Fabrizio Padula

Testicular FasL is expressed by sperm cells

The testis is the main source of Fas ligand (FasL) mRNA in rodents; it is generally believed that this molecule, expressed on bordering somatic Sertoli cells, bestows an immune-privileged status in the testis by eliminating infiltrating inflammatory Fas-bearing leukocytes. Our results demonstrate that the attribution of testicular expression of FasL to Sertoli cells is erroneous and that FasL transcription instead occurs in meiotic and postmeiotic germ cells, whereas the protein is only displayed on mature spermatozoa. These findings point to a significant role of the Fas system in the biology of mammalian reproduction.

Toll-like receptor 3 triggers apoptosis of human prostate cancer cells through a PKC-α-dependent mechanism

Toll-like receptors (TLRs) are known to play a key role in the innate immune system particularly in inflammatory response against invading pathogens. Recent reports strongly indicate that they play important roles in cancer cells. Prostate cancer represents one of the most common cancer for which no cure is available once metastatic and androgen refractory. Since TLR3 has been recently suggested as a possible therapeutic target in some cancer cell lines, we studied TLR3 expression and functionality in two human prostate cancer cell lines, LNCaP and PC3. We report that both cell lines express TLR3 and that the TLR3 agonist poly (I:C) activates mitogen-activated protein kinases and induces inhibition of proliferation as well as caspase-dependent apoptosis. By using pharmacological and genetic approaches, we demonstrate the involvement of TLR3 in poly (I:C)-induced effects. We also show that a novel interferon-independent pathway involving protein kinase C (PKC)-alpha activation, upstream of p38 and c-jun N-terminal kinase, is responsible for poly (I:C) pro-apoptotic effects on LNCaP cells. To our knowledge, this is the first report describing a role of PKC-alpha in poly (I:C)-mediated apoptosis. The comprehension of the mechanisms underlying TLR3-mediated apoptosis can contribute tools to develop new agonists useful for the treatment of prostate cancer.

Histone Deacetylase Inhibitor Valproic Acid Enhances the Cytokine-Induced Expansion of Human Hematopoietic Stem Cells

Ex vivo amplification of human hematopoietic stem cells (HSC) without loss of their self-renewing potential represents an important target for transplantation, gene and cellular therapies. Valproic acid is a safe and widely used neurologic agent that acts as a potent inhibitor of histone deacetylase activities. Here, we show that valproic acid addition to liquid cultures of human CD34+ cells isolated from cord blood, mobilized peripheral blood, and bone marrow strongly enhances the ex vivo expansion potential of different cytokine cocktails as shown by morphologic, cytochemical, immunophenotypical, clonogenic, and gene expression analyses. Notably, valproic acid highly preserves the CD34 positivity after 1 week (range, 40-89%) or 3 weeks (range, 21-52%) amplification cultures with two (Flt3L + thrombopoietin) or four cytokines (Flt3L + thrombopoietin + stem cell factor + interleukin 3). Moreover, valproic acid treatment increases histone H4 acetylation levels at specific regulatory sites on HOXB4, a transcription factor gene with a key role in the regulation of HSC self-renewal and AC133, a recognized marker gene for stem cell populations. Overall, our results relate the changes induced by valproic acid on chromatin accessibility with the enhancement of the cytokine effect on the maintenance and expansion of a primitive hematopoietic stem cell population. These findings underscore the potentiality of novel epigenetic approaches to modify HSC fate in vitro.

Sequential valproic acid/all-trans retinoic acid treatment reprograms differentiation in refractory and high-risk acute myeloid leukemia

Epigenetic alterations of chromatin due to aberrant histone deacetylase (HDAC) activity and transcriptional silencing of all-trans retinoic acid (ATRA) pathway are events linked to the pathogenesis of acute myeloid leukemia (AML) that can be targeted by specific treatments. A pilot study was carried out in eight refractory or high-risk AML patients not eligible for intensive therapy to assess the biological and therapeutic activities of the HDAC inhibitor valproic acid (VPA) used to remodel chromatin, followed by the addition of ATRA, to activate gene transcription and differentiation in leukemic cells. Hyperacetylation of histones H3 and H4 was detectable at therapeutic VPA serum levels (>or=50 microg/mL) in blood mononuclear cells from seven of eight patients. This correlated with myelomonocytic differentiation of leukemic cells as revealed by morphologic, cytochemical, immunophenotypic, and gene expression analyses. Differentiation of the leukemic clone was proven by fluorescence in situ hybridization analysis showing the cytogenetic lesion +8 or 7q- in differentiating cells. Hematologic improvement, according to established criteria for myelodysplastic syndromes, was observed in two cases. Stable disease and disease progression were observed in five and one cases, respectively. In conclusion, VPA-ATRA treatment is well tolerated and induces phenotypic changes of AML blasts through chromatin remodeling. Further studies are needed to evaluate whether VPA-ATRA treatment by reprogramming differentiation of the leukemic clone might improve the response to chemotherapy in leukemia patients.

