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M Robledo

Publications and source records attributed to M Robledo.

At least 19 recordsLinked to original sources
PubMed2006

Differential mitogenic signaling in insulin receptor-deficient fetal pancreatic beta-cells.

Insulin receptor (IR) may play an essential role in the development of beta-cell mass in the mouse pancreas. To further define the function of this signaling system in beta-cell development, we generated IR-deficient beta-cell lines. Fetal pancreata were dissected from mice harboring a floxed allele of the insulin receptor (IRLoxP) and used to isolate islets. These islets were infected with a retrovirus to express simian virus 40 large T antigen, a strategy for establishing beta-cell lines (beta-IRLoxP). Subsequently, these cells were infected with adenovirus encoding cre recombinase to delete insulin receptor (beta-IR(-/-)). beta-Cells expressed insulin and Pdx-1 mRNA in response to glucose. In beta-IRLoxP beta-cells, p44/p42 MAPK and phosphatidylinositol 3 kinase pathways, mammalian target of rapamycin (mTOR), and p70S(6)K phosphorylation and beta-cell proliferation were stimulated in response to insulin. Wortmannin or PD98059 had no effect on insulin-mediated mTOR/p70S(6)K signaling and the corresponding mitogenic response. However, the presence of both inhibitors totally impaired these signaling pathways and mitogenesis in response to insulin. Rapamycin completely blocked insulin-activated mTOR/p70S(6)K signaling and mitogenesis. Interestingly, in beta-IR(-/-) beta-cells, glucose failed to stimulate phosphatidylinositol 3 kinase activity but induced p44/p42 MAPKs and mTOR/p70S(6)K phosphorylation and beta-cell mitogenesis. PD98059, but not wortmannin, inhibited glucose-induced mTOR/p70S(6)K signaling and mitogenesis in those cells. Finally, rapamycin blocked glucose-mediated mitogenesis of beta-IR(-/-) cells. In conclusion, independently of glucose, insulin can mediate mitogenesis in fetal pancreatic beta-cell lines. However, in the absence of the insulin receptor, glucose induces beta-cell mitogenesis.

Animals↗
PubMed2005

Germline homozygous mutations at codon 804 in the RET protooncogene in medullary thyroid carcinoma/multiple endocrine neoplasia type 2A patients.

The effect of mutations at codon 804 in the RET protooncogene is disputed. Some studies have suggested that the V804L mutation causes the low penetrance multiple endocrine neoplasia type 2 syndrome, with late onset and relatively indolent course, whereas others have reported that V804L and V804M have an aggressive potential. In this paper, we report three apparently unrelated medullary thyroid carcinoma cases homozygous for these mutations. To clarify the phenotypic heterogeneity associated with these mutations, we compare the clinical data and age of diagnosis among these three homozygous patients, six other heterozygous cases from the same populations, and other homozygous and heterozygous subjects reported previously. The data are consistent with a model in which codon 804 mutations have low penetrance, the developing of medullary thyroid carcinoma being associated with a second germline or somatic mutation. The activity and (in the case of somatic mutations) timing of these other genetic alterations in the RET gene may explain the wide clinical variability associated with germline mutations at codon 804.

Adult↗
PubMed2005

Epigenetic analysis of HIC1, CASP8, FLIP, TSP1, DCR1, DCR2, DR4, DR5, KvDMR1, H19 and preferential 11p15.5 maternal-allele loss in von Hippel-Lindau and sporadic phaeochromocytomas.

Phaeochromocytoma is a neural-crest-derived tumour that may be a feature of several familial cancer syndromes including von Hippel-Lindau (VHL) disease, multiple endocrine neoplasia type 2 (MEN 2), neurofibromatosis type 1 (NF1) and germline succinate dehydrogenase subunit (SDHB and SDHD) mutations. However the somatic genetic and epigenetic events that occur in phaeochromocytoma tumourigenesis are not well defined. Epigenetic events including de novo promoter methylation of tumour-suppressor genes are frequent in many human neoplasms. As neuroblastoma and phaeochromocytoma are both neural-crest-derived tumours, we postulated that some epigenetic events might be implicated in both tumour types and wished to establish how somatic epigenetic alterations compared in VHL-associated and sporadic phaeochromocytomas. We identified frequent aberrant methylation of HIC1 (82%) and CASP8 (31%) in phaeochromocytoma, but both genes were significantly more methylated in VHL phaeochromocytomas than in sporadic cases. Of four tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors analysed, DR4 was most commonly methylated (41%; compared with DcR2 (26%), DcR1 (23%) and DR5 (10%)). Gene methylation patterns in phaeochromocytoma and neuroblastoma did not differ significantly suggesting overlapping mechanisms of tumourigenesis. We also investigated the role of 11p15.5-imprinted genes in phaeochromocytoma. We found that in 10 sporadic and VHL phaeochromocytomas with 11p15.5 allele loss, the patterns of methylation of 11p15.5-differentially methylated regions were consistent with maternal, rather than, paternal chromosome loss in all cases (P<0.001). This suggests that 11p15.5-imprinted genes may be implicated in the pathogenesis of both familial (germline VHL and SDHD mutations) and sporadic phaeochromocytomas.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Complex cytogenetic abnormalities including telomeric associations and MEN1 mutation in a pediatric ependymoma.

