Armando Felsani | Consiglio Nazionale delle Ricerche (CNR) (original) (raw)

Papers by Armando Felsani

Research paper thumbnail of Myc down-regulation induces apoptosis in M14 melanoma cells by increasing p27kip1 levels

Oncogene, 2001

In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the ... more In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the melanoma pathogenesis. We have previously demonstrated that treatment of melanoma cells with c-myc antisense oligodeoxynucleotides can inhibit cell proliferation and activate apoptosis. To gain insight into the mechanisms activated by Myc down-regulation, we have now developed an experimental model that allows modulating Myc protein expression in melanoma cells. This was achieved by originating stable melanoma cell clones expressing ecdysone-inducible c-myc antisense RNA. We show that the induction of c-myc antisense RNA in M14 melanoma cells leads to an inhibition of cell proliferation characterized by accumulation of cells in the G 1 phase of the cell cycle (up to 80%) and activation of apoptosis (50%). These data are associated with an increase of p27 kip1 levels and a signi®cant reduction of the cdk2associated kinase activity. In addition, we show that an ectopic overexpression of p27 kip1 in this experimental model can enhance the apoptotic rate. Our results indicate that down-regulation of Myc protein induces a G 1 arrest and activates apoptosis by increasing p27 kip1 content in melanoma cells, that are known to be defective for the p16-cyclinD/cdk4-pRb G 1 checkpoint. This is particularly relevant for identifying new therapeutic strategies based on the re-establishment of the apoptotic pathways in cancer cells. Oncogene (2001) 20, 2814 ± 2825.

Research paper thumbnail of Poly(a)-containing rna in neuroblastoma. Immature and differentiated cells in culture

European Journal of Biochemistry, 1977

We have analysed the poly(A)-containing RNA from neuroblastoma cells at two different development... more We have analysed the poly(A)-containing RNA from neuroblastoma cells at two different developmental states : either as round, immature neuroblasts grown in suspension, or as differentiated cells exhibiting the morphological properties of mature neurons, when attached to a culture dish.

Research paper thumbnail of Changes in the pattern of poly(A)-containing RNA during terminal differentiation in neuroblastoma cells

Research paper thumbnail of Complexity of polysomal polyadenylated rna in undifferen- tiated and differentiated neuroblastoma cells

European Journal of Biochemistry, 1978

Research paper thumbnail of Complexity of polysomal polyadenylated rna in mouse whole brain and cortex

Research paper thumbnail of Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

European Journal of Biochemistry, 1983

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single an... more A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase 11, sized on a preparative agarose gel and inserted in the PstI site of pBR 322 by the dG . dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18s and 26s transcribed sequences were located by S1 protection mapping.

Research paper thumbnail of Mouse Genes Coding for

Proceedings of The National Academy of Sciences, 1989

By using the zinc-finger region of human cIHF.10 cDNA as a probe at low-stringency hybridization ... more By using the zinc-finger region of human cIHF.10 cDNA as a probe at low-stringency hybridization conditions, several individual phages from a mouse skeletal muscle cDNA library have been isolated. The amino acid sequences of the "zinc-finger" domains derived from the DNA sequences of three cDNA clones are shown. The expression of the corresponding mRNAs in three cell lines (NIl 3T3, F9 teratocarcinoma, and C2 myoblast cells) at different stages of differentiation and in eight adult mouse tissues has been analyzed. The transcription of these genes is induced during the in vitro differentiation of the cell lines tested. These three genes are widely and evenly expressed in adult mouse tissues, with the remarkable exception of one that is expressed predominately in the testis.

Research paper thumbnail of New Technologies Used in the Study of Human Melanoma

International Review of Cytology-a Survey of Cell Biology, 2007

Research paper thumbnail of New Technologies Used in the Study of Human Melanoma

International Review of Cytology-a Survey of Cell Biology, 2007

Research paper thumbnail of The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells

Oncogene, 2002

Differential gene expression of cell lines derived from a malignant melanoma or its autologous ly... more Differential gene expression of cell lines derived from a malignant melanoma or its autologous lymph node metastasis using cDNA arrays indicated down-regulation of PRSS11, a gene encoding the serine protease HtrA1, a homolog of the Escherichia coli protease HtrA, in the metastatic line. Stable PRSS11 overexpression in the metastatic cell line strongly inhibited proliferation, chemoinvasion and Nm23-H1 protein expression in vitro, as well as cell growth in vivo in nu/nu mice. A polyclonal anti-HtrA1 serum demonstrated a significantly higher expression in primary melanomas when compared to unrelated metastatic lesions in a human melanoma tissue array, and down-modulation of HtrA1 expression in autologous lymph node melanoma metastases in seven out of 11 cases examined. These results suggest that down-regulation of PRSS11 and HtrA1 expression may represent an indicator of melanoma progression.

