Haploinsufficiency of the lysosomal sialidase NEU1 results in a model of pleomorphic rhabdomyosarcoma in mice.

Rhabdomyosarcoma, the most common pediatric sarcoma, has no effective treatment for the pleomorphic subtype. Still, what triggers transformation into this aggressive phenotype remains poorly understood. Here we used Ptch1/ETV7 mice with enhanced incidence of rhabdomyosarcoma to generate a model of pleomorphic rhabdomyosarcoma driven by haploinsufficiency of the lysosomal sialidase neuraminidase 1.

Physical Therapy and Sedation While on Extracorporeal Membrane Oxygenation for COVID-19-Associated Acute Respiratory Distress Syndrome.

Objectives: This study aimed to determine whether patients on extracorporeal membrane oxygenation (ECMO) with coronavirus disease 2019 (COVID-19) achieved lower rates of physical therapy participation and required more sedation than those on ECMO without COVID-19.

Design: Retrospective, observational, matched-cohort study.

Setting: Bicenter academic quaternary medical centers.

Requirement for antiapoptotic MCL-1 during early erythropoiesis.

Although BCL-xL is critical to the survival of mature erythrocytes, it is still unclear whether other antiapoptotic molecules mediate survival during earlier stages of erythropoiesis. Here, we demonstrate that erythroid-specific Mcl1 deletion results in embryonic lethality beyond embryonic day 13.5 as a result of severe anemia caused by a lack of mature red blood cells (RBCs).

Efficacy and toxicity of a virus-directed enzyme prodrug therapy purging method: preclinical assessment and application to bone marrow samples from neuroblastoma patients.

Autologous stem cell transplantation is used to rescue cancer patients from myelosuppression caused by high-dose chemotherapy. However, autologous grafts often contain tumor cells that can contribute directly to relapse. Current purging methods are useful when fewer than 1% tumor cells contaminate the bone marrow, and patients with tumor burdens of >1% are considered ineligible for chemotherapy that necessitates stem cell rescue.

Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis.

Phage that display a surface peptide with the NGR sequence motif home selectively to tumor vasculature in vivo. A drug coupled to an NGR peptide has more potent antitumor effects than the free drug [W. Arap et al.

The influence of recombinant human insulin-like growth factor-I (rhIGF-I) on cell growth and cytotoxicity of drugs in childhood rhabdomyosarcoma cell lines and xenograft models.

Purpose: Recombinant human insulin-like growth factor I (rhIGF-I) has been reported to ameliorate vincristine-induced neuropathy, the dose-limiting side effect of this antimitotic anticancer drug. However, rhIGF-I also might have adverse effects, as has been shown in vitro, where it stimulates growth of cancer cells and protects them from cytotoxicity of anticancer drugs. The influence of rhIGF-I on the cytotoxicity of vincristine has not yet been studied.

Wild-type p53 can induce p21 and apoptosis in neuroblastoma cells but the DNA damage-induced G1 checkpoint function is attenuated.

p53 is a tumor suppressor protein important in the regulation of apoptosis. Because p53 functions as a transcription factor, cellular responses depend upon activity of p53 localized in the nucleus. Cytoplasmic sequestration of p53 has been proposed as a mechanism by which the function of this protein can be suppressed, particularly in tumor types such as neuroblastoma in which the frequency of mutations of p53 is low.

c-Maf induces monocytic differentiation and apoptosis in bipotent myeloid progenitors.

The transcriptional mechanisms that drive colony-forming unit granulocyte-macrophage (CFU-GM) myeloid progenitors to differentiate into cells of either the granulocytic or monocytic lineage are not fully understood. We have shown that the c-Maf and c-Myb transcription factors physically interact in myeloid cells to form inhibitory complexes that hinder transactivation of c-Myb target genes through direct binding to Myb consensus sites. These complexes arise in a developmentally regulated pattern, peaking at the promyelocyte stage, or in cell model systems, appearing soon after the induction of monocytic differentiation.

Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells.

The mammalian target of rapamycin (mTOR) has been shown to link growth factor signaling and posttranscriptional control of translation of proteins that are frequently involved in cell cycle progression. However, the role of this pathway in cell survival has not been demonstrated. Here, we report that rapamycin, a specific inhibitor of mTOR kinase, induces G1 cell cycle arrest and apoptosis in two rhabdomyosarcoma cell lines (Rh1 and Rh30) under conditions of autocrine cell growth.

A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2.

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc.

Enforced expression of the GATA-2 transcription factor blocks normal hematopoiesis.

The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells.

In vivo selection of retrovirally transduced hematopoietic stem cells.

One of the main impediments to effective gene therapy of blood disorders is the resistance of human hematopoietic stem cells to stable genetic modification. We show here that a small minority of retrovirally transduced stem cells can be selectively enriched in vivo, which might be a way to circumvent this obstacle. We constructed two retroviral vectors containing an antifolate-resistant dihydrofolate reductase cDNA transcriptionally linked to a reporter gene.

The chimeric E2A-HLF transcription factor abrogates p53-induced apoptosis in myeloid leukemia cells.

Leukemic lymphoblasts expressing the E2A-HLF oncoprotein possess wild-type p53 genes, but do not undergo apoptosis in response to DNA damage. Experimentally, E2A-HLF prevents apoptosis due to growth factor deprivation or gamma-irradiation in interleukin-3 (IL-3)-dependent murine pro-B cells. To directly test the chimeric protein's ability to abrogate p53-mediated cell death, we used mouse myeloid leukemia cells (M1p53tsval) that constitutively express a temperature-sensitive (ts) mutant p53 gene and undergo apoptosis when p53 assumes an active wild-type configuration.