Ependymomas are neuroectodermal tumors of the brain and spinal cord. Some recurrent cytogenetic aberrations have been reported in these tumors, including alterations involving chromosomes 22, 6, and 11. However, consistent molecular alterations have not been identified in ependymal tumors. We studied a recurrent ependymoma in a 3-year-old patient by standard cytogenetic and molecular analysis of TP53 and MEN1 genes. In the present case, we found many of the cytogenetic features previously described as being recurrent in ependymomas, including unstable telomeric alterations. Furthermore, we detected a novel acquired heterozygous mutation in the MEN1 gene. The chromosomal instability produced by the telomeric alterations and the mutation in the MEN1 gene could be important events in the tumorigenesis of ependymomas.

Base Sequence↗
PubMed2001

Retinoic acid receptor alpha1 variants, RARalpha1DeltaB and RARalpha1DeltaBC, define a new class of nuclear receptor isoforms.

Retinoic acid (RA) binds and activates retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers, which regulate the transcription of genes that have retinoic acid response elements (RARE). The RAR isotypes (alpha, beta and gamma) are comprised of six regions designated A-F. Two isoforms of RARalpha, 1 and 2, have been identified in humans, which have different A regions generated by differential promoter usage and alternative splicing. We have isolated two new splice variants of RARalpha1 from human B lymphocytes. In one of these variants, exon 2 is juxtaposed to exon 5, resulting in an altered reading frame and a stop codon. This variant, designated RARalpha1DeltaB, does not code for a functional receptor. In the second variant, exon 2 is juxtaposed to exon 6, maintaining the reading frame. This isoform, designated RARalpha1DeltaBC, retains most of the functional domains of RARalpha1, but omits the transactivation domain AF-1 and the DNA-binding domain. Consequently, it does not bind nor transactivate RARE on its own. Nevertheless, RARalpha1DeltaBC interacts with RXRalpha and, as an RXRalpha/RARalpha1DeltaBC heterodimer, transactivates the DR5 RARE upon all-trans-RA binding. The use of RAR- and RXR-specific ligands shows that, whereas transactivation of the DR5 RARE through the RXRalpha/RARalpha1 heterodimer is mediated only by RAR ligands, transactivation through the RXRalpha/RARalpha1DeltaBC heterodimer is mediated by RAR and RXR ligands. Whilst RARalpha1 has a broad tissue distribution, RARalpha1DeltaBC has a more heterogeneous distribution, but with significant expression in myeloid cells. RARalpha1DeltaBC is an infrequent example of a functional nuclear receptor which deletes the DNA-binding domain.

Alternative Splicing↗
PubMed2001

Multiplex-polymerase chain reaction assay for the detection of prognostically significant translocations in acute lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: The presence of specific chromosomal translocations in acute lymphoblastic leukemias (ALL) plays an important role in determining the prognosis of the patients. Our aim is to develop a highly sensitive and specific method to screen simultaneously for the four most frequent translocations in ALL: t(9;22), t(1;19), t(4;11), t(12;21). DESIGN AND METHODS: Our approach uses a multiplex-polymerase chain reaction (PCR) method, which involves two rounds of PCR using fluorescence-labeled nested primers. The chimeric transcripts resulting from these translocations can be identified by agarose gel electrophoresis or by fluorescence analysis. To validate this method we carried out the analysis in 42 pediatric ALL samples previously studied by cytogenetic and fluorescent in situ hybridization (FISH) techniques. RESULTS: In all samples with a known translocation detected by cytogenetic or FISH techniques, the same translocation was identified by the multiplex-PCR assay. Moreover, with this method we detected rearrangements in five patients in clinical remission and in two patients at diagnosis for whom karyotypes were normal and rearrangements had not been detected. The application of this multiplex-PCR assay was also useful in cases without cytogenetic results. INTERPRETATION AND CONCLUSIONS: These results show that the multiplex-PCR method allows reliable, sensitive and rapid detection of the prognostically significant translocations in ALL. We believe that this assay combined with cytogenetic analysis should be the strategy of choice for the initial diagnostic phase of acute lymphoblastic leukemia, and that it could be used not only at diagnosis but also to follow-up these alterations in remission samples without previous controls.