Research paper thumbnail of The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells

Oncogene, 2002

Differential gene expression of cell lines derived from a malignant melanoma or its autologous ly... more Differential gene expression of cell lines derived from a malignant melanoma or its autologous lymph node metastasis using cDNA arrays indicated down-regulation of PRSS11, a gene encoding the serine protease HtrA1, a homolog of the Escherichia coli protease HtrA, in the metastatic line. Stable PRSS11 overexpression in the metastatic cell line strongly inhibited proliferation, chemoinvasion and Nm23-H1 protein expression in vitro, as well as cell growth in vivo in nu/nu mice. A polyclonal anti-HtrA1 serum demonstrated a significantly higher expression in primary melanomas when compared to unrelated metastatic lesions in a human melanoma tissue array, and down-modulation of HtrA1 expression in autologous lymph node melanoma metastases in seven out of 11 cases examined. These results suggest that down-regulation of PRSS11 and HtrA1 expression may represent an indicator of melanoma progression.

Research paper thumbnail of Oligopeptides impairing the Myc-Max heterodimerization inhibit lung cancer cell proliferation by reducing Myc transcriptional activity

Journal of Cellular Physiology, 2007

Deregulated CMYC gene causes cell transformation and is often correlated with tumor progression a... more Deregulated CMYC gene causes cell transformation and is often correlated with tumor progression and a worse clinical outcome of cancer patients. The transcription factor Myc functions by heterodimerizing with its partner, Max. As a strategy to inhibit Myc activity, we have synthesized three small peptides corresponding to segments of the leucine zipper (LZ) region of Max. The purpose of these peptides is to occupy the site of recognition between Myc and Max located in the LZ and inhibit-specific heterodimerization between these proteins. We have used the synthesized oligopeptides in two lung cancer cell lines with different levels of Myc expression. Results demonstrate that: (i) the three peptides resulted equally effective in competing the interaction between Myc and Max in vitro; (ii) they were efficiently internalized into the cells and significantly inhibited cell growth in the cells showing the highest Myc expression; (iii) one specific peptide, only nine aminoacids long, efficiently impaired the transcriptional activity of Myc in vivo, showing a more stable interaction with this protein. Our results are relevant to the development of novel anti-tumoral therapeutic strategies, directed to Myc-overexpressing tumors. J. Cell. Physiol. 210: 72–80, 2007. © 2006 Wiley-Liss, Inc.

Research paper thumbnail of Oligopeptides impairing the Myc-Max heterodimerization inhibit lung cancer cell proliferation by reducing Myc transcriptional activity

Journal of Cellular Physiology, 2007

Deregulated CMYC gene causes cell transformation and is often correlated with tumor progression a... more Deregulated CMYC gene causes cell transformation and is often correlated with tumor progression and a worse clinical outcome of cancer patients. The transcription factor Myc functions by heterodimerizing with its partner, Max. As a strategy to inhibit Myc activity, we have synthesized three small peptides corresponding to segments of the leucine zipper (LZ) region of Max. The purpose of these peptides is to occupy the site of recognition between Myc and Max located in the LZ and inhibit-specific heterodimerization between these proteins. We have used the synthesized oligopeptides in two lung cancer cell lines with different levels of Myc expression. Results demonstrate that: (i) the three peptides resulted equally effective in competing the interaction between Myc and Max in vitro; (ii) they were efficiently internalized into the cells and significantly inhibited cell growth in the cells showing the highest Myc expression; (iii) one specific peptide, only nine aminoacids long, efficiently impaired the transcriptional activity of Myc in vivo, showing a more stable interaction with this protein. Our results are relevant to the development of novel anti-tumoral therapeutic strategies, directed to Myc-overexpressing tumors. J. Cell. Physiol. 210: 72–80, 2007. © 2006 Wiley-Liss, Inc.

