Armando Felsani

Human p300 Protein Is a Coactivator for the Transcription Factor MyoD

Human p300 protein is a cellular target of adenoviral E1A oncoprotein and a potential transcriptional coactivator. Both p300 and Rb family protein-binding regions of E1A are required for the repression of muscle gene expression, which is regulated by MyoD family transactivators. This implies that p300 is involved in MyoD-dependent transactivation. We show that the repression of MyoD-mediated E box (MyoD consensus) reporter activity by E1A is correlated with its interaction with p300, indicating that p300 participates in MyoD-dependent transactivation. In addition, p300 is able to interact both in vivo and in vitro with MyoD through a portion at the carboxyl-terminal cysteine/histidine-rich domain and associates with the components of the basal transcriptional complex through its two separate transactivation domains at the amino and carboxyl termini. Consistent with its role as a coactivator, p300 potentiates MyoD-activated transcription.

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

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.

Retinoblastoma family proteins as key targets of the small DNA virus oncoproteins.

RB, the most investigated tumor suppressor gene, is the founder of the RB family of growth/tumor suppressors, which comprises also p107 (RBL1) and Rb2/p130 (RBL2). The protein products of these genes, pRb, p107 and pRb2/p130, respectively, are also known as ‘pocket proteins’, because they share a ‘pocket’ domain responsible for most of the functional interactions characterizing the activity of this family of cellular factors. The interest in these genes and proteins springs essentially from their ability to regulate negatively cell cycle processes and for their ability to slow down or abrogate neoplastic growth. The pocket domain of the RB family proteins is dramatically hampered in its functions by the interference of a number of proteins produced by the small DNA viruses. In the last two decades, the ‘viral hypothesis’ of cancer has received a considerable renewed impulse from the notion that small DNA viruses, such as Adenovirus, Human papillomavirus (HPV) and Polyomavirus, produce factors that can physically interact with major cellular regulators and alter their function. These viral proteins (oncoproteins) act as multifaceted molecular devices that have evolved to perform very specific tasks. Owing to these features, viral oncoproteins have been widely employed as invaluable experimental tools for the identification of several key families of regulators, particularly of the cell cycle homeostasis. Adenovirus early-region 1A (E1A) is the most widely investigated small DNA tumor virus oncoprotein, but relevant interest in human oncology is raised by the E1A-related E7 protein from transforming HPV strains and by Polyomavirus oncoproteins, particularly large and small T antigens from Simian virus 40, JC virus and BK virus.

Critical Role Played by Cyclin D3 in the MyoD-Mediated Arrest of Cell Cycle during Myoblast Differentiation

ABSTRACT During the terminal differentiation of skeletal myoblasts, the activities of myogenic factors regulate not only tissue-specific gene expressions but also the exit from the cell cycle. The induction of cell cycle inhibitors such as p21 and pRb has been shown to play a prominent role in the growth arrest of differentiating myoblasts. Here we report that, at the onset of differentiation, activation by MyoD of the Rb, p21, and cyclin D3 genes occurs in the absence of new protein synthesis and with the requirement of the p300 transcriptional coactivator. In differentiated myocytes, cyclin D3 also becomes stabilized and is found nearly totally complexed with unphosphorylated pRb. The detection of complexes containing cyclin D3, cdk4, p21, and PCNA suggests that cdk4, along with PCNA, may get sequestered into high-order structures held together by pRb and cyclin D3. Cyclin D3 up-regulation and stabilization is inhibited by adenovirus E1A, and this correlates with the ability of E1A to promote pRb phosphorylation; conversely, the overexpression of cyclin D3 in differentiated myotubes counteracts the E1A-mediated reactivation of DNA synthesis. These results indicate that cyclin D3 critically contributes to the irreversible exit of differentiating myoblasts from the cell cycle.

MyoD stimulates RB promoter activity via the CREB/p300 nuclear transduction pathway

ABSTRACT 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.

Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer.

