RESUMO
The colorectal cell line HCA-7 expresses surface human leucocyte antigen-A*0201 (HLA-A*0201), but lacks expression of HLA-A*0101 whilst the normal B-cell line (EVA-1224), derived from the same individual, expresses both surface HLA-A1 and HLA-A2. Amplification refractory mutation system-polymerase chain reaction analysis, using sequence-specific primers, suggested that HCA-7 has a mutation in a 7 base pair (bp) cytosine repeat sequence located at the beginning of Exon 4 (bp 621-627). Cloning and sequencing revealed HCA-7 to have eight cytosine residues in this repeat sequence. In contrast, EVA-1224 contained only 7 cytosines. Analysis of the mRNA for HLA-A*010 using reverse trancriptase-polymerase chain reaction (RT-PCR), with an allele-specific 5' primer in exon 2 (bp 253-271) and a series of 3' primers in exons 3, 4 and 7 and in the 3'untranslated region, revealed that HCA-7 contained a shortened message terminating in the region of the exon 3/4 boundary. The insertion of an extra cytosine in this region, which is only two bases from the exon 3/4 splice site, is presumed to lead to a splicing defect between exons 3 and 4 resulting in the lack of expression of a functional HLA-A*0101 product. HCA-7 is mismatch repair (MMR) defective due to lack of expression of hMLH1 resulting from hypermethylation of the promoter region. The consequential increase in errors in single-nucleotide repeat stretches of DNA can account for the HLA-A*0101 mutation. This has probably then been selected for in the tumour to enable escape from immune attack against an HLA-A*0101-restricted tumour-specific determinant that has also arisen as a result of the tumour being MMR defective.
Assuntos
Neoplasias Colorretais/genética , Antígenos HLA-A/genética , Mutação , Regiões 3' não Traduzidas , Alelos , Pareamento Incorreto de Bases , Linhagem Celular Tumoral , Clonagem Molecular , Citosina , DNA/metabolismo , Primers do DNA/genética , Reparo do DNA , Éxons , Frequência do Gene , Antígeno HLA-A1 , Antígeno HLA-A2 , Humanos , Focalização Isoelétrica , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Radioimmunoguided surgery (RIGS) has been known as a sophisticated tool to detect micrometastasis intraoperatively. A preclinical model of RIGS was designed to test the possible clinical applicability of the biparatopic antibody in detecting colorectal cancer. The biparatopic antibody was constructed using two anti-carcinoembryonic antigen (CEA)-specific antibodies, T84.66 and PR1A3, reacting against two different epitopes. (125)I-labeled biparatopic antibody was introduced via the principal colonic arteries at the end of operation in 10 operable patients with colon cancer. After 24 h, the radioactivities of the tumors and lymph nodes were counted using the gamma-detecting probe. The radioactivity count was performed ex vivo. The accurate detection in the primary tumors and metastatic lymph nodes were 100 and 88.7% respectively. False-positive detections occurred in 24 of 256 lymph nodes (9.4%), whereas false-negative detections occurred in 5 of them (2%). The most frequent cause of false-positive detection was dissociated radionuclides trapped in the lymphatic tissues. False-negative detections occurred mainly from weak targeting by radiolabeled antibody, probably due to weak expression of tumor CEA. Conclusively, as most detection errors appear to be reduced within 3 days in vivo, the biparatopic antibody can efficiently be applied to the clinical RIGS, thereby facilitating accurate detection and removal of occult cancer foci in colorectal cancer.
