Biomedical subjects

S Carillo

Publications and source records attributed to S Carillo.

14 recordsLinked to original sources

PubMed2005

High plasma proteasome levels are detected in patients with metastatic malignant melanoma.

BACKGROUND: Proteasomes, nonlysosomal proteolytic structures, are implicated in cell growth and differentiation. An abnormal expression has been described in haematopoietic malignancies and in some solid tumours. OBJECTIVES: To study the plasma proteasome levels in patients with malignant melanoma (MM) using an enzyme-linked immunosorbent assay (ELISA) technique, and to compare them with the values obtained in a normal population and in patients with severe psoriasis or chronic idiopathic urticaria (CIU). METHODS: Plasma proteasome level was measured using a sandwich ELISA test in normal donors (n = 14), and in patients with stage I/II (n = 13), stage III (n = 6) and stage IV (n = 10) MM, severe psoriasis (n = 13) and CIU (n = 6). Tissue proteasome expression was also detected by immunohistology using a monoclonal antibody in paraffin-embedded samples of normal tissue, psoriasis skin and MM. RESULTS: In normal donors, mean +/- SEM plasma proteasome concentration was 2138 +/- 221 ng mL(-1). Patients with stages III and IV MM exhibited a significantly higher value (3373 +/- 470 ng mL(-1) and 8931 +/- 1232 ng mL(-1), respectively). Values in patients with stage I/II MM and CIU were not significantly different from those in normal volunteers. Patients with severe psoriasis also exhibited increased values (3398 +/- 374 ng mL(-1)) but to a lesser extent than in patients with stage IV MM. There was a significant correlation of proteasome levels with serum lactate dehydrogenase in the MM group. Tissue expression as demonstrated by immunohistochemistry paralleled these findings. The strongest expression was seen on MM slides and to a lesser extent in psoriasis samples, the weakest expression being observed in normal skin. CONCLUSIONS: Proteasomes are strongly expressed in cutaneous MM; high levels of circulating proteasomes are detected in patients with metastatic MM with a high melanoma burden, and at a lesser extent in psoriatic patients, which suggests proteasomes represent a marker more of nonspecific inflammation than of early cancer.

Adult↗

PubMed2001

Plasma proteasome level is a potential marker in patients with solid tumors and hemopoietic malignancies.

BACKGROUND: Proteasomes are nonlysosomal proteolytic structures that have been implicated in cell growth and differentiation. Abnormal expression levels of proteasomes have been described in tumor cells, and proteasomes can be detected and measured in plasma. The objective of this study was to characterize differences in proteasome levels between normal, healthy donors and patients with neoplastic disease and to correlate the findings with clinical status and other biologic markers of disease spread. METHODS: Plasma proteasome levels were measured using a sandwich enzyme-linked immunosorbent assay in normal donors (n = 73 donors) and in patients with solid tumors (n = 20 patients), acute leukemia (n = 35 patients), myeloproliferative (n = 37 patients) and myelodysplastic (n = 19 patients) syndromes, chronic lymphocytic leukemia (n = 44 patients), non-Hodgkin lymphoma (n =104 patients), Hodgkin disease (n = 14 patients), other lymphoid disorders (n = 17 patients), and multiple myeloma (n = 27 patients). RESULTS: In the normal donors, the plasma proteasome concentration was 2356 ng/mL +/- 127 ng/mL. Patients with solid tumors exhibited a significantly higher value (7589 ng/mL +/- 2124 ng/mL), similar to the patients with myeloproliferative (4099 ng/mL +/- 498 ng/mL) and myelodysplastic (2922 ng/mL +/- 322 ng/mL) syndromes. Patients with lymphoproliferative disorders, in contrast, had significantly lower values than normal donors (1751 ng/mL +/- 107 ng/mL), except those in aggressive phase of the disease. This low level persisted in patients who were in complete remission. Proteasome levels decreased during the initial phase of treatment. Although there was a significant correlation with serum lactic dehydrogenase levels, frequent discrepancies were noted. There was no correlation with C-reactive protein or beta2-microglobulin levels, even in the group of patients with multiple myeloma. CONCLUSIONS: The plasma proteasome level is a potential new tool for the monitoring of patients with neoplastic disease. It is not correlated solely with cell lysis and may be involved in the pathophysiology of disease progression.

