33 results on '"Dombkowski, D."'
Search Results
2. Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney–Bone Marrow Transplantation to Induce Transplantation Tolerance
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Sprangers, B., DeWolf, S., Savage, T. M., Morokata, T., Obradovic, A., LoCascio, S. A., Shonts, B., Zuber, J., Lau, S. P., Shah, R., Morris, H., Steshenko, V., Zorn, E., Preffer, F. I., Olek, S., Dombkowski, D. M., Turka, L. A., Colvin, R., Winchester, R., Kawai, T., and Sykes, M.
- Abstract
We examined tolerance mechanisms in patients receiving HLA‐mismatched combined kidney–bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high‐throughput sequencing of T cell receptor‐β hypervariable regions of DNAfrom peripheral blood regulatory T cells (Tregs) and CD4 non‐Tregs revealed marked early enrichment of Tregs (CD3+CD4+CD25highCD127lowFoxp3+) in blood that resulted from peripheral proliferation (Ki67+), possibly new thymic emigration (CD31+), and, in one tolerant subject, conversion from non‐Tregs. Among recovering conventional T cells, central memory CD4+and CD8+cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptorsequencing were detected in the peripheral blood and were not donor‐reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia‐driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells. In patients receiving HLA‐mismatched combined kidney–bone marrow transplantation to induce transplantation tolerance, polychromatic flow cytometry and high‐throughput T cell receptor sequencing reveal an early enrichment of regulatory T cells, probably arising from new thymic emigration and lymphopenia‐driven peripheral proliferation, though the T cell receptors detected are likely from circulating T cells in the allograft microvasculature rather than infiltrating T cells.
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- 2017
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3. Human ovarian cancer stem/progenitor cells are stimulated by doxorubicin but inhibited by Mullerian inhibiting substance
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Meirelles, K., Benedict, Lance Mitchell, Dombkowski, D., Pepin, David, Preffer, Frederic Ira, Teixeira, J, Tanwar, P, Young, Robert Henry, MacLaughlin, D. T., Donahoe, Patricia, and Wei, X.
- Abstract
Women with late-stage ovarian cancer usually develop chemotherapeutic-resistant recurrence. It has been theorized that a rare cancer stem cell, which is responsible for the growth and maintenance of the tumor, is also resistant to conventional chemotherapeutics. We have isolated from multiple ovarian cancer cell lines an ovarian cancer stem cell-enriched population marked by CD44, CD24, and Epcam (3+) and by negative selection for Ecadherin (Ecad−) that comprises less than 1% of cancer cells and has increased colony formation and shorter tumor-free intervals in vivo after limiting dilution. Surprisingly, these cells are not only resistant to chemotherapeutics such as doxorubicin, but also are stimulated by it, as evidenced by the significantly increased number of colonies in treated 3+Ecad− cells. Similarly, proliferation of the 3+Ecad− cells in monolayer increased with treatment, by either doxorubicin or cisplatin, compared with the unseparated or cancer stem cell-depleted 3−Ecad+ cells. However, these cells are sensitive to Mullerian inhibiting substance (MIS), which decreased colony formation. MIS inhibits ovarian cancer cells by inducing G1 arrest of the 3+Ecad− subpopulation through the induction of cyclin-dependent kinase inhibitors. 3+Ecad− cells selectively expressed LIN28, which colocalized by immunofluorescence with the 3+ cancer stem cell markers in the human ovarian carcinoma cell line, OVCAR-5, and is also highly expressed in transgenic murine models of ovarian cancer and in other human ovarian cancer cell lines. These results suggest that chemotherapeutics may be stimulative to cancer stem cells and that selective inhibition of these cells by treating with MIS or targeting LIN28 should be considered in the development of therapeutics.
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- 2012
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4. Mullerian inhibiting substance preferentially inhibits stem/progenitors in human ovarian cancer cell lines compared with chemotherapeutics
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Wei, X., Dombkowski, D., Meirelles, K., Pieretti-Vanmarcke, R., Szotek, P. P., Chang, H, Preffer, Frederic Ira, Mueller, Peter Raff, Teixeira, J, MacLaughlin, D. T., and Donahoe, Patricia
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anthrapyrazolone ,chemotherapy resistance ,stem/progenitor cell-enriched populations - Abstract
Cancer stem cells are proposed to be tumor-initiating cells capable of tumorigenesis, recurrence, metastasis, and drug resistance, and, like somatic stem cells, are thought to be capable of unlimited self-renewal and, when stimulated, proliferation and differentiation. Here we select cells by expression of a panel of markers to enrich for a population with stem cell-like characteristics. A panel of eight was initially selected from 95 human cell surface antigens as each was shared among human ovarian primary cancers, ovarian cancer cell lines, and normal fimbria. A total of 150 combinations of markers were reduced to a panel of three—CD44, CD24, and Epcam—which selected, in three ovarian cancer cell lines, those cells which best formed colonies. Cells expressing CD44, CD24, and Epcam exhibited stem cell characteristics of shorter tumor-free intervals in vivo after limiting dilution, and enhanced migration in invasion assays in vitro. Also, doxorubicin, cisplatin, and paclitaxel increased this enriched population which, conversely, was significantly inhibited by Müllerian inhibiting substance (MIS) or the MIS mimetic SP600125. These findings demonstrate that flow cytometry can be used to detect a population which shows differential drug sensitivity, and imply that treatment of patients can be individualized to target both stem/progenitor cell enriched and nonenriched subpopulations. The findings also suggest that this population, amenable to isolation by flow cytometry, can be used to screen for novel treatment paradigms, including biologic agents such as MIS, which will improve outcomes for patients with ovarian cancer.
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- 2010
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5. The kinase DYRK1A reciprocally regulates the differentiation of Th17 and regulatory T cells.
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Khor B, Gagnon JD, Goel G, Roche MI, Conway KL, Tran K, Aldrich LN, Sundberg TB, Paterson AM, Mordecai S, Dombkowski D, Schirmer M, Tan PH, Bhan AK, Roychoudhuri R, Restifo NP, O'Shea JJ, Medoff BD, Shamji AF, Schreiber SL, Sharpe AH, Shaw SY, and Xavier RJ
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- Animals, Cell Culture Techniques, Harmine pharmacology, Mice, Mice, Inbred C57BL, Mice, Knockout, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases genetics, Protein-Tyrosine Kinases antagonists & inhibitors, Protein-Tyrosine Kinases genetics, Dyrk Kinases, Cell Differentiation immunology, Homeostasis immunology, Protein Serine-Threonine Kinases metabolism, Protein-Tyrosine Kinases metabolism, T-Lymphocytes, Regulatory metabolism, Th17 Cells metabolism
- Abstract
The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity.
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- 2015
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6. Differential regulation of myeloid leukemias by the bone marrow microenvironment.
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Krause DS, Fulzele K, Catic A, Sun CC, Dombkowski D, Hurley MP, Lezeau S, Attar E, Wu JY, Lin HY, Divieti-Pajevic P, Hasserjian RP, Schipani E, Van Etten RA, and Scadden DT
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- Animals, Female, Genes, abl, Humans, Leukemia, Myeloid genetics, Male, Mice, Mice, Inbred BALB C, Mice, Inbred NOD, Mice, SCID, Mice, Transgenic, Oncogene Proteins, Fusion genetics, Parathyroid Hormone metabolism, Signal Transduction, Stem Cell Niche, Transforming Growth Factor beta1 metabolism, Tumor Microenvironment, Bone Marrow metabolism, Bone Marrow pathology, Leukemia, Myeloid metabolism, Leukemia, Myeloid pathology, Neoplastic Stem Cells metabolism, Neoplastic Stem Cells pathology
- Abstract
Like their normal hematopoietic stem cell counterparts, leukemia stem cells (LSCs) in chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML) are presumed to reside in specific niches in the bone marrow microenvironment (BMM) and may be the cause of relapse following chemotherapy. Targeting the niche is a new strategy to eliminate persistent and drug-resistant LSCs. CD44 (refs. 3,4) and interleukin-6 (ref. 5) have been implicated previously in the LSC niche. Transforming growth factor-β1 (TGF-β1) is released during bone remodeling and plays a part in maintenance of CML LSCs, but a role for TGF-β1 from the BMM has not been defined. Here, we show that alteration of the BMM by osteoblastic cell-specific activation of the parathyroid hormone (PTH) receptor attenuates BCR-ABL1 oncogene-induced CML-like myeloproliferative neoplasia (MPN) but enhances MLL-AF9 oncogene-induced AML in mouse transplantation models, possibly through opposing effects of increased TGF-β1 on the respective LSCs. PTH treatment caused a 15-fold decrease in LSCs in wild-type mice with CML-like MPN and reduced engraftment of immune-deficient mice with primary human CML cells. These results demonstrate that LSC niches in CML and AML are distinct and suggest that modulation of the BMM by PTH may be a feasible strategy to reduce LSCs, a prerequisite for the cure of CML.
