Dale G. Schaar

miR-320 targets transferrin receptor 1 (CD71) and inhibits cell proliferation

OBJECTIVE MicroRNAs (miRNAs) have been implicated in complex vertebrate developmental systems, such as hematopoiesis, and may play an integral role in the development of human cancers. Based on these observations, we investigated the contribution of miRNAs to acute myelogenous leukemia cell lineage-specific differentiation. MATERIALS AND METHODS To facilitate the identification of miRNAs and their targets relevant to leukemic cell differentiation, changes miRNA expression were analyzed in the human leukemia cell line HL-60, which historically has been utilized to study lineage-specific changes in response to the differentiation agent 12-O-tetradecanoylphorbol-13-acetate (TPA). RESULTS Using this approach, we have identified a panel of TPA-induced miRNAs that are expressed coincident with HL-60 stereotypic morphological changes characteristic of monocytic differentiation. The transferrin receptor 1(TfR-1; CD71), whose surface expression is downregulated during TPA-mediated HL-60 cell differentiation, has been identified as a target of the TPA-induced miRNA miR-320. Cell culture experiments indicate that enforced miR-320 expression can suppress TfR-1 expression and cell proliferation. CONCLUSION TPA induces the expression of several miRNAs in HL-60 cells, one such miRNA (miR-320) contributes to downregulation of TfR-1 surface expression characteristically seen during HL-60 monocytic differentiation. Moreover, TfR-1-targeting miRNAs, such as miR-320, may have potential as novel therapeutic agents for cancer due to their inhibitory effects on cell proliferation.

Differentiation-associated miR-22 represses Max expression and inhibits cell cycle progression.

Differentiation agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) engage cell signaling pathways that activate downstream transcriptional programs necessary for cell differentiation. Recent evidence has indicated microRNAs (miRNAs) are an integral part of these transcriptional programs, which target key proteins and impact cell growth thereby facilitating changes required for differentiation. To further investigate the role of miRNAs in cell growth and differentiation, we focused on miR-22, a miRNA induced by TPA in the HL-60 leukemia cell line model of monocytic differentiation. TPA-induced miR-22 transcription was found to be downstream of the protein kinase c (PKC)-extracellular regulated kinase (ERK) signaling module, a pathway central to the growth and differentiation of many different cell types. Enforced miR-22 expression inhibited the growth of several different cancer cell lines, causing an accumulation of cells in the G1 phase of the cell cycle. The mechanism of miR-22s inhibitory effects involves targeting of the obligate c-Myc binding partner Max. Enforced miR-22 expression presumably lowers Max levels available for Myc binding, which differentially influenced the transcription of downstream targets of the Myc-Max complex. Our study provides additional support for miRNAs targeting key cellular regulatory microcircuits such as those governed by the Myc-Max transcriptional complex as well as their being active participants in cell growth and differentiation.

Multiple Astrocyte Transcripts Encode Nigral Trophic Factors in Rat and Human

The recent discovery of glial cell line-derived neurotrophic factor (GDNF) identified a novel trophin that selectively increases survival of substantia nigra dopaminergic neurons, which degenerate in Parkinsons disease. Our previous studies indicated that GDNF RNA can be amplified from cultured rat nigral type 1 astrocytes and from rat striatum in vivo, implying local as well as target trophic support. The current study establishes the regional pattern of GDNF RNA expression in adult human brain. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the highest expression of GDNF mRNA in the human caudate, with low levels in the putamen and no detectable message in the nigra, suggesting that GDNF is a target-derived factor in humans. We also report the isolation of two additional GDNF-related cDNAs, termed astrocyte-derived trophic factors (ATF), which apparently result from differential RNA processing. Sequence analysis of rat ATF-1 revealed a 78-bp deletion corresponding to a loss of 26 amino acids within the prepro region of the predicted GDNF protein. The RNA processing events responsible for ATF-1 formation in rat brain are conserved in humans; we report the isolation of a full-length human ATF-1 homologue. We identified a second alternative transcript, human ATF-2; the transcript encodes a protein which differs in its first 18 amino acids from the predicted mature GDNF and ATF-1 proteins and shares the terminal 115 residues with the other two forms. To begin assessing the biologic significance of multiple transcript expression we characterized the actions of COS-expressed GDNF and ATF-1 cDNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel structure of the human GDNF gene.

