[Show abstract][Hide abstract] ABSTRACT: Ferritin is best known as the key molecule in intracellular iron storage, and is involved in several metabolic processes such as cell proliferation, differentiation and neoplastic transformation. We have recently demonstrated that the shRNA silencing of the ferritin heavy subunit (FHC) in a melanoma cell line is accompanied by a consistent modification of gene expression pattern leading to a reduced potential in terms of proliferation, invasiveness, and adhesion ability of the silenced cells. In this study we sought to define the repertoire of genes whose expression might be affected by FHC during the hemin-induced differentiation of the erythromyeloid cell line K562. To this aim, gene expression profiling was performed in four different sets of cells: i) wild type K562; ii) sh-RNA FHC-silenced K562; iii) hemin-treated wild-type K562; iv) hemin-treated FHC-silenced K562. Statistical analysis of the gene expression data, performed by two-factor ANOVA, identified three distinct classes of transcripts: a) Class 1, including 657 mRNAs whose expression is modified exclusively during hemin-induced differentiation of K562 cells, independently from the FHC relative amounts; b) Class 2, containing a set of 70 mRNAs which are consistently modified by hemin and FHC-silencing; c) Class 3, including 128 transcripts modified by FHC-silencing but not by hemin. Our data indicate that FHC may function as a modulator of gene expression during erythroid differentiation and add new findings to the knowledge of the complex gene network modulated during erythroid differentiation.
[Show abstract][Hide abstract] ABSTRACT: Objective
The molecular aspects involved in human implantation include many elements that were first discovered due to their role in cancer cell metastasis. Periostin, a cell adhesion protein that allows the maintenance of cancer stem cells, may influence implantation. The objective of this experimental case–control study was to investigate tissue and serum expression of periostin during pregnancy, and evaluate the potential role of periostin in endometrial receptivity and embryo implantation.
Forty-five subjects were included in the final analysis: 15 women who had experienced spontaneous pregnancy loss were enrolled as cases, and 30 healthy pregnant women awaiting voluntary pregnancy termination were enrolled as controls. For both cases and controls, trophoblastic and decidual tissues were collected at 12 weeks of gestation. Periostin expression in decidual and trophoblastic tissues was evaluated by immunohistochemical staining and reverse transcription polymerase chain reaction in cases and controls, and periostin serum levels were analysed by Western blotting assays in cases, controls and non-pregnant female volunteers.
Periostin mRNA and protein levels were higher in decidual and trophoblastic tissues from women undergoing voluntary pregnancy termination compared with women who had experienced spontaneous pregnancy loss. This finding was also reflected at serum level.
Periostin may be a serum marker of good endometrial receptivity and embryo quality, and predictive of pregnancy evolution.
European journal of obstetrics, gynecology, and reproductive biology 01/2013; 175(1). DOI:10.1016/j.ejogrb.2013.12.027 · 1.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ferritin, the major intracellular iron-storage protein, is made of 24 subunits of two types, H and L. Besides regulating intracellular iron homeostasis, it has been found that ferritin, in particular the H subunit (FHC), is involved in different biological events such as cell differentiation and pathologic states (i.e., neurodegeneration and cancer). This study is aimed at investigating the whole-cell proteome of FHC-expressing and sh-RNA-silenced human metastatic melanoma cells (MM07(m)) in the attempt to identify and classify the highest number of proteins directly or indirectly controlled by the FHC. We identified about 200 differentially expressed proteins and classified them in clusters on the basis of their functions, as proteins involved in metabolic processes, cell adhesion, migration, and proliferation processes. Some of them have captured our attention because of their involvement in metabolic pathways related to tumor progression and metastasis. In vitro assays confirmed that the FHC-silenced MM07(m) cells are characterized by a decreased growth activity, a reduced invasiveness, and a reduced cell adhesion capability. Moreover, nude mice (CD1 nu/nu), subcutaneously injected with FHC-silenced MM07(m) cells, showed a remarkable 4-fold reduction of their tumor growth capacity compared to those who received the FHC-unsilenced MM07(m) counterpart. In conclusion, these data indicate that gene silencing technology, coupled to proteomic analysis, is a powerful tool for a better understanding of H ferritin signaling pathways and lend support to the hypothesis that specific targeting of this gene might be an attractive and potentially effective strategy for the management of metastatic melanoma.