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- 2013
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7. Densely interconnected transcriptional circuits control cell states in human hematopoiesis.
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Novershtern N, Subramanian A, Lawton LN, Mak RH, Haining WN, McConkey ME, Habib N, Yosef N, Chang CY, Shay T, Frampton GM, Drake AC, Leskov I, Nilsson B, Preffer F, Dombkowski D, Evans JW, Liefeld T, Smutko JS, Chen J, Friedman N, Young RA, Golub TR, Regev A, and Ebert BL
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- Gene Expression Profiling, Humans, Gene Expression Regulation, Gene Regulatory Networks, Hematopoiesis, Transcription Factors metabolism
- Abstract
Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed., (Copyright © 2011 Elsevier Inc. All rights reserved.)
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- 2011
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8. Normal ovarian surface epithelial label-retaining cells exhibit stem/progenitor cell characteristics.
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Szotek PP, Chang HL, Brennand K, Fujino A, Pieretti-Vanmarcke R, Lo Celso C, Dombkowski D, Preffer F, Cohen KS, Teixeira J, and Donahoe PK
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- Animals, Bromodeoxyuridine, Cell Proliferation, Estrous Cycle, Female, Green Fluorescent Proteins, Mice, Epithelial Cells cytology, Ovary cytology, Regeneration, Stem Cells cytology
- Abstract
Ovulation induces cyclic rupture and regenerative repair of the ovarian coelomic epithelium. This process of repeated disruption and repair accompanied by complex remodeling typifies a somatic stem/progenitor cell-mediated process. Using BrdU incorporation and doxycycline inducible histone2B-green fluorescent protein pulse-chase techniques, we identify a label-retaining cell population in the coelomic epithelium of the adult mouse ovary as candidate somatic stem/progenitor cells. The identified population exhibits quiescence with asymmetric label retention, functional response to estrous cycling in vivo by proliferation, enhanced growth characteristics by in vitro colony formation, and cytoprotective mechanisms by enrichment for the side population. Together, these characteristics identify the label-retaining cell population as a candidate for the putative somatic stem/progenitor cells of the coelomic epithelium of the mouse ovary.
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- 2008
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9. Regulatory T-cell recovery in recipients of haploidentical nonmyeloablative hematopoietic cell transplantation with a humanized anti-CD2 mAb, MEDI-507, with or without fludarabine.
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Shaffer J, Villard J, Means TK, Alexander S, Dombkowski D, Dey BR, McAfee S, Ballen KK, Saidman S, Preffer FI, Sachs DH, Spitzer TR, and Sykes M
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- Antibodies, Monoclonal, Humanized, Antigens, CD analysis, Antigens, Differentiation analysis, CD2 Antigens immunology, CTLA-4 Antigen, Forkhead Transcription Factors analysis, Histocompatibility Testing, Humans, Interferon-gamma genetics, Transforming Growth Factor beta genetics, Transplantation Chimera, Vidarabine pharmacology, Antibodies, Monoclonal pharmacology, Hematopoietic Stem Cell Transplantation, T-Lymphocytes, Regulatory physiology, Vidarabine analogs & derivatives
- Abstract
Objective: We have evaluated T-cell reconstitution and reactivity in patients receiving nonmyeloablative haploidentical hematopoietic cell transplantation (HCT) protocols involving an anti-CD2 monoclonal antibody (MEDI 507) to treat chemorefractory hematopoietic malignancies., Methods: Three cohorts of four patients each and one cohort of six patients received one of four Medi-507-based regimens, all of which included cyclophosphamide, thymic irradiation, and a short posttransplantation course of cyclosporine., Results: Following marked T-cell depletion, initially recovering CD4 and CD8 T cells were mainly memory-type cells. A high percentage of CD4 T cells expressed high levels of CD25 in recipients of all protocols, except the only protocol to include fludarabine, early post-HCT. CD25 expression varied inversely with T-cell concentrations in blood. CD25(high) CD4 T cells expressed Foxp3 and cytotoxic T-lymphocyte-associated protein 4, indicating that they were regulatory T cells (Treg)., Conclusions: Fludarabine treatment prevents Treg enrichment after haploidentical nonmyeloablative stem cell transplantation, presumably by depleting recipient Tregs. In vitro analyses of allorecognition were consistent with a cytokine-mediated rejection process in one case and in another provided proof of principle that mixed chimerism achieved without graft-vs-host disease induces donor- and recipient-specific tolerance. More reliable achievement of this outcome could provide a promising strategy for organ allograft tolerance induction.
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- 2007
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10. Adult mouse myometrial label-retaining cells divide in response to gonadotropin stimulation.
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Szotek PP, Chang HL, Zhang L, Preffer F, Dombkowski D, Donahoe PK, and Teixeira J
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- Adipocytes cytology, Adipocytes drug effects, Animals, Bacterial Proteins metabolism, Cell Differentiation drug effects, Cell Division drug effects, Cell Proliferation drug effects, Female, Humans, Integrases metabolism, Luminescent Proteins metabolism, Mice, Myocytes, Smooth Muscle cytology, Myocytes, Smooth Muscle drug effects, Phenotype, Receptors, Peptide metabolism, Receptors, Transforming Growth Factor beta, Stromal Cells cytology, Stromal Cells drug effects, beta Catenin deficiency, Aging physiology, Bromodeoxyuridine metabolism, Chorionic Gonadotropin pharmacology, Myometrium cytology, Myometrium drug effects
- Abstract
Conditional deletion of beta-catenin in the Müllerian duct mesenchyme results in a degenerative uterus characterized by replacement of the myometrial smooth muscle with adipose tissue. We hypothesized that the mouse myometrium houses somatic smooth muscle progenitor cells that are hormonally responsive and necessary for remodeling and regeneration during estrous cycling and pregnancy. We surmise that the phenotype observed in beta-catenin conditionally deleted mice is the result of dysregulation of these progenitor cells. The objective of this study was to identify the mouse myometrial smooth muscle progenitor cell and its niche, define the surface marker phenotype, and show a functional response of these cells to normal myometrial cycling. Uteri were labeled with 5-bromo-2'-deoxyuridine (BrdU) and chased for up to 14 weeks. Myometrial label-retaining cells (LRCs) were observed in the myometrium and stroma throughout the chase period. After 12 weeks, phenotypic analysis of the LRCs by immunofluorescence demonstrated that the majority of LRCs colocalized with alpha-smooth muscle actin, estrogen receptor-alpha, and beta-catenin. Flow cytometry of myometrial cells identified a myometrial Hoechst 33342 effluxing "side population" that expresses MISRII-Cre-driven YFP. Functional response of LRCs was investigated by human chorionic gonadotropin stimulation of week 12 chase mice and demonstrated sequential proliferation of LRCs in the endometrial stroma, followed by the myometrium. These results suggest that conventional myometrial regeneration and repair is executed by hormonally responsive stem or progenitor cells derived from the Müllerian duct mesenchyme. Disclosure of potential conflicts of interest is found at the end of this article.
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- 2007
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11. Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness.