MicroRNA-128-2 targets the transcriptional repressor E2F5 enhancing mutant p53 gain of function

p53 mutations have profound effects on non-small-cell lung cancer (NSCLC) resistance to chemotherapeutic treatments. Mutant p53 proteins are usually expressed at high levels in tumors, where they exert oncogenic functions. Here we show that p53R175H, a hotspot p53 mutant, induces microRNA (miRNA)-128-2 expression. Mutant p53 binds to the putative promoter of miR128-2 host gene, ARPP21, determining a concomitant induction of ARPP21 mRNA and miR-128-2. miR-128-2 expression in lung cancer cells inhibits apoptosis and confers increased resistance to cisplatin, doxorubicin and 5-fluorouracyl treatments. At the molecular level, miR-128-2 post-transcriptionally targets E2F5 and leads to the abrogation of its repressive activity on p21waf1 transcription. p21waf1 protein localizes to the cytoplasmic compartment, where it exerts an anti-apoptotic effect by preventing pro-caspase-3 cleavage. This study emphasizes miRNA-128-2 role as a master regulator in NSCLC chemoresistance.

TNF-alpha and IFN-gamma regulate expression and function of the Fas system in the seminiferous epithelium

Sertoli cells have long been considered to be involved in the regulation of the immune response in the testis. More recently, the Fas system has been implicated in the maintenance of the immune privilege in the testis as well as in the regulation of germ cell apoptosis. However, the control of Fas and Fas ligand (FasL) expression in the testis remains unknown. In the present study, we demonstrate that cultured mouse Sertoli cells constitutively express a low level of membrane-bound Fas protein, but not a soluble form of Fas. Sertoli cells stimulated with TNF-α and IFN-γ markedly increase the expression of both soluble and membrane-bound Fas in a dose-dependent manner. The up-regulated membrane-bound Fas protein is functionally active because it induces a significant level of Sertoli cell death in the presence of Neuro-2a FasL+ effector cells. Interestingly, the soluble form of Fas, which is induced by the same cytokines but has an antiapoptotic effect, is also functional. In fact, conditioned media from TNF-α-stimulated Sertoli cell cultures inhibit Neuro-2a FasL+-induced cell death. Taken together, our data suggest a possible regulatory role of TNF-α and IFN-γ on Fas-mediated apoptosis in the testis through disruption of the balance between different forms of Fas.

Critical Role of c-Myc in Acute Myeloid Leukemia Involving Direct Regulation of miR-26a and Histone Methyltransferase EZH2

Increased expression or aberrant activation of c-Myc plays an important role in leukemogenesis. Here, we show that in acute myeloid leukemia (AML), c-Myc directly controls the expression of EZH2, a component of the Polycomb repressive complex 2, and miR-26a. miR-26a is downregulated in primary blasts from AML patients and, during myeloid differentiation of AML cells, is induced together with a decrease in c-Myc and Ezh2 levels. Previously, EZH2 was shown to be regulated by miR-26a at the translational levels in lymphomas. However, we demonstrate that in AML, the variation of EZH2 mainly depends on c-Myc transcriptional control. We also show that enforced expression of miR-26a in AML cells is able to inhibit cell cycle progression by downregulating cyclin E2 expression. In addition, increased levels of miR-26a potentiate the antiproliferative effects of 1,25-dihydroxyvitamin D(3) (VitD) and stimulate myeloid differentiation. Our results identify new molecular targets of c-Myc in AML and highlight miR-26a attractiveness as a therapeutic target in leukemia.

