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Biomedical subjects

R Maestro

Publications and source records attributed to R Maestro.

At least 19 recordsLinked to original sources
PubMed2006

Twist is substrate for caspase cleavage and proteasome-mediated degradation.

Twist is a member of the basic helix-loop-helix family of transcription factors. An aberrant Twist expression has been found in diverse types of cancer, including sarcomas, carcinomas and lymphomas, supporting a role for Twist in tumor progression. Twist is known to be essential for mesodermal development. However, since a prolonged Twist expression results in a block of muscle, cartilage and bone differentiation, Twist has to be excluded from somites during late embryogenesis for terminal differentiation to occur. This implies that Twist expression must be target of a tight control. Here we provide evidence that Twist undergoes post-transcriptional regulation. Twist is substrate for cleavage by caspases during apoptosis and its cleavage results in ubiquitin-mediated proteasome degradation. Our findings suggest that Twist post-transcriptional regulation may play an important role in tissue determination and raise the possibility that alterations in the protein turnover may account for Twist overexpression observed in tumors.

Animals↗
PubMed2005

Caspase activation and apoptosis in response to proteasome inhibitors.

Several studies have indicated that proteasome inhibitors (PIs) are promising anticancer agents. We have discovered that PIs have the unique ability to activate effector caspases through a mitochondrial Bcl-2 inhibitable but caspase-9 independent pathway. Stabilization of released Smac induced by blockade of the proteasome could explain the apoptosome-independent cell death induced by PIs. In fact, Smac/DIABLO critically supports this PIs-dependent caspase activation. By using a new assay, we confirm that at a single cell level both Smac and PIs can activate caspases in the absence of the apoptosome. Moreover, we have observed two PIs-induced kinetics of caspase activation, with caspase-9 being still required for the rapid caspase activation in response to mitochondrial depolarization, but dispensable for the slow DEVDase activation. In summary, our data indicate that PIs can activate downstream caspases at least in part through Smac/DIABLO stabilization.

Animals↗
PubMed2000

Coordinated expression and amplification of the MDM2, CDK4, and HMGI-C genes in atypical lipomatous tumours.

Atypical lipomatous tumours (ALTs) represent a distinctive subset of mesenchymal neoplasms featuring mature adipocytic differentiation. Most ALTs are characterized cytogenetically by the presence of supernumerary ring and/or long marker chromosomes derived from the chromosomal region 12q13-15. The 12q13-15 chromosome region contains several genes which may play an important role in human tumorigenesis. A series of ALTs was analysed by investigating the MDM2, CDK4, and HMGI-C genes and their proteins. The study was extended to a series of ordinary lipomas, to determine whether the immunohistochemical investigation of these gene products might play any diagnostic role. Cytogenetic analysis revealed the presence of various cytogenetic aberrations involving the 12q13-15 region in 11/18 (61%) lipomas and of ring chromosomes in all ALTs. Overexpression of mdm2 protein was observed in 6/12 (50%) atypical lipomatous tumours. All lipomas were mdm2-negative. cdk4 overexpression was present in 100% of ALTs. Weak cdk4 immunopositivity was detected in 2/18 (11%) ordinary lipomas in a minority of cells. HMGI-C immunopositivity was observed in 10/12 (83%) ALTs. Positive immunoreactivity was also observed in 8/18 (44%) lipomas. Southern blot analysis revealed amplification of the CDK4 and MDM2 genes in 3/5 ALTs analysed. HMGI-C was amplified in 3/5 cases and was deleted in one case. Mutation analysis of the CDK4 gene did not demonstrate any mutation. These data support the hypothesis that ordinary lipomas may form a molecular genetic and morphological continuum with ALT. At one end of the spectrum are lipomas characterized by 12q13-15 rearrangements and HMGI-C activation and at the other end are ALTs with ring chromosomes, 12q13-15 amplification with overrepresentation of the HMGI-C, CDK4 or MDM2 genes, and aberrant cdk4, mdm2, and HMGI-C protein expression. These findings not only provide insights into the molecular pathogenesis of lipomatous tumours, but also indicate that the immunohistochemical analysis of mdm2 and cdk4 may help to increase diagnostic accuracy.

