Biomedical subjects

Xavier Dolcet

Publications and source records attributed to Xavier Dolcet.

13 recordsLinked to original sources

PubMed2006

PIK3CA gene mutations in endometrial carcinoma: correlation with PTEN and K-RAS alterations.

Alterations in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway are common in endometrial carcinoma. Inactivation of the tumor suppressor gene PTEN leads to a constitutively active PI3K pathway, which plays a role in the early steps of endometrial tumorigenesis. Other alterations in the PI3K/AKT pathway are mutations in the PIK3CA gene, which encode the p110alpha catalytic subunit of PI3K. PIK3CA mutations cluster to the helical (exon 9) and the kinase (exon 20) domains of the gene. In endometrial carcinomas, PIK3CA mutations have been found to coexist frequently with PTEN mutations, but it is not clear whether they occur in cells with monoallelic or biallelic inactivation of PTEN. In the present study we have evaluated PIK3CA mutational status in a series of 33 endometrial carcinomas, previously screened for microsatellite instability and mutations in PTEN, K-RAS, and CTNNB-1. The tumors were also evaluated for loss of heterozygosity on 10q23 and hypermethylation of the promoter region of PTEN/psiPTEN to assess the monoallelic or biallelic inactivation status of PTEN. PIK3CA mutations were detected in 8 (24%) of the 33 cases. Seven mutations were located in exon 20 and 1 in exon 9. PTEN alterations were found in 19 cases (57%). Biallelic inactivation of PTEN was demonstrated in 11 tumors, whereas 8 tumors exhibited alteration in only 1 of the 2 alleles. PIK3CA mutations coexisted with monoallelic alterations of PTEN in 4 cases (2 mutations and 2 allelic imbalances), with biallelic PTEN inactivation in 1 case (mutation and promoter methylation), and 3 tumors showed PIK3CA mutations in association with wild-type PTEN. PIK3CA mutations did not correlate with microsatellite instability or mutations in CTNNB-1. However, PIK3CA and K-RAS mutations (8 cases) were mutually exclusive alterations. In summary, the results confirm that PIK3CA mutations are frequent in endometrial carcinoma and support the hypothesis that PIK3CA mutations may have an additive effect to PTEN monoallelic inactivation in endometrial carcinoma.

Class I Phosphatidylinositol 3-Kinases↗

PubMed2006

Proteasome inhibitors induce death but activate NF-kappaB on endometrial carcinoma cell lines and primary culture explants.

Proteasome inhibitors are currently used as chemotherapeutic drugs because of their ability to block NF-kappaB, a transcription factor constitutively activated in many different types of human cancer. In the present study, we demonstrate that proteasome inhibitors induce cell death in endometrial carcinoma cell lines and primary explants but, instead of blocking NF-kappaB, they increase its transcriptional activity. Proteasome inhibitors induce phosphorylation of IKK alpha/beta, phosphorylation and degradation of IkappaB alpha, and phosphorylation of the p65 NF-kappaB subunit on serine 536. Proteasome inhibitor-induced NF-kappaB activity can be blocked by a non-degradable form of IkappaB alpha or dominant negative forms of either IKK alpha or IKK beta. Lentiviral delivery of shRNAs to either IKK alpha or IKK beta cause blockade of NF-kappaB transcriptional activity and inhibit phosphorylation of p65 on serine 536, but has no effect on IkappaB alpha degradation. These results suggest a role for p65 phosphorylation in proteasome inhibitor-induced NF-kappaB activation. Accordingly, siRNA knockdown of p65 inhibits proteasome inhibitor-induced NF-kappaB transcriptional activity. Our results demonstrate that proteasome inhibitors, including bortezomib, induce cell death on endometrial carcinoma cells and primary explants. However, they activate NF-kappaB instead of blocking its transcriptional potential. Therefore, the concept that proteasome inhibitors are blockers of NF-kappaB activation should be carefully examined in particular cell types.

Antineoplastic Agents↗

PubMed2006

Antiproliferative effect of STI571 on cultured human cutaneous melanoma-derived cell lines.