Research paper thumbnail of Interaction between the pRb2/p130 C-terminal domain and the N-terminal portion of cyclin D3

Journal of Cellular Biochemistry, 1999

An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using th... more An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using the yeast two-hybrid system. Further analysis restricted the epitope responsible for the binding within the 74 N-terminal amino acids of cyclin D3, independent of the LXCXE amino acid motif present in the D-type cyclin N-terminal region. In a coprecipitation assay in T98G cells, a human glioblastoma cell line, the C-terminal domain of pRb2/p130 was able to interact solely with cyclin D3, while the corresponding portion of pRb interacted with either cyclin D3 or cyclin D1. In T98G cells, endogenous cyclin D3-associated kinase activity showed a clear predisposition to phosphorylate preferentially the C-terminal domain of pRb2/p130, rather than that of pRb. This propensity was also confirmed in LAN-5 human neuroblastoma cells, where phosphorylation of the pRb2/p130 C-terminal domain and expression of cyclin D3 also decreased remarkably in the late neural differentiation stages.

Research paper thumbnail of Interaction between the pRb2/p130 C-terminal domain and the N-terminal portion of cyclin D3

Journal of Cellular Biochemistry, 1999

An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using th... more An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using the yeast two-hybrid system. Further analysis restricted the epitope responsible for the binding within the 74 N-terminal amino acids of cyclin D3, independent of the LXCXE amino acid motif present in the D-type cyclin N-terminal region. In a coprecipitation assay in T98G cells, a human glioblastoma cell line, the C-terminal domain of pRb2/p130 was able to interact solely with cyclin D3, while the corresponding portion of pRb interacted with either cyclin D3 or cyclin D1. In T98G cells, endogenous cyclin D3-associated kinase activity showed a clear predisposition to phosphorylate preferentially the C-terminal domain of pRb2/p130, rather than that of pRb. This propensity was also confirmed in LAN-5 human neuroblastoma cells, where phosphorylation of the pRb2/p130 C-terminal domain and expression of cyclin D3 also decreased remarkably in the late neural differentiation stages.

Research paper thumbnail of Mechanisms of defence from Fe(II) toxicity in human intestinal Caco-2 cells

Toxicology in Vitro, 2009

Iron is used to cure iron-deficient anaemia but can also be toxic to the intestine. Fe(II) toxici... more Iron is used to cure iron-deficient anaemia but can also be toxic to the intestine. Fe(II) toxicity was investigated using differentiated human intestinal Caco-2 cells treated with 15 and 50 lM of Fe(II)/ascorbate for 2 h (acute phase), and followed for 24 h after iron removal and replacement of complete culture medium (late phase). During the acute phase damage to tight junctions occurred as demonstrated by an increase in cell monolayer permeability and by partial delocalization of the tight junction protein claudin 4 from the plasma membrane to an intracellular compartment. At the end of the late phase, cells treated with 15 lM Fe(II) showed full restoration of claudin 4 localization to the plasma membrane and their tight junction permeability returned to values close to those of control cells. Conversely, cells treated with 50 lM Fe(II) showed sustained and irreversible damage to the tight junctions, accompanied by apoptosis and necrosis. Activation of NF-jB occurred at both Fe(II) concentrations after 30 min of Fe(II) treatment, followed, at the end of the acute phase, by a strong induction of mRNA coding for heat shock protein 70 and metallothionein 2A. Our results indicate that intestinal cells respond to iron toxicity by strongly activating two genes involved in cell response to stress, although the outcome in terms of cell survival is different depending on the dose of treatment, namely almost complete restoration of epithelial permeability and cell survival at 15 lM Fe(II), and progressive and irreversible cytotoxicity leading to apoptosis and necrosis at 50 lM Fe(II).

Research paper thumbnail of Mechanisms of defence from Fe(II) toxicity in human intestinal Caco-2 cells

Toxicology in Vitro, 2009

Iron is used to cure iron-deficient anaemia but can also be toxic to the intestine. Fe(II) toxici... more Iron is used to cure iron-deficient anaemia but can also be toxic to the intestine. Fe(II) toxicity was investigated using differentiated human intestinal Caco-2 cells treated with 15 and 50 lM of Fe(II)/ascorbate for 2 h (acute phase), and followed for 24 h after iron removal and replacement of complete culture medium (late phase). During the acute phase damage to tight junctions occurred as demonstrated by an increase in cell monolayer permeability and by partial delocalization of the tight junction protein claudin 4 from the plasma membrane to an intracellular compartment. At the end of the late phase, cells treated with 15 lM Fe(II) showed full restoration of claudin 4 localization to the plasma membrane and their tight junction permeability returned to values close to those of control cells. Conversely, cells treated with 50 lM Fe(II) showed sustained and irreversible damage to the tight junctions, accompanied by apoptosis and necrosis. Activation of NF-jB occurred at both Fe(II) concentrations after 30 min of Fe(II) treatment, followed, at the end of the acute phase, by a strong induction of mRNA coding for heat shock protein 70 and metallothionein 2A. Our results indicate that intestinal cells respond to iron toxicity by strongly activating two genes involved in cell response to stress, although the outcome in terms of cell survival is different depending on the dose of treatment, namely almost complete restoration of epithelial permeability and cell survival at 15 lM Fe(II), and progressive and irreversible cytotoxicity leading to apoptosis and necrosis at 50 lM Fe(II).