Adult↗
PubMed1999

A proinflammatory cytokine inhibits p53 tumor suppressor activity.

p53 has a key role in the negative regulation of cell proliferation, in the maintenance of genomic stability, and in the suppression of transformation and tumorigenesis. To identify novel regulators of p53, we undertook two functional screens to isolate genes which bypassed either p53-mediated growth arrest or apoptosis. In both screens, we isolated cDNAs encoding macrophage migration inhibitory factor (MIF), a cytokine that was shown previously to exert both local and systemic proinflammatory activities. Treatment with MIF overcame p53 activity in three different biological assays, and suppressed its activity as a transcriptional activator. The observation that a proinflammatory cytokine, MIF, is capable of functionally inactivating a tumor suppressor, p53, may provide a link between inflammation and tumorigenesis.

Animals↗
PubMed1999

Twist is a potential oncogene that inhibits apoptosis.

Oncogene activation increases susceptibility to apoptosis. Thus, tumorigenesis must depend, in part, on compensating mutations that protect from programmed cell death. A functional screen for cDNAs that could counteract the proapoptotic effects of the myc oncogene identified two related bHLH family members, Twist and Dermo1. Both of these proteins inhibited oncogene- and p53-dependent cell death. Twist expression bypassed p53-induced growth arrest. These effects correlated with an ability of Twist to interfere with activation of a p53-dependent reporter and to impair induction of p53 target genes in response to DNA damage. An underlying explanation for this observation may be provided by the ability of Twist to reduce expression of the ARF tumor suppressor. Thus, Twist may affect p53 indirectly through modulation of the ARF/MDM2/p53 pathway. Consistent with a role as a potential oncoprotein, Twist expression promoted colony formation of E1A/ras-transformed mouse embryo fibroblasts (MEFs) in soft agar. Furthermore, Twist was inappropriately expressed in 50% of rhabdomyosarcomas, a tumor that arises from skeletal muscle precursors that fail to differentiate. Twist is known to block myogenic differentiation. Thus, Twist may play multiple roles in the formation of rhabdomyosarcomas, halting terminal differentiation, inhibiting apoptosis, and interfering with the p53 tumor-suppressor pathway.

Animals↗
PubMed1999

Loss of heterozygosity at 10q in tumors of the upper respiratory tract is associated with poor prognosis.

Frequent loss of a specific chromosomic region in cancers is often associated with inactivation of a tumor-suppressor gene. The long arm of chromosome 10 is deleted in several types of tumor, among them squamous-cell carcinomas of the head and neck (HNSCC). To determine the role of 10q deletions in the tumorigenesis of the upper respiratory tract, 47 HNSCCs were examined for loss of heterozygosity (LOH) at 10q: 43% of the cases analyzed showed LOH at 10q, and 2 distinct hot spots of deletion were identified, at 10q22-23 and 10q25-26. The possible involvement of pTEN/MMAC1, a tumor-suppressor gene mapped at 10q23, was also evaluated. No mutation, homozygous deletion or loss of expression of pTEN/MMAC1 was detected, indicating that inactivation of this gene plays a minor role in HNSCC development. Interestingly, the frequency of deletion at 10q was greater in invasive carcinoma than in adjacent carcinoma in situ, and a significant association between LOH and poor prognosis was observed. Taken together, our results suggest the presence in the long arm of chromosome 10 of (a) tumor-suppressor gene(s) other than pTEN/MMAC1 and presumably involved in the malignant progression of tumors of the upper respiratory tract. Int. J. Cancer (Pred. Oncol.) 84:432-436, 1999.

Blotting, Southern↗
PubMed1998

Microsatellite instability in squamous cell carcinomas of the head and neck related to field cancerization phenomena.

Patients affected by squamous cell carcinoma of the head and neck (HNSCC) show frequent occurrence of multiple cancers and widespread precancerous lesions in the mucosa of the upper respiratory tract, a phenomenon known as field cancerization. In this study, we investigated the role of genetic instability in the development of HNSCC and in particular in tumour multiplicity phenomena of the upper respiratory tract. For this purpose, we analysed microsatellite instability (MI) and loss of heterozygosity (LOH) at 20 loci mapping on five chromosomal arms in 67 HNSCC patients, 45 of whom had a single cancer and 22 had multiple primary tumours. The possible involvement of the hMLH1 gene in genetic instability and as a potential target of 3p21 deletion phenomena in head and neck cancers was also investigated. Our data indicate that mismatch repair-related genetic instability plays a minor role in the carcinogenesis of HNSCC and in tumour multiplicity of the head and neck region. Moreover, our results exclude a role for the hMLH1 gene as a determinant of MI and as a specific gene target of deletion at 3p21 in HNSCC. We conclude that presumably other genetic mechanisms, such as those hypothesized for MI-negative hereditary non-polyposis colorectal cancer patients, may play a major role in the carcinogenesis of the mucosa of the upper respiratory tract.