Glial cell line-derived neurotrophic factor (GDNF) is the most potent known survival factor for substantia nigra neurons, which degenerate in Parkinsons disease, for spinal motoneurons, which die in Lou Gehrigs disease (ALS), and for Purkinje neurons, the critical outflow cells of the cerebellum. Moreover, targeted deletion of the GDNF gene results in renal dysgenesis and abnormal development of the enteric nervous system. GDNF mRNA is expressed in a complex temporospatial pattern in the central nervous system and the periphery, consistent with these observations. To begin elucidating mechanisms regulating the pattern of expression of GDNF, we have cloned the human gene, and characterized the promoter. The promoter is highly GC rich, and lacks canonical CCAT-box and TATA-box motifs. It contains more than 12 binding sites for known transcription factors. These cis-elements have the potential to interact with factors regulating constitutive expression (Sp1) and developmental expression (bHLH). Moreover, the promoter contains sites for binding transcription factors which respond to environmental signals, including CREB, AP2, Zif/268, NFkB, and MRE-BP. Combinatorial actions of these transcription factors may account for the extraordinarily complex expression patterns of the GDNF gene. Importantly, we demonstrate that the hGDNF gene utilizes a promoter distinct from that identified in the rodent GDNF gene, a finding with ramifications for Parkinsons disease and ALS research.

A phase 2 randomized multicenter study of 2 extended dosing schedules of oral ezatiostat in low to intermediate-1 risk myelodysplastic syndrome

Ezatiostat is a glutathione analog prodrug glutathione S‐transferase P1‐1 (GSTP1‐1) inhibitor. This study evaluated 2 extended dose schedules of oral ezatiostat in 89 heavily pretreated patients with low to intermediate‐1 risk myelodysplastic syndrome (MDS).

Antineoplastic Effects of Partially HLA-Matched Irradiated Blood Mononuclear Cells in Patients With Renal Cell Carcinoma

PURPOSE Vaccines, cytokines, and other biologic-based therapies are being developed as antineoplastic agents. Many of these agents are designed to induce an autologous immune response directed against the malignancy. In contrast, hematopoietic stem-cell transplantation is being developed as a form of allogeneic immunotherapy. This study tests the tolerance and antineoplastic activity of sequential infusions of partially HLA-matched allogeneic blood mononuclear cells (obtained from relatives) when administered outside of the context of a hematopoietic stem-cell transplantation. The cells are irradiated to prevent graft-versus-host disease. PATIENTS AND METHODS Fifteen patients with relapsed or refractory malignancies for which no standard therapy was available were enrolled onto a clinical trial designed to assess the tolerability and antineoplastic effects of irradiated partially HLA-matched blood mononuclear cells obtained from relatives. RESULTS There was disease regression in three patients with metastatic renal cell carcinoma during treatment. There was disease progression in six patients with metastatic renal cell carcinoma and two patients with metastatic melanoma during treatment. There was no change in disease state in several other patients. CONCLUSION Irradiated allogeneic blood mononuclear cells administered outside the context of hematopoietic stem-cell transplantation may induce disease responses in patients with relapsed or refractory malignancies. Transfusion of irradiated allogeneic blood mononuclear cells should be developed further as a novel therapeutic antineoplastic approach.

The identification of a novel cDNA preferentially expressed in the olfactory-limbic system of the adult rat

To analyze cell-specific brain gene expression, we have developed a PCR-based subtractive hybridization cloning method utilizing trace starting material, allowing isolation of novel genes expressed under specific conditions. Our previous studies indicated that local substantia nigra (SN) type 1 astrocytes elaborate an array of trophic molecules which support the survival of SN dopaminergic neurons. Therefore, the current study focused on astrocyte gene expression utilizing a type 1 astrocyte-enriched cDNA library. We report initial characterization of a novel cDNA, designated AT1-46, that is preferentially expressed in the olfactory-limbic system of the adult rat brain. Although AT1-46 is expressed widely in the periphery, it is regulated both developmentally and in a cell-specific fashion in the brain. Structurally, AT1-46 is predicted to encode a highly alpha-helical molecule with several domains of potential coiled coil formation, and exhibits a 28% amino acid sequence identity with the intermediate filament-associated protein, trichohyalin.

