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Biomedical subjects

A Sacchi

Publications and source records attributed to A Sacchi.

At least 37 records · Page 2Linked to original sources
PubMed2000

Physical and functional interaction between p53 mutants and different isoforms of p73.

p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro and in vivo with p73 alpha, beta, gamma, and delta. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.

Breast Neoplasms↗
PubMed2000

Insulin receptor substrate-1, p70S6K, and cell size in transformation and differentiation of hemopoietic cells.

After an initial burst of cell proliferation, the type 1 insulin-like growth factor receptor (IGF-IR) induces granulocytic differentiation of 32D IGF-IR cells, an interleukin-3-dependent murine hemopoietic cell line devoid of insulin receptor substrate-1 (IRS-1). The combined expression of the IGF-IR and IRS-1 (32D IGF-IR/IRS-1 cells) inhibits IGF-I-mediated differentiation, and causes malignant transformation of 32D cells. Because of the role of IRS-1 in changing the fate of 32D IGF-IR cells from differentiation (and subsequent cell death) to malignant transformation, we have looked for differences in IGF-IR signaling between 32D IGF-IR and 32D IGF-IR/IRS-1 cells. In this report, we have focused on p70(S6K), which is activated by the IRS-1 pathway. We find that the ectopic expression of IRS-1 and the inhibition of differentiation correlated with a sustained activation of p70(S6K) and an increase in cell size. Phosphorylation in vivo of threonine 389 and, to a lesser extent, of threonine 421/serine 424 of p70(S6K) seemed to be a requirement for inhibition of differentiation. A role of IRS-1 and p70(S6K) in the alternative between transformation or differentiation of 32D IGF-IR cells was confirmed by findings that inhibition of p70(S6K) activation or IRS-1 signaling, by rapamycin or okadaic acid, induced differentiation of 32D IGF-IR/IRS-1 cells. We have also found that the expression of myeloperoxidase mRNA (a marker of differentiation, which sharply increases in 32D IGF-IR cells), does not increase in 32D IGF-IR/IRS-1 cells, suggesting that the expression of IRS-1 in 32D IGF-IR cells causes the extinction of the differentiation program initiated by the IGF-IR, while leaving intact its proliferation program.

Animals↗
PubMed2000

Cooperative transformation of 32D cells by the combined expression of IRS-1 and V-Ha-Ras.

32D cells expressing v-Ha-Ras fail to show a transformed phenotype. Since Ras requires an active IGF-1R for transformation of fibroblasts, we asked whether expression of IRS-1 or Shc (two of the major substrates of the IGF-1R) could co-operate with oncogenic Ras in transforming 32D cells. We find that IRS-1, but not Shc, in combination with v-Ha-Ras generates a fully transformed phenotype in 32D cells. 32D cells expressing both IRS-1 and v-Ha-Ras (32D/IRS1/Ras) survive and proliferate in the absence of IL-3, do not undergo granulocytic differentiation in the presence of G-CSF and form tumors in nu/nu and syngeneic mice. In contrast, 32D cells expressing singly IRS-1 or v-Ha-Ras exhibit only a block in differentiation capacity. Over-expression of Shc proteins, by itself, promotes differentiation of 32D cells. Concomitant expression of IRS-1 and v-Ha-Ras synergistically phosphorylates ERK-1 and ERK-2 whereas a MEK inhibitor rapidly induces death of 32D/IRS1/Ras transformed cells. Furthermore, transformed 32D/IRS1/Ras cells display high levels of PI3-K activation and undergo rapid apoptosis when exposed to PI3-K inhibitors. The data indicate that: (1) a fully transformed phenotype in 32D cells is generated when a block in differentiation (v-Ha-Ras) is coupled with another differentiation block (IRS-1); (2) PI3-K and MAPK activity are required for the survival of transformed cells; (3) the signals generated by IRS-1 and oncogenic Ras converge on ERK and PI3-K resulting in high levels of activation.

Androstadienes↗
PubMed2000

Cooperative signaling between alpha(6)beta(4) integrin and ErbB-2 receptor is required to promote phosphatidylinositol 3-kinase-dependent invasion.