Mouse Sertoli Cells Display Phenotypical and Functional Traits of Antigen-Presenting Cells in Response to Interferon Gamma

Abstract The testis is regarded as an immunologically privileged site because it tolerates either autoantigenic germ cells or allografts. Because the blood testis barrier represents an incomplete immunological barrier, we have explored whether Sertoli cells, the somatic cells of the seminiferous epithelium, might play an active role in immune evasion. We report data indicating that B7-H1(officially known as CD274)-mediated co-inhibition, an immunomodulatory mechanism based on cell-cell interaction, can be activated in Sertoli cell-lymphocyte cocultures. We have found that, in response to interferon gamma (IFNG), mouse Sertoli cells strongly up-regulate the negative co-stimulatory ligand B7-H1 but remain devoid of positive co-stimulatory molecules. Blockade of B7-H1 on the Sertoli cell surface resulted in enhanced proliferation of CD8+ T cells cocultured with Sertoli cells. Moreover, IFNG-stimulated Sertoli cells were found to express, concurrent with B7-H1, MHC class II. Therefore, we have hypothesized that Sertoli cells could function as nonprofessional tolerogenic antigen-presenting cells by inducing enrichment in regulatory T cells (Tregs) in a mixed T lymphocyte population. Interestingly, we found that coculturing T cells with Sertoli cells can indeed induce an increase in CD4+CD25+(officially known as IL2RA)FOXP3+ Tregs and a decrease in CD4+CD25− T cells, suggesting Sertoli cell-mediated Treg conversion; this process was found to be B7-H1-independent. Altogether these data show that Sertoli cells are potentially capable of down-regulating the local immune response, on one hand by directly inhibiting CD8+ T cell proliferation through B7-H1 and, on the other hand, by inducing an increase in Tregs that might suppress other bystander T cells.

The microRNA-26a target E2F7 sustains cell proliferation and inhibits monocytic differentiation of acute myeloid leukemia cells.

Blocks in genetic programs required for terminal myeloid differentiation and aberrant proliferation characterize acute myeloid leukemia (AML) cells. 1,25-Dihydroxy-vitamin D3 (VitD3) arrests proliferation of AML cells and induces their differentiation into mature monocytes. In a previous study, we showed that miR-26a was induced upon VitD3-mediated monocytic differentiation. Here, we identify E2F7 as a novel target of miR-26a. We show that E2F7 significantly promotes cell cycle progression and inhibits monocytic differentiation of AML cells. We also demonstrate that E2F7 binds the cyclin-dependent kinase inhibitor p21CIP1/WAF1 (cyclin-dependent kinase inhibitor 1A) promoter repressing its expression. Moreover, interfering with E2F7 expression results in inhibition of c-Myc (v-myc myelocytomatosis viral oncogene homolog) transcriptional activity. This leads to the downregulation of c-Myc transcriptional target miR-17-92 cluster, whose expression has a well-defined role in contributing to block monocytic differentiation and sustain AML cell proliferation. Finally, we show that the expression of E2F7 is upregulated in primary blasts from AML patients. Thus, these findings indicate that the newly identified miR-26a target E2F7 might have an important role in monocytic differentiation and leukemogenesis.

FLIP is expressed in mouse testis and protects germ cells from apoptosis

AbstractApoptosis control in adult testis is crucial to achieve normal spermatogenesis. In this study c-FLIP, an apoptosis-modulating protein, was investigated. In Western blot and immunohistochemical analyses, the 55 KDa c-FLIP long isoform (c-FLIPL) was found to be expressed strongly in spermatocytes and spermatids, at low levels in spermatogonia and at almost undetectable levels in Sertoli cells. This expression pattern was confirmed by Northern blot analyses. Further experiments carried out on GC-1spg germ cell line revealed that reducing c-FLIPL expression increases Fas-dependent apoptosis. Conversely, restoring c-FLIPL expression reduces this response to control levels. Caspase-10 expression was found to match c-FLIPL expression pattern; further, caspase-10 activation upon anti-Fas treatment inversely correlated with c-FLIPL expression. Finally, TUNEL staining of seminiferous tubules incubated with anti-Fas antibody showed that apoptosis occurs mostly in basally located germ cells, indicating that such cells, expressing low levels of c-FLIPL, are sensitive to Fas-mediated apoptosis.These data indicate for the first time that c-FLIPL might control germ cell apoptosis and caspase activity in the adult testis.