Assuntos
Anticorpos , Antígeno Carcinoembrionário/análise , Carcinoma/cirurgia , Neoplasias do Colo/cirurgia , Epitopos , Radioimunodetecção/métodos , Autorradiografia , Antígeno Carcinoembrionário/imunologia , Carcinoma/diagnóstico , Neoplasias do Colo/diagnóstico , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Imuno-Histoquímica , Metástase Linfática/diagnóstico , Pessoa de Meia-IdadeRESUMO
Expression of the homeobox protein CDX1 is lost or reduced in a significant proportion of colorectal carcinomas (CRCs) but the underlying mechanism for this is unclear. We have demonstrated absence of CDX1 mRNA expression in 7 of 37 CRC cell lines and shown that all 7 cell lines have a methylated CDX1 promoter. Twenty-five cell lines showed both CDX1 mRNA expression and an unmethylated CDX1 promoter. The five remaining cell lines had a partially methylated CDX1 promoter and all expressed CDX1 mRNA; when treated with the demethylating agent, 5-aza-2'-deoxycytidine, these five cell lines all showed increased CDX1 expression. No mutations were found in the promoter and coding regions of CDX1. One polymorphism was demonstrated in each of the promoter, 5' UTR, and coding region of exon 1 of CDX1, but there were no associations between CDX1 mRNA expression and different polymorphic genotypes. Similarly, there was no association between CDX1 mRNA expression and loss of heterozygosity at the CDX1 locus. In conclusion, absence or reduction of CDX1 expression in CRC seems to be primarily regulated by promoter methylation and is probably selected for because of its impact on the differentiation of colonocytes.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA , Proteínas de Homeodomínio/genética , Perda de Heterozigosidade , Mutação , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , DNA de Neoplasias , Humanos , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido NucleicoRESUMO
Loss of chromosome 18q21 is well documented in colorectal cancer, and it has been suggested that this loss targets the DCC, DPC4/SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-beta signaling pathway, in colorectal cancer has been highlighted, although the frequency of SMAD4 mutations appears much lower than that of 18q21 loss. We set out to investigate allele loss, mutations, protein expression, and cytogenetics of chromosome 18 copy number in a collection of 44 colorectal cancer cell lines of known status with respect to microsatellite instability (MSI). Fourteen of thirty-two MSI(-) lines showed loss of SMAD4 protein expression; usually, one allele was lost and the other was mutated in one of a number of ways, including deletions of various sizes, splice site changes, and missense and nonsense point mutations (although no frameshifts). Of the 18 MSI(-) cancers with retained SMAD4 expression, four harbored missense mutations in the 3' part of the gene and showed allele loss. The remaining 14 MSI(-) lines had no detectable SMAD4 mutation, but all showed allele loss at SMAD4 and/or DCC. SMAD4 mutations can therefore account for about 50-60% of the 18q21 allele loss in colorectal cancer. No MSI(+) cancer showed loss of SMAD4 protein or SMAD4 mutation, and very few had allelic loss at SMAD4 or DCC, although many of these MSI(+) lines did carry TGFBIIR changes. Although SMAD4 mutations have been associated with late-stage or metastatic disease, our combined molecular and cytogenetic data best fit a model in which SMAD4 mutations occur before colorectal cancers become aneuploid/polyploid, but after the MSI(+) and MSI(-) pathways diverge. Thus, MSI(+) cancers may diverge first, followed by CIN(+) (chromosomal instability) cancers, leaving other cancers to follow a CIN(-)MSI(-) pathway.
Assuntos
Adenocarcinoma/genética , Cromossomos Humanos Par 18/genética , Neoplasias Colorretais/genética , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Repetições de Microssatélites , Proteínas de Neoplasias/genética , Transativadores/genética , Western Blotting , Transformação Celular Neoplásica/genética , Deleção Cromossômica , Análise Mutacional de DNA , Proteínas de Ligação a DNA/biossíntese , Deleção de Genes , Perfilação da Expressão Gênica , Genes DCC , Humanos , Perda de Heterozigosidade , Mutação , Proteínas de Neoplasias/biossíntese , Ploidias , Polimorfismo Conformacional de Fita Simples , Transdução de Sinais , Proteína Smad4 , Fatores de Tempo , Transativadores/biossíntese , Fator de Crescimento Transformador beta/fisiologia , Células Tumorais CultivadasRESUMO
Locomotion of colorectal-carcinoma cells was tested in order to establish whether it might be affected by carcino-embryonic antigen (CEA). CEA production, cell growth and DNA ploidy were measured in 22 colorectal-carcinoma cell lines. A cell-invasion assay was adapted using a transfilter chamber, the lower surface of which was coated with various substrates in the amount of 5 microgram/filter (CEA, type-IV collagen, laminin). Cells infiltrated into the lower surface of the filter were counted over 9-microscope fields (x400). All cell lines produced CEA, 9 producing more than 100 ng/ml medium. Of the total, 8 cell lines were diploid and 14 were aneuploid. Invasiveness, measured by the number of infiltrated cells, was highest in CEA-coated filters, and next highest in type-IV-collagen- and laminin-coated filters, in descending order (p < 0.001-0.05). Invasiveness of each cell line was closely correlated with 2 substrates. Poorly differentiated or advanced-stage tumors were more invasive than well-differentiated or early-stage tumors (p < 0.001-0. 05). However, invasiveness was not associated with DNA ploidy or CEA production. CEA may function as a chemo-attractant as well as an adhesion molecule in colorectal-carcinoma cell lines. In addition, adhesion to CEA appears to be related to type-IV collagen and laminin.