Standard antineoplastic treatment for metastatic melanoma is ineffective in the large majority of patients. Therefore, alternative approaches need to be investigated. STI571 is a new antineoplastic compound, which selectively inhibits the tyrosine kinase activity of ABL, c-Kit and platelet-derived growth factor receptor (PDGFR). Melanoma may express all of these proteins. The aim of this study was to investigate whether STI571 inhibits the in-vitro growth of melanoma cells. Nineteen cell lines were obtained from four primary and 15 metastatic melanomas of cutaneous origin. The percentages of positive cells for the putative targets of STI571 were as follows: ABL, 41-100%; c-Kit, 8-97%; PDGFR-alpha, 41-98%; PDGFR-beta, 51-99%. 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium (MTT) and viability assays showed that STI571 clearly inhibits the proliferation of eight of the 19 (42.1%) cell lines. No relationship could be established between the expression of c-Kit, ABL, PDGFR-alpha or PDGFR-beta and the response of cell lines to STI571. Our study shows, for the first time, an antiproliferative effect of STI571 on human melanoma cell lines of cutaneous origin, raising the possibility of the future clinical use of STI571. The identification of the target of STI571 in human cutaneous melanoma cells would allow the selection of patients who could benefit from this treatment.

Antineoplastic Agents↗

PubMed2005

The contribution of apoptosis-inducing factor, caspase-activated DNase, and inhibitor of caspase-activated DNase to the nuclear phenotype and DNA degradation during apoptosis.

We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIF-independent process.

Apoptosis↗

PubMed2005

NF-kB in development and progression of human cancer.

The nuclear factor kB (NF-kB) comprises a family of transcription factors involved in the regulation of a wide variety of biological responses. NF-kB plays a well-known function in the regulation of immune responses and inflammation, but growing evidences support a major role in oncogenesis. NF-kB regulates the expression of genes involved in many processes that play a key role in the development and progression of cancer such as proliferation, migration and apoptosis. Aberrant or constitutive NF-kB activation has been detected in many human malignancies. In recent years, numerous studies have focused on elucidating the functional consequences of NF-kB activation as well as its signaling mechanisms. NF-kB has turned out to be an interesting therapeutic target for treatment of cancer.

Apoptosis↗

PubMed2005

NF-kappaB signalling regulates the growth of neural processes in the developing PNS and CNS.

The proper growth and elaboration of neural processes is essential for the establishment of a functional nervous system during development and is an integral feature of neural plasticity throughout life. Nuclear factor-kappa B (NF-kappaB) is classically known for its ubiquitous roles in inflammation, immune and stress-related responses and regulation of cell survival in all tissues, including the nervous system. NF-kappaB participation in other cellular processes remains poorly understood. Here we report a mechanism for controlling the growth of neural processes in developing peripheral and central neurons involving the transcription factor NF-kappaB. Inhibiting NF-kappaB activation with super-repressor IkappaB-alpha, BAY 11 7082 (IkappaB-alpha phosphorylation inhibitor) or N-acetyl-Leu-Leu-norleucinal (proteosomal degradation inhibitor), or inhibiting NF-kappaB transcriptional activity with kappaB decoy DNA substantially reduced the size and complexity of the neurite arbors of sensory neurons cultured with brain-derived neurotrophic factor while having no effect on their survival. NF-kappaB exerted this effect during a restricted period of development following the phase of naturally occurring neuronal death when the processes and connections of the remaining neurons are extensively modified and refined. Inhibiting NF-kappaB activation or NF-kappaB transcriptional activity in layer 2 pyramidal neurons in postnatal somatosensory cortical slices reduced dendritic arbor size and complexity. This function of NF-kappaB has important implications for neural development and may provide an explanation for reported involvement of NF-kappaB in learning and memory.

Animals↗

PubMed2005

FLIP is frequently expressed in endometrial carcinoma and has a role in resistance to TRAIL-induced apoptosis.

The FLICE-inhibitory protein (FLIP) plays a key role in the regulation of apoptosis triggered by death ligands. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in some types of tumor but not in others. To assess the possible role of FLIP in apoptosis resistance in endometrial carcinoma, we performed an immunohistochemical study on a tissue microarray composed of 95 endometrial carcinomas. We found positive signals in 43% of the cases, as well as a significant difference in FLIP expression between stage I and II tumors. Moreover, we observed that endometrial carcinoma cell lines Ishikawa and KLE did not undergo apoptosis after TRAIL treatment. Cotreatment of these cells with the inhibitor of transcription actinomycin D resulted in a dramatic decrease in cell viability and induced activation of caspase-8. These events coincided with downregulation of FLIP mRNA and protein. Inhibitors of caspase-8 or overexpression of FLIP completely blocked apoptosis induced by actinomycin D plus TRAIL cotreatment. More importantly, downregulation of endogenous FLIP expression by specific siRNAs sensitized endometrial carcinoma cells to TRAIL-induced apoptosis in the absence of actinomycin D. Taken together, our results suggest for the first time a critical role for FLIP in the regulation apoptosis triggered by TRAIL in endometrial carcinoma cells.