Research paper thumbnail of pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

Molecular and Cellular Biology, 2007

The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the proces... more The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that cyclin D3 is nearly totally associated with hypophosphorylated pRb in differentiated myotubes, whereas Rb ؊/؊ myocytes fail to accumulate the cyclin D3 protein despite normal induction of cyclin D3 mRNA. Here we report that pRb promotes cyclin D3 protein accumulation in differentiating myoblasts by preventing cyclin D3 degradation. We show that cyclin D3 displays rapid turnover in proliferating myoblasts, which is positively regulated through glycogen synthase kinase 3␤ (GSK-3␤)-mediated phosphorylation of cyclin D3 on Thr-283. We describe a novel interaction between pRb and cyclin D3 that maps to the C terminus of pRb and to a region of cyclin D3 proximal to the Thr-283 residue and provide evidence that the pRb-cyclin D3 complex formation in terminally differentiated myotubes hinders the access of GSK-3␤ to cyclin D3, thus inhibiting Thr-283 phosphorylation. Interestingly, we observed that the ectopic expression of a stabilized cyclin D3 mutant in C2 myoblasts enhances musclespecific gene expression; conversely, cyclin D3-null embryonic fibroblasts display impaired MyoD-induced myogenic differentiation. These results indicate that the pRb-dependent accumulation of cyclin D3 is functionally relevant to the process of skeletal muscle cell differentiation.

Research paper thumbnail of In vitro and in vivo tumor growth inhibition by a p16-mimicking peptide in p16INK4A-defective, pRb-positive human melanoma cells

Journal of Cellular Physiology, 2005

The cell cycle regulatory pathway responsible for the control of the late-G1 checkpoint is found ... more The cell cycle regulatory pathway responsible for the control of the late-G1 checkpoint is found recurrently altered in human malignant melanoma, often due to lack of functional p16 or pRb (pRb-1) proteins. Here we examined the ability of p16-derived peptides to mimic p16 function in two exemplary human melanoma cell lines: the p16-defective, pRb-positive A375M cells and p16-positive, pRb-defective A2058 cells. The synthetic p16-mimicking peptides strongly induced apoptosis in p16−, pRb+ A375M cells in vitro, while they had significantly less activity on p16+, pRb− A2058 cells. The most active p16-mimicking peptide, p16-AP9, also potently inhibited in vivo growth of the A375M melanoma. Treated tumors showed a threefold smaller volume (P < 0.025) and a significant reduction of the mitotic index and of PCNA expression. Growth of A2058 cells in vivo was not affected by treatment with the p16-mimicking peptide. Our results demonstrate that p16-mimicking peptides can induce apoptosis in vitro and that can inhibit tumor growth in vivo in p16-defective, pRb-expressing human melanoma cells, suggesting that p16-mimicking peptides can represent a promising tool for targeted therapy in selected cancer phenotypes. © 2004 Wiley-Liss, Inc.

Research paper thumbnail of MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway

Molecular and Cellular Biology, 2003

The induction of RB gene transcription by MyoD is a key event in the process of skeletal muscle d... more The induction of RB gene transcription by MyoD is a key event in the process of skeletal muscle differentiation, because elevated levels of the retinoblastoma protein are essential for myoblast cell cycle arrest as well as for the terminal differentiation and survival of postmitotic myocytes. We previously showed that MyoD stimulates transcription from the RB promoter independently of direct binding to promoter sequences. Here we demonstrate that stimulation by MyoD requires a cyclic AMP-responsive element (CRE) in the RB promoter, bound by the transcription factor CREB in differentiating myoblasts. We also show that both the CREB protein level and the level of phosphorylation of the CREB protein at Ser-133 rapidly increase at the onset of muscle differentiation and that both remain high throughout the myogenic process. Biochemical and functional evidence indicates that in differentiating myoblasts, MyoD becomes associated with CREB and is targeted to the RB promoter CRE in a complex also containing the p300 transcriptional coactivator. The resulting multiprotein complex stimulates transcription from the RB promoter. These and other observations strongly suggest that MyoD functions by promoting the efficient recruitment of p300 by promoter-bound, phosphorylated CREB.