Alleles↗
PubMed1997

Overexpression of CDC25A and CDC25B in head and neck cancers.

The deregulation of several cell cycle-related genes participates in neoplastic transformation. Cell cycle progression is driven by cyclin-dependent kinases, which are positively regulated by association with cyclins and negatively regulated by binding to inhibitory subunits. The activity of cyclin-dependent kinases is also regulated by the phosphorylation status, which is controlled by the antagonistic action of wee1 kinase and CDC25 phosphatases. Three CDC25 genes are present in human cells: CDC25A, CDC25B, and CDC25C. These three genes function at different phases of the cell cycle. Whereas CDC25A and CDC25B are expressed throughout the cell cycle, with peak expression in G1 for CDC25A and in both G1-S-phase and G2 for CDC25B, CDC25C is predominantly expressed in G2. Several lines of evidence suggest a role for CDC25s as oncogenes. CDC25A and CDC25B cooperate with Ha-ras or loss of Rb1 in the oncogenic transformation of rodent fibroblasts. Moreover, they are transcriptional targets of c-myc, and CDC25A in particular plays an important role as a mediator of myc functions. On the basis of the evidence that CDC25 phosphatases can act as oncogenes, we analyzed the expression of CDC25A, CDC25B, and CDC25C genes in 20 squamous cell carcinomas of the head and neck by quantitative reverse transcription-PCR. Our results show that whereas CDC25C is expressed at a low level with no relevant differences between neoplastic tissue and normal mucosa, CDC25A and CDC25B are overexpressed in a large fraction of tumors.

Carcinoma, Squamous Cell↗
PubMed1997

Human non-Hodgkin's lymphomas overexpress a wild-type form of p53 which is a functional transcriptional activator of the cyclin-dependent kinase inhibitor p21.

A large fraction of non-Hodgkin's lymphomas (NHLs) accumulate a wild-type form of the p53 tumor suppressor protein at the nuclear level. In normal cells, p53 induction is associated with a temporary cell growth arrest at the G1-S boundary of the cell cycle. This activity of p53 as a G1 checkpoint molecule is strictly dependent on its ability to induce the transcription of the inhibitor of the cyclin dependent kinase, p21. To verify the functionality of the wild-type p53 protein accumulated in NHL cells, 70 cases were comparatively analyzed for p53 and p21 expression and status of the respective genes. Overexpression of the wt p53 protein was associated with the accumulation of p21, indicating that p53 is functional with respect to p21 induction in these tumors. The coaccumulation of p53 with Ki-67 antigen indicates that wt p53-positive cells and p21-positive cells, as well, are actively proliferative elements, supporting the notion that p53-induced, p21-mediated growth arrest is somehow overridden in NHL cells. No p21 mutation or particular allele variant was shown to correlate with p21 protein accumulation, thus excluding a role for p21 structural abnormalities. Taken together, our data suggest the existence in NHL of a peculiar mechanism of functional inactivation of the p53 G1 checkpoint pathway occurring downstream of the CDK inhibitor p21.

Cell Division↗
PubMed1997

p16/CDKN2 and CDK4 gene mutations in sporadic melanoma development and progression.

The p16/CDKN2(MTS1) gene encoding for the p16 inhibitor of cyclin D/CDK4 complexes is frequently mutated and deleted in a large fraction of melanoma cell lines, and p16 germline mutations have also been observed in familial melanomas. Moreover, a CDK4 gene mutation, responsible for a functional resistance of CDK4 kinase to p16 inhibitory activity, has been described to occur in some cases of familial melanoma. These data strongly support the idea that deregulation of the CDK4/cyclin D pathway, via CDKN2 or CDK4 mutations, is of biological significance in the development of melanoma. To shed light on the role of these alterations in the development and progression of sporadic melanoma, 12 primary melanomas and 9 corresponding metastases were analyzed for CDKN2 and CDK4 gene mutations. Of the 12 primary melanomas analyzed, 4 showed the presence of mutational inactivation of the p 16 protein and 2 carried silent mutations. No metastases showed the presence of CDKN2 mutations, indicating that mutations of this cyclin-dependent kinase inhibitor is not common in the progression of sporadic melanoma. On the other hand, the absence, in the metastases, of the CDKN2 mutation detected in the corresponding primary tumors suggests that 9p21 homozygous deletion may play a major role in the metastatic spreading of this type of tumor. None of the cases analyzed showed the presence of an Arg24Cys mutation, which functionally protects CDK4 from p16 inhibition. This indicates that CDK4 mutation plays a minor role in the development and progression of sporadic melanoma.