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Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, Dinulescu DM, Connolly D, Foster R, Dombkowski D, Preffer F, Maclaughlin DT, and Donahoe PK
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- Animals, Anti-Mullerian Hormone, Antineoplastic Agents pharmacology, Benzimidazoles pharmacology, CHO Cells, Cell Line, Tumor, Cricetinae, Female, Fluorescent Dyes pharmacology, Humans, Mice, Signal Transduction, Verapamil pharmacology, Gene Expression Regulation, Neoplastic, Glycoproteins pharmacology, Ovarian Neoplasms drug therapy, Ovarian Neoplasms metabolism, Stem Cells cytology, Testicular Hormones pharmacology
- Abstract
The recent identification of "side population" (SP) cells in a number of unrelated human cancers and their normal tissue sources has renewed interest in the hypothesis that cancers may arise from somatic stem/progenitor cells. The high incidence of recurrence attributable to multidrug resistance and the multiple histologic phenotypes indicative of multipotency suggests a stem cell-like etiology of ovarian cancer. Here we identify and characterize SP cells from two distinct genetically engineered mouse ovarian cancer cell lines. Differential efflux of the DNA-binding dye Hoechst 33342 from these cell lines defined a human breast cancer-resistance protein 1-expressing, verapamil-sensitive SP of candidate cancer stem cells. In vivo, mouse SP cells formed measurable tumors sooner than non-SP (NSP) cells when equal numbers were injected into the dorsal fat pad of nude mice. The presence of Mullerian Inhibiting Substance (MIS) signaling pathway transduction molecules in both SP and NSP mouse cells led us to investigate the efficacy of MIS against these populations in comparison with traditional chemotherapies. MIS inhibited the proliferation of both SP and NSP cells, whereas the lipophilic chemotherapeutic agent doxorubicin more significantly inhibited the NSP cells. Finally, we identified breast cancer-resistance protein 1-expressing verapamil-sensitive SPs in three of four human ovarian cancer cell lines and four of six patient primary ascites cells. In the future, individualized therapy must incorporate analysis of the stem cell-like subpopulation of ovarian cancer cells when designing therapeutic strategies for ovarian cancer patients.
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- 2006
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12. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size.
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Stier S, Ko Y, Forkert R, Lutz C, Neuhaus T, Grünewald E, Cheng T, Dombkowski D, Calvi LM, Rittling SR, and Scadden DT
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- Angiopoietin-1 analogs & derivatives, Angiopoietin-1 metabolism, Animals, Apoptosis drug effects, Apoptosis physiology, Bone Marrow physiology, Calcium-Binding Proteins, Hematopoiesis genetics, Hematopoietic Stem Cells cytology, Intercellular Signaling Peptides and Proteins, Jagged-1 Protein, Membrane Proteins metabolism, Mice, Osteoblasts cytology, Osteopontin, Parathyroid Hormone administration & dosage, Parathyroid Hormone metabolism, Serrate-Jagged Proteins, Sialoglycoproteins administration & dosage, Sialoglycoproteins deficiency, Signal Transduction drug effects, Signal Transduction physiology, Hematopoiesis physiology, Hematopoietic Stem Cells physiology, Osteoblasts physiology, Sialoglycoproteins metabolism
- Abstract
Stem cells reside in a specialized niche that regulates their abundance and fate. Components of the niche have generally been defined in terms of cells and signaling pathways. We define a role for a matrix glycoprotein, osteopontin (OPN), as a constraining factor on hematopoietic stem cells within the bone marrow microenvironment. Osteoblasts that participate in the niche produce varying amounts of OPN in response to stimulation. Using studies that combine OPN-deficient mice and exogenous OPN, we demonstrate that OPN modifies primitive hematopoietic cell number and function in a stem cell-nonautonomous manner. The OPN-null microenvironment was sufficient to increase the number of stem cells associated with increased stromal Jagged1 and Angiopoietin-1 expression and reduced primitive hematopoietic cell apoptosis. The activation of the stem cell microenvironment with parathyroid hormone induced a superphysiologic increase in stem cells in the absence of OPN. Therefore, OPN is a negative regulatory element of the stem cell niche that limits the size of the stem cell pool and may provide a mechanism for restricting excess stem cell expansion under conditions of niche stimulation.
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- 2005
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13. Flowing cells through pulsed electric fields efficiently purges stem cell preparations of contaminating myeloma cells while preserving stem cell function.
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Craiu A, Saito Y, Limon A, Eppich HM, Olson DP, Rodrigues N, Adams GB, Dombkowski D, Richardson P, Schlossman R, Choi PS, Grogins J, O'Connor PG, Cohen K, Attar EC, Freshman J, Rich R, Mangano JA, Gribben JG, Anderson KC, and Scadden DT
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- Animals, Blood, Bone Marrow, Cell Size, Humans, Leukapheresis, Mice, Mice, Inbred NOD, Transplantation, Autologous methods, Bone Marrow Purging methods, Cell Separation methods, Electroporation, Hematopoietic Stem Cell Transplantation methods, Hematopoietic Stem Cells cytology, Multiple Myeloma pathology
- Abstract
Autologous stem cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Here we show that flowing hematopoietic cells through pulsed electric fields (PEFs) effectively purges myeloma cells without sacrificing functional stem cells. Electric fields can induce irreversible cell membrane pores in direct relation to cell diameter, an effect we exploit in a flowing system appropriate for clinical scale. Multiple myeloma (MM) cell lines admixed with human bone marrow (BM) or peripheral blood (PB) cells were passed through PEFs at 1.35 kV/cm to 1.4 kV/cm, resulting in 3- to 4-log tumor cell depletion by flow cytometry and 4.5- to 6-log depletion by tumor regrowth cultures. Samples from patients with MM gave similar results by cytometry. Stem cell engraftment into nonobese diabetic-severe combined immunodeficient (NOD/SCID)/beta2m-/- mice was unperturbed by PEFs. Flowing cells through PEFs is a promising technology for rapid tumor cell purging of clinical progenitor cell preparations.
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- 2005
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14. Matrix metalloproteinase-9-deficient dendritic cells have impaired migration through tracheal epithelial tight junctions.
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Ichiyasu H, McCormack JM, McCarthy KM, Dombkowski D, Preffer FI, and Schneeberger EE
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- Animals, Cells, Cultured, Chemokine CCL19, Chemokine CCL3, Chemokine CCL4, Chemokines, CC metabolism, Claudin-1, Dipeptides metabolism, Electric Impedance, Epithelial Cells cytology, Lung anatomy & histology, Lung immunology, Lung metabolism, Macrophage Inflammatory Proteins metabolism, Matrix Metalloproteinase 9 genetics, Membrane Proteins metabolism, Mice, Mice, Knockout, Occludin, Protease Inhibitors metabolism, Receptors, CCR5 metabolism, Receptors, CCR7, Receptors, Chemokine metabolism, Respiratory Mucosa metabolism, Cell Movement physiology, Dendritic Cells metabolism, Epithelial Cells metabolism, Matrix Metalloproteinase 9 metabolism, Respiratory Mucosa cytology, Tight Junctions metabolism, Trachea anatomy & histology
- Abstract
When sampling inhaled antigens, dendritic cells (DC) must penetrate the tight junction (TJ) barrier while maintaining the TJ seal. In matrix metalloproteinase (MMP)-9-deficient mice, in vivo experiments suggest that migration of DC into air spaces is impaired. To examine the underlying mechanisms, we established a well-defined in vitro model using mouse tracheal epithelial cells and mouse bone marrow DC (BMDC). Transmigration was elicited with either macrophage inflammatory protein (MIP)-1alpha or MIP-3beta in a time-dependent manner. Control MMP-9(+/+) BMDC cultured with granulocyte macrophage-colony-stimulating factor for 7 d showed a 30-fold greater transepithelial migration toward MIP-3beta than MIP-1alpha, indicating a more mature DC phenotype. MMP-9(-/-) BMDC as well as MMP-9(+/+) BMDC in the presence of the MMP inhibitor GM6001, although showing a similar preference for MIP-3beta, were markedly impaired in their ability to traverse the epithelium. Expression levels of CCR5 and CCR7, however, were similar in both MMP-9(-/-) and MMP-9(+/+) BMDC. Expression of the integral TJ proteins, occludin and claudin-1, were examined in BMDC before and after transepithelial migration. Interestingly, occludin but not claudin-1 was degraded following transepithelial migration in both MMP-9(-/-) and control BMDC. In addition, there was a > 2-fold increase in claudin-1 expression in MMP-9(-/-) as compared with control BMDC. These observations indicate that occludin and claudin-1 are differentially regulated and suggest that the lack of MMP-9 may affect claudin-1 turnover.
