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1.
Mol Ther ; 32(2): 372-383, 2024 Feb 07.
Article in English | MEDLINE | ID: mdl-38053334

ABSTRACT

Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.


Subject(s)
Epidermolysis Bullosa Simplex , Humans , Epidermolysis Bullosa Simplex/genetics , Epidermolysis Bullosa Simplex/therapy , Epidermolysis Bullosa Simplex/metabolism , Alleles , CRISPR-Cas Systems , Keratinocytes/metabolism , Mutation , Stem Cells/metabolism
2.
Ann Oncol ; 27(10): 1867-73, 2016 10.
Article in English | MEDLINE | ID: mdl-27484801

ABSTRACT

BACKGROUND: The aim of this work was to evaluate the impact of (and relative contribution of) tumor-related and immune-related diversity of HER2-positive disease on the response to neoadjuvant chemotherapy plus anti-HER2 agents. PATIENTS AND METHODS: The CherLOB phase II study randomized 121 HER2-positive breast cancer patients to neoadjuvant chemotherapy plus trastuzumab, lapatinib or both. Tumor samples from diagnostic core biopsy were centralized. Tumor-infiltrating lymphocytes (TILs) were evaluated on H&E slides. Intrinsic subtyping was carried out using the research-based 50-gene prediction analysis of a microarray (PAM50) subtype predictor. Immune-related gene signatures were also evaluated. RESULTS: Continuous Str-TILs and It-TILs were significantly associated with pCR [OR 1.03, 95% CI 1.02-1.05 (P < 0.001) and OR 1.09, 95% CI 1.04-1.15 (P < 0.001) for Str-TILs and It-TILs, respectively]. According to PAM50, the subtype distribution was as follows: HER2-enriched 26.7%, Luminal A 25.6%, Luminal B 16.3%, Basal-like 14% and Normal-like 17.4%. The highest rate of pCR was observed for the HER2-enriched subtype (50%), followed by Basal-like, Luminal B and Luminal A (χ(2) test, P = 0.026). Immune gene signatures significantly associated with pCR in univariate analyses were identified: most of them maintained a significant association with pCR in multivariate analyses corrected for PAM50 subtypes, whereas TILs did not. CONCLUSIONS: In this study, both tumor-related and immune-related features contribute to the modulation of pCR after neoadjuvant chemotherapy plus anti-HER2 agents. Immune signatures rather than TILs added significant prediction of pCR beyond PAM50 intrinsic subtypes.


Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/immunology , Receptor, ErbB-2/immunology , Adult , Aged , Biopsy, Large-Core Needle , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Humans , Lapatinib , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/pathology , Microarray Analysis , Middle Aged , Neoadjuvant Therapy , Prognosis , Quinazolines/administration & dosage , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-2/genetics , Receptors, Estrogen/genetics , Trastuzumab/administration & dosage
3.
Br J Dermatol ; 173(3): 731-8, 2015 Sep.
Article in English | MEDLINE | ID: mdl-25913354

ABSTRACT

BACKGROUND: Epidermolysis bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available for diagnosing EB involve immunohistochemistry of biopsy samples and transmission electron microscopy followed by single-candidate gene Sanger sequencing (SS), which are labour-intensive and expensive clinical pathways. OBJECTIVES: According to the recently published recommendations for the diagnosis and treatment of EB, the assessment of the mutational landscape is now a fundamental step for developing a comprehensive diagnostic path. We aimed to develop a customized, cost-effective amplicon panel for the complete and accurate sequencing of all the pathogenic genes already identified in EB, and to minimize the processing time required for the execution of the test and to refine the analysis pipeline to achieve cost-effective results from the perspective of a routine laboratory set-up. Next-generation sequencing (NGS) via the parallel ultra-deep sequencing of many genes represents a proper method for reducing the processing time and costs of EB diagnostics. MATERIALS AND METHODS: We developed an EB disease-comprehensive AmpliSeq panel to accomplish the NGS on an Ion Torrent Personal Genome Machine platform. The panel was performed on 10 patients with known genetic diagnoses and was then employed in eight family trios with unknown molecular footprints. RESULTS: The panel was successful in finding the causative mutations in all 10 patients with known mutations, fully confirming the SS data and providing proof of concept of the sensitivity, specificity and accuracy of this procedure. In addition to being consistent with the clinical diagnosis, it was also effective in the trios, identifying all of the variants, including ones that the SS missed or de novo mutations. CONCLUSIONS: The NGS and AmpliSeq were shown to be an effective approach for the diagnosis of EB, resulting in a cost- and time-effective 72-h procedure.


