselves and/or with elements of their environment. The possibility is also discussed that evolutionary relics of a complete cell death system may operate in unicellular parasites with functions other than inducing cell death. Section 2 reviews data on the mechanisms of host-cell PCD and the consequences of this phenomenon in host defence and pathogenesis. Infectious agents, from viruses to parasites, can either delay or induce apoptosis of different types of host cells. Apoptosis following lymphocyte polyclonal activation and stimulation of peripheral T lymphocytes, as a result of the engagement of specific counter-receptor systems, is of special interest for defining host immunocompetence and mechanisms of immunopathology.

Apoptosis and apoptotic mimicry: the Leishmania connection

Different death-styles have been described in unicellular organisms. In most cases they evolve with phenotypic features similar to apoptotic death of animal cells, such as phosphatidylserine (PS) exposure, oligonucleosomal DNA fragmentation, and loss of mitochondrial transmembrane potential, hinting that similar mechanisms operate in both situations. However, the biochemical pathways underlying death in unicellular organisms are still unclear. Host recognition of PS exposed on the surface of unicellular parasites is an important feature of the process of infection and progression of the disease. Here, we discuss data showing that entirely different mechanisms of PS exposure co-exist during the life-cycle of Leishmania amazonensis: in the case of promastigotes, a sub-population dies by apoptosis; in the case of amastigotes, the entire population exposes PS, not necessarily followed by apoptotic death. This phenomenon has been called apoptotic mimicry. The elusive caspase-like activities described in protozoa are also discussed.

Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma.

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non‐neoplastic tissue were obtained from six patients. A custom‐built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down‐regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave‐one‐out patient cross‐validation. Among the down‐regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein‐coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P < 0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P < 0.05), were confirmed in clear cell RCC by Real‐time RT‐PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells.

Malignant transformation in melanocytes is associated with increased production of procoagulant microvesicles

Shedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte-derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plasma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs produced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagulation-related protumoural responses.

Apoptotic cell and phagocyte interplay: recognition and consequences in different cell systems

Cell death by apoptosis is characterized by specific biochemical changes, including the exposure of multiple ligands, expected to tag the dying cell for prompt recognition by phagocytes. In non-pathological conditions, an efficient clearance is assured by the redundant interaction between apoptotic cell ligands and multiple receptor molecules present on the engulfing cell surface. This review concentrates on the molecular interactions operating in mammalian and non-mammalian systems for apoptotic cell recognition, as well as on the consequences of their signaling. Furthermore, some cellular models where the exposure of the phosphatidylserine (PS) phospholipid, a classical hallmark of the apoptotic phenotype, is not followed by cell death will be discussed.