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- 2004
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15. Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells.
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Kuhlencordt PJ, Rosel E, Gerszten RE, Morales-Ruiz M, Dombkowski D, Atkinson WJ, Han F, Preffer F, Rosenzweig A, Sessa WC, Gimbrone MA Jr, Ertl G, and Huang PL
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- Animals, Cell Adhesion, Cell Division physiology, Cell Membrane metabolism, Cells, Cultured, Endothelium, Vascular cytology, Endothelium, Vascular drug effects, Endothelium, Vascular enzymology, Interleukin-1 pharmacology, Mice, Mice, Knockout, Nitric Oxide metabolism, Nitric Oxide Synthase Type II, Nitric Oxide Synthase Type III, P-Selectin metabolism, Perfusion, Reactive Oxygen Species metabolism, Tumor Necrosis Factor-alpha pharmacology, Endothelium, Vascular physiology, Nitric Oxide Synthase physiology
- Abstract
The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N(omega)-monomethyl-l-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation.
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- 2004
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16. Global expression analysis of gene regulatory pathways during endocrine pancreatic development.
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Gu G, Wells JM, Dombkowski D, Preffer F, Aronow B, and Melton DA
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- Animals, Cell Differentiation genetics, Cell Differentiation physiology, Female, Green Fluorescent Proteins, In Situ Hybridization, Islets of Langerhans cytology, Islets of Langerhans growth & development, Luminescent Proteins genetics, Mice, Mice, Inbred ICR, Mice, Transgenic, Pregnancy, Protein Serine-Threonine Kinases genetics, Protein-Tyrosine Kinases genetics, Reverse Transcriptase Polymerase Chain Reaction, Transcription, Genetic, DNA-Binding Proteins, Embryonic and Fetal Development genetics, Gene Expression Regulation, Developmental physiology, Islets of Langerhans embryology, Transcription Factors
- Abstract
To define genetic pathways that regulate development of the endocrine pancreas, we generated transcriptional profiles of enriched cells isolated from four biologically significant stages of endocrine pancreas development: endoderm before pancreas specification, early pancreatic progenitor cells, endocrine progenitor cells and adult islets of Langerhans. These analyses implicate new signaling pathways in endocrine pancreas development, and identified sets of known and novel genes that are temporally regulated, as well as genes that spatially define developing endocrine cells from their neighbors. The differential expression of several genes from each time point was verified by RT-PCR and in situ hybridization. Moreover, we present preliminary functional evidence suggesting that one transcription factor encoding gene (Myt1), which was identified in our screen, is expressed in endocrine progenitors and may regulate alpha, beta and delta cell development. In addition to identifying new genes that regulate endocrine cell fate, this global gene expression analysis has uncovered informative biological trends that occur during endocrine differentiation.
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- 2004
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17. Ephrin receptor, EphB4, regulates ES cell differentiation of primitive mammalian hemangioblasts, blood, cardiomyocytes, and blood vessels.
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Wang Z, Cohen K, Shao Y, Mole P, Dombkowski D, and Scadden DT
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- Animals, Base Sequence, Blood Cells cytology, Blood Vessels cytology, Cell Differentiation genetics, Cell Differentiation physiology, Cell Line, DNA Primers genetics, Gene Expression, Mesoderm cytology, Mice, Mice, Knockout, Myocytes, Cardiac cytology, Receptor, EphB4 deficiency, Receptor, EphB4 genetics, Signal Transduction, Pluripotent Stem Cells cytology, Receptor, EphB4 physiology
- Abstract
Differentiation of pluripotent embryonic stem (ES) cells is associated with expression of fate-specifying gene products. Coordinated development, however, must involve modifying factors that enable differentiation and growth to adjust in response to local microenvironmental determinants. We report here that the ephrin receptor, EphB4, known to be spatially restricted in expression and critical for organized vessel formation, modifies the rate and magnitude of ES cells acquiring genotypic and phenotypic characteristics of mesodermal tissues. Hemangioblast, blood cell, cardiomyocyte, and vascular differentiation was impaired in EphB4-/- ES cells in conjunction with decreased expression of mesoderm-associated, but not neuroectoderm-associated, genes. Therefore, EphB4 modulates the response to mesoderm induction signals. These data add differentiation kinetics to the known effects of ephrin receptors on mammalian cell migration and adhesion. We propose that modifying sensitivity to differentiation cues is a further means for ephrin receptors to contribute to tissue patterning and organization.
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- 2004
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18. NK cell recovery, chimerism, function, and recognition in recipients of haploidentical hematopoietic cell transplantation following nonmyeloablative conditioning using a humanized anti-CD2 mAb, Medi-507.
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Koenecke C, Shaffer J, Alexander SI, Preffer F, Dombkowski D, Saidman SL, Dey B, McAfee S, Spitzer TR, and Sykes M
- Subjects
- Cytotoxicity, Immunologic, Haplotypes, Histocompatibility Antigens Class I physiology, Histocompatibility Testing, Host vs Graft Reaction, Humans, Receptors, Immunologic immunology, Receptors, KIR, Antibodies, Monoclonal therapeutic use, CD2 Antigens immunology, Hematopoietic Stem Cell Transplantation, Killer Cells, Natural immunology, Transplantation Chimera, Transplantation Conditioning
- Abstract
Objective: Natural killer (NK) cells kill allogeneic cells that lack a class I MHC ligand for clonally distributed killer inhibitory receptors (KIR). Following HLA-mismatched hematopoietic cell transplantation (HCT), donor NK cells might mediate graft-vs-host (GVH) reactions that promote donor chimerism and mediate anti-tumor effects. Additionally, recipient NK cells might mediate donor marrow rejection. We have developed a nonmyeloablative approach to haploidentical HCT involving recipient treatment with a T cell-depleting mAb, Medi-507, that can achieve donor engraftment and mixed hematopoietic chimerism without graft-vs-host disease (GVHD). Donor lymphocyte infusions (DLI) are later administered in an effort to achieve graft-vs-leukemia/lymphoma (GVL) effects without GVHD. It is unknown whether NK cell "tolerance" develops in human mixed chimeras., Methods: We have addressed these issues in 12 patients receiving Medi-507-based nonmyeloablative haploidentical HCT., Results: NK cells recovered relatively early, despite the presence of circulating anti-CD2 mAb, but the majority of initially recovering cells lacked CD2 expression. These NK cells showed a reduced capacity, compared to those from normal donors, to kill class I-deficient targets. No association was detected between KIR mismatches in the host-vs-graft (HVG) or GVH direction and graft or tumor outcomes in this small series. NK cell chimerism did not correlate with chimerism in other lineages in mixed chimeras. NK cell tolerance to the host was not observed in a patient with full donor chimerism. One patient developed NK cell reactivity against donor-derived lymphoblast targets after loss of chimerism, despite the absence of an HVG KIR mismatch., Conclusion: Our results do not show an impact of NK cells on the outcome of nonmyeloablative, even T cell-depleted, HCT across haplotype barriers using an anti-CD2 mAb. Our data also raise questions about the applicability of observations made with NK cell clones to the bulk NK cell repertoire in humans.
- Published
- 2003
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19. Early host CD8 T-cell recovery and sensitized anti-donor interleukin-2-producing and cytotoxic T-cell responses associated with marrow graft rejection following nonmyeloablative allogeneic bone marrow transplantation.