Subject(s)
Epidermolysis Bullosa/diagnosis , Mutation/genetics , Sequence Analysis, DNA/methods , Cell Adhesion Molecules/genetics , Collagen Type VII/genetics , Cost-Benefit Analysis , DNA/genetics , Epidermolysis Bullosa/economics , Epidermolysis Bullosa/genetics , Female , Heterozygote , Humans , Keratin-5/genetics , Male , Sequence Analysis, DNA/economics , Kalinin
4.
J Exp Med ; 178(2): 381-9, 1993 Aug 01.
Article in English | MEDLINE | ID: mdl-8340750

ABSTRACT

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation, but so far no definite function has been attributed to the product of this oncogene. To tackle this problem, the c-fes protooncogene expression has been inhibited in HL60 cells, and fresh leukemic promyelocytes of acute promyelocytic leukemia have been induced to differentiate with retinoic acid (RA) and dimethylsulfoxide (DMSO). Inhibition was obtained by incubating the cells with a specific c-fes antisense oligodeoxynucleotide. It was observed that the cells, rather than differentiating, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. This process was inhibited by granulocyte and granulocyte/macrophage colony-stimulating factor, but not by interleukin 3 (IL-3), IL-6, or stem cell factor. Our present results demonstrate that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of the c-fes gene product and treatment with RA-DMSO, is due to activation of programmed cell death. It is concluded that a possible role of the c-fes gene product is to exert an antiapoptotic effect during granulocytic differentiation.


Subject(s)
Apoptosis/genetics , Gene Expression/drug effects , Granulocytes/cytology , Oligonucleotides, Antisense/pharmacology , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins/genetics , Apoptosis/drug effects , Base Sequence , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Dimethyl Sulfoxide/pharmacology , Humans , Molecular Sequence Data , Proto-Oncogene Proteins c-fes , Tretinoin/pharmacology
5.
Cell Death Differ ; 15(1): 94-104, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17932502

ABSTRACT

Skeletal muscle fibers form in overlapping, but distinct phases that depend on the generation of temporally different lineages of myogenic cells. During primary myogenesis (E10.5-E12.5 in the mouse), embryonic myoblasts fuse homotypically to generate primary fibers, whereas during later development (E14.5-E17.5), fetal myoblasts differentiate into secondary fibers. How these myogenic waves are regulated remains largely unknown. Studies have been hampered by the lack of markers which would distinguish embryonic from fetal myoblast populations. We show here that the homeobox gene Arx is strongly expressed in differentiating embryonic muscle, downstream of myogenic basic helix-loop-helix (bHLH) genes. Its expression progressively decreases during development. When overexpressed in the C2C12 myogenic cell line, Arx enhances differentiation. Accordingly, it stimulates the transcriptional activity from the Myogenin promoter and from multimerized E-boxes when co-expressed with MyoD and Mef2C in CH310T1/2. Furthermore, Arx co-immunoprecipitates with Mef2C, suggesting that it participates in the transcriptional regulatory network acting in embryonic muscle. Finally, embryonic myoblasts isolated from Arx-deficient embryos show a delayed differentiation in vivo together with an enhanced clonogenic capacity in vitro. We propose here that Arx acts as a novel positive regulator of embryonic myogenesis by synergizing with Mef2C and MyoD and by establishing an activating loop with Myogenin.