Child↗
PubMed2000

Identification by comparative genomic hybridization of genetic changes involved in tumoral progression of a T-cell non-Hodgkin lymphoma.

Comparative genomic hybridization (CGH) was used to detect chromosomal imbalances in tumor DNA from two relapsed samples obtained in stages II and IV of a T-cell non-Hodgkin lymphoma in order to identify genetic mechanisms involved in tumor progression of this neoplasm. With conventional cytogenetic techniques (CCT), a complex hyperdiploid karyotype was obtained in stage IV. Using CGH analysis, a normal profile was observed in stage II, whereas gains of 6p11.2, 7q11.2, 7q21-->q32, 7q34, 10p13, Xp11.4, and loss of 4q33-->qter chromosomal regions were detected in stage IV.

Humans↗
PubMed2000

Hypermethylation of P16ink4a and P15ink4b genes as a marker of disease in the follow-up of non-Hodgkin's lymphomas.

The hypermethylation of p16ink4a and p15ink4b genes have been described as an inactivating mechanism alternative to deletions and mutations that accounts for a relatively high proportion of cancers, including non-Hodgkin's lymphomas (NHLs). To investigate whether detection of abnormal methylation could have clinical applications in the management and follow-up of lymphomas, we have analysed the behaviour and evolution of p16ink4a and p15ink4b methylation in 13 NHL cases undergoing chemotherapy. All cases were also analysed for the presence of monoclonal rearrangements of immunoglobulin or T-cell receptor genes. Six patients showed methylation in at least one of these genes at diagnosis, whereas in two other cases methylation appeared during the treatment. The other five cases were always unmethylated. Methylation was detected when any histological or molecular evidence of disease was present, suggesting a good correlation between methylation and disease. In some cases, we were able to detect methylation in patients at complete remission and without evidence of monoclonal cell population, indicating a high sensitivity of the PCR to detect methylation. These results suggest that p16ink4a and p15ink4b methylation could be good markers of disease and could be helpful in identifying lymphoma patients at risk of relapse.

Adult↗
PubMed2000

Molecular analysis of the BRCA1 and BRCA2 genes in 32 breast and/or ovarian cancer Spanish families.

It is estimated that about 5-10% of breast cancer cases may be due to inherited predisposition. Until now, two main susceptibility genes have been identified: BRCA1 and BRCA2. The first linkage and mutational studies suggested that mutations in these two genes would account for the majority of high-risk breast cancer families, but recent studies show how the proportion of families due to BRCA1 or BRCA2 mutations strongly depends on the population and the types of family analyzed. It is now clear that, in the context of families with a modest cancer profile, which are the most commonly found in the clinical practice, the percentage of mutations found is much lower than that suggested by the first studies. In the present study, we analyze a group of 32 Spanish families, which contained at least three cases of female breast cancer (at least one of them diagnosed before the age of 50 years), for the presence of mutations in the BRCA genes. The total proportion of mutations was low (25%), although the percentage of mutations in the BRCA1 and BRCA2 genes was higher, considering the breast and ovarian cancer families and the male breast cancer families respectively. Our results are in agreement with the idea that a great proportion of moderate-risk cancer families could be due to low penetrance susceptibility genes distinct from BRCA1 or BRCA2.

Adult↗
PubMed1999

Multiple hereditary infundibulocystic basal cell carcinomas: a genodermatosis different from nevoid basal cell carcinoma syndrome.