Adult↗

PubMed2000

The sensitivity of c-Jun and c-Fos proteins to calpains depends on conformational determinants of the monomers and not on formation of dimers.

Milli- and micro-calpains are ubiquitous cytoplasmic cysteine proteases activated by calcium. They display a relatively strict specificity for their substrates which they usually cleave at only a limited number of sites. Motifs responsible for recognition by calpains have not been characterized yet, and recently a role for PEST motifs in this process has been ruled out. c-Fos and c-Jun transcription factors are highly sensitive to calpains in vitro. They thus provide favourable protein contexts for studying the structural requirements for recognition and degradation by these proteases. Using in vitro degradation assays and site-directed mutagenesis, we report here that susceptibility to calpains is primarily determined by conformational determinants of the monomers and not by the quaternary structure of c-Fos and c-Jun proteins. The multiple cleavage sites borne by both proteins can be divided into at least two classes of sensitivity, the most sensitive ones being easily visualized in the presence of rate-limiting amounts of calpains. One site located at position 90-91 in c-Fos protein is extremely sensitive. However, efficient proteolysis did not have any strict dependence on the nature of the amino acids on either side of the scissile bond in the region extending from P2 to P'2. The structural integrity of the monomers is not crucial for recognition by calpains. Rather, sensitive sites can be recognized independently and their recognition is dependent on the local conformation of peptide regions that may span several tens of amino acids and maybe more in the case of the identified c-Fos hypersensitive site.

Amino Acid Sequence↗

PubMed2000

[The proteasome and malignant hemopathies].

Proteasomes are the main non lysosomal proteolytic structures of the cells. They correspond to the major system eliminating abnormal proteins, short half-life proteins and proteins controlling the cell cycle. They are essential for the production of peptides subsequently presented by the MHC-I. They are formed by a proteolytic core (the 20S proteasome) made of 4 rings of 7 proteic subunits associated with regulatory complexes (namely the 19S complex forming the 26S proteasome). Using classical cell biology techniques (cytometry, immunofluorescence microscopy, Western blot) our group has particularly studied the proteasome expression of leukaemic cell lines (U937 and CCRF-CEM) during in vitro differentiation induced by PMA and Vitamin D plus retinoïc acid. During differentiation, the level of proteasome expression and its localization vary. The various monoclonal antibodies used provided different patterns according to the different subunits. There was a general trend to a disappearance of nuclear proteasome and to a decrease in their cytoplasmic expression. In contrast, proteosomal antigens were increased on the cell membrane and in culture supernatants. We derived an ELISA test to measure plasma proteasome concentrations. Preliminary results showed differences between patients with haemopoietic malignancies or solid tumors and normal donors. Proteasome levels varied under treatment. They were correlated with LDH levels. Taken together, these results argue in favor of a role for cellular proteasomes in malignant differentiation process, and emphasize the qualitative changes in proteasome expression. Plasma proteasomes do not only reflect tumor cell mass and could play a role in addition to their proteolytic activity. They seem to be a relevant tool for diagnosis, prognosis and therapeutic monitoring.

Biomarkers, Tumor↗

PubMed1999

Are there multiple proteolytic pathways contributing to c-Fos, c-Jun and p53 protein degradation in vivo?

The c-Fos and c-Jun oncoproteins and the p53 tumor suppressor protein are short-lived transcription factors. Several catabolic pathways contribute to their degradation in vivo. c-Fos and c-Jun are thus mostly degraded by the proteasome, but there is indirect evidence that, under certain experimental/physiological conditions, calpains participate in their destruction, at least to a limited extent. Lysosomes have also been reported to participate in the destruction of c-Fos. Along the same lines, p53 is mostly degraded following the ubiquitin/proteasome pathway and calpains also seem to participate in its degradation. Moreover, c-Fos, c-Jun and p53 turnovers are regulated upon activation of intracellular signalling cascades. All taken together, these observations underline the complexity of the mechanisms responsible for the selective destruction of proteins within cells.