Complementation of growth factor receptor-dependent mitogenic signaling by a truncated type I phosphatidylinositol 4-phosphate 5-kinase.

Substitution of phenylalanine for tyrosine at codon 809 (Y809F) of the human colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) impairs ligand-stimulated tyrosine kinase activity, prevents induction of c-MYC and cyclin D1 genes, and blocks CSF-1-dependent progression through the G1 phase of the cell cycle. We devised an unbiased genetic screen to isolate genes that restore the ability of CSF-1 to stimulate growth in cells that express mutant CSF-1R (Y809F). This screen led us to identify a truncated form of the murine type Ibeta phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta).

Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF.

The INK4a tumor suppressor locus encodes p16INK4a, an inhibitor of cyclin D-dependent kinases, and p19ARF, an alternative reading frame protein that also blocks cell proliferation. Surprisingly, mice lacking p19ARF but expressing functional p16INK4a develop tumors early in life. Their embryo fibroblasts (MEFs) do not senesce and are transformed by oncogenic Ha-ras alone.

Retroviral-mediated transfer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo.

We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene.

Cancer-associated mutations at the INK4a locus cancel cell cycle arrest by p16INK4a but not by the alternative reading frame protein p19ARF.

The INK4a gene, one of the most frequently disrupted tumor suppressor loci in human cancer, encodes two unrelated proteins, p16INK4a and p19ARF, each of which is capable of inducing cell cycle arrest. Splicing of alternative first exons (1 alpha vs. 1 beta) to a common second exon within INK4a generates mRNAs in which exon 2 sequences are translated in two different reading frames.

The Fanconi anemia complementation group C protein corrects DNA interstrand cross-link-specific apoptosis in HSC536N cells.

Fanconi anemia (FA) cells are hypersensitive to cytotoxicity, cell cycle arrest, and chromosomal aberrations induced by DNA cross-linking agents, such as mitomycin C (MMC) and nitrogen mustard (HN2). Although MMC hypersensitivity is complemented in a subset of FA cells (complementation group C [FA-C]) by wild-type FAC cDNA, the cytoprotective mechanism is unknown. In the current study, we tested the hypothesis that FAC protein functions in the suppression of DNA interstand cross-link (ISC)-induced cell cycle arrest and apoptosis.

DNA index of glial tumors in children. Correlation with tumor grade and prognosis.

Background: Although DNA index (DI) has prognostic significance in a variety of pediatric malignancies, there are few data regarding its utility in central nervous system (CNS) tumors. We have previously shown that patients with hyperdiploid medulloblastoma have a significantly better survival than those whose tumors are diploid. Here, we examine the effect of DI and tumor grade on the progression free survival (PFS) of 57 patients with a variety of glial neoplasms.

Reversal of apoptosis by the leukaemia-associated E2A-HLF chimaeric transcription factor.

The E2A-HLF (for hepatic leukaemia factor) fusion gene, formed by action of the t(17;19) (q22;p13) chromosomal translocation, drives the leukaemic transformation of early B-cell precursors, but the mechanism of this activity remains unknown. Here we report that human leukaemia cells carrying the translocation t(17;19) rapidly died by apoptosis when programmed to express a dominant-negative suppressor of the fusion protein E2A-HLF, indicating that the chimaeric oncoprotein probably affects cell survival rather than cell growth. Moreover, when introduced into murine pro-B lymphocytes, the oncogenic E2A-HLF fusion protein reversed both interleukin-3-dependent and p53-mediated apoptosis.

Inhibition of cell proliferation by the Mad1 transcriptional repressor.

Mad1 is a basic helix-loop-helix-leucine zipper protein that is induced upon differentiation of a number of distinct cell types. Mad1 dimerizes with Max and recognizes the same DNA sequences as do Myc:Max dimers. However, Mad1 and Myc appear to have opposing functions.

Cytokines suppress apoptosis independent of increases in reactive oxygen levels.

Antioxidants suppress apoptosis induced by diverse stimuli in many cells, including immune cells, suggesting that reactive oxygen species (ROS) are common mediators of apoptosis. We evaluated the potential role for ROS in the apoptosis of myeloid progenitors following withdrawal of survival factors and in the apoptosis triggered by enforced c-myc expression, two model systems of programmed cell death. ROS are potential mediators of these cell deaths, as low concentrations of H2O2 (0.

Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest.

The INK4a (MTS1, CDKN2) gene encodes an inhibitor (p16INK4a) of the cyclin D-dependent kinases CDK4 and CDK6 that blocks them from phosphorylating the retinoblastoma protein (pRB) and prevents exit from the G1 phase of the cell cycle. Deletions and mutations involving INK4a occur frequently in cancers, implying that p16INK4a, like pRB, suppresses tumor formation. An unrelated protein (p19ARF) arises in major part from an alternative reading frame of the mouse INK4a gene, and its ectopic expression in the nucleus of rodent fibroblasts induces G1 and G2 phase arrest.

Cloning and characterization of murine p16INK4a and p15INK4b genes.

Progression through the G1 phase of the cell cycle is regulated in part by the D-type cyclin-dependent kinases, cdk4 and cdk6. Genes encoding two specific inhibitors of these kinases, human p16(INK4a/MTS1) and p15(INK4b/MTS2), map to a region of common cytogenetic abnormalities on chromosome 9p21. The murine cognates of these genes were isolated and identified as mouse p16INK4a and p15INK4b based on their homology to their human counterparts and their selective transcriptional induction by SV40T-antigen and TGF-beta, respectively.