RESUMEN
The ability to detect changes in RNA expression in single cells would greatly enhance understanding of the molecular basis of biological responses to positive and negative growth regulators. To this end, we compared expression of RNA encoding the receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, leukemia inhibitory factor (LIF) and stem cell factor (SCF) in populations of primitive hematopoietic progenitors (lineage marker negative, Lin(-), and Lin(-) c-Kit(+)) by RT-PCR and in situ RT-PCR. Both Lin(-) and Lin(-) c-Kit(+) progenitors expressed all receptors by RT-PCR. However, RT-PCR could not distinguish between the possibility that all cells expressed growth factor receptor RNA, or the possibility that only a proportion of cells expressed RNA. Therefore, we used in situ RT-PCR to examine growth factor receptor mRNA expression in individual cells. In contrast to RT-PCR, we observed that only 40-80% of Lin(-) cells and 75-100% of Lin(-) c-Kit(+) cells were positive for expression of the growth factor receptor subunits, demonstrating that not all cells were receptor positive. We found that in situ RT-PCR could also be used to measure induction or repression of receptor RNA expression in these cell populations. Specifically, the percentage of cells expressing IL-6alpha receptor RNA decreased from 88% positive in freshly harvested cells to 9% in Lin(-) c-Kit(+) cells cultured in IL-3 for 18 h. Thus, in situ RT-PCR can be used to detect and quantify the number of individual cells that express growth factor receptor mRNA, and may also be useful to measure changes in expression of other endogenous genes or genes introduced by transfection and gene therapy vectors.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , ARN/análisis , ARN/genética , Receptores de Factores de Crecimiento/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Actinas/genética , Animales , Secuencia de Bases , Cartilla de ADN/genética , Expresión Génica , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas c-kit/genética , Receptores de Citocinas/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Interleucina-3/genética , Receptores de Interleucina-6/genética , Receptores OSM-LIFRESUMEN
The inability of adenovirus to infect primitive hematopoietic cells presents an obstacle to the use of adenovirus vectors for gene transfer to these cell types. Therefore, expanding the tropism of adenovirus vectors to unique cell surface antigens would be an important development for gene therapy protocols. In this study, we sought to redirect infection of adenovirus vectors to primitive human hematopoietic cells that universally express the c-Kit receptor on their cell surface. To accomplish this, a vector was constructed by covalently linking biotin molecules to recombinant adenovirus, followed by addition of the biotinylated ligand for the c-Kit receptor, stem cell factor (SCF), through an avidin bridge. Gene transfer was directed specifically to c-Kit-positive hematopoietic cell lines, resulting in up to a 2,440-fold increase in luciferase expression with frequencies equivalent to recombinant virus infection of permissive cells. Substitution of biotinylated antibodies directed against c-Kit, CD34 (binds L-selectin), and CD44 (hyaluronate receptor) receptors for biotinylated SCF resulted in 50-, 8-, and 260-fold increases in reporter gene expression, respectively, demonstrating that infection also could be redirected through antibody-antigen interactions and through antigens other than growth factor receptors. The versatility of this vector was demonstrated further by infection of primary T cells with vectors targeted with antibodies to CD44 (resting and activated T cells) and biotinylated IL-2 (activated T cells only). Taken together, directly biotinylated adenovirus vectors represent a versatile and efficient method for redirection of virus infection to specific cells.