Subject(s)
Genes, Homeobox , Homeodomain Proteins/metabolism , Muscle Development , Muscle, Skeletal/embryology , Myoblasts, Skeletal/metabolism , Transcription Factors/metabolism , Animals , Cell Differentiation , Cell Line , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , MEF2 Transcription Factors , Mice , Mice, Mutant Strains , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , MyoD Protein/metabolism , Myoblasts, Skeletal/cytology , Myogenic Regulatory Factors/metabolism , Myogenin/metabolism , Transcription Factors/genetics
6.
Leukemia ; 32(2): 438-449, 2018 02.
Article in English | MEDLINE | ID: mdl-28745329

ABSTRACT

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.


Subject(s)
Bone Marrow/metabolism , Bone Marrow/pathology , Fibrosis/metabolism , Fibrosis/pathology , Osteopontin/metabolism , Proto-Oncogene Proteins c-maf/metabolism , Antigens, CD34/metabolism , Cell Proliferation/physiology , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Humans , Megakaryocytes/metabolism , Megakaryocytes/pathology , Myeloproliferative Disorders/metabolism , Myeloproliferative Disorders/pathology , Polycythemia Vera/metabolism , Polycythemia Vera/pathology , Primary Myelofibrosis/metabolism , Primary Myelofibrosis/pathology , Thrombocythemia, Essential/metabolism , Thrombocythemia, Essential/pathology
7.
Cell Death Differ ; 13(10): 1686-96, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16456583

ABSTRACT

Upregulation of specific transcription factors is a generally accepted mechanism to explain the commitment of hematopoietic stem cells along precise maturation lineages. Based on this premise, transduction of primary hematopoietic stem/progenitor cells with viral vectors containing the investigated transcription factors appears as a suitable experimental model to identify such regulators. Although MafB transcription factor is believed to play a role in the regulation of monocytic commitment, no demonstration is, to date, available supporting this function in normal human hematopoiesis. To address this issue, we retrovirally transduced cord blood CD34+ hematopoietic progenitors with a MafB cDNA. Immunophenotypic and morphological analysis of transduced cells demonstrated the induction of a remarkable monomacrophage differentiation. Microarray analysis confirmed these findings and disclosed the upregulation of macrophage-related transcription factors belonging to the AP-1, MAF, PPAR and MiT families. Altogether our data allow to conclude that MafB is a key regulator of human monocytopoiesis.


Subject(s)
Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , MafB Transcription Factor/genetics , MafB Transcription Factor/metabolism , Monocytes/cytology , Monocytes/metabolism , Antigens, CD34/metabolism , Cell Line , Colony-Forming Units Assay , DNA, Complementary/genetics , Fetal Blood/cytology , Fetal Blood/immunology , Fetal Blood/metabolism , Gene Expression Profiling , Hematopoietic Stem Cells/immunology , Humans , In Vitro Techniques , Infant, Newborn , MafB Transcription Factor/antagonists & inhibitors , Monocytes/immunology , Myelopoiesis , Oligonucleotide Array Sequence Analysis , RNA, Small Interfering/genetics , Retroviridae/genetics , Transduction, Genetic , Up-Regulation
8.
Leukemia ; 20(11): 1978-88, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16990782

ABSTRACT

Acute promyelocytic leukemia (APL) is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. Gene expression profiles of APL cells obtained from 16 patients were compared to eight samples of CD34+-derived normal promyelocytes. Malignant promyelocytes showed widespread changes in transcription in comparison to their normal counterpart and 1020 differentially expressed genes were identified. Discriminating genes include transcriptional regulators (FOS, JUN and HOX genes) and genes involved in cell cycle and DNA repair. The strong upregulation in APL of some transcripts (FLT3, CD33, CD44 and HGF) was also confirmed at protein level. Interestingly, a trend toward a transcriptional repression of genes involved in different DNA repair pathways was found in APL and confirmed by real-time polymerase chain reactor (PCR) in a new set of nine APLs. Our results suggest that both inefficient base excision repair and recombinational repair might play a role in APLs development. To investigate the expression pathways underlying the development of APL occurring as a second malignancy (sAPL), we included in our study eight cases of sAPL. Although both secondary and de novo APL were characterized by a strong homogeneity in expression profiling, we identified a small set of differentially expressed genes that discriminate sAPL from de novo cases.