Assuntos
Antígeno Carcinoembrionário/fisiologia , Fatores Quimiotáticos , Quimiotaxia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/fisiopatologia , Divisão Celular , Colágeno , Humanos , Cinética , Laminina , Metástase Linfática , Invasividade Neoplásica , Estadiamento de Neoplasias , Células Tumorais CultivadasRESUMO
Novel peptide antigens complexed with human leukocyte antigen (HLA) and beta 2-microglobulin (beta 2M) molecules are presented at the cell surface to cytotoxic T lymphocytes (CTLs), provoking lysis of the antigen-presenting cell [1]. In tumor cells, genetically altered or abnormally expressed proteins provide a source of peptides that can be presented to CTLs; the resulting anti-tumour CTL responses may provide part of the body's defence against cancer. Disabling mutations in the HLA and beta 2M proteins required for peptide presentation allow a tumour cell to escape destruction by CTLs. Cells with deficient DNA mismatch repair have high spontaneous mutation rates [2] and produce many altered proteins that are a potential source of numerous unique peptides. Mutator tumour cells might therefore be particularly vulnerable to immune surveillance and CTL attack. Mutator phenotypes [3,4] and loss of beta 2M (or HLA) expression [5,6] are both relatively common among sporadic colorectal tumours. We have compared the frequency of beta 2M mutations in sporadic colorectal and other tumours with and without a mutator phenotype. Mutations were more frequent among colorectal tumours with the microsatellite instability indicative of a defect in DNA mismatch repair. The inactivating beta 2M mutations were predominantly frameshifts, which is consistent with the underlying mismatch repair defects. Evasion of immune surveillance by acquiring beta 2M mutations therefore occurs at high frequency in tumour cells with a mutator phenotype due to defective DNA mismatch repair.
Assuntos
Neoplasias Colorretais/genética , Reparo do DNA , DNA de Neoplasias , Microglobulina beta-2/genética , Neoplasias Colorretais/metabolismo , Mutação da Fase de Leitura , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
The technique of single-strand conformation polymorphism (SSCP) was used to screen a series of 37 established colorectal cell lines, 22 fresh tumor samples, and 22 normal DNA samples for mutations in the beta 2-microglobulin gene. Exon 1 (including the leader peptide sequence) and exon 2 were screened separately. Six of 37 colorectal cell lines and 1 of 22 fresh tumors were shown to contain mutations, whereas no mutations were detected in the normal DNA samples. Sequencing of these mutations showed that an 8-bp CT repeat in the leader peptide sequence was particularly variable, since 3 of the cell lines and one fresh tumor sample have deletions in this region. In the related cell lines, DLD-1 and HCT-15, two similar mutations were identified, a C-->A substitution in codon 10 and a G-->T mutation in the splice sequence of intron 1. Expression of beta 2-microglobulin was examined using a series of monoclonal antibodies in an ELISA system. Reduced expression correlated with a mutation in one allele of beta 2-microglobulin, whereas loss of expression was seen in instances where a line was homozygous for a mutation or heterozygous for two mutations.
Assuntos
Neoplasias Colorretais/genética , Mutação , Microglobulina beta-2/genética , Sequência de Aminoácidos , Sequência de Bases , DNA de Neoplasias , Éxons , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Genético , Células Tumorais CultivadasRESUMO
A system devised for tissue typing the human leukocyte antigen-A (HLA-A) locus from genomic DNA by the polymerase chain reaction (PCR) has been used to investigate abnormalities of HLA expression in a panel of 30 cell lines derived from colorectal adenocarcinomas, by comparison of the HLA-A locus genotype with surface expression of HLA. Eleven cell lines gave single HLA-A locus specificity on PCR typing, suggesting that loss of HLA alleles is a common abnormality. In one of these cell lines the loss of an HLA-A locus allele was confirmed by comparison with DNA from a lymphoblastoid B cell line derived from the same patient. In three cell lines, loss of expression of an HLA-A locus determinant was observed in spite of the presence of the relevant allele in genomic DNA. Three cell lines showed absent HLA expression associated with failure to express beta 2-microglobulin. These data indicate that at least three independent mechanisms were involved in the loss of HLA expression on the colorectal tumor cell lines.