Biomarkers, Tumor↗
PubMed1997

All-trans, 13-cis and 9-cis retinoic acids induce a fully reversible growth inhibition in HNSCC cell lines: implications for in vivo retinoic acid use.

Retinoids are a group of vitamin A analogues that have shown promise as chemopreventive and therapeutic agents in many types of malignancy and have been entered in clinical trials with some successful results. To better understand the mechanism that mediates retinoid action and the anti-proliferative effects, we treated 7 human oral squamous-cell carcinoma (SCC) cell lines (FADU, HEp-2, CCL-17, SCC-9, SCC-15, SCC-25 and HN-212) with 10(-6) M of all-trans retinoic acid (ATRA), 9-cis and 13-cis retinoic acid (RA) in continuous for different periods of time. We assessed the extent of growth inhibition, the stability of the anti-proliferative effect and the mRNA expression levels (by RT-PCR) of RA receptors (RARs), retinoid X receptors alpha (RXR alpha) and cytosolic RA-binding proteins (CRBP I and CRABP II) in treated cells compared with controls. The data obtained showed that all 3 RAs were able to inhibit the cellular growth of the tested cell lines, although to a different extent. The cis compounds were able to inhibit the proliferation of all cell lines, whereas ATRA was ineffective in inhibiting the proliferation of the CCL-17 cell line, which was naturally resistant to ATRA concentrations in the range between 10(-5) and 10(-6) M. All inhibitory effects were completely reversible since all cell lines restored their normal growth proliferation within few days after drug removal. RT-PCR analysis of the receptor and cell binding protein status of control and treated cells showed a good correlation between growth inhibition and induction of, or increase in, the expression levels of RAR beta in RA-treated cells. No differences were observed in RAR alpha and RXR alpha mRNA expression levels between control and treated cells. CRBP I, CRABP II and RAR gamma mRNA levels increased in some treated cell lines but not in all.

Alitretinoin↗
PubMed1997

Molecular abnormalities of the p53 pathway in dedifferentiated liposarcoma.

Dedifferentiated liposarcoma represents a distinct subtype of liposarcoma and is characterized by the presence of abrupt transition from well-differentiated liposarcoma to high-grade pleomorphic sarcoma (mostly MFH-like). A key role for p53 in tumour progression of this subset of liposarcomas has been suggested on the basis of p53 immunopositivity. A series of 14 dedifferentiated liposarcomas has been investigated by analysing the p53 gene and protein together with the p53-related molecules p21Waf1 and mdm2, to verify whether the p53 pathway is involved in the development and progression of this tumour type. The results indicate that the p53 gene is rarely involved in dedifferentiated liposarcoma (7 per cent of cases analysed) and that low percentages of p53 immunopositivity are still compatible with integrity of the p53 gene. This concept is also supported by the observed preservation of p21Waf1 immunoreactivity in all but the p53-mutated cases. By contrast, mdm2 overexpression emerges as the most frequent abnormality in dedifferentiated liposarcoma (57 and 78 per cent of cases in well-differentiated and high-grade areas, respectively).

Cell Differentiation↗
PubMed1996

Chromosome 13q deletion mapping in head and neck squamous cell carcinomas: identification of two distinct regions of preferential loss.

Heal and neck squamous cell carcinomas show frequent cytogenetic alterations involving the long arm of chromosome 13. To define the extent of 13q deletions and to identify the minimal areas of chromosome loss, 48 primary squamous cell carcinomas of the head and neck were analyzed for loss of heterozygosity using 11 different polymorphic loci. About 67% of the tumors displayed loss of genetic material at 13q. Most of the cases showed loss of the entire long arm of the chromosome. However, the presence of partial deletions in 10 cases provided evidence of the existence of two preferential sites of chromosome loss at 13q32-ter and 13q14.2-q14.3. The colocalization of the 13q14 minimal region of deletion with the retinoblastoma (RB) gene, which has been proposed as an oncosuppressor in diverse tumor types, prompted us to verify the involvement of this gene in the development of head and neck cancer. No significant variation in RB protein or RB mRNA expression was detected, thus excluding a role for such a gene in the genesis of this type of tumor. Taken together, our data suggest the existence of two new tumor suppressor genes (one close to and one distal to RB), which play a role in the development and/or progression of head and neck squamous cell carcinomas.