Subject(s)
DNA Repair/genetics , Granulocyte Precursor Cells/pathology , Granulocyte Precursor Cells/physiology , Leukemia, Promyelocytic, Acute/genetics , Leukemia, Promyelocytic, Acute/pathology , Adult , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, CD34/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/metabolism , Cluster Analysis , Female , Flow Cytometry , Gene Expression Regulation, Leukemic , Humans , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Immunophenotyping , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Sialic Acid Binding Ig-like Lectin 3 , Transcription, Genetic , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/metabolism
9.
Leukemia ; 20(10): 1751-8, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16932344

ABSTRACT

Acute myeloid leukemia (AML) blasts are immature committed myeloid cells unable to spontaneously undergo terminal maturation, and characterized by heterogeneous sensitivity to natural differentiation inducers. Here, we show a molecular signature predicting the resistance or sensitivity of six myeloid cell lines to differentiation induced in vitro with retinoic acid or vitamin D. The identified signature was further validated by TaqMan assay for the prediction of response to an in vitro differentiation assay performed on 28 freshly isolated AML blast populations. The TaqMan assay successfully predicts the in vitro resistance or responsiveness of AML blasts to differentiation inducers. Furthermore, performing a meta-analysis of publicly available microarray data sets, we also show the accuracy of our prediction on known phenotypes and suggest that our signature could become useful for the identification of patients eligible for new therapeutic strategies.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/genetics , Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/genetics , Tretinoin/pharmacology , Acute Disease , Cell Differentiation/drug effects , Cell Line, Tumor , Cluster Analysis , Databases, Factual , Gene Expression Regulation, Leukemic/drug effects , Humans , Leukemia, Myeloid/pathology , Meta-Analysis as Topic , Oligonucleotide Array Sequence Analysis , Predictive Value of Tests , Reverse Transcriptase Polymerase Chain Reaction , Vitamin D/pharmacology , Vitamins/pharmacology
10.
Cell Death Differ ; 12(12): 1588-600, 2005 Dec.
Article in English | MEDLINE | ID: mdl-15947790

ABSTRACT

In spite of their apparently restricted differentiation potentiality, hematopoietic precursors are plastic cells able to trans-differentiate from a maturation lineage to another. To better characterize this differentiation plasticity, we purified CD14- and CD14+ myeloid precursors generated by 'in vitro' culture of human CD34+ hematopoietic progenitors. Morphological analysis of the investigated cell populations indicated that, as expected, they consisted of granulocyte and monocyte precursors, respectively. Treatment with differentiation inducers revealed that CD14- cells were bipotent granulo-monocyte precursors, while CD14+ cells appeared univocally committed to a terminal macrophage maturation. Flow cytometry analysis demonstrated that the conversion of granulocyte precursors to the mono-macrophage maturation lineage occurs through a differentiation transition in which the granulocyte-related myeloperoxidase enzyme and the monocyte-specific CD14 antigen are co-expressed. Expression profiling evidenced that the observed trans-differentiation process was accompanied by a remarkable upregulation of the monocyte-related MafB transcription factor.


Subject(s)
Antigens, CD34/immunology , Cell Differentiation/physiology , Hematopoietic Stem Cells/physiology , Lipopolysaccharide Receptors/immunology , RNA, Messenger/metabolism , Antigens, Differentiation/metabolism , Cell Lineage , Cells, Cultured , Flow Cytometry , Granulocytes/cytology , Granulocytes/immunology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Humans , MafB Transcription Factor/metabolism , Monocytes/cytology , Monocytes/immunology
11.
Cancer Res ; 50(18): 5825-8, 1990 Sep 15.
Article in English | MEDLINE | ID: mdl-1697501

ABSTRACT

The steady state levels of mRNAs codying for the ribosomal proteins S6, S11, and S14 have been evaluated in quiescent and proliferating human fibroblasts and in resting and proliferating human peripheral blood mononuclear cells. It was found that the amounts of ribosomal protein mRNA are very similar and are not increased by serum or mitogen stimulation. The constitutive expression of these genes appears to be coordinately regulated and it is not modified after protein synthesis inhibition by cycloheximide. The ribosomal protein mRNA was also assayed in 15 different populations of human leukemic blast cells. In these populations the abundance of each ribosomal protein mRNA is remarkably different from the other. The results of our present experiments indicate that the expression of the three ribosomal protein genes undergoes independent noncoordinated changes in the large majority of the leukemic populations studied.