Assuntos
Neoplasias Colorretais/imunologia , Citotoxicidade Imunológica , Antígenos HLA/análise , Imunoterapia , Antígenos de Superfície/análise , Mapeamento Cromossômico , Neoplasias Colorretais/patologia , Genoma , Genótipo , Antígenos HLA-A/genética , Antígeno HLA-A2/análise , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
A system devised for tissue typing the HLA-A locus by PCR from genomic DNA has been used to investigate abnormalities of HLA expression in a panel of 30 colorectal tumor cell lines, by comparing the HLA-A locus genotype with surface expression of HLA. Three cell lines showed complete lack of HLA expression associated with failure to express beta 2-microglobulin. In two other cell lines, loss of expression of HLA-A2 was observed, in spite of the presence of the gene in genomic DNA. Eleven cell lines gave a single HLA-A locus specificity on PCR typing. In one of these cell lines we have demonstrated the loss of an HLA-A locus gene in the tumor cell by comparison with DNA from a lymphoblastoid B-cell line derived from the same patient. These data indicate that at least three independent mechanisms were involved in the loss of HLA expression on the colorectal tumor cell lines.
Assuntos
Neoplasias do Colo/imunologia , Neoplasias Colorretais/imunologia , DNA de Neoplasias/genética , Antígenos HLA-A/genética , Teste de Histocompatibilidade , Sequência de Bases , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Expressão Gênica , Genótipo , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Células Tumorais Cultivadas , Microglobulina beta-2/genéticaRESUMO
The ubiquitous nature of the Alu sequence throughout the human genome forms the basis of an assay we present here for analyzing the human chromosome content of human x rodent somatic cell hybrids. A human-specific Alu primer was used both to amplify sequences and to 32P label the products in a polymerase chain reaction (PCR) technique. Unlabeled inter-Alu PCR products from two series of human x rodent hybrids were used to prepare dot blots which were probed with labeled inter-Alu products prepared from between 10(3) and 10(4) hybrid cells. In the first series we demonstrate that a labeled inter-Alu probe from the hybrid DL18ts, containing a single chromosome 18, on a dot blot hybridized only with those inter-Alu products containing chromosome 18. Similar specificity for human chromosome 5 was shown when a Southern blot of the PCR products was hybridized with a probe made from the hybrid HHW 213, which contains only chromosome 5p. Using a dot blot from a second series of control hybrids, 15 of which contained single human chromosomes, hybridization of a labeled probe from the hybrid 18X4-1 was shown to react specifically with the controls that expressed chromosome 18. Application of the technique reported here allows simple and rapid characterization of the human chromosome content in human x rodent hybrids.
Assuntos
Cromossomos Humanos , Células Híbridas , Sequências Repetitivas de Ácido Nucleico , Animais , Sequência de Bases , Southern Blotting , Cricetinae , DNA/isolamento & purificação , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Moldes GenéticosRESUMO
The iron-binding proteins lactoferrin (LF) and H-ferritin have been implicated in the negative regulation of myelopoiesis in vitro and in vivo. The present studies evaluated the functional activity of affinity-purified LF from polymorphonuclear neutrophils (PMN) of patients with chronic myelogenous leukemia (CML) and LF/H-ferritin-cell interactions in a nonleukemic patient with LF deficiency with normal levels of circulating blood leukocytes. Affinity-purified CML-PMN-LF was found to be qualitatively deficient as a suppressor of the release of colony-stimulating factors from mononuclear blood cells, adding to previous information from our group documenting defective LF-cell interactions in CML. LF was detected by immunoradiometric assay in PMN of the patient with LF deficiency, but at a much lower level than normal. This LF was found, however, to be active as a suppressor molecular against the patient's cells and normal donor cells. Patient cells were as responsive as normal cells to effects of purified milk LF. Decreased LF levels in this patient were associated with increased levels of monocyte H-ferritin inhibitory activity, consistent with the known suppressive effects in vitro of LF on H-ferritin release from monocytes. Patient marrow hematopoietic progenitor cells were as responsive as progenitors from normal donors to suppression by purified H-ferritin and prostaglandin E1. These results are consistent with a role of LF and H-ferritin in the control of myelopoiesis in this patient.