BACKGROUND: Infundibulocystic basal cell carcinoma is a recently described distinctive clinicopathologic variant of basal cell carcinoma. Histopathologic differential diagnosis among infundibulocystic basal cell carcinoma, trichoepithelioma, and basaloid follicular hamartoma has generated controversy in the literature. OBSERVATIONS: Members of 2 families with multiple infundibulocystic basal cell carcinomas are described. Each patient showed multiple papular lesions, mostly located on the face. No patient showed palmar pits or jaw cysts. Forty-two cutaneous lesions from 5 patients were studied histopathologically. Thirty-nine lesions were infundibulocystic basal cell carcinomas. This clinicopathologic variant of basal cell carcinoma consists of a relatively well-circumscribed basaloid neoplasm composed of buds and cords of neoplastic cells arranged in anastomosing fashion and with scant stroma. Some of the neoplastic cords contain tiny infundibular cysts filled by cornified cells with abundant melanin. Linkage analysis in family 2 was performed using polymorphic markers (D9S196, D9S280, D9S287, and D9S180), and the affected members shared the same haplotype. Loss of heterozygosity analysis was performed in 2 affected members of this family from whom tumoral DNA was available, and although these individuals were constitutively heterozygous for D9S196, they did not show loss of heterozygosity for this marker in their neoplasms. CONCLUSIONS: Multiple hereditary infundibulocystic basal cell carcinomas represent a distinctive genodermatosis different from multiple hereditary trichoepitheliomas and nevoid basal cell carcinoma syndrome. We propose clinical and histopathologic criteria to distinguish infundibulocystic basal cell carcinoma from trichoepithelioma, basaloid follicular hamartoma, and folliculocentric basaloid proliferation.

Adult↗
PubMed1999

Prevalence of BRCA1 and BRCA2 Jewish mutations in Spanish breast cancer patients.

We screened the 185delAG and 5382insC (BRCA1) and the 6174delT (BRCA2) mutation in 298 Spanish women with breast cancer. Two women (one with Sephardic ancestors) presented the 185delAG mutation and the same haplotype reported in Ashkenazim with this mutation. This suggests a common origin of the 185delAG in both Sephardic and Ashkenazi populations.

Adult↗
PubMed1999

Genetic and clinical analysis in 10 Spanish patients with multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN 1) is characterised by the combination of tumours of the parathyroid, endocrine pancreas and anterior pituitary glands. In 1988 the MEN 1 gene was mapped to chromosome 11q13 and it was cloned in 1997. This gene contains 10 exons and extends across 9 Kb of genomic DNA; it encodes for a product of 610 amino acid named menin whose function is unknown. We have studied 10 unrelated MEN 1 kindreds by a complete sequencing analysis of the entire gene; mutations were identified in nine of them: five deletions, one insertion, two nonsense mutation and a complex alteration consisting of a deletion and an insertion that can be explained by a hairpin loop model. Two of the mutations have been previously described; the other seven were novel, and they were scattered throughout the coding sequence of the gene. As in previous series, no correlation was found between phenotype and genotype.

Base Sequence↗
PubMed1999

[Prevalence of Factor V Leiden and the G20210A mutation of the prothrombin gene in a random group of patients with thrombotic episodes].

UNLABELLED: Factor V Leiden and G20210A mutation of the prothrombin gene have been described as risk factors in thrombophilic pathologies. Our objective has been to know the prevalence of these two mutations in a group of patients with thrombophilic pathology and to compare it with its prevalence in a control group of Spanish population. PATIENTS AND METHODS: 64 patients were divided in two groups. First, 39 patients with deep venous thrombosis (DVT): 24 with an unique episode of DVT; 11 with more than one episode; 3 with DVT and pulmonary thromboembolism, and one with DVT and more than one episode of cerebral thrombosis. Second, 25 patients with other pathologies, such as pulmonary thromboembolism (9 patients), acute cerebrovascular accident (10 patients) and 6 who came to our Department because there were some carrier in their families. The 20210A allele was analyzed in 37 of the 64 patients. Some of the 64 patients had haematological determinations of the activated protein C resistance (APC resistance). As well, 103 unrelated subjects with unknown thrombotic pathologies were analyzed. RESULTS: We have found a prevalence of factor V Leiden in the group of patients of 14.1% (9 carriers in 64 patients, all of them in the first group of 39 patients with DVT) versus 1% in the control group (1 carrier in 103 controls). On the other hand, the difference between the prevalence of the 20210A allele was not statistically significant between the group of patients and the control group (2.7% vs 2.9%). In 75% of the patients no haematological results of APC resistance were obtained, generally because they were with anticoagulant treatment, and in 11.1% of the carriers the result of the determination was considered as ambiguous or false negative. CONCLUSION: Factor V Leiden is well established as risk factor in the thrombophilic pathology, but more studies are needed to know the meaning of the G20210A mutation of the prothrombin gene.

Alleles↗