Subject(s)
Gene Expression , Leukemia/genetics , Ribosomal Proteins/genetics , Blast Crisis/genetics , Cell Differentiation , Fibroblasts/metabolism , Humans , Leukemia/pathology , Leukocytes, Mononuclear/metabolism , RNA/analysis , Ribosomal Protein S6
12.
Cancer Res ; 52(1): 11-6, 1992 Jan 01.
Article in English | MEDLINE | ID: mdl-1727370

ABSTRACT

The relative abundance of primary transcript and mature mRNA of the c-myc, calcyclin, S14 ribosomal protein, and rRNA genes was determined densitometrically after reverse transcriptase-polymerase chain reaction and Northern blotting analysis in resting and mitogen-stimulated lymphocytes, proliferating and terminally differentiated HL-60 cells, and leukemic blast cells. Transcription and processing of c-myc and rRNA gene transcripts increased proportionally after mitogen stimulation, whereas these processes were independent of cell cycling status in the case of the S14 gene. Normal lymphocytes showed an unexpectedly large amount of primary transcript of the calcyclin gene, whereas the corresponding mRNA was undetectable. The abundance of c-myc, calcyclin, and S14 mRNA in terminally differentiated HL-60 cells decreased sharply, compared to their proliferating counterparts. This decrease reflected post-transcriptional modulation, since the abundance of precursor remained essentially unchanged. After HL-60 differentiation, the 32S rRNA levels remained relatively high, whereas the 45S primary transcript almost disappeared. Leukemic blast cells displayed highly variable precursor/mRNA ratios of c-myc, calcyclin, and S14 genes but consistently high ratios of 32S to 45S RNA, suggesting that the cleavage rate of the 32S rRNA was sharply reduced in these cells, resulting in an accumulation of this molecule. These results suggest the importance of efficient processing of primary transcript to generate adequate functional mRNA, thus regulating gene expression. Furthermore, in terminally differentiated cells and leukemic blast cells a stabilization of the primary transcript without efficient processing can be observed. The role of the stabilization of the primary transcript in terminal differentiation is further supported by the results of run-off transcription, indicating a sharp decrease of c-myc and calcyclin transcription rate in retinoic acid/dimethyl sulfoxide-treated HL-60 cells.


Subject(s)
Calcium-Binding Proteins/genetics , Cell Cycle Proteins , Gene Expression Regulation, Leukemic/genetics , Proto-Oncogene Proteins c-myc/genetics , RNA, Messenger/analysis , Ribosomal Proteins/genetics , S100 Proteins , Acute Disease , Base Sequence , Blotting, Northern , Cell Differentiation , Cell Division , DNA Probes , Humans , Leukemia, Myeloid/genetics , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/genetics , S100 Calcium Binding Protein A6
13.
Cancer Res ; 59(15): 3803-11, 1999 Aug 01.
Article in English | MEDLINE | ID: mdl-10446999