Asunto(s)
Adenovirus Humanos/genética , Antígenos CD/fisiología , Vectores Genéticos , Proteínas Proto-Oncogénicas c-kit/fisiología , Factor de Células Madre/genética , Linfocitos T/fisiología , Anticuerpos Monoclonales/farmacología , Antígenos CD/genética , Antígenos CD34/genética , Antígenos CD34/fisiología , Biotinilación , Línea Celular , Células Cultivadas , Citomegalovirus , Proteínas Fluorescentes Verdes , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/fisiología , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Recombinantes de Fusión/biosíntesis , Recombinación Genética , TransfecciónRESUMEN
While the majority of purified pluripotential hematopoietic stem cells (PHSC) express c-Kit, the receptor for steel factor, we have phenotypically and functionally separated a distinct class of PHSC that does not express c-Kit. In contrast to c-Kit-positive (c-Kit(pos)) PHSC, the c-Kit-negative (c-Kit(neg)) PHSC do not proliferate in response to multiple hematopoietic growth factors in vitro and do not radioprotect or form macroscopic spleen colonies (CFU-s) when transplanted into lethally irradiated recipients. However, the c-Kit(neg) PHSC show delayed or slow reconstitution kinetics when cotransplanted with radioprotective bone marrow cells. c-Kit(neg) PHSCs cells can give rise to c-Kit(pos) cells with CFU-s activity, radioprotective activity, and PHSC activity. Thus, constitutive hematopoiesis is maintained by c-Kit(pos) PHSCS cells that are recruited from a more primitive quiescent c-Kit(neg) PHSC population, which represents a critical developmental stage in definitive hematopoiesis.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Proteínas Proto-Oncogénicas c-kit/fisiología , Animales , Células de la Médula Ósea/fisiología , Línea Celular , Separación Celular , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la PolimerasaRESUMEN
To develop a rapid preclinical in vivo model to study gene transfer into human hematopoietic progenitor cells, MO-7e cells (CD-34+, c-kit+) were infected with multidrug resistance (MDR1)-containing retroviruses and then transplanted into nonobese diabetic severe combined immunodeficient mice (NOD SCID). MO-7e cells infected with a retrovirus encoding the human MDR1 cDNA showed integration, transcription, and expression of the transfered MDR1 gene. This resulted in a 20-fold increase in the resistance of MO-7e cells to paclitaxel in vitro. The expression of the MDR1 gene product was stable over a 6-month period in vitro without selection in colchicine. MO-7e and MDR1-infected MO-7e cells were transplanted into NOD SCID mice to determine whether MDR1 could confer drug resistance in vivo. A sensitive polymerase chain reaction method specific for human sequences was developed to quantitate the level of human cell engraftment in NOD SCID bone marrow (BM) cells. The percentage of human DNA in BM cells from MO-7e-transplanted mice was 10.9% and decreased to 0.7% in mice treated with paclitaxel. The percentage of human DNA in infected-MO-7e transplanted mice was 7.6% and that level was unchanged in mice treated with paclitaxel. These results show that expression of the MDR1 gene in human hematopoietic progenitor cells can confer functional drug resistance in an in vivo model.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Resistencia a Múltiples Medicamentos/genética , Células Madre Hematopoyéticas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Animales , Técnicas de Transferencia de Gen , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/patología , Humanos , Ratones , Ratones SCID , RetroviridaeRESUMEN
The pathogenesis of progressive spastic paraparesis [HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP)], a serious consequence of human T-cell leukemia virus type I (HTLV-I) infection, is unclear. T and B lymphocytes can be naturally infected by HTLV-I, but the susceptibility to HTLV-I infection of other cell types that could contribute to the pathogenesis of HAM/TSP has not been determined. We found that a human monocyte cell line (THP-1), primary human peripheral blood monocytes, and isolated microglial cells but not astrocytes or oligodendroglial cells derived from adult human brain were infected by HTLV-I in vitro. Infection with HTLV-I enhanced the secretion of interleukin 6 in human microglial cell-enriched cultures but did not stimulate the release of interleukin 1 from monocytes or microglial cells. Tumor necrosis factor alpha production was stimulated by HTLV-I infection of monocytes and microglial cells and could be enhanced by suboptimal amounts of lipopolysaccharide. Since both tumor necrosis factor alpha and interleukin 6 have been implicated in inflammatory demyelination and gliosis, our findings suggest that human microglial cells and monocytes infected with and activated by HTLV-I could play a role in the pathogenesis of HAM/TSP.