Assuntos
Fatores Estimuladores de Colônias/metabolismo , Ferritinas/farmacologia , Lactoferrina/deficiência , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Neutrófilos/química , Alprostadil/farmacologia , Cromatografia de Afinidade , Ensaio de Unidades Formadoras de Colônias , Depressão Química , Dinoprostona/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Lactoferrina/sangue , Lactoferrina/isolamento & purificação , Lactoferrina/farmacologia , Contagem de Leucócitos , Leucócitos Mononucleares/metabolismo , Taxa Secretória/efeitos dos fármacosRESUMO
Purified recombinant human heavy-chain (acidic) ferritin (rHF) was assessed in vivo in mice for effects on the proliferation (percentage of cells in S-phase) and absolute numbers of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells in the femur and spleen and on the nucleated cells in the marrow, spleen, and blood. rHF significantly decreased cycling rates and absolute numbers of marrow and splenic hematopoietic progenitors and marrow and blood nucleated cellularity. These effects were apparent in BDF1, C3H/Hej and DBA/2 mice and were dose dependent, time related, and reversible. Suppressive effects were noted within three hours for progenitor cell cycling, within 24 hours for progenitor cell numbers, and within 48 hours for circulating neutrophils. Additionally, hematopoietic progenitor cells in DBA/2 mice infected with the polycythemia-inducing strain of the Friend virus complex (FVC-P) were insensitive to the in vivo administration of rHF. These studies demonstrate activity of rHF in vivo on myelopoiesis of normal but not FVC-P-infected mice. Since rHF suppresses hematopoietic progenitor cell proliferation from normal donors in vitro and from normal mice in vitro and in vivo but does not suppress progenitor cells from patients with leukemia in vitro or from mice with FVC-P-infection in vitro or in vivo, rHF may be useful as a candidate adjunct molecule for the protection of normal hematopoietic progenitor cells during chemotherapy.
Assuntos
Medula Óssea/fisiologia , Ferritinas/farmacologia , Inibidores do Crescimento/farmacologia , Hematopoese/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Animais , Contagem de Células/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Vírus da Leucemia Murina de Friend , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos DBA , Neutrófilos/efeitos dos fármacos , Policitemia/patologiaRESUMO
Synergistic interactions in the regulation of myelopoiesis have been noted in vitro and in vivo and are discussed. Moreover, data is presented to highlight such synergistic interactions in vitro and in vivo. It is shown that purified recombinant human B-cell stimulating factor-1/interleukin-4 (rh BSF-1/IL-4) synergizes with rh Granulocyte (G)-Colony Stimulating Factor (CSF), but not with rh Granulocyte-Macrophage (GM)-CSF, rh IL-3, or rh Macrophage CSF (CSF-1) to enhance colony formation in vitro by normal human bone marrow cells. This synergism is restricted to granulocyte progenitors. Also, it is shown that rh G-CSF or rh CSF-1 enhance the proliferation of granulocyte-macrophage progenitor cells (CFU-GM) in vivo in mice pretreated with human lactoferrin, and when added together these preparations of CSF act synergistically. It is apparent that a true understanding of how myeloid blood cell production is regulated requires insight into how molecules collaborate with or antagonise one another.
Assuntos
Fatores Estimuladores de Colônias/farmacologia , Células-Tronco Hematopoéticas/citologia , Interleucina-3/farmacologia , Interleucinas/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Sinergismo Farmacológico , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interleucina-4 , Camundongos , Camundongos EndogâmicosRESUMO
The gene product of the viral proto-oncogene v-fms, associated with the feline sarcoma virus (SM-FeSV), is a glycoprotein of 170 kd with associated tyrosine kinase activity. The murine c-fms proto-oncogene produces a similar glycoprotein of 165 kd and has been implicated as a similar, if not identical, molecule to the cell surface receptor of the hematopoietic growth factor CSF-1. Employing a v-fms probe in an in situ hybridization assay we examined the expression of c-fms transcripts in a population of purified granulocyte-macrophage progenitor cells (CFU-GM). Using this approach, which requires only 10-20 thousand cells, we demonstrate that 53% of the cells in the purified CFU-GM containing fraction (FR-28) express c-fms transcripts. In the presence of natural purified CSF-1, 51% +/- 3% of FR-28 cells form colonies or clusters in a semi-solid culture assay system. 94% of the colonies and clusters stimulated by CSF-1 were morphologically macrophage in nature. In addition, 84% +/- 4% of FR-28 cells formed colonies and clusters when stimulated with pokeweed mitogen spleen cell conditioned medium (PWMSCM) in a similar in vitro assay (35% granulocyte, 29% macrophage and 39% mixed granulocyte/macrophage colonies). These results indicate there is a correlation between the responsiveness of CFU-GM progenitor cells to CSF-1 and the expression of c-fms RNA.