Nuclear Factor-κB Modulation in Patients Undergoing Induction Chemotherapy for Acute Myelogenous Leukemia

Purpose: Nuclear factor-κB (NF-κB) is constitutively expressed in many acute myelogenous leukemia (AML) cells and AML stem cells. Ex vivo treatment of AML cells with inhibitors of NF-κB results in diminished AML cell survival and enhances the cytotoxic effects of chemotherapeutic agents. The purpose of this study was to determine if standard anti-inflammatory agents modulate AML cell nuclear NF-κB when administered in conjunction with induction chemotherapy. Experimental Design: Patients with newly diagnosed AML were treated with dexamethasone, choline magnesium trisalicylate, or both for 24 hours prior to and 24 hours following initiation of standard induction chemotherapy. AML cell nuclear NF-κB was measured at baseline, 24, and 48 hours. Results: Choline magnesium trisalicylate ± dexamethasone decreased nuclear NF-κB, whereas dexamethasone alone was associated with an increase in nuclear NF-κB in AML cells. Conclusions: These results show the feasibility of NF-κB modulation in conjunction with induction chemotherapy for patients with AML using inexpensive readily available medications. A follow-up study to determine the effects of NF-κB modulation on clinical end points is warranted.

PDCD2 functions in cancer cell proliferation and predicts relapsed leukemia

PDCD2 is an evolutionarily conserved eukaryotic protein with unknown function. The Drosophila PDCD2 ortholog Zfrp8 has an essential function in fly hematopoiesis. Zfrp8 mutants exhibit marked lymph gland hyperplasia that results from increased proliferation of partially differentiated hemocytes, suggesting Zfrp8 may participate in cell growth. Based on the above observations we have focused on the role of PDCD2 in human cancer cell proliferation and hypothesized that aberrant PDCD2 expression may be characteristic of human malignancies. We report that PDCD2 is highly expressed in human acute leukemia cells as well as in normal hematopoietic progenitors. PDCD2 knockdown in cancer cells impairs their proliferation, but not viability relative to parental cells, supporting the notion that PDCD2 overexpression facilitates cancer cell growth. Prospective analysis of PDCD2 in acute leukemia patients indicates PDCD2 RNA expression correlates with disease status and is a significant predictor of clinical relapse. PDCD2’s role in cell proliferation and its high expression in human malignancies make it an attractive, novel potential molecular target for new anti-cancer therapies.

A Pilot study of allogeneic cellular therapy for patients with advanced hematologic malignancies

Allogeneic hematopoietic stem cell transplantation provides curative therapy for some patients with advanced hematologic malignancies. Disease response after allogeneic transplant is, at least in part, mediated by donor immune cells. In this report we describe a cellular therapy using haploidentical peripheral blood stem cells administered after very low dose total body irradiation (TBI) (100cGy). The donor cells were anticipated to be rejected, so no graft-versus-host (GVHD) prophylaxis was used. Patients with persistent disease beyond 8 weeks could be further treated with infusions of irradiated haploidentical donor cells. Of the 10 patients enrolled in the study, durable engraftment of allogeneic cells was seen in one patient. Two patients with resistant relapsed acute myelogenous leukemia (AML) had a disease response. Analysis of T cell reactivity from one patient who achieved a complete response but did not have durable engraftment of donor cells indicated that disease response was associated with the generation of host-derived anti-leukemic cytotoxic CD8+ T cells that reacted with an AML-associated proteinase 3 epitope. Results from this patient suggest that allogeneic therapy induced a host anti-tumor response associated with cytotoxic T cells reactive with a low affinity self-antigen.