Asunto(s)
Infecciones por HTLV-I/microbiología , Virus Linfotrópico T Tipo 1 Humano/crecimiento & desarrollo , Monocitos/microbiología , Neuroglía/microbiología , Encéfalo/microbiología , Células Cultivadas , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Técnicas In Vitro , Interleucina-6/biosíntesis , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Individuals infected with HIV may be asymptomatic for years before progressing to overt AIDS. Since HIV can latently infect monocytoid cell lines, we examined whether HIV latency occurs in monocytes in vivo. Freshly isolated monocytes from asymptomatic seropositive individuals examined before and after culture were positive for HIV DNA, but not RNA, as measured by polymerase chain reaction, showing that HIV latency occurs in monocytes in vivo. Coculture of these latently infected monocytes with Con A-activated T cells from HIV-negative normal donors stimulated 90% of the patients' samples and latently infected THP-1 to produce infectious virus. Neither Con A, resting T cells, nor T cell supernatants induced virus. Plasma membranes from activated T cells stimulated HIV production, suggesting cell contact induces factor(s) in monocytes to overcome latency. Thus, monocytes in AIDS patients harbor latent HIV inducible during an immune response, leading to T cell infection and viral-induced pathology.
Asunto(s)
Seropositividad para VIH/microbiología , VIH-1/crecimiento & desarrollo , Monocitos/microbiología , Activación Viral , Línea Celular , ADN Viral/análisis , VIH-1/genética , Humanos , Activación de Linfocitos , ARN Viral/análisis , Linfocitos T/inmunologíaRESUMEN
Treatment of HL-60 cells, a human promyelocytic leukemia cell line, with the vitamin A derivative retinoic acid (RA) for 7 days resulted in a dose-dependent decrease in proliferation and increase in granulocytic differentiation. The role of transforming growth factor-beta 1 (TGF-beta 1), a protein with pleiotropic effects on the proliferation and differentiation of various cell types, was examined during RA-induced differentiation of HL-60 cells. Although TGF-beta 1 alone had little effect on proliferation or differentiation of HL-60 cells, addition of TGF-beta 1 to HL-60 cells treated with a suboptimum concentration of RA (1.0 nmol/L) resulted in a marked decrease in proliferation with no effect on granulocytic differentiation. Studies of the mechanism of RA-induced TGF-beta sensitivity showed that although untreated HL-60 cells expressed low levels of TGF-beta 1 binding proteins on the cell surface, the levels were increased in a dose-dependent manner after RA treatment. Maximum induction was achieved after treatment with 10 nmol/L RA and consisted predominantly of the 65-Kd TGF-beta 1 receptor type. Moreover, RA treatment also resulted in a dose-dependent increase in both TGF-beta 1 steady-state mRNA expression and production of active TGF-beta with maximum induction at 10 nmol/LRA. RA treatment of HL-60 cells had no effect on TGF-beta 2 and TGF-beta 3 mRNA expression. These data suggest that the effects of RA may be mediated by a TGF-beta 1-mediated autocrine antiproliferative loop during differentiation of HL-60 cells.
Asunto(s)
Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Receptores de Superficie Celular/genética , Factor de Crecimiento Transformador beta/metabolismo , Tretinoina/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Relación Dosis-Respuesta a Droga , Regulación Leucémica de la Expresión Génica/fisiología , Hematopoyesis/efectos de los fármacos , Hematopoyesis/fisiología , Humanos , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento Transformadores beta , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/fisiología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patología , Células Tumorales Cultivadas/ultraestructuraRESUMEN
In THP-1 monocytoid cells infected with HIV, viral expression can be regulated in several ways: (a) latency (no viral expression); (b) restricted expression (chronic low-level viral expression with little or no detectable virus released); and (c) continuous production. In cells with restricted HIV expression, nuclear factor(s) were found that blocked tat-associated DNA binding complex formation, suggesting that initiation of transcription was negatively regulated. Also, viral particles were seen budding into and accumulating within intracytoplasmic vacuoles with little virus released, suggesting multiple levels of regulation. These cells with restricted expression had no detectable viral antigens on the cell surface and were not lysed by IL-2-activated large granular lymphocytes. However, they could cause viral-mediated T cell cytolysis in cell-cell assays, suggesting viral transmission through cell contact. In addition, cells with latent HIV were identified and could still produce infectious virus after 5-azacytidine exposure 10 mo later. LPS and other treatments could increase viral production in cells with restricted but not latent expression, suggesting they occur by distinct mechanisms. These infected cells provide a reservoir for viral transmission to uninfected T cells that itself is not detected by immune surveillance mechanisms.