ABSTRACT

Different types of acute myeloid leukemia blast cells were induced to differentiate in vitro with all-trans-retinoic acid (ATRA) and vitamin D3 (VD). M0/M1 leukemic cells are not sensitive to differentiating agents, whereas M3 leukemic cells are induced to undergo granulocytic differentiation after ATRA treatment but are not sensitive to VD. M2 leukemic blast cells behave differently because they undergo monocytic differentiation with both the differentiation inducers. To gain some insight into the maturation of M2-type leukemic cells, we studied the molecular mechanisms underlying monocytic differentiation induced by ATRA and VD in spontaneous M2 blast cells as well as in Kasumi-1 cells (an acute myeloid leukemia M2-type cell line). Our results indicate that ATRA as well as VD efficiently increases the nuclear abundance of VD receptor (VDR) and promotes monocytic differentiation. VDR is functionally active in ATRA-treated Kasumi-1 cells because it efficiently heterodimerizes with retinoid X receptor, binds to a DR3-type vitamin D-responsive element, and activates the transcription of a vitamin D-responsive element-regulated reporter gene. Consistent with these findings, VD-responsive genes are induced by ATRA treatment of Kasumi-1 cells, suggesting that the genetic program underlying monocytic differentiation is activated. The molecular mechanism by which ATRA increases the nuclear abundance of a functional VDR is still unknown, but our data clearly indicate that the M2 leukemic cell context is only permissive of monocytic differentiation.


Subject(s)
Calcitriol/pharmacology , Leukemia, Myeloid, Acute/pathology , Monocytes/cytology , Neoplastic Stem Cells/metabolism , Receptors, Calcitriol/physiology , Receptors, Retinoic Acid/metabolism , Transcription Factors/metabolism , Tretinoin/pharmacology , Acute Disease , Cell Differentiation/drug effects , Cell Lineage , Cell Nucleus/metabolism , DNA/metabolism , Dimerization , Gene Expression Regulation, Leukemic/drug effects , HL-60 Cells/metabolism , Humans , Leukemia, Myeloid/pathology , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neoplastic Stem Cells/pathology , Promoter Regions, Genetic/drug effects , Protein Multimerization , Receptors, Calcitriol/genetics , Recombinant Fusion Proteins/physiology , Regulatory Sequences, Nucleic Acid , Retinoic Acid Receptor alpha , Retinoid X Receptors , Transcription, Genetic , Tumor Cells, Cultured
14.
Cancer Res ; 50(7): 1988-91, 1990 Apr 01.
Article in English | MEDLINE | ID: mdl-2180559

ABSTRACT

The abundance of the mRNAs of two growth-related genes, vimentin and c-myc, and that of the corresponding proteins have been studied in unstimulated and phytohemagglutinin-stimulated lymphocytes as well as in 18 populations of leukemic blast cells. The quantitative assay was carried out by densitometric scanning of Northern and Western blots. In normal lymphocytes the mRNA and the protein of both genes were almost undetectable. The phytohemagglutinin stimulation led to a sharp increase of the mRNA and the proteins of vimentin and c-myc. The increase was followed by a progressive fall of the gene products. The rate of decrease of the two mRNAs was similar to that of the corresponding proteins. In some leukemic populations very similar amounts of the vimentin protein were accompanied by amounts of the mRNA differing at least 25 times. Not unlikely, very similar amounts of p62c-myc corresponded to mRNA abundances differing at least 16 times. The coordinated biogenesis of both messenger RNAs and proteins, which occurs in mitogen-stimulated lymphocytes, is substituted, in approximately 30% of the leukemic blast cell populations, by molecular events leading to the accumulation of an excess of mRNA.


Subject(s)
Blast Crisis/genetics , Leukemia/genetics , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , Vimentin/genetics , Blast Crisis/pathology , Blotting, Northern , Blotting, Western , Cell Division , Gene Expression , Humans , Leukemia/pathology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-myc , Vimentin/metabolism
15.
Cell Death Dis ; 7: e2263, 2016 06 16.
Article in English | MEDLINE | ID: mdl-27310872