Research paper thumbnail of Myc down-regulation induces apoptosis in M14 melanoma cells by increasing p27kip1 levels

Oncogene, 2001

In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the ... more In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the melanoma pathogenesis. We have previously demonstrated that treatment of melanoma cells with c-myc antisense oligodeoxynucleotides can inhibit cell proliferation and activate apoptosis. To gain insight into the mechanisms activated by Myc down-regulation, we have now developed an experimental model that allows modulating Myc protein expression in melanoma cells. This was achieved by originating stable melanoma cell clones expressing ecdysone-inducible c-myc antisense RNA. We show that the induction of c-myc antisense RNA in M14 melanoma cells leads to an inhibition of cell proliferation characterized by accumulation of cells in the G 1 phase of the cell cycle (up to 80%) and activation of apoptosis (50%). These data are associated with an increase of p27 kip1 levels and a signi®cant reduction of the cdk2associated kinase activity. In addition, we show that an ectopic overexpression of p27 kip1 in this experimental model can enhance the apoptotic rate. Our results indicate that down-regulation of Myc protein induces a G 1 arrest and activates apoptosis by increasing p27 kip1 content in melanoma cells, that are known to be defective for the p16-cyclinD/cdk4-pRb G 1 checkpoint. This is particularly relevant for identifying new therapeutic strategies based on the re-establishment of the apoptotic pathways in cancer cells. Oncogene (2001) 20, 2814 ± 2825.

Research paper thumbnail of Poly(a)-containing rna in neuroblastoma. Immature and differentiated cells in culture

European Journal of Biochemistry, 1977

We have analysed the poly(A)-containing RNA from neuroblastoma cells at two different development... more We have analysed the poly(A)-containing RNA from neuroblastoma cells at two different developmental states : either as round, immature neuroblasts grown in suspension, or as differentiated cells exhibiting the morphological properties of mature neurons, when attached to a culture dish.

Research paper thumbnail of Changes in the pattern of poly(A)-containing RNA during terminal differentiation in neuroblastoma cells

Research paper thumbnail of Complexity of polysomal polyadenylated rna in undifferen- tiated and differentiated neuroblastoma cells

European Journal of Biochemistry, 1978

Research paper thumbnail of Complexity of polysomal polyadenylated rna in mouse whole brain and cortex

Research paper thumbnail of Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

European Journal of Biochemistry, 1983

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single an... more A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase 11, sized on a preparative agarose gel and inserted in the PstI site of pBR 322 by the dG . dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18s and 26s transcribed sequences were located by S1 protection mapping.

Research paper thumbnail of Mouse Genes Coding for

Proceedings of The National Academy of Sciences, 1989

By using the zinc-finger region of human cIHF.10 cDNA as a probe at low-stringency hybridization ... more By using the zinc-finger region of human cIHF.10 cDNA as a probe at low-stringency hybridization conditions, several individual phages from a mouse skeletal muscle cDNA library have been isolated. The amino acid sequences of the "zinc-finger" domains derived from the DNA sequences of three cDNA clones are shown. The expression of the corresponding mRNAs in three cell lines (NIl 3T3, F9 teratocarcinoma, and C2 myoblast cells) at different stages of differentiation and in eight adult mouse tissues has been analyzed. The transcription of these genes is induced during the in vitro differentiation of the cell lines tested. These three genes are widely and evenly expressed in adult mouse tissues, with the remarkable exception of one that is expressed predominately in the testis.

Research paper thumbnail of New Technologies Used in the Study of Human Melanoma

International Review of Cytology-a Survey of Cell Biology, 2007

Research paper thumbnail of New Technologies Used in the Study of Human Melanoma

International Review of Cytology-a Survey of Cell Biology, 2007

Research paper thumbnail of The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells

Oncogene, 2002

Differential gene expression of cell lines derived from a malignant melanoma or its autologous ly... more Differential gene expression of cell lines derived from a malignant melanoma or its autologous lymph node metastasis using cDNA arrays indicated down-regulation of PRSS11, a gene encoding the serine protease HtrA1, a homolog of the Escherichia coli protease HtrA, in the metastatic line. Stable PRSS11 overexpression in the metastatic cell line strongly inhibited proliferation, chemoinvasion and Nm23-H1 protein expression in vitro, as well as cell growth in vivo in nu/nu mice. A polyclonal anti-HtrA1 serum demonstrated a significantly higher expression in primary melanomas when compared to unrelated metastatic lesions in a human melanoma tissue array, and down-modulation of HtrA1 expression in autologous lymph node melanoma metastases in seven out of 11 cases examined. These results suggest that down-regulation of PRSS11 and HtrA1 expression may represent an indicator of melanoma progression.