We previously demonstrated that beta(4) integrin subunit overexpression increases in vitro invasiveness of NIH3T3 cells that have been transformed by ErbB-2 oncogene. We used this model to identify domains within the large beta(4) cytoplasmic domain that are involved in the interaction of alpha(6)beta(4) with ErbB-2, invasion, and phosphatidylinositol 3-kinase (PI3K) activation. For this purpose, we expressed deletion mutants of beta(4) that lacked either all or portions of the beta(4) cytoplasmic domain in NIH3T3/ErbB-2 cells. We also used an ecto-domain mutant in which most of the extracellular domain of beta(4) was replaced with a c-Myc tag. These transfectants were examined for their ability to invade Matrigel and their ability to activate PI3K, as well as for the ability of alpha(6)beta(4) to co-immunoprecipitate with ErbB-2. The results obtained revealed that a region of the beta(4) cytoplasmic domain between amino acids 854 and 1183 is critical for the ability of alpha(6)beta(4) integrin to increase invasion. Interestingly, the extracellular domain of beta(4) is not necessary for alpha(6)beta(4) to stimulate invasion. The association of alpha(6)beta(4) with ErbB-2 is dependent upon the beta(4) cytoplasmic domain and can occur in the absence of alpha(6)beta(4) heterodimerization. Finally, we observed strong activation of PI3K with beta(4) wild type and with those beta(4) deletion mutants that were able to stimulate invasion upon the expression in NIH3T3/ErbB-2 cells. In conclusion, our results establish that there is cooperation between alpha(6)beta(4) and ErbB-2 in promoting PI3K-dependent invasion and implicate a specific region of the beta(4) cytoplasmic domain (amino acids 854-1183) in this event.

3T3 Cells↗
PubMed2000

Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13).

The clinical use of tumor necrosis factor alpha (TNF) as an anticancer drug is limited to local treatments because of its dose-limiting systemic toxicity. We show here that murine TNF fused with CNGRC peptide (NGR-TNF), an aminopeptidase N (CD13) ligand that targets activated blood vessels in tumors, is 12-15 times more efficient than murine TNF in decreasing the tumor burden in lymphoma and melanoma animal models, whereas its toxicity is similar. Similarly, human NGR-TNF induced stronger antitumor effects than human TNF, even with 30 times lower doses. Coadministration of murine NGR-TNF with a CNGRC peptide or an anti-CD13 antibody markedly decreased its antitumor effects. Tumor regression, induced by doses of murine NGR-TNF lower than the LD50, was accompanied by protective immunity. In contrast, no cure was induced by TNF at any dose. These results suggest that targeted delivery of TNF to CD13 may enhance its immunotherapeutic properties. Moreover, these findings reveal the potential of tumor homing peptides to generate a new class of recombinant cytokines that compared to immunocytokines have a simpler structure, could be easier to produce and are potentially less immunogenic.

Animals↗
PubMed2000

Long-term fate of terminally differentiated skeletal muscle cells following E1A-initiated cell cycle reactivation.

We have previously shown that E1A reactivates the cell cycle in 'irreversibly' growth arrested, terminally differentiated (TD) cells. The molecular events following E1A-mediated reactivation of TD skeletal muscle cells have been extensively investigated. However, the long-term fate of the reactivated cells has not been directly determined. In this paper, E1A is used to reactivate TD myotubes derived from established cell lines or primary myoblasts. We show that the reactivated muscle cells continue proliferating beyond the end of the first cell cycle and progress through at least a second one. Experiments performed with an inducible E1A/estrogen receptor chimera indicate that the reactivated cell cycle is self-sustained, since E1A is exclusively necessary to reactivate TD cells, but is dispensable for both the continuation of the first cycle and the progression into the following one. Finally, we report that E1A-mediated reactivation of muscle cells results in apoptotic cell death that can be delayed by the antiapoptotic, adenoviral E1B 55 kDa oncogene.

Adenovirus E1A Proteins↗
PubMed2000

Development of a murine orthotopic model of leukemia: evaluation of TP53 gene therapy efficacy.

The onco-suppressor gene TP53 has potential use in the gene therapy of many human cancers including leukemias. The latter indication derived from numerous experimental reports of p53-mediated suppressing effects on human and murine leukemia cells in vitro. However, few in vivo experiments have been performed, and those that have used a subcutaneous injection of p53-transduced leukemia cells. Thus, we developed an orthotopic leukemia model in adult, syngenic mice to evaluate the feasibility of TP53-mediated therapeutic approaches. We found that among other cells, v-src-transformed 32D myeloid progenitors induce leukemia when injected intravenously in syngenic mice. The resulting malignancy resembles the clinical manifestations of human acute myeloid leukemia because it is characterized by a massive invasion of bone marrow compartments, splenomegaly, generalized lymphadenopathy, and a macroscopic or microscopic infiltration of the kidneys, liver, and lungs. When these 32Dv-src cells were infected with a TP53-recombinant retrovirus before intravenous injection, we found a decreased mortality and, in those animals that develop leukemia, a drastic reduction of the generalized organ infiltration, suggesting that exogenous TP53 expression might be used for ex vivo bone marrow purging from leukemia cells.