Animals↗

PubMed1997

Decreased susceptibility to calpains of v-FosFBR but not of v-FosFBJ or v-JunASV17 retroviral proteins compared with their cellular counterparts.

The c-Fos and c-Jun transcription factors are rapidly turned over in vivo. One of the multiple pathways responsible for their breakdown is probably initiated by calpains, which are cytoplasmic calcium-dependent cysteine proteases. The c-fos gene has been transduced by two murine oncogenic retroviruses called Finkel-Biskis-Jenkins murine sarcoma virus (FBJ-MSV) and Finkel-Biskis-Reilly murine sarcoma virus (FBR-MSV); c-jun has been transduced by the chicken avian sarcoma virus 17 (ASV17) retrovirus. Using an in vitro degradation assay, we show that the mutated v-FosFBR, but not v-FosFBJ or v-JunASV17, is resistant to calpains. This property raises the interesting possibility that decreased sensitivity to calpains might contribute to the tumorigenic potential of FBR-MSV by allowing greater accumulation of the protein that it encodes in infected cells. It has also been demonstrated that resistance to cleavage by calpains does not result from mutations that have accumulated in the Fos moiety of the viral protein but rather from the addition of atypical peptide motifs at its both ends. This observation raises the interesting possibility that homologous regions in viral and cellular Fos either display slightly different conformations or are differentially accessible to interacting proteins.

Avian Sarcoma Viruses↗

PubMed1997

Proteolysis by calpains: a possible contribution to degradation of p53.

p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

Animals↗

PubMed1996

PEST motifs are not required for rapid calpain-mediated proteolysis of c-fos protein.

Cytoplasmic degradation of c-fos protein is extremely rapid. Under certain conditions, it is a multi-step process initiated by calcium-dependent and ATP-independent proteases called calpains. PEST motifs are peptide regions rich in proline, glutamic acid/aspartic acid and serine/threonine residues, commonly assumed to constitute built-in signals for rapid recognition by intracellular proteases and particularly by calpains. Using a cell-free degradation assay and site-directed mutagenesis, we report here that the three PEST motifs of c-fos are not required for rapid cleavage by calpains. Testing the susceptibility of PEST motif-bearing and non-bearing transcription factors including GATA1, GATA3, Myo D, c-erbA, Tal-1 and Sry, demonstrates that PEST sequences are neither necessary nor sufficient for specifying degradation of other proteins by calpains. This conclusion is strengthened by the observation that certain proteins, reportedly known to be cleavable by calpains, are devoid of PEST motifs.

Base Sequence↗

PubMed1995

c-fos mRNA instability determinants present within both the coding and the 3' non coding region link the degradation of this mRNA to its translation.

The instability of oncogenic mRNA such as c-fos mRNA is controlled in cis by sequences present in both the coding and the 3' untranslated regions (3'UTR). The latter contains AU-rich elements (ARE) which, depending on the cellular context, mediate either their rapid degradation or inhibit their translation. These observations, along with the known increase of the life spans of many unstable mRNA promoted by inhibitors of protein synthesis, raise the possibility that both processes are linked. To investigate further the putative involvement of translation in both coding region and ARE-mediated rapid decay of c-fos mRNA, we designed an expression vector based on the use of the ferritin mRNA iron regulatory element (IRE). The latter structure links translation to intracellular iron concentration when inserted at the proper location within the 5'UTR. Rapid degradation of a beta-globin/c-fos 3'UTR construct was prevented by Desferrioxamine, an iron chelator, and facilitated by ferric ammonium citrate or hemin, while stability of other mRNAs not containing the IRE or the ARE were unchanged. The same conclusion was reached when the stability of a c-fos mRNA devoid of ARE was assessed in function of iron availability.

Adenine↗

PubMed1995

Ubiquitinylation is not an absolute requirement for degradation of c-Jun protein by the 26 S proteasome.