ABSTRACT

Mu-protocadherin (MUCDHL) is an adhesion molecule predominantly expressed by colorectal epithelial cells which is markedly downregulated upon malignant transformation. Notably, treatment of colorectal cancer (CRC) cells with mesalazine lead to increased expression of MUCDHL, and is associated with sequestration of ß-catenin on the plasma membrane and inhibition of its transcriptional activity. To better characterize the causal relationship between ß-catenin and MUCDHL expression, we performed various experiments in which CRC cell lines and normal colonic organoids were subjected to culture conditions inhibiting (FH535 treatment, transcription factor 7-like 2 siRNA inactivation, Wnt withdrawal) or stimulating (LiCl treatment) ß-catenin activity. We show here that expression of MUCDHL is negatively regulated by functional activation of the ß-catenin signaling pathway. This finding was observed in cell culture systems representing conditions of physiological stimulation and upon constitutive activation of ß-catenin in CRC. The ability of MUCDHL to sequester and inhibit ß-catenin appears to provide a positive feedback enforcing the effect of ß-catenin inhibitors rather than serving as the primary mechanism responsible for ß-catenin inhibition. Moreover, MUCDHL might have a role as biomarker in the development of CRC chemoprevention drugs endowed with ß-catenin inhibitory activity.


Subject(s)
Cadherins/genetics , Colonic Neoplasms/genetics , Enterocytes/metabolism , Gene Expression Regulation, Neoplastic , beta Catenin/genetics , Caco-2 Cells , Cadherin Related Proteins , Cadherins/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Enterocytes/drug effects , Enterocytes/pathology , Feedback, Physiological , HCT116 Cells , Humans , Lithium Chloride/pharmacology , Primary Cell Culture , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Sulfonamides/pharmacology , Tissue Culture Techniques , Transcription Factor 7-Like 2 Protein/antagonists & inhibitors , Transcription Factor 7-Like 2 Protein/genetics , Transcription Factor 7-Like 2 Protein/metabolism , Wnt Signaling Pathway/drug effects , beta Catenin/metabolism
16.
Cell Death Differ ; 2(3): 155-62, 1995 Jul.
Article in English | MEDLINE | ID: mdl-17180038

ABSTRACT

The main purpose of this report is to provide a review of the present knowledge on the structure, function, and possible regulatory role of c-fes in the genetic programs underlying the proliferation and differentiation of hematopoietic myeloid cells. Fes encodes a non-receptor tyrosine kinase that is highly expressed in immature and differentiated cells of the granulocytic and mono-macrophagic lineages. It is therefore possible that c-fes is involved in the signal transduction of myeloid cell differentiation, even if the specific substrates phosphorylated by this protooncogene are only poorly characterised. Several experimental models have been established to evaluate the role of c-fes in myeloid differentiation, in particular: the differentiation capacity of HL60 cells lacking the p92(c-fes) protein, the transfection of c-fes gene into K562 cells and transgenic animals overexpressing c-fes. The results obtained point to the importance of c-fes in myeloid cells, since it appears to be involved in granulocytic maturation as an antiapoptotic gene, and in macrophagic maturation as a regulatory gene.

17.
Cell Death Differ ; 9(11): 1185-95, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12404117

ABSTRACT

By high density oligonucleotide microarrays we have studied the expression profile of proliferating and VD treated HL60 cells and the molecular phenotype of VD monocytes and that of CD14+ peripheral monocytes has been compared. The results indicate that important changes in functional categories of the differentially expressed genes underlie the differentiation transition from myeloblasts to monocytes. This differential gene expression pattern leads to an increased expression of mRNAs involved in surface and external activities since many of the VD induced genes belong to ligand binding, receptors, cell surface antigens, defense/immunity and adhesion molecules functional categories. The results also indicate that the molecular phenotypes of monocytes and VD induced cells diverge for a small but significant set of defense related genes. Particularly, class II MHC genes are not expressed in these cells. Furthermore, the high levels of expression of these genes induced by serum treatment of monocytes are decreased by VD.


Subject(s)
Cell Differentiation/drug effects , Cholecalciferol/pharmacology , Gene Expression Profiling , Monocytes/drug effects , Cell Differentiation/immunology , Down-Regulation , Humans , Monocytes/immunology , Up-Regulation
18.
Cell Death Differ ; 4(6): 497-505, 1997 Aug.
Article in English | MEDLINE | ID: mdl-16465271