- Author
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Kraus AB, Shaffer J, Toh HC, Preffer F, Dombkowski D, Saidman S, Colby C, George R, McAfee S, Sackstein R, Dey B, Spitzer TR, and Sykes M
- Subjects
- CD8-Positive T-Lymphocytes cytology, Hematologic Neoplasms immunology, Hematologic Neoplasms therapy, Humans, Transplantation Conditioning, Transplantation, Homologous, Bone Marrow Transplantation, CD8-Positive T-Lymphocytes immunology, Graft Rejection, Interleukin-2 biosynthesis
- Abstract
Objective: We developed a nonmyeloablative conditioning regimen for allogeneic bone marrow transplantation (BMT) followed by donor lymphocyte infusions (DLI) for treatment of chemotherapy refractory malignancies. Although the majority of patients who receive this regimen achieve lasting mixed or full allogeneic chimerism, approximately 30% show initial mixed chimerism followed by loss of the donor graft. These patients recover host hematopoiesis without significant cytopenias. To assess the role of immunologic rejection in graft loss, we compared T-cell recovery and in vitro alloresponses in six patients who lost their marrow graft to that in 16 concurrent patients with sustained donor chimerism., Patients and Methods: Conditioning included pretransplant cyclophosphamide (150-200 mg/kg), thymic irradiation (700 cGy), and pre- and post-transplant equine antithymocyte globulin (ATG; ATGAM). HLA-identical related donor BMT was followed by DLI at approximately day 35 in patients without graft-vs-host disease., Results: The group with transient chimerism showed significantly increased circulating host T-cell (median 416 cells/mm(3) vs 10 cells/mm(3), p<0.05) and CD8 T-cell numbers (354 cells/mm(3) vs 71 cells/mm(3), p<0.05) compared to the group with stable mixed or full donor chimerism within the first 100 days post-BMT. All DLI recipients who lost chimerism following DLI had greater than 80% recipient T cells at the time of DLI, whereas those with persistent chimerism had <60% host T cells. Graft rejection was associated with the development of a sensitized anti-donor bulk cytotoxic T-lymphocyte (CTL) response in 4 of 6 evaluated patients, compared to only 1 of 10 evaluated patients with sustained chimerism (p<0.05). Additionally, 3 of 5 evaluated transient chimeras showed high anti-donor CTL precursor frequencies in limiting dilution assays, and 3 of 4 evaluated transient chimeras showed high anti-donor interleukin-2 (IL-2)-producing T-helper (T(H)) cell frequencies. High anti-donor T(H) or cytotoxic T-lymphocyte precursors were not detected in sustained chimeras., Conclusion: These data indicate that loss of chimerism in patients receiving this nonmyeloablative regimen is due to immune-mediated rejection. This rejection appears to bemediated by recovering recipient cytolytic CD8(+) cells as well as IL-2-producing recipient T(H) cells. These data are the first to demonstrate sensitization of recipient anti-donor IL-2-producing cells in association with human marrow allograft rejection.
- Published
- 2003
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20. P2Y-like receptor, GPR105 (P2Y14), identifies and mediates chemotaxis of bone-marrow hematopoietic stem cells.
- Author
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Lee BC, Cheng T, Adams GB, Attar EC, Miura N, Lee SB, Saito Y, Olszak I, Dombkowski D, Olson DP, Hancock J, Choi PS, Haber DA, Luster AD, and Scadden DT
- Subjects
- ADP-ribosyl Cyclase analysis, ADP-ribosyl Cyclase 1, Amino Acid Sequence, Animals, Antigens, CD analysis, Antigens, CD34 analysis, COS Cells, Cell Cycle, Cell Lineage, Cell Separation, Cells, Cultured, Colony-Forming Units Assay, Culture Media, Conditioned, Flow Cytometry, Hematopoietic Stem Cells chemistry, Humans, Immunophenotyping, Liver chemistry, Liver embryology, Membrane Glycoproteins, Mice, Molecular Sequence Data, Receptors, Purinergic P2 analysis, Receptors, Purinergic P2 chemistry, Receptors, Purinergic P2 genetics, Receptors, Purinergic P2Y, Resting Phase, Cell Cycle, Transfection, Chemotaxis, Hematopoiesis, Hematopoietic Stem Cells physiology, Receptors, G-Protein-Coupled, Receptors, Purinergic P2 physiology
- Abstract
Hematopoiesis in mammals undergoes a developmental shift in location from fetal liver to bone marrow accompanied by a gradual transition from highly proliferative to deeply quiescent stem cell populations. P2Y receptors are G-protein-coupled nucleotide receptors participating in vascular and immune responses to injury. We identified a P2Y-like receptor for UDP-conjugated sugars, GPR105 (P2Y14), with restricted expression on primitive cells in the hematopoietic lineage. Anti-GPR105 antibody selectively isolated a subset of hematopoietic cells within the fetal bone marrow, but not in the fetal liver, that was enriched for G0 cell cycle status and for in vitro stem-cell-like multipotential long-term culture capability. Conditioned media from bone marrow stroma induced receptor activation and chemotaxis that was sensitive to G alpha i and anti-receptor antibody inhibition. GPR105 is a G-protein-coupled receptor identifying a quiescent, primitive population of hematopoietic cells restricted to bone marrow. It mediates primitive cell responses to specific hematopoietic microenvironments and extends the known immune system functions of P2Y receptors to the stem cell level. These data suggest a new class of receptors participating in the regulation of the stem cell compartment.
- Published
- 2003
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21. Heterologous cells cooperate to augment stem cell migration, homing, and engraftment.
- Author
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Adams GB, Chabner KT, Foxall RB, Weibrecht KW, Rodrigues NP, Dombkowski D, Fallon R, Poznansky MC, and Scadden DT
- Subjects
- Animals, Bone Marrow Cells cytology, CD4-Positive T-Lymphocytes, CD8-Positive T-Lymphocytes physiology, CD8-Positive T-Lymphocytes ultrastructure, Chemokine CXCL12, Chemokines, CXC physiology, Cytoskeleton ultrastructure, Hematopoietic Stem Cells metabolism, Humans, Lymphocyte Depletion adverse effects, Lymphocyte Depletion methods, Mice, Mice, Knockout, Receptors, CXCR4 physiology, Signal Transduction, Transplantation, Heterologous, CD8-Positive T-Lymphocytes transplantation, Cell Communication, Chemotaxis, Cord Blood Stem Cell Transplantation methods, Graft Survival, Hematopoietic Stem Cells cytology
- Abstract
T-lymphocyte depletion of bone marrow grafts compromises engraftment, suggesting a facilitating mechanism provided by the T cells that has been shown to associate with CD8(+) but not CD4(+) T cells. Explanations for this phenomenon have focused on immune targeting of residual host cells or cytokine production. We provide evidence for an alternative mechanism based on cooperative effects on cell motility. We observed that engraftment of CD34(+) cells in a beta(2)-microglobulin-deficient nonobese diabetic/severe combined immunodeficiency (beta(2)m(-/-) NOD/SCID) mouse model paralleled clinical observations in humans, with an enhancing effect noted from the addition of CD8(+) cells but not CD4(+) cells. This correlated with CD8(+) augmentation of CD34(+) cell homing to the bone marrow in vivo and CD8(+) cell-associated increases of CD34(+) cell transmigration through a bone marrow endothelial cell line in vitro. The cooperative interaction was not sensitive to brefeldin A inhibition of protein secretion. However, cytochalasin D-induced inhibition of CD8(+) cytoskeletal rearrangements abrogated CD34(+) transendothelial migration and impaired CD34(+) cell homing in vivo. CD8(+) cells did not migrate in tandem with CD34(+) cells or alter endothelial barrier integrity; rather, they affected phosphotyrosine-mediated signaling in CD34(+) cells in response to the chemokine stromal derived factor-1alpha (SDF-1alpha). These data demonstrate cell-cell cooperativity between different cell types in mediating chemotactic events and provide one potential explanation for the clinically observed effect of CD8(+) cells on bone marrow transplantation. This modification of cell migration by neighboring cells provides broad possibilities for combinatorial effects between cells of different types to influence cell localization.