Degradation of rapidly turned over cellular proteins is commonly thought to be energy dependent, to require tagging of protein substrates by multi-ubiquitin chains, and to involve the 26 S proteasome, which is the major neutral proteolytic activity in both the cytosol and the nucleus. The c-Jun oncoprotein is very unstable in vivo. Using cell-free degradation assays, we show that ubiquitinylation, along with other types of tagging, is not an absolute prerequisite for ATP-dependent degradation of c-Jun by the 26 S proteasome. This indicates that a protein may bear intrinsic structural determinants allowing its selective recognition and breakdown by the 26 S proteasome. Moreover, taken together with observations by different groups, our data point to the notion of the existence of multiple degradation pathways operating on c-Jun.

Animals↗

PubMed1994

Mouse JunD negatively regulates fibroblast growth and antagonizes transformation by ras.

As NIH 3T3 fibroblasts become quiescent, the level of c-Jun protein decreases while JunD accumulates. When resting cells are stimulated with fresh serum, nuclear-localized JunD is rapidly degraded, followed by resynthesis of both c-Jun and JunD later in G1. Overexpression of JunD results in slower growth and an increase in the percentage of cells in G0/G1 while c-Jun overexpression produces larger S/G2 and M phase populations. In addition, JunD partially suppresses transformation by an activated ras gene whereas c-Jun cooperates with ras to transform cells. These data indicate that two closely related transcription factors can function in an opposing manner.

Amino Acid Sequence↗

PubMed1994

Differential sensitivity of FOS and JUN family members to calpains.

Degradation of c-fos protein (c-FOS) in the cytoplasm is very rapid in vivo and constitutes a crucial regulation of the nuclear steady-state level through the control of the amount of full-length molecules available for nuclear transport. Using cytoplasmic extracts from various origins, we report herein that c-FOS degradation can be initiated in a calcium-dependent manner which involves cysteine proteases called milli- and micro-calpain. Interestingly, FOS-B, a member of the fos multigene family, as well as all members of the jun family (JUN-B, c-JUN and JUN-D) are also sensitive to calpains albeit to different extents. FRA-2, which is a c-FOS-related protein, is resistant to micro- but not to milli-calpain whereas FRA-1, another member of the fos family, is resistant to both proteases. Given the fact that a work by others (Hiraï et al., 1991b) suggests that calpains can be involved in c-FOS and c-JUN degradation in vivo, our observations raises the possibility of a novel contribution to the regulation of AP-1 transcription complex activity through a differential control of the steady-state level of some of its components that involves calpains.

Animals↗

PubMed1992

The efficiency of cell targeting by recombinant retroviruses depends on the nature of the receptor and the composition of the artificial cell-virus linker.

Using streptavidin-bound antibodies specific for both viral and cell membrane epitopes, we have reported previously that human cells may be infected by murine ecotropic retroviruses through an interaction with major histocompatibility complex class I and class II antigens, and thus have demonstrated that cell targeting by recombinant retroviruses is feasible. We report here that (i) growth factor or hormone receptors, such as those for epidermal growth factor (EGF) and insulin, can also mediate infection of human cells; (ii) a biotinylated cytokine or hormone can substitute for the anti-cell antibody in bispecific antibody complexes, thus extending the versatility of the method; (iii) although yields are low in our assay, infection efficiency clearly appears to depend upon the biochemical composition of molecular bridges because bi-functional antibody complexes are more efficient than cytokine-antibody complexes in the case of the EGF receptor. Finally, our study indicates that different cell membrane molecules are not equally efficient in allowing infection of human cells because targeting of the transferrin, high density lipoprotein and galactose receptors, as well as that of various membrane glycoconjugates, by murine ecotropic retroviruses did not lead to the establishment of a proviral state.

DNA, Recombinant↗

PubMed1992

Cell targeting by murine recombinant retroviruses.

Cell targeting by murine retroviruses carrying recombinant genes would have numerous applications such as immortalization of under-represented cell types from complex cellular mixtures, increasing therapeutical yields in the case of gene therapy and delivery of specific genes at any location and at any moment of the life-time of living animals. To this aim, we are currently developing techniques that allow binding of viral particles to specific cell membrane markers different from the natural receptors. We have shown that biochemical bi-specific molecular adaptors able to bind both viral particles and cell surface molecules may reveal appropriate for piloting viruses toward specific cell types. More recently, we have undertaken the genetic engineering of the retroviral envelope glycoprotein in order to modify its natural tropism. This approach is discussed below.

Defective Viruses↗