Journal of Proteome Research 11/2011; 10(12):5444-53. DOI:10.1021/pr200705z · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human DNA mismatch repair (MMR) is involved in the removal of DNA base mismatches that arise either during DNA replication or are caused by DNA damage. In this study, we show that the activation of the MMR component hMLH1 in response to doxorubicin (DOX) treatment requires the presence of BRCA1 and that this phenomenon is mediated by an ATM/ATR dependent phosphorylation of the hMLH1 Ser-406 residue. BRCA1 is an oncosuppressor protein with a central role in the DNA damage response and it is a critical component of the ATM/ATR mediated checkpoint signaling. Starting from a previous finding in which we demonstrated that hMLH1 is able to bind to BRCA1, in this study we asked whether BRCA1 might be the bridge for ATM/ATR dependent phosphorylation of the hMLH1 molecular partner. We found that: (i) the negative modulation of BRCA1 expression is able to produce a remarkable reversal of hMLH1 stabilization, (ii) BRCA1 is required for post-translational modification produced by DOX treatment on hMLH1 which is, in turn, attributed to the ATM/ATR activity, (iii) the serine 406 phosphorylatable residue is critical for hMLH1 activation by ATM/ATR via BRCA1. Taken together, our data lend support to the hypothesis suggesting an important role of this oncosuppressor as a scaffold or bridging protein in DNA-damage response signaling via downstream phosphorylation of the ATM/ATR substrate hMLH1.
The international journal of biochemistry & cell biology 08/2011; 43(12):1754-63. DOI:10.1016/j.biocel.2011.08.011 · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Periostin (POSTN), an osteoblast-specific secreted protein known to be associated with cell adhesion activity for bone formation and development by the epithelial cell-derived tumors, leads to a significant enhancement in angiogenesis and tumorigenesis. At present, little is known about the mechanisms underlying its transcriptional control either in physiological or neoplastic conditions. In this study we demonstrate that the ability of the human POSTN promoter to drive transcription mostly depends on the activity of YingYang-1 (YY1) zinc finger transcription factor. YY1, whose regulatory role in biology includes, besides transcriptional control, also chromatin remodeling, DNA damage repair and tumorigenesis, acts as a strong negative modulator of the POSTN expression. We retain that the identification of the functional role of YY1 in the transcriptional control of the human POSTN gene adds new insights in the studies focused on gene expression in normal and transformed cells.
[Show abstract][Hide abstract] ABSTRACT: Periostin (POSTN), an osteoblast-specific secreted protein involved in cell adhesion activity for bone formation and development by the epithelial cell-derived tumors, leads to a significant enhancement in angiogenesis and tumorigenesis. Despite the complex correlation of dysregulated periostin expression levels both in physiological and neoplastic conditions, very little is known about the mechanisms underlying its transcriptional control. Here, we report the initial characterization of the human POSTN gene promoter and show that its activity in HeLa cells depends on the binding of YingYang-1 (YY1) zinc finger transcription factor. We mapped, by ChIP and EMSA analysis, the functional YY1-binding site on human POSTN gene promoter at position - 604. In order to define the functional role of YY1 binding on the expression of the human periostin gene, we performed RNA interference experiments to knock down endogenous YY1 and analyzed the effects on periostin mRNA levels. We found, by RT-PCR experiments, a consistent and reproducible increase of POSTN transcript in the HeLa cells silenced for YY1. Conversely, over-expression of YY1 results in a significant down regulation of POSTN gene when compared to control HeLa cells. Next, ChIP and sequential ChIP showed that the repression observed on POSTN gene expression is not solely ascribable to the YY1 activity. In fact, immunoprecipitation experiments revealed that YY1 interact with BRCA1 to form a complex that co-ordinately represses POSTN expression via the functional YY1-binding site. These findings shed light in the transcriptional control of the POSTN gene expression and provide evidences supporting the potential role of this protein in BRCA1-dependent tumorigenesis.
Biochemistry for tomorrow's medicine. 36th FEBS Congress, Turin, Italy; 06/2011
[Show abstract][Hide abstract] ABSTRACT: Ferritin, the major intracellular iron-storage protein, is made of 24 subunits of two types, H and L. Besides regulating intracellular iron homeostasis, it has been found that ferritin, in particular H subunit (FHC), is involved in different biological events such as cell differentiation and pathologic states such as neurodegeneration and cancer. This study is aimed at investigating the wholecell proteome of the FHC-silenced MM07m cells, a cell line established from a supraclavicular lymph node metastasis, in
comparison with that of the wild-type counterpart, in the attempt to identify and classify the highest number of proteins directly or indirectly controlled by the FHC. More than 500 peptides were identified and classified in clusters on the basis of their functions, as proteins involved in metabolic processes, cell adhesion, migration
and proliferation processes. Some of them have captured our attention because of their involvement in metabolic processes related to tumor progression and metastasis. Among them: galectin- 1, a family of proteins that is essential in tumor angiogenesis, whose overexpression is often related to invasiveness; cathepsin B, that is involved in cancer metastasis by facilitating cell invasiveness and migration; integrin-a5, which is known to play a critical role in cell surface-mediated signaling and adhesion functions. In vitro assays have confirmed that the FHC-silenced MM07m cells are characterized by a decreased growth activity,
by a reduced invasiveness and a reduced cell adhesion capability. Moreover nude mice (CD1 nu/nu), subcutaneously-injected with FHC-silenced MM07m cells, have shown a remarkable 4-fold
reduction of their tumor growth capacity compared to those who received the FHC-unsilenced MM07m counterpart. In conclusion, the coupling of gene silencing technology with the proteomic analysis appears as an original and very promising approach to identify the molecular patterns in which the silenced gene might be involved.