- Published
- 2003
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22. Notch1 activation increases hematopoietic stem cell self-renewal in vivo and favors lymphoid over myeloid lineage outcome.
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Stier S, Cheng T, Dombkowski D, Carlesso N, and Scadden DT
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- Animals, Bone Marrow Transplantation physiology, Cell Division, Cell Line, Colony-Forming Units Assay, Gene Expression Regulation physiology, Genes, RAG-1, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Humans, Kidney, Mice, Mice, Inbred C57BL, Mice, Knockout, Models, Animal, Morphogenesis, Receptor, Notch1, Transfection, Hematopoietic Stem Cells cytology, Membrane Proteins genetics, Receptors, Cell Surface, Transcription Factors
- Abstract
Hematopoietic stem cells sequentially pass through a series of decision points affecting self-renewal or lineage-specific differentiation. Notch1 receptor is a known modulator of lineage-specific events in hematopoiesis that we assessed in the context of in vivo stem cell kinetics. Using RAG-1(-/-) mouse stems cells, we documented increased stem cell numbers due to decreased differentiation and enhanced stem cell self-renewal induced by Notch1. Unexpectedly, preferential lymphoid over myeloid lineage commitment was noted when differentiation occurred. Therefore, Notch1 affects 2 decision points in stem cell regulation, favoring self-renewal over differentiation and lymphoid over myeloid lineage outcome. Notch1 offers an attractive target for stem cell manipulation strategies, particularly in the context of immunodeficiency and acquired immunodeficiency syndrome.
- Published
- 2002
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23. Lineage-negative side-population (SP) cells with restricted hematopoietic capacity circulate in normal human adult blood: immunophenotypic and functional characterization.
- Author
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Preffer FI, Dombkowski D, Sykes M, Scadden D, and Yang YG
- Subjects
- Animals, Antigens, CD34 immunology, Biomarkers, Blood Cells cytology, Dendritic Cells cytology, Dendritic Cells immunology, Female, Hematopoietic Stem Cells cytology, Humans, Immunophenotyping, Killer Cells, Natural cytology, Killer Cells, Natural immunology, Mice, Mice, SCID, T-Lymphocytes cytology, T-Lymphocytes immunology, Tissue Transplantation, Antigens, Surface immunology, Blood Cells immunology, Cell Differentiation immunology, Cell Lineage immunology, Hematopoiesis immunology, Hematopoietic Stem Cells immunology
- Abstract
Side-population (SP) cells are a recently described rare cell population detected in selected tissues of various mammalian species, but not yet described in the peripheral circulation. In the present study, we have identified for the first time SP cells in lineage-negative adult human blood and have provided an extensive functional and immunophenotypic characterization of these cells. Adult peripheral blood was depleted of mature leukocyte cell types by density gradient and immunomagnetic separation. The SP cell population was identified by its characteristic Hoechst 33342 profile. Immunophenotypic analysis revealed that blood SP cells expressed high levels of CD45, CD59, CD43, CD49d, CD31, and integrin markers and lacked CD34. Highly purified SP cells were put into cobblestone area-forming cell (CAFC), long-term culture-initiating cell (LTC-IC), and liquid cell culture assays; repopulating assays were performed utilizing nonobese diabetic/severe combined immunodeficient mice. Circulating SP cells were shown to exhibit verapamil sensitivity and a low growth rate. LTC-IC, CAFC, and engraftment assays indicated that circulating SP cells had lost the multipotentiality described in murine bone marrow SP cells. However, outgrowth of mature cell types from liquid cell culture suggests the presence of common lymphoid (T and natural killer) and dendritic cell precursor(s) within circulating SP cell populations. The absence of SP cell growth in the LTC-IC, CAFC, and repopulating assays might be intrinsic to the tissue source (marrow versus blood) or species (mouse versus human) tested. Thus, human blood SP cells, although rare, may serve as a source of selected leukocyte progenitor cells. The immunophenotype of circulating SP cells may provide clues to their seeding and homing capacity.
- Published
- 2002
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24. Mullerian inhibiting substance inhibits ovarian cell growth through an Rb-independent mechanism.
- Author
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Ha TU, Segev DL, Barbie D, Masiakos PT, Tran TT, Dombkowski D, Glander M, Clarke TR, Lorenzo HK, Donahoe PK, and Maheswaran S
- Subjects
- Animals, Anti-Mullerian Hormone, Blood Proteins metabolism, Calmodulin-Binding Proteins metabolism, Cell Differentiation, Cyclin-Dependent Kinase Inhibitor p16 metabolism, Female, Humans, Ligands, Male, Mice, Ovarian Neoplasms pathology, Ovary drug effects, Retinoblastoma-Like Protein p130, Tumor Cells, Cultured, Glycoproteins, Growth Inhibitors pharmacology, Ovary pathology, Proteins, Rubidium metabolism, Testicular Hormones pharmacology
- Abstract
Müllerian inhibiting substance (MIS), a transforming growth factor-beta family member, causes regression of the Müllerian duct in male embryos. MIS overexpression in transgenic mice ablates the ovary, and MIS inhibits the growth of ovarian cancer cell lines in vitro, suggesting a key role for this hormone in postnatal development of the ovary. This report describes a mechanism for MIS-mediated growth inhibition in both a human epithelial ovarian cancer cell line and a cell line derived from normal ovarian surface epithelium, which is the origin of human epithelial ovarian cancers. MIS-treated cells accumulated in the G(1) phase of the cell cycle and subsequently underwent apoptosis. MIS up-regulated the cyclin-dependent kinase inhibitor p16 through an MIS type II receptor-mediated mechanism and inhibited growth in the absence of detectable or inactive Rb protein. Prolonged treatment with MIS down-regulated the Rb-related protein p130 and increased the Rb family-regulated transcription factor E2F1, overexpression of which inhibited growth. These findings demonstrate that p16 is required for MIS-mediated growth inhibition in ovarian epithelial cells and tumor cells and suggest that up-regulation of E2F1 also plays a role in this process.
- Published
- 2000
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25. Stem cell repopulation efficiency but not pool size is governed by p27(kip1).
- Author
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Cheng T, Rodrigues N, Dombkowski D, Stier S, and Scadden DT
- Subjects
- Animals, Bone Marrow Cells cytology, Cell Cycle, Cells, Cultured, Colony-Forming Units Assay, Crosses, Genetic, Cyclin-Dependent Kinase Inhibitor p27, Cyclin-Dependent Kinases antagonists & inhibitors, Female, Flow Cytometry, Heterozygote, Homozygote, Mice, Mice, Inbred Strains, Mice, Knockout, Microtubule-Associated Proteins deficiency, Microtubule-Associated Proteins genetics, Cell Cycle Proteins, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells physiology, Microtubule-Associated Proteins physiology, Tumor Suppressor Proteins
- Abstract
Sustained blood cell production requires preservation of a quiescent, multipotential stem cell pool that intermittently gives rise to progenitors with robust proliferative potential. The ability of cells to shift from a highly constrained to a vigorously active proliferative state is critical for maintaining stem cells while providing the responsiveness necessary for host defense. The cyclin-dependent kinase inhibitor (CDKI), p21(cip1/waf1) (p21) dominates stem cell kinetics. Here we report that another CDKI, p27(kip1) (p27), does not affect stem cell number, cell cycling, or self-renewal, but markedly alters progenitor proliferation and pool size. Therefore, distinct CDKIs govern the highly divergent stem and progenitor cell populations. When competitively transplanted, p27-deficient stem cells generate progenitors that eventually dominate blood cell production. Modulating p27 expression in a small number of stem cells may translate into effects on the majority of mature cells, thereby providing a strategy for potentiating the impact of transduced cells in stem cell gene therapy.
- Published
- 2000
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26. Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants.