Animals↗
PubMed2000

Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells.

In mammals, molecular mechanisms and factors involved in the tight regulation of telomerase expression and activity are still largely undefined. In this study, we provide evidence for a role of estrogens and their receptors in the transcriptional regulation of hTERT, the catalytic subunit of human telomerase and, consequently, in the activation of the enzyme. Through a computer analysis of the hTERT 5'-flanking sequences, we identified a putative estrogen response element (ERE) which was capable of binding in vitro human estrogen receptor alpha (ERalpha). In vivo DNA footprinting revealed specific modifications of the ERE region in ERalpha-positive but not ERalpha-negative cells upon treatment with 17beta-estradiol (E2), indicative of estrogen-dependent chromatin remodelling. In the presence of E2, transient expression of ERalpha but not ERbeta remarkably increased hTERT promoter activity, and mutation of the ERE significantly reduced this effect. No telomerase activity was detected in human ovary epithelial cells grown in the absence of E2, but the addition of the hormone induced the enzyme within 3 h of treatment. The expression of hTERT mRNA and protein was induced in parallel with enzymatic activity. This prompt estrogen modulation of telomerase activity substantiates estrogen-dependent transcriptional regulation of the hTERT gene. The identification of hTERT as a target of estrogens represents a novel finding which advances the understanding of telomerase regulation in hormone-dependent cells and has implications for a potential role of hormones in their senescence and malignant conversion.

3T3 Cells↗
PubMed2000

Effects of exogenous p53 transduction in thyroid tumor cells with different p53 status.

Recovery of p53 function in undifferentiated thyroid carcinoma cells carrying an altered p53 gene is able to modify cell tumorigenic properties. It is not known whether such an effect may also be achieved in thyroid cancer cells expressing wild-type p53, as in the majority of differentiated thyroid carcinomas. Effects of p53 transduction in a thyroid carcinoma cell line (FRO) exhibiting a wild-type endogenous p53 gene, in comparison to a cell line (WRO) exhibiting mutant p53, were investigated by using an inducible chimeric construct containing human p53 complementary DNA fused to the ligand binding domain of the estrogen receptor (p53ER). FRO cells were unaffected by exogenous p53 expression in terms of both proliferation and viability. On the contrary, p53 reexpression in WRO cells containing hemizygous mutated p53 allele caused a strong growth inhibition due to cell accumulation in the G1 phase of the cell cycle. In addition, exogenous p53 did not influence FRO cell behavior in response to TSH treatment or modify cell resistance to the chemotherapeutic agent, doxorubicin. Our results indicate that exogenous expression of wild-type p53 affects thyroid tumorigenic properties only in cells carrying an altered p53, whereas it is ineffective in cells expressing wild-type p53 activity. Therefore, the endogenous p53 status seems to be a major determinant for the effectiveness of a p53-based gene therapy for thyroid cancer.

Antibiotics, Antineoplastic↗
PubMed2000

Caspase inhibition reveals functional cooperation between p55- and p75-TNF receptors in cell necrosis.

TNF-induced caspase activation is critically involved in both apoptosis and protection from cell necrosis. We have investigated the roles of the p55- and p75-TNF receptors (TNFR1 and TNFR2) in the induction of mouse L-M cell death in the presence of a caspase inhibitor (zVAD-fmk) and a transcription inhibitor (actinomycin D), i.e. under conditions in which protective pathways requiring caspase activation and protein synthesis were blocked. Cytometric analysis after TNF treatment showed that apoptosis was inhibited, while necrosis was highly activated. In contrast, apoptosis was observed in cells treated with TNF and actinomycin D alone. Stimulation of TNFR1 was sufficient to induce either cell necrosis or apoptosis, even when we blocked endogenous TNF with an anti-murine TNF antibody. Experiments based on the use of receptor-agonist and antagonist antibodies also showed that TNFR2 contributes to cell necrosis and apoptosis. Simultaneous stimulation of TNFR2 and TNFR1 with specific agonists indicated that TNFR2 functionally cooperates with TNFR1 to potentiate the response indirectly, by inducing endogenous TNF cytotoxicity. Caspase inhibitors enhanced the cytotoxic effect of endogenous TNF, suggesting that TNFR2 modulation can regulate the global necrotic response to TNF. TNFR2 modulation could play an important role in determining the response to TNF in pathophysiological conditions characterized by caspase down-regulation and local TNF production.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Research on heterocyclic compounds, XLI. 2-Phenylimidazo