Research paper thumbnail of The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells

Oncogene, 2002

Differential gene expression of cell lines derived from a malignant melanoma or its autologous ly... more Differential gene expression of cell lines derived from a malignant melanoma or its autologous lymph node metastasis using cDNA arrays indicated down-regulation of PRSS11, a gene encoding the serine protease HtrA1, a homolog of the Escherichia coli protease HtrA, in the metastatic line. Stable PRSS11 overexpression in the metastatic cell line strongly inhibited proliferation, chemoinvasion and Nm23-H1 protein expression in vitro, as well as cell growth in vivo in nu/nu mice. A polyclonal anti-HtrA1 serum demonstrated a significantly higher expression in primary melanomas when compared to unrelated metastatic lesions in a human melanoma tissue array, and down-modulation of HtrA1 expression in autologous lymph node melanoma metastases in seven out of 11 cases examined. These results suggest that down-regulation of PRSS11 and HtrA1 expression may represent an indicator of melanoma progression.

Research paper thumbnail of Oligopeptides impairing the Myc-Max heterodimerization inhibit lung cancer cell proliferation by reducing Myc transcriptional activity

Journal of Cellular Physiology, 2007

Deregulated CMYC gene causes cell transformation and is often correlated with tumor progression a... more Deregulated CMYC gene causes cell transformation and is often correlated with tumor progression and a worse clinical outcome of cancer patients. The transcription factor Myc functions by heterodimerizing with its partner, Max. As a strategy to inhibit Myc activity, we have synthesized three small peptides corresponding to segments of the leucine zipper (LZ) region of Max. The purpose of these peptides is to occupy the site of recognition between Myc and Max located in the LZ and inhibit-specific heterodimerization between these proteins. We have used the synthesized oligopeptides in two lung cancer cell lines with different levels of Myc expression. Results demonstrate that: (i) the three peptides resulted equally effective in competing the interaction between Myc and Max in vitro; (ii) they were efficiently internalized into the cells and significantly inhibited cell growth in the cells showing the highest Myc expression; (iii) one specific peptide, only nine aminoacids long, efficiently impaired the transcriptional activity of Myc in vivo, showing a more stable interaction with this protein. Our results are relevant to the development of novel anti-tumoral therapeutic strategies, directed to Myc-overexpressing tumors. J. Cell. Physiol. 210: 72–80, 2007. © 2006 Wiley-Liss, Inc.

Research paper thumbnail of Oligopeptides impairing the Myc-Max heterodimerization inhibit lung cancer cell proliferation by reducing Myc transcriptional activity

Journal of Cellular Physiology, 2007

Deregulated CMYC gene causes cell transformation and is often correlated with tumor progression a... more Deregulated CMYC gene causes cell transformation and is often correlated with tumor progression and a worse clinical outcome of cancer patients. The transcription factor Myc functions by heterodimerizing with its partner, Max. As a strategy to inhibit Myc activity, we have synthesized three small peptides corresponding to segments of the leucine zipper (LZ) region of Max. The purpose of these peptides is to occupy the site of recognition between Myc and Max located in the LZ and inhibit-specific heterodimerization between these proteins. We have used the synthesized oligopeptides in two lung cancer cell lines with different levels of Myc expression. Results demonstrate that: (i) the three peptides resulted equally effective in competing the interaction between Myc and Max in vitro; (ii) they were efficiently internalized into the cells and significantly inhibited cell growth in the cells showing the highest Myc expression; (iii) one specific peptide, only nine aminoacids long, efficiently impaired the transcriptional activity of Myc in vivo, showing a more stable interaction with this protein. Our results are relevant to the development of novel anti-tumoral therapeutic strategies, directed to Myc-overexpressing tumors. J. Cell. Physiol. 210: 72–80, 2007. © 2006 Wiley-Liss, Inc.

Research paper thumbnail of Interaction between the pRb2/p130 C-terminal domain and the N-terminal portion of cyclin D3

Journal of Cellular Biochemistry, 1999

An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using th... more An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using the yeast two-hybrid system. Further analysis restricted the epitope responsible for the binding within the 74 N-terminal amino acids of cyclin D3, independent of the LXCXE amino acid motif present in the D-type cyclin N-terminal region. In a coprecipitation assay in T98G cells, a human glioblastoma cell line, the C-terminal domain of pRb2/p130 was able to interact solely with cyclin D3, while the corresponding portion of pRb interacted with either cyclin D3 or cyclin D1. In T98G cells, endogenous cyclin D3-associated kinase activity showed a clear predisposition to phosphorylate preferentially the C-terminal domain of pRb2/p130, rather than that of pRb. This propensity was also confirmed in LAN-5 human neuroblastoma cells, where phosphorylation of the pRb2/p130 C-terminal domain and expression of cyclin D3 also decreased remarkably in the late neural differentiation stages.