- Author
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Eppich HM, Foxall R, Gaynor K, Dombkowski D, Miura N, Cheng T, Silva-Arrieta S, Evans RH, Mangano JA, Preffer FI, and Scadden DT
- Subjects
- ADP-ribosyl Cyclase, ADP-ribosyl Cyclase 1, Antigens, CD34 analysis, Antigens, Differentiation analysis, Electricity, Hematopoietic Stem Cells immunology, Humans, Membrane Glycoproteins, NAD+ Nucleosidase analysis, Tumor Cells, Cultured, Antigens, CD, Bone Marrow Purging, Cell Separation methods, Hematopoietic Stem Cells cytology
- Abstract
Purging of tumor cells and selection of stem cells are key technologies for enabling stem cell transplantation and stem cell gene therapy. Here we report a strategy for cell selection based on physical properties of the cells. Exposing cells to an external pulsed electric field (PEF) increases the natural potential difference across the cell membrane until a critical threshold is reached and pore formation occurs, resulting in fatal perturbation of cell physiology. Attaining this threshold is a function of the applied field intensity and cell size, with larger cells porated at lower field intensities than smaller cells. Since hematopoietic stem cells are smaller than other hematopoietic cells and tumor cells, we found that exposure of peripheral blood mononuclear cells (PBMCs) to PEFs caused stepwise elimination of monocytes without affecting the function of smaller lymphocyte populations. Mobilized peripheral blood exposed to PEFs was enriched for CD34+/CD38- cells and stem cell function was preserved. Furthermore, PEF treatment was able to selectively purge blood preparations of tumor cells and eradicate transplantable tumor.
- Published
- 2000
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27. Hematopoietic stem cell quiescence maintained by p21cip1/waf1.
- Author
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Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, Sykes M, and Scadden DT
- Subjects
- Animals, Antimetabolites pharmacology, Blood Cell Count, Bone Marrow Transplantation, Cell Count, Cell Death, Cell Differentiation, Cell Division, Coculture Techniques, Colony-Forming Units Assay, Cyclin-Dependent Kinase Inhibitor p21, Cyclins genetics, Female, Fluorouracil pharmacology, Hematopoietic Stem Cells drug effects, Hematopoietic Stem Cells physiology, Homeostasis, Male, Mice, Mice, Inbred Strains, Cell Cycle, Cyclins physiology, Hematopoiesis, Hematopoietic Stem Cells cytology
- Abstract
Relative quiescence is a defining characteristic of hematopoietic stem cells, while their progeny have dramatic proliferative ability and inexorably move toward terminal differentiation. The quiescence of stem cells has been conjectured to be of critical biologic importance in protecting the stem cell compartment, which we directly assessed using mice engineered to be deficient in the G1 checkpoint regulator, cyclin-dependent kinase inhibitor, p21cip1/waf1 (p21). In the absence of p21, hematopoietic stem cell proliferation and absolute number were increased under normal homeostatic conditions. Exposing the animals to cell cycle-specific myelotoxic injury resulted in premature death due to hematopoietic cell depletion. Further, self-renewal of primitive cells was impaired in serially transplanted bone marrow from p21-/- mice, leading to hematopoietic failure. Therefore, p21 is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion. Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death.
- Published
- 2000
- Full Text
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28. Accumulation of macrophages with dendritic cell characteristics in the pulmonary response to Listeria.
- Author
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Kradin RL, Sakamoto H, Preffer FI, Dombkowski D, Springer KM, and Leary CP
- Subjects
- Animals, Antigen-Presenting Cells immunology, Female, Inflammation Mediators metabolism, Lung immunology, Nitric Oxide metabolism, Rats, Rats, Inbred Lew, T-Lymphocytes immunology, Dendritic Cells immunology, Listeriosis immunology, Macrophages, Alveolar immunology, Pneumonia, Bacterial immunology
- Abstract
Pulmonary immunity reflects a balance between proinflammatory and immunosuppressive factors in the lung. To determine the immune activities of exudate macrophages in the pulmonary immune response, Lewis rats were injected intratracheally with heat-killed Listeria (HKL), labeled ex vivo with the lipophilic dye PKH-26. At 24 h, macrophages from bronchoalveolar lavage fluid were purified on the basis of their surface membrane expression of RMA, a macrophage-specific antigen, which is brightly expressed by resident alveolar macrophages but dimly expressed by monocytes. Pulmonary macrophages were analyzed for uptake of PKH-26-HKL, and RMA(bright/dim) macrophages sorted by FACS were compared for cytokine expression, nitric oxide (NO) release, and APC activities. RMA(bright) macrophages were OX-62(-), B7(-), and factor XIIIa(-); they were the dominant mediators of phagocytosis when low doses of HKL were administered intratracheally but did not support the proliferation of T lymphocytes. RMA(dim) exudate macrophages were OX-62(+), B7(+), and factor XIIIa(+). They expressed more IL-1 and TNF, but less nitric oxide, than did RMA(bright) macrophages; they were excellent APCs for T cell responses. We conclude that a subset of RMA(dim) exudate macrophages shows phenotypic and functional evidence of dendritic cell differentiation.
- Published
- 2000
29. Human ovarian cancer, cell lines, and primary ascites cells express the human Mullerian inhibiting substance (MIS) type II receptor, bind, and are responsive to MIS.
- Author
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Masiakos PT, MacLaughlin DT, Maheswaran S, Teixeira J, Fuller AF Jr, Shah PC, Kehas DJ, Kenneally MK, Dombkowski DM, Ha TU, Preffer FI, and Donahoe PK
- Subjects
- Adult, Aged, Aged, 80 and over, Amino Acid Sequence, Animals, Anti-Mullerian Hormone, Ascites genetics, Ascites pathology, COS Cells, Cell Division drug effects, Cystadenocarcinoma genetics, Female, Fetus, Growth Inhibitors metabolism, Humans, Male, Middle Aged, Molecular Sequence Data, Mullerian Ducts, Ovarian Neoplasms genetics, Peptide Fragments chemistry, Peptide Fragments immunology, Rats, Receptors, Transforming Growth Factor beta, Recombinant Proteins metabolism, Testicular Hormones metabolism, Testis embryology, Testis metabolism, Transfection, Tumor Cells, Cultured, Cystadenocarcinoma pathology, Glycoproteins, Growth Inhibitors pharmacology, Ovarian Neoplasms pathology, Receptors, Peptide genetics, Receptors, Peptide metabolism, Testicular Hormones pharmacology
- Abstract
Six human ovarian cancer cell lines and samples of ascites cells isolated from 27 patients with stage III or IV ovarian papillary serous cystadenocarcinoma were studied individually to test whether recombinant human Mullerian inhibiting substance (rhMIS) acts via its receptor. To do these experiments, we scaled up production of rhMIS and labeled it successfully with biotin for binding studies, cloned the human MIS type II receptor for mRNA detection, and raised antibodies to an extracellular domain peptide for protein detection. These probes were first tested on the human ovarian cancer cell lines and then applied to primary ovarian ascites cells. rhMIS inhibited colony growth of five of six cell lines that expressed the human MIS type II receptor mRNA by Northern analysis while not inhibiting receptor-negative COS cells. Flow cytometry performed on MIS-sensitive ovarian cancer cell lines demonstrated specific and saturable binding of rhMIS (Kd = 10.2 nM). Ascites cells from 15 of 27 or 56% of patients tested bound biotinylated MIS (MIS-biotin) and, of the 11 that grew in soft agarose, 9 of 11 or 82% showed statistically significant inhibition of colony formation. Of the 15 patients who bound biotinylated MIS, mRNA was available for analysis from 9, and 8 of 9 expressed MIS type II receptor mRNA by reverse transcription-PCR, showing a statistically significant correlation, compared with binding, by chi2 analysis (P = 0.025). Solid ovarian cancers were positive for the MIS type II receptor protein by immunohistochemical staining, which colocalized with staining for antibody to CA-125 (OC-125). Thus, the detection of the MIS type I receptor by flow cytometry may be a useful predictor of therapeutic response to MIS and may be a modality to rapidly choose patients with late-stage ovarian cancer for treatment with MIS.