Adenocarcinoma↗

PubMed2005

Immunohistochemical analysis of PTEN in endometrial carcinoma: a tissue microarray study with a comparison of four commercial antibodies in correlation with molecular abnormalities.

The tumor suppressor gene PTEN/MMAC1 is located on chromosome 10q23.3. Inactivation of PTEN, either by mutations, deletions, or promoter hypermethylation, has been identified in a wide variety of tumors. Inactivation of the two alleles of PTEN is required, because it is a tumor suppressor gene. Immunohistochemical staining may be an effective screening method to demonstrate the absence of the protein in tumors exhibiting PTEN inactivation. We studied a tissue microarray, constructed from paraffin-embedded blocks of 95 endometrial carcinomas, 38 of them previously evaluated for alterations in PTEN. We also studied cell blocks obtained from one PTEN-defective endometrial cancer cell line, after transfection with either a plasmid encoding wild-type PTEN or the empty vector. The tumor samples were tested with four different anti-PTEN commercial antibodies: a polyclonal antibody, the monoclonal antibody 28H6, the monoclonal antibody 10P03, and the monoclonal antibody 6.H2.1. Results were correlated with the presence of abnormalities in PTEN, as well as with the immunohistochemical expression of phosphorylated AKT. Antibody 28H6 produced a predominant nuclear staining, while the other three antibodies produced a predominant cytoplasmic staining. There was no significant correlation between the results obtained with the four antibodies. The monoclonal antibody 6.H2.1 was the only one that exhibited a correlation with the presence of molecular alterations in PTEN, and a statistically significant association with immunostaining for phosphorylated AKT (r=-0.249, P=0.037). The monoclonal antibody 10P03 exhibited an association with phospho-AKT that did not have statistical significance. Both 6.H2.1 and 10P03 antibodies stained PTEN-transfected cells, and were negative in the PTEN-deficient cell line blocks. The polyclonal antibody and the monoclonal antibody 28H6 produced positive staining in PTEN-deficient cell line blocks, suggesting nonspecific staining. The results indicate that monoclonal antibody 6.H2.1 may be a suitable alternative for tumors with inactivation of PTEN.

Antibodies, Monoclonal↗

PubMed2005

Survivin expression in endometrial carcinoma: a tissue microarray study with correlation with PTEN and STAT-3.

Evasion of apoptotic cell death plays a key role in cancer development. Survivin is a member of the inhibitor of apoptosis proteins, which also has a role in the control of cell division. Survivin may be overexpressed in some tumors and has been suggested to be related to PTEN, beta-catenin, p53 [all of them frequently abnormal in endometrial carcinomas (ECs)], and STAT-3. A tissue microarray was constructed from paraffin-embedded blocks of 95 ECs, previously studied for microsatellite instability and for alterations in PTEN, k-RAS, and CTNNB-1. Immunohistochemical evaluation included 1) survivin, 2) markers of cell proliferation and apoptosis (Ki67-MIB1 and M 30-neoepitope cytokeratin 18), and 3) proteins involved in cell signaling pathways (PTEN, phospho-AKT, beta-catenin, p53, and STAT-3). Survivin expression was frequent in ECs (75.95%) but did not show any statistical significant correlation with histological type and grade, stage, overall survival, or mitotic and apoptotic indexes. Survivin expression had a statistical significant correlation with decreased PTEN expression (r = -0.383, p = 0.001), increased phospho-AKT (r = 0.70, p < 0.001), and positive STAT-3 immunostaining (r = 0.6, p < 0.001). Survivin expression did not show statistical correlation with either beta-catenin or p53 alterations. The results suggest that increased survivin expression is frequent in ECs and may be dependent on STAT-3 and PI3 K/AKT activation. Because PTEN abnormalities are very frequent in ECs, the results from this study indicate that PTEN may interfere with the process of apoptosis and cell proliferation by promoting survivin expression.

Apoptosis↗

PubMed2004

The death receptor antagonist FAIM promotes neurite outgrowth by a mechanism that depends on ERK and NF-kapp B signaling.

Fas apoptosis inhibitory molecule (FAIM) is a protein identified as an antagonist of Fas-induced cell death. We show that FAIM overexpression fails to rescue neurons from trophic factor deprivation, but exerts a marked neurite growth-promoting action in different neuronal systems. Whereas FAIM overexpression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells. FAIM overexpression promoted NF-kappa B activation, and blocking this activation by using a super-repressor I kappa B alpha or by carrying out experiments using cortical neurons from mice that lack the p65 NF-kappa B subunit prevented FAIM-induced neurite outgrowth. The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras-ERK pathway. Finally, we show that FAIM interacts with both Trk and p75 neurotrophin receptor NGF receptors in a ligand-dependent manner. These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras-ERK pathway and NF-kappa B.