Research paper thumbnail of Interaction between the pRb2/p130 C-terminal domain and the N-terminal portion of cyclin D3

Journal of Cellular Biochemistry, 1999

An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using th... more An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using the yeast two-hybrid system. Further analysis restricted the epitope responsible for the binding within the 74 N-terminal amino acids of cyclin D3, independent of the LXCXE amino acid motif present in the D-type cyclin N-terminal region. In a coprecipitation assay in T98G cells, a human glioblastoma cell line, the C-terminal domain of pRb2/p130 was able to interact solely with cyclin D3, while the corresponding portion of pRb interacted with either cyclin D3 or cyclin D1. In T98G cells, endogenous cyclin D3-associated kinase activity showed a clear predisposition to phosphorylate preferentially the C-terminal domain of pRb2/p130, rather than that of pRb. This propensity was also confirmed in LAN-5 human neuroblastoma cells, where phosphorylation of the pRb2/p130 C-terminal domain and expression of cyclin D3 also decreased remarkably in the late neural differentiation stages.

Research paper thumbnail of Mechanisms of defence from Fe(II) toxicity in human intestinal Caco-2 cells

Toxicology in Vitro, 2009

Iron is used to cure iron-deficient anaemia but can also be toxic to the intestine. Fe(II) toxici... more Iron is used to cure iron-deficient anaemia but can also be toxic to the intestine. Fe(II) toxicity was investigated using differentiated human intestinal Caco-2 cells treated with 15 and 50 lM of Fe(II)/ascorbate for 2 h (acute phase), and followed for 24 h after iron removal and replacement of complete culture medium (late phase). During the acute phase damage to tight junctions occurred as demonstrated by an increase in cell monolayer permeability and by partial delocalization of the tight junction protein claudin 4 from the plasma membrane to an intracellular compartment. At the end of the late phase, cells treated with 15 lM Fe(II) showed full restoration of claudin 4 localization to the plasma membrane and their tight junction permeability returned to values close to those of control cells. Conversely, cells treated with 50 lM Fe(II) showed sustained and irreversible damage to the tight junctions, accompanied by apoptosis and necrosis. Activation of NF-jB occurred at both Fe(II) concentrations after 30 min of Fe(II) treatment, followed, at the end of the acute phase, by a strong induction of mRNA coding for heat shock protein 70 and metallothionein 2A. Our results indicate that intestinal cells respond to iron toxicity by strongly activating two genes involved in cell response to stress, although the outcome in terms of cell survival is different depending on the dose of treatment, namely almost complete restoration of epithelial permeability and cell survival at 15 lM Fe(II), and progressive and irreversible cytotoxicity leading to apoptosis and necrosis at 50 lM Fe(II).

Research paper thumbnail of Mechanisms of defence from Fe(II) toxicity in human intestinal Caco-2 cells

Toxicology in Vitro, 2009

Iron is used to cure iron-deficient anaemia but can also be toxic to the intestine. Fe(II) toxici... more Iron is used to cure iron-deficient anaemia but can also be toxic to the intestine. Fe(II) toxicity was investigated using differentiated human intestinal Caco-2 cells treated with 15 and 50 lM of Fe(II)/ascorbate for 2 h (acute phase), and followed for 24 h after iron removal and replacement of complete culture medium (late phase). During the acute phase damage to tight junctions occurred as demonstrated by an increase in cell monolayer permeability and by partial delocalization of the tight junction protein claudin 4 from the plasma membrane to an intracellular compartment. At the end of the late phase, cells treated with 15 lM Fe(II) showed full restoration of claudin 4 localization to the plasma membrane and their tight junction permeability returned to values close to those of control cells. Conversely, cells treated with 50 lM Fe(II) showed sustained and irreversible damage to the tight junctions, accompanied by apoptosis and necrosis. Activation of NF-jB occurred at both Fe(II) concentrations after 30 min of Fe(II) treatment, followed, at the end of the acute phase, by a strong induction of mRNA coding for heat shock protein 70 and metallothionein 2A. Our results indicate that intestinal cells respond to iron toxicity by strongly activating two genes involved in cell response to stress, although the outcome in terms of cell survival is different depending on the dose of treatment, namely almost complete restoration of epithelial permeability and cell survival at 15 lM Fe(II), and progressive and irreversible cytotoxicity leading to apoptosis and necrosis at 50 lM Fe(II).