Base Sequence↗
PubMed1996

Immunophenotypic and molecular analyses of acquired immune deficiency syndrome-related and Epstein-Barr virus-associated lymphomas: a comparative study.

Limited information is current available on the molecular and immunophenogenotypic characteristics of CD30-positive anaplastic large cell (ALC) lymphomas occurring in human immunodeficiency virus (HIV)-infected individuals. To address this issue, the authors have undertaken a combined analysis of these lymphomas in a comparison with other Epstein-Barr virus (EBV)-associated tumors in the setting of HIV infection. Twenty-one AIDS-related lymphomas, including five CD30-positive ALC and 11 small noncleaved cell (SNCC) lymphomas, and five Hodgkin's disease (HD) specimens were characterized regarding the immunophenogenotypic features, the frequency and subtype distribution of EBV (as defined by in situ hybridization [ISH], Southern blot, and a polymerase chain reaction [PCR] amplification of the EBV nuclear antigen-2 [EBNA-2] region) antigen expression (latent membrane protein-1 [LMP-1], EBNA-2, and for alterations of the tumor suppressor gene p53. Combined immunophenotypic and immunogenotypic analyses showed a derivation from anomalously matured B cells in four of five CD30-positive ALC lymphomas, whereas SNCC showed features of mature B cells; no evidence of immunoglobulin or TCR gene rearrangement could be obtained in HD cases. Combined ISH and Southern blot analyses revealed that EBV was more strictly associated with HD (five of five) and CD30-positive ALC lymphomas (four of five) than with SNCC lymphomas (four of 11). EBV-positive samples from CD30-positive ALC lymphomas carried type 1 EBV (two of two specimens tested), whereas both EBV subtypes were observed in SNCC lymphomas and HD samples. All three forms of viral latent gene expression were found in the EBV positive CD30-positive ALC lymphomas. SNCC specimens did not express LMP-1 or EBNA-2, whereas HD specimens expressed LMP-1 (four of five tested) but no EBNA-2. Immunostaining for ZEBRA was consistently negative. HHV-6 DNA sequences were detected by PCR in one SNCC of the 19 specimens analyzed. Three out of five CD30-positive ALC lymphoma specimens and six of 10 SNCC showed nuclear staining for p53. No mutation was detected in any of the three CD30-positive Alc lymphoma analyzed, whereas an aberrant SSCP pattern was found in all the four SNCC samples tested. At variance with SNCC lymphomas, AIDS-related B-cell CD30- positive ALC lymphomas are strictly associated with EBV infection and may also express the broad lymphoblastoid cell line-like (LMP-1-positive, EBNA-2-positive) pattern, and lack p53 genetic lesions. Unlike EBV, HHV-6 probably does not represent a relevant factor involved in the pathogenesis of CD30-positive ALC and other HIV related lymphomas.

Adult↗
PubMed1996

Tumor suppressor genes and related molecules in leiomyosarcoma.

Soft tissue sarcomas represent a heterogeneous group of mesenchymal malignancies, and the majority of the previous scientific studies that have analyzed the occurrence of cell cycle regulators aberrations within soft tissue sarcomas have dealt with broad categories of different tumors. As a consequence, data concerning single classes of sarcomas are very limited. The authors analyze herein a histologically homogeneous series of 23 cases of leiomyosarcoma of the deep soft tissue. The p53 pathway was studied by investigating the p53 gene and protein, MDM2 protein, and p21waf1 protein. The Rb-cyclin D pathway was analyzed by studying the Rb gene and protein, p16MTS1/INK4A gene and protein, cyclin D1Prad1/bcl1 and cyclin D3 proteins. Aberrations of the p53 pathway were observed in about 16 percent of cases and were limited to the p53 gene. Such a finding contrasts with the higher rates of p53/MDM2 abnormalities reported in other types of sarcomas such as liposarcoma. Interestingly, abnormalities involving the Rb-cyclin D pathway were detected in about 90 percent of cases. The Rb-cyclin D pathway therefore emerges as the preferred target for molecular abnormalities in this subset of soft tissue sarcomas.

Base Sequence↗