Animals↗

PubMed2004

HGF regulates the development of cortical pyramidal dendrites.

Although hepatocyte growth factor (HGF) and its receptor tyrosine kinase MET are widely expressed in the developing and mature central nervous system, little is known about the role of MET signaling in the brain. We have used particle-mediated gene transfer in cortical organotypic slice cultures established from early postnatal mice to study the effects of HGF on the development of dendritic arbors of pyramidal neurons. Compared with untreated control cultures, exogenous HGF promoted a highly significant increase in dendritic growth and branching of layer 2 pyramidal neurons, whereas inactivation of endogenous HGF with function-blocking, anti-HGF antibody caused a marked reduction in size and complexity of the dendritic arbors of these neurons. Furthermore, pyramidal neurons transfected with an MET dominant-negative mutant receptor likewise had much smaller and less complex dendritic arbors than did control transfected neurons. Our results indicate that HGF plays a role in regulating dendritic morphology in the developing cerebral cortex.

Animals↗

PubMed2004

Abnormalities in the NF-kappaB family and related proteins in endometrial carcinoma.

The NF-kappaB family of transcription factors regulates a wide variety of cellular processes including cell growth, differentiation, and apoptosis. A tissue microarray was constructed from paraffin wax-embedded blocks from 95 endometrial carcinomas (EC), previously studied for microsatellite instability, as well as for alterations in PTEN, k-RAS and beta-catenin. Immunohistochemical evaluation included members of the NF-kappaB (p50, p65, p52, c-Rel, Rel-B) and the IkappaB (IkappaBalpha, IkappaBbeta, IkappaBepsilon, Bcl-3) families, as well as putative targets of NF-kappaB such as Flip, Bcl-xL, Cyclin D1, and oestrogen and progesterone receptors. Results were correlated with the clinical and pathological data. Nuclear immunostaining for members of the NF-kappaB family was frequent in EC (p50, 20%; p65, 16.5-21.9%; p52, 9.3%; c-Rel, 48.9%; Rel-B, 15.7%); and it correlated with negativity for members of the IkappaB family in some cases. There was a statistically significant association between immunoreaction for p50 and p65 (p = 0.006), suggesting activation of the so-called 'classic form' of NF-kappaB, similar to that described in breast cancer. Bcl-3 nuclear immunostaining was detected in 60.7% of cases. The vast majority of p52-positive tumours showed Bcl-3 nuclear immunoreaction (p = 0.038). Immunostaining for putative targets of NF-kappaB was as follows: Bcl-xL, 76.2% (p = 0.001); Flip 43.0%; Cyclin D1, 64.79%. p65 immunostaining correlated with increased immunoreaction for steroid hormone receptors. No correlation was found between NF-kappaB nuclear pattern and the presence of microsatellite instability, or alterations in PTEN, k-RAS, or beta-catenin. These results suggest that the NF-kappaB and IkappaB families of genes may be important in endometrial carcinogenesis, by controlling apoptosis and cell proliferation.

Adenocarcinoma↗

PubMed2004

HGF promotes survival and growth of maturing sympathetic neurons by PI-3 kinase- and MAP kinase-dependent mechanisms.

Hepatocyte growth factor (HGF) is a pleiotrophic factor whose many functions include promoting neuronal survival and growth. Hitherto, these effects have been observed in the presence of other neurotrophic factors like NGF and CNTF, and this requirement for an accessory factor has made it difficult to elucidate the signaling pathways that mediate its survival and growth-enhancing effects. Here, we show that HGF promotes the survival of mature sympathetic neurons of the superior cervical ganglion (SCG) grown at low density in defined medium lacking other neurotrophic factors. This effect was first clearly observed in cultures established from postnatal day 20 (P20) mice and became maximal by P40. HGF also enhanced the growth of neurite arbors from neurons throughout postnatal development and in the adult. HGF treatment resulted in phosphorylation of Akt and ERK1/ERK2. Preventing Akt activation with the phosphatidylinositol-3 (PI-3) kinase inhibitor LY294002 blocked the HGF survival response, and inhibition of ERK activation with the MEK inhibitors PD98059 or U0126 reduced the HGF survival response and the neurite growth-promoting effects of HGF. These results indicate that HGF promotes the survival and growth of maturing sympathetic neurons by both PI-3 kinase- and MAP kinase-dependent mechanisms.

Animals↗