The human intestinal Caco‐2 cell line has been extensively used as a model of the intestinal barrier. However, it is widely reported in literature that culture‐related conditions, as well as the different Caco‐2 cell lines utilized in different laboratories, often lead to problems of reproducibility making difficult to compare results. We developed a new cell‐maintenance protocol in which Caco‐2 cells were subcultured at 50% of confluence instead of 80% of confluence, as usually suggested. Using this new protocol, Caco‐2 cells retained a higher proliferation potential resulting in a cell population, which, on reaching confluence, was able to differentiate almost synchronously, forming a more homogeneous and polarized cell monolayer, as compared to that obtained using a high cell growing density. This comparison has been done by analyzing the gene expression and the structural characteristics of the 21‐days differentiated monolayers by microarrays hybridization and by confocal microscopy. We then investigated if these differences could also modify the effects of toxicants on 21‐days‐differentiated cells. We analyzed the 2 h‐acute toxicity of CuCl2 in terms of actin depolymerization and metallothionein 2A (MT2A) and heat shock protein 70 (HSPA1A) genes induction. Copper treatment resulted in different levels of actin depolymerization and gene expression induction in relationship with culture protocol, the low‐density growing cells showing a more homogeneous and stronger response. Our results suggest that cell growing density could influence a number of morphological and physiological properties of differentiated Caco‐2 cells and these effects must be taken in account when these cells are used as intestinal model. J. Cell. Physiol. 226: 1531–1543, 2011.

Good Caco-2 cell culture practices.

The human Caco-2 cells differentiate spontaneously in culture forming monolayers of mature intestinal enterocytes which have been used as a model of the intestinal barrier for in vitro toxicology studies. Reproducibility problems often reported in literature have been generally ascribed to different culture-related conditions, such as the type of animal serum used, the supplements added to the culture media, the passage number and the source of cell clones. The Caco-2 cell culture protocol here described has been recently optimized in our laboratory, producing a homogeneous and highly polarized monolayer of cells which display many of the characteristics of the intestinal enterocytes. This protocol differs from standard protocols mainly because Caco-2 cells are subcultured when they reach just 50% of confluence, instead of 80%, retaining a high proliferation potential. When this cell population is seeded at high density on filter inserts differentiates almost synchronously and much more homogenously.

Identification of genes down-regulated during melanoma progression: a cDNA array study.

Abstract: In order to identify genes relevant for melanoma development, we carried out cDNA array experiments employing an in vitro model of human melanoma progression, consisting of two cell lines: one, LP, derived from a primary melanoma and the other, LM, from its metastatic supraclavicular lymph node. Basic cDNA array data identified 26 genes as down‐regulated in the LM cell line. Northern blot analysis confirmed an effective transcriptional down‐regulation for five out of 13 genes analyzed. The products of these five genes belong to different functional protein types, such as transcription and translation regulators (Edg‐2, eIF‐3 p110, and RNPL/RBM3), extracellular communicators (PRSS11) and members of the major histocompatibility complex (β2‐microglobulin). Some previously described differences in expression patterns, such as loss of HLA I, were confirmed by our array data. In addition, we identified and validated for the first time the reduced expression level of several genes during melanoma progression. In particular, reduced Edg‐2 gene product expression was also confirmed in a group of 50 primary melanomas and unrelated metastases. In conclusion, comparative hybridization by means of cDNA arrays assisted in identifying a series of novel progression‐associated changes in gene expression, confirming, at the same time, a number of previously described results.

Signaling by exosomal microRNAs in cancer

A class of small non-coding RNAs, the microRNAs (miRNAs), have recently attracted great attention in cancer research since they play a central role in regulation of gene-expression and miRNA aberrant expression is found in almost all types of human cancer. The discovery of circulating miRNAs in body fluids and the finding that they are often tumor specific and can be detected early in tumorigenesis has soon led to the evaluation of their possible use as cancer biomarkers and treatment-response predictors. The evidence that tumor cells communicate via the secretion and delivery of miRNAs packed into tumor-released microvesicles has prompted to investigate miRNA contribution as signaling molecules to the establishment and maintenance of the tumor microenvironment and the metastatic niche in cancer. In this review we highlight the recent advances on the role of exosomal miRNAs as mediators of cancer cell-to-cell communication.

Myc down-regulation induces apoptosis in M14 melanoma cells by increasing p27 kip1 levels

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 G1 phase of the cell cycle (up to 80%) and activation of apoptosis (50%). These data are associated with an increase of p27kip1 levels and a significant reduction of the cdk2-associated kinase activity. In addition, we show that an ectopic overexpression of p27kip1 in this experimental model can enhance the apoptotic rate. Our results indicate that down-regulation of Myc protein induces a G1 arrest and activates apoptosis by increasing p27kip1 content in melanoma cells, that are known to be defective for the p16-cyclinD/cdk4-pRb G1 checkpoint. This is particularly relevant for identifying new therapeutic strategies based on the re-establishment of the apoptotic pathways in cancer cells.