- Published
- 1999
30. Intrinsic human immunodeficiency virus type 1 resistance of hematopoietic stem cells despite coreceptor expression.
- Author
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Shen H, Cheng T, Preffer FI, Dombkowski D, Tomasson MH, Golan DE, Yang O, Hofmann W, Sodroski JG, Luster AD, and Scadden DT
- Subjects
- Acquired Immunodeficiency Syndrome therapy, Adult, Antigens, CD34 analysis, Genetic Therapy, Hematopoietic Stem Cells physiology, Humans, RNA, Messenger analysis, Receptors, CCR5 genetics, Receptors, CXCR4 genetics, CD4 Antigens analysis, HIV-1 physiology, Hematopoietic Stem Cells virology, Receptors, CCR5 analysis, Receptors, CXCR4 analysis
- Abstract
Interactions of human immunodeficiency virus type 1 (HIV-1) with hematopoietic stem cells may define restrictions on immune reconstitution following effective antiretroviral therapy and affect stem cell gene therapy strategies for AIDS. In the present study, we demonstrated mRNA and cell surface expression of HIV-1 receptors CD4 and the chemokine receptors CCR-5 and CXCR-4 in fractionated cells representing multiple stages of hematopoietic development. Chemokine receptor function was documented in subsets of cells by calcium flux in response to a cognate ligand. Productive infection by HIV-1 via these receptors was observed with the notable exception of stem cells, in which case the presence of CD4, CXCR-4, and CCR-5, as documented by single-cell analysis for expression and function, was insufficient for infection. Neither productive infection, transgene expression, nor virus entry was detectable following exposure of stem cells to either wild-type HIV-1 or lentivirus constructs pseudotyped in HIV-1 envelopes of macrophage-tropic, T-cell-tropic, or dualtropic specificity. Successful entry into stem cells of a vesicular stomatitis virus G protein-pseudotyped HIV-1 construct demonstrated that the resistance to HIV-1 was mediated at the level of virus-cell membrane fusion and entry. These data define the hematopoietic stem cell as a sanctuary cell which is resistant to HIV-1 infection by a mechanism independent of receptor and coreceptor expression that suggests a novel means of cellular protection from HIV-1.
- Published
- 1999
- Full Text
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31. Differential sensitivity of p53(-) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay.
- Author
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Powell SN, DeFrank JS, Connell P, Eogan M, Preffer F, Dombkowski D, Tang W, and Friend S
- Subjects
- Animals, Cell Cycle drug effects, Cell Survival drug effects, Cell Survival radiation effects, Cells, Cultured, Dose-Response Relationship, Radiation, Embryo, Mammalian, Fibroblasts drug effects, Fibroblasts radiation effects, G2 Phase drug effects, Mice, Mice, Knockout, Mitosis drug effects, Mitosis radiation effects, X-Rays, Caffeine pharmacology, Cell Cycle radiation effects, G2 Phase radiation effects, Genes, p53, Radiation-Sensitizing Agents pharmacology
- Abstract
Most drug discovery efforts have focused on finding new DNA-damaging agents to kill tumor cells preferentially. An alternative approach is to find ways to increase tumor-specific killing by modifying tumor-specific responses to that damage. In this report, we ask whether cells lacking the G1-S arrest in response to X-rays are more sensitive to X-ray damage when treated with agents that override G2-M arrest. Mouse embryonic fibroblasts genetically matched to be (+) or (-) p53 and rat embryonic fibroblasts (+) or (-) for wild-type p53 function were irradiated with and without caffeine, a known checkpoint inhibitor. At low doses (500 microM), caffeine caused selective radiosensitization in the p53(-) cells. At this low dose (where no effect was seen in p53(+) cells), the p53(-) cells showed a 50% reduction in the size of the G2-M arrest. At higher doses (2 mM caffeine), where sensitization was seen in both p53(+) and p53(-) cells, the radiosensitization and the G2-M override were more pronounced in the p53(-) cells. The greater caffeine-induced radiosensitization in p53(-) cells suggests that p53, already shown to control the G1-S checkpoint, may also influence aspects of G2-M arrest. These data indicate an opportunity for therapeutic gain by combining DNA-damaging agents with compounds that disrupt G2-M arrest in tumors lacking functional p53.
- Published
- 1995
32. Cell proliferation kinetics in human tumor xenografts measured with iododeoxyuridine labeling and flow cytometry: a study of heterogeneity and a comparison between different methods of calculation and other proliferation measurements.
- Author
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Perez LA, Dombkowski D, Efird J, Preffer F, and Suit HD
- Subjects
- Animals, Carcinoma, Squamous Cell pathology, Cell Count, Colorectal Neoplasms pathology, Flow Cytometry, Glioma pathology, Head and Neck Neoplasms pathology, Humans, Idoxuridine, Kinetics, Male, Mathematics, Mice, Mice, Nude, Neurilemmoma pathology, Specific Pathogen-Free Organisms, Tumor Cells, Cultured, Cell Division genetics, DNA, Neoplasm analysis, Neoplasm Transplantation pathology, Transplantation, Heterologous
- Abstract
The influence of overall treatment time in the results of fractionated radiation treatment was initially established in experimental tumors and, subsequently, in the clinic. The availability of techniques (antibodies against halogenated thymidine analogues and flow cytometry) which permit determinations of the duration of the synthesis phase, the labeling index, and the tumor potential doubling time (Tpot) in a short period of time and requiring only a small biopsy of tumor tissue, has expanded interest in the relationship between tumor cell proliferation and response to irradiation. A valuable tool in the study of this relationship are human tumor xenografts. Previous studies have shown a substantial intratumoral heterogeneity in the determinations of Tpot. Different methods of calculation of the kinetic parameters have been published. We have conducted a heterogeneity analysis and an evaluation of the different calculation methods in order to define the validity of Tpot as a proliferation rate measurement in human tumor xenografts. Results show the intertumoral variability in Tpot [between different types of human tumor xenografts systems (coefficient of variation = 88.2%)] to be greater than mean intratumoral variation (coefficient of variation = 30.8%); this suggests that this variation is potentially adequate to serve as a predictor of response. The diverse calculation methods provided significantly different absolute values but not different tumor ranking, probably because the time interval between labeling and sampling was maintained, for all the samples, between 6 and 8 h. Our study has found significant differences between the labeling index and the S-phase fraction determined with the DNA profile in 9 out of 10 tumor types. No correlation was found between the DNA index of the tumors in this series and their proliferation rate.
- Published
- 1995
33. Transfection of rat embryo cells with mutant p53 increases the intrinsic radiation resistance.
- Author
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Pardo FS, Su M, Borek C, Preffer F, Dombkowski D, Gerweck L, and Schmidt EV
- Subjects
- Animals, Cell Cycle, Cells, Cultured, Embryo, Mammalian cytology, Mutation, Rats, Rats, Sprague-Dawley, Transfection, Genes, p53 physiology, Radiation Tolerance
- Abstract
Dominant oncogenic sequences have been shown to modulate the intrinsic radiation sensitivity of cells of both human and murine tumor cell lines. Whether transfection with candidate tumor-suppressor genes can modulate intrinsic radiation sensitivity is unknown. The data presented here demonstrate that transfection of rat embryo cells with a mutant p53 allele can increase the intrinsic radiation resistance of cells in vitro. First, transfection with mutant p53 resulted in transformed cellular morphology. Second, the transfected clone and the corresponding pooled population of transfected clones were more resistant to ionizing radiation in vitro. Last, analyses of the parameters of cell kinetics suggested that the radiobiological effects were unlikely to be due to altered parameters of cell kinetics at the time of irradiation, suggesting that mutant p53 altered the intrinsic radiation resistance of transfected cells by a more direct mechanism. Further experimentation will be necessary to develop a mechanistic approach for the study of these alterations.
- Published
- 1994
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