ABSTRACT

Although VDR is expressed in all the acute myeloid leukemia cell populations studied, most of these leukemias do not exhibit any phenotypic response when exposed to VD. To determine whether VD resistance is related to an altered VDR function, we performed an analysis of VDR expression, phosphorylation, DNA binding capacity and transactivation activity in several leukemic myeloid cell lines arrested at different levels of maturation. Our results indicate that VD induces a clear phenotypic effect, i.e. terminal monocytic differentiation, only in leukemic cells of M2/M3 (intermediate myeloblasts) and M5 (monoblasts) types but not in erythroid precursor cells, early leukemic myeloblasts (M0/M1 type) and promyelocytes (M3 type). VDR expression and function are evident in all the nuclear extracts obtained from the different myeloid cell lines after 12 h of VD treatment, but VD activation of monocytic differentiation is limited to a narrow differentiation window characterized by the M2 type myeloid cellular context.

19.
Cell Death Differ ; 8(1): 70-82, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11313705

ABSTRACT

Although all-trans retinoic acid (ATRA) can restore the differentiation capacity of leukemic promyelocytes, early leukemic myeloblasts are conversely not responsive to ATRA induced granulocytic differentiation. To assess whether this resistance to ATRA is related to an impaired function of the Retinoic Acid Receptor alpha (RARalpha), we performed an analysis of RARalpha expression and transactivation activity, in several myeloid leukemic cell lines, representative of different types of spontaneous acute myeloid leukemias. Our results indicate that a functionally active RARalpha nuclear receptor is expressed in all the analyzed cell lines, regardless of their differentiation capacity following exposure to ATRA. The observation that ATRA treatment is able to induce the expression of retinoic acid target genes, in late- but not in early-myeloblastic leukemic cells, raises the possibility that the differentiation block of these cells is achieved through a chromatin mediated mechanism. Acetylation is apparently not involved in this process, since the histone deacetylase inhibitor trichostatin A, is not able to restore the differentiation capacity of early leukemic myeloblasts. Further investigation is needed to clarify whether myeloid transcription factors, distinct to RARalpha, play a role in the resistance of these cells to ATRA treatment.


Subject(s)
Leukemia, Myeloid/metabolism , Receptors, Cytoplasmic and Nuclear/biosynthesis , Receptors, Retinoic Acid/metabolism , Tretinoin/pharmacology , Alkaline Phosphatase/pharmacology , Blotting, Northern , Blotting, Western , Cell Differentiation/drug effects , Cell Nucleus/chemistry , Cell Nucleus/metabolism , DNA/metabolism , Dimerization , Enhancer Elements, Genetic , Flow Cytometry , Gene Expression/drug effects , Genetic Vectors/genetics , Genetic Vectors/metabolism , Genetic Vectors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/pathology , Macrophage-1 Antigen/biosynthesis , Phosphorylation/drug effects , Receptors, Retinoic Acid/genetics , Retinoic Acid Receptor alpha , Retinoid X Receptors , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation/physiology , Tumor Cells, Cultured
20.
Leukemia ; 4(10): 688-93, 1990 Oct.
Article in English | MEDLINE | ID: mdl-2170780

ABSTRACT

The hybridization to a complementary RNA (cRNA) probe both in situ and in solution was used to assay tiny amounts of mRNA of the lactoferrin (LF) and myeloperoxidase (MPO) genes in normal bone marrow cells and in acute and chronic lymphoid leukemias. Evidence is reported that this technique is much more sensitive than the standard Northern blot technique. The LF mRNA was detectable in three of seven cases of acute lymphoblastic leukemia (ALL) and in three of seven cases of chronic lymphocytic leukemia (CLL). Four cases of ALL were also positive when tested with the MPO cRNA. It is apparent from these results that myeloid specific mRNA, different from MPO, may be detected in leukemic cells with lymphoid phenotype using a method more sensitive than the Northern blot technique. Whether or not the molecular events observed in these cell populations reflect events physiologically occurring rather than a deregulation of gene expression associated to leukemogenesis remains to be established.


Subject(s)
Genes , Lactoferrin/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Nucleic Acid Hybridization , Peroxidase/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , RNA, Messenger/analysis , Bone Marrow/metabolism , Genetic Techniques , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , RNA Probes
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