Asunto(s)
VIH-1/fisiología , Monocitos/microbiología , Replicación Viral , Antígenos Virales/análisis , Línea Celular , Supervivencia Celular , ADN Viral/análisis , VIH-1/genética , VIH-1/ultraestructura , Humanos , Microscopía Electrónica , Monocitos/citología , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Oncogenes carried by retroviruses can alter the growth properties of many cell types. We examined the molecular mechanism by which a retrovirus containing one or a combination of oncogenes can transform and immortalize hematopoietic cells. Murine fetal liver cells were used as an enriched source of early hematopoietic cell progenitors; the cells were infected with a series of recombinant murine retroviruses capable of expressing the avian v-myc, v-H-ras and v-raf oncogenes. Three factor-independent cell lines were obtained: FL-ras/myc, FL-J2 (v-raf/v-myc) and FL-myc, a unique cell line generated using a single oncogene. Cytochemical, morphologic and phenotypic analyses indicated that these cell lines were of the monocyte lineage. Southern and Northern blot analyses revealed that the three cell lines had integrated viral DNA and were expressing the mRNA transcripts corresponding to these viral oncogenes. To examine the mechanism of factor independence, supernatants from these cell lines were tested for CSF-1 activity. Supernatants from FL-myc and FL-ras/myc cells were shown to contain CSF-1 activity and Northern blot analysis of the three cell lines revealed the presence of mRNA transcripts for the CSF-1 and c-fms genes. It is possible that the growth factor independence of these cell lines is related to the development of autocrine-induced proliferation.
Asunto(s)
Transformación Celular Neoplásica , Transformación Celular Viral , Genes Virales , Oncogenes , Retroviridae/genética , Animales , Línea Celular , Factores Estimulantes de Colonias/biosíntesis , Células Madre Hematopoyéticas/microbiología , Immunoblotting , Hígado/citología , Ratones , Fenotipo , ARN Viral/biosíntesis , Transcripción GenéticaRESUMEN
We describe procedures for the large-scale production of equine infectious anemia virus (EIAV) and for the isolation of the four major non-glycosylated virion proteins, designated p26, p15, p11, and p9. Comparisons of the purified proteins by peptide mapping procedures and by enzyme-linked immunosorbent assays demonstrated the unrelatedness of the four proteins. The characteristic properties of each purified protein were examined by determining isoelectric points and amino acid compositions. We found that EIAV p26 and p9 focus at pI values of 6.2 and 5.0, respectively, and that these proteins contain no unusual amino acids. In contrast, EIAV p15 reproducibly displayed a heterogeneous isoelectric focusing pattern, with major pI values ranging from 5.7 to 8.3. This charge variation evidently correlated with different levels of phosphorylated serine or threonine or both, which could be detected by an amino acid analysis of purified p15. EIAV p11 apparently focused at a pI of greater than 10, reflecting its high content of basic amino acids. Moreover, localization experiments indicated that all four nonglycosylated proteins constitute the internal components of the virus, with all of the virion p11 closely associated with the viral RNA genome. Thus, our results demonstrated that EIAV, a lentivirus, contains structural polypeptides which are analogous to the structural polypeptides described previously in prototype C oncoviruses.