The synthesis of a group of 2-phenylimidazo[1,2-b]pyridazine-3-acetic esters and acids is described. The structures of the new compounds are supported by 1H-NMR spectra. These compounds were tested in vivo for their anti-inflammatory, analgesic and ulcerogenic activity. All new compounds showed remarkable anti-inflammatory action in the carrageenan rat paw oedema (one third of that for indomethacin) but no significant analgesic activity in the acetic acid writhing test together with negligible ulcerogenic action, and were also found to be lacking inhibitory activity on cyclooxygenase in vitro.

Journal Article↗
PubMed1999

The role of wild-type p53 in the differentiation of primary hemopoietic and muscle cells.

Experiments previously performed on 32D and C2C12 cell lines indicated that wild type p53 (wtp53) protein has a role in granulocyte and myotube differentiation. Since these are immortal cells, we asked whether the inhibition of differentiation induced by the expression of dominant-negative p53 (dnp53) proteins was dependent on the immortalization-determined microenvironment. Thus, we evaluated the effects produced by interfering with the endogenous p53 gene in murine primary hemopoietic and muscle cells. Expression of dnp53 protein reduced the differentiation of bone marrow cells into granulocytes and macrophages. Moreover, p53 activation was measurable during the differentiation process of primary myoblasts, while interference with this activation led to a consistent slow down of terminal differentiation. Since the impairment of the differentiation was not accompanied by alterations in the cell cycle withdrawal and in the rate of apoptosis which are coupled with these types of differentiation, the data here reported support a specific role for p53 in the differentiation process. However, the difference in the intensity of inhibition between immortal and primary cells, i. e., complete versus slow down, respectively, suggests that the immortalization process might render the cells more sensitive to the loss of wtp53 activity for the differentiation process.

Animals↗
PubMed1999

E2F activates late-G1 events but cannot replace E1A in inducing S phase in terminally differentiated skeletal muscle cells.

We have previously shown that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. Current models imply that much or all of this E1A activity is mediated by the release of the E2F transcription factors from pocket-protein control. In contrast, we show here that overexpression of E2F-1, E2F-2 and E2F-4, or a chimeric E2F-4 tethered to a nuclear localization signal cannot reactivate postmitotic skeletal muscle cells (myotubes). This is not due to lack of transcriptional activity, as demonstrated on both a reporter construct and a number of endogenous target genes. Although cyclin E was strongly overexpressed in E2F-transduced myotubes, it lacked associated kinase activity, possibly explaining the inability of the myotubes to enter S phase and accumulate cyclin A. Although E2F is not sufficient to trigger DNA synthesis in myotubes, its activity is necessary even in the presence of E1A, as dominant-negative DP-1 mutants inhibit E1A-mediated cell cycle reentry. Our data show that, to reactivate myotubes, E1A must exert other functions, in addition to releasing E2F. They also establish mouse myotubes as an experimental system uniquely suited to study the most direct E2F functions in the absence of downstream cell cycle effects.

Adenovirus E1A Proteins↗
PubMed1999

Tumor pretargeting with avidin improves the therapeutic index of biotinylated tumor necrosis factor alpha in mouse models.

The clinical use of tumor necrosis factor alpha (TNF) as an anticancer drug is limited to local or locoregional administration because of dose-limiting systemic toxicity. We investigated in animal models whether the therapeutic index of systemically administered human or murine TNF can be increased by tumor pretargeting strategies based on the biotin-avidin system. Pretargeting of s.c. mouse WEHI-164 fibrosarcoma and RMA lymphoma genetically engineered to express the Thy 1.1 antigen on the cell membrane was achieved by i.p. injection of a biotinylated anti-Thy 1.1 antibody and avidin. This pretreatment increased the antitumor activity of systemically administered biotin-TNF conjugates by at least 5-fold. In contrast, pretargeting did not increase the toxicity of biotin-TNF, as judged by animal survival and weight loss after treatment. Ex vivo analysis of tumor cells 24 h after treatment showed that biotin-TNF persisted for several hours on the surface of pretargeted tumors, but not when avidin was omitted. The potentiation of the antitumor effects was related primarily to indirect mechanisms, involving a host-mediated response. The results indicate that tumor pretargeting improves the antitumor activity of TNF. Tumor pretargeting with avidin, which is currently used to increase the uptake of radioactive-labeled biotin in patients, could represent a new strategy for improving the therapeutic index of TNF.