Research paper thumbnail of pRb-Dependent Cyclin D3 Protein Stabilization Is Required for Myogenic Differentiation

Molecular and Cellular Biology, 2007

The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the proces... more The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that cyclin D3 is nearly totally associated with hypophosphorylated pRb in differentiated myotubes, whereas Rb ؊/؊ myocytes fail to accumulate the cyclin D3 protein despite normal induction of cyclin D3 mRNA. Here we report that pRb promotes cyclin D3 protein accumulation in differentiating myoblasts by preventing cyclin D3 degradation. We show that cyclin D3 displays rapid turnover in proliferating myoblasts, which is positively regulated through glycogen synthase kinase 3␤ (GSK-3␤)-mediated phosphorylation of cyclin D3 on Thr-283. We describe a novel interaction between pRb and cyclin D3 that maps to the C terminus of pRb and to a region of cyclin D3 proximal to the Thr-283 residue and provide evidence that the pRb-cyclin D3 complex formation in terminally differentiated myotubes hinders the access of GSK-3␤ to cyclin D3, thus inhibiting Thr-283 phosphorylation. Interestingly, we observed that the ectopic expression of a stabilized cyclin D3 mutant in C2 myoblasts enhances musclespecific gene expression; conversely, cyclin D3-null embryonic fibroblasts display impaired MyoD-induced myogenic differentiation. These results indicate that the pRb-dependent accumulation of cyclin D3 is functionally relevant to the process of skeletal muscle cell differentiation.

Research paper thumbnail of In vitro and in vivo tumor growth inhibition by a p16-mimicking peptide in p16INK4A-defective, pRb-positive human melanoma cells

Journal of Cellular Physiology, 2005

The cell cycle regulatory pathway responsible for the control of the late-G1 checkpoint is found ... more The cell cycle regulatory pathway responsible for the control of the late-G1 checkpoint is found recurrently altered in human malignant melanoma, often due to lack of functional p16 or pRb (pRb-1) proteins. Here we examined the ability of p16-derived peptides to mimic p16 function in two exemplary human melanoma cell lines: the p16-defective, pRb-positive A375M cells and p16-positive, pRb-defective A2058 cells. The synthetic p16-mimicking peptides strongly induced apoptosis in p16−, pRb+ A375M cells in vitro, while they had significantly less activity on p16+, pRb− A2058 cells. The most active p16-mimicking peptide, p16-AP9, also potently inhibited in vivo growth of the A375M melanoma. Treated tumors showed a threefold smaller volume (P < 0.025) and a significant reduction of the mitotic index and of PCNA expression. Growth of A2058 cells in vivo was not affected by treatment with the p16-mimicking peptide. Our results demonstrate that p16-mimicking peptides can induce apoptosis in vitro and that can inhibit tumor growth in vivo in p16-defective, pRb-expressing human melanoma cells, suggesting that p16-mimicking peptides can represent a promising tool for targeted therapy in selected cancer phenotypes. © 2004 Wiley-Liss, Inc.

Research paper thumbnail of MyoD Stimulates RB Promoter Activity via the CREB/p300 Nuclear Transduction Pathway

Molecular and Cellular Biology, 2003

The induction of RB gene transcription by MyoD is a key event in the process of skeletal muscle d... more The induction of RB gene transcription by MyoD is a key event in the process of skeletal muscle differentiation, because elevated levels of the retinoblastoma protein are essential for myoblast cell cycle arrest as well as for the terminal differentiation and survival of postmitotic myocytes. We previously showed that MyoD stimulates transcription from the RB promoter independently of direct binding to promoter sequences. Here we demonstrate that stimulation by MyoD requires a cyclic AMP-responsive element (CRE) in the RB promoter, bound by the transcription factor CREB in differentiating myoblasts. We also show that both the CREB protein level and the level of phosphorylation of the CREB protein at Ser-133 rapidly increase at the onset of muscle differentiation and that both remain high throughout the myogenic process. Biochemical and functional evidence indicates that in differentiating myoblasts, MyoD becomes associated with CREB and is targeted to the RB promoter CRE in a complex also containing the p300 transcriptional coactivator. The resulting multiprotein complex stimulates transcription from the RB promoter. These and other observations strongly suggest that MyoD functions by promoting the efficient recruitment of p300 by promoter-bound, phosphorylated CREB.