Animals↗
PubMed1999

Down-regulation of cyclin B1 gene transcription in terminally differentiated skeletal muscle cells is associated with loss of functional CCAAT-binding NF-Y complex.

The observation that cyclin B1 protein and mRNAs are down-regulated in terminally differentiated (TD) C2C12 cells, suggested us to investigate the transcriptional regulation of the cyclin B1 gene in these cells. Transfections of cyclin B1 promoter constructs indicate that two CCAAT boxes support cyclin B1 promoter activity in proliferating cells. EMSAs demonstrate that both CCAAT boxes are recognized by the trimeric NF-Y complex in proliferating but not in TD cells. Transfecting a dominant-negative mutant of NF-YA we provide evidence that NF-Y is required for maximal promoter activity. Addition of recombinant NF-YA to TD C2C12 nuclear extracts restores binding activity in vitro, thus indicating that the loss of NF-YA in TD cells is responsible for the lack of the NF-Y binding to the CCAAT boxes. Consistent with this, we found that the NF-YA protein is absent in TD C2C12 cells. In conclusion, our data demonstrate that NF-Y is required for cyclin B1 promoter activity. We also demonstrate that cyclin B1 expression is regulated at the transcriptional level in TD C2C12 cells and that the switch-off of cyclin B1 promoter activity in differentiated cells depends upon the loss of a functional NF-Y complex. In particular the loss of NF-YA protein is most likely responsible for its inactivation.

Animals↗
PubMed1999

Growth and differentiation signals by the insulin-like growth factor 1 receptor in hemopoietic cells are mediated through different pathways.

The type 1 insulin-like growth factor receptor (IGF-IR) plays an important role in the growth of cells both in vivo and in vitro. The IGF-IR is also capable of inducing differentiation in a number of cell types, raising the question of how the same receptor can send two seemingly contradictory signals, one for growth and one for differentiation. Using 32D cells, which are murine hemopoietic cells, we show that the activated IGF-IR can induce differentiation along the granulocytic pathway in a manner similar to the granulocyte colony-stimulating factor. We find that one of the major substrates of the IGF-IR, the insulin receptor substrate-1 inhibits IGF-I-mediated differentiation of 32D cells. In the absence of insulin receptor substrate-1, functional impairment of another major substrate of the IGF-IR, the Shc proteins, is associated with a decrease in the extent of differentiation. Although the end points of the respective pathways remain to be defined, these results show for the first time that IGF-I-mediated growth or differentiation of hemopoietic cells may depend on a balance between two of its substrates.

Animals↗
PubMed1999

Photoadaptation of two members of the Chlorophyta (Scenedesmus and Chlorella) in laboratory and outdoor cultures: changes in chlorophyll fluorescence quenching and the xanthophyll cycle.

The role of the xanthophyll cycle in the adaptation of two chlorococcal algae Scenedesmus quadricauda and Chlorella sorokiniana to high irradiance was studied under laboratory and outdoor conditions. We wished to elucidate whether the xanthophyll cycle plays a key role in dissipating the excesses of absorbed light, as in higher plants, and to characterise the relationship between chlorophyll fluorescence parameters and the content of xanthophyll-cycle pigments. The xanthophyll cycle was found to be operative in both species; however, its contribution to overall non-photochemical quenching (NPQ) could only be distinguished in Scenedesmus (15-20% of total NPQ). The Scenedesmus cultures showed a larger pool of xanthophyll-cycle pigments than Chlorella, and lower sensitivity to photoinhibition as judged from the reduction of maximum quantum yield of photosystem II. In general, both algae had a larger xanthophyll-cycle pool when grown outdoors than in laboratory cultures. Comparing the two species, Scenedesmus exhibited a higher capacity to adapt to high irradiance, due to an effective quenching mechanism and high photosynthetic capacity; in contrast, Chlorella represents a species with a larger antennae system, less-efficient quenching and lower photosynthetic performance. Non-photochemical quenching (NPQ) induced through the xanthophyll cycle can, to a limited extent, represent a regulatory factor in diluted algal cultures grown in outdoor solar photobioreactors, as well as in natural algal phytoplankton populations exposed transiently to high irradiance. However, it does not play an appreciable role in dense, well-mixed microalgal suspensions.

Adaptation, Physiological↗