Assuntos
Fatores Estimuladores de Colônias/fisiologia , Granulócitos/citologia , Macrófagos/citologia , Proteínas Proto-Oncogênicas/fisiologia , Células-Tronco/citologia , Animais , Autorradiografia , Camundongos , Hibridização de Ácido Nucleico , Receptor de Fator Estimulador de Colônias de MacrófagosRESUMO
The gene product of the viral proto-oncogene v-fms, associated with the feline sarcoma virus (SM-FeSV), is a glycoprotein of 170 kd with associated tyrosine kinase activity. The murine c-fms proto-oncogene produces a similar glycoprotein of 165 kd and has been implicated as a similar, if not identical, molecule to the cell surface receptor of the hematopoietic growth factor CSF-1. Employing a v-fms probe in an in situ hybridization assay we examined the expression of c-fms transcripts in a population of purified granulocyte-macrophage progenitor cells (CFU-GM). Using this approach, which requires only 10-20 thousand cells, we demonstrate that 53% of the cells in the purified CFU-GM containing fraction (FR-28) express c-fms transcripts. In the presence of natural purified CSF-1, 51% +/- 3% of FR-28 cells form colonies or clusters in a semi-solid culture assay system. 94% of the colonies and clusters stimulated by CSF-1 were morphologically macrophage in nature. In addition, 84% +/- 4% of FR-28 cells formed colonies and clusters when stimulated with pokeweed mitogen spleen cell conditioned medium (PWMSCM) in a similar in vitro assay (35% granulocyte, 29% macrophage and 39% mixed granulocyte/macrophage colonies). These results indicate there is a correlation between the responsiveness of CFU-GM progenitor cells to CSF-1 and the expression of c-fms RNA.
Assuntos
Fatores Estimuladores de Colônias/fisiologia , Granulócitos/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Macrófagos/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Divisão Celular , Ensaio de Unidades Formadoras de Colônias , Regulação da Expressão Gênica , Técnicas In Vitro , Camundongos , Receptor de Fator Estimulador de Colônias de Macrófagos , Receptores de Fator Estimulador de Colônias , Transcrição GênicaRESUMO
Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with 125I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with 125I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor with an approximate Ka of 10(8)-10(9) M-1. CFU-GM expressed an average of 3783 +/- 4 receptors per cell; normal bone marrow cells, 1518 +/- 242 receptors per cell; and peritoneal exudate cells, 2025 +/- 216 receptors per cell. Affinity crosslinking studies demonstrated that 125I-labeled GM-CSF bound specifically to two species of Mr 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The Mr 70,000 species is thought to be a proteolytic fragment of the intact Mr 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.
Assuntos
Fatores Estimuladores de Colônias/metabolismo , Granulócitos/metabolismo , Substâncias de Crescimento/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Ligação Competitiva , Medula Óssea/metabolismo , Linhagem Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Granulócitos/citologia , Células-Tronco Hematopoéticas/citologia , Cinética , Macrófagos/citologia , Camundongos , Receptores de Fator Estimulador de ColôniasRESUMO
Purified iron-saturated human milk lactoferrin (LF) and purified recombinant murine interleukin-3 (IL-3) were assessed in vivo for their effects on replication of spleen focus forming viruses (SFFV) in spleens of DBA/2 mice injected with the polycythemia-inducing strain of the Friend virus complex. LF and IL-3, inoculated 2 hr prior to the administration of the polycythemia-inducing strain of the Friend virus complex, respectively decreased and increased the replication of SFFV in mice as assessed by the spleen focus forming unit assay in primary and secondary DBA/2 mice. Since virus infectivity is associated with the DNA synthetic phase of the cell cycle and it has been shown elsewhere that LF decreases and IL-3 increases the percent of hematopoietic progenitor cells in S-phase in vivo, the results suggest that the opposing actions of LF and IL-3 on replication of SFFV may reflect the actions of these molecules on cycling of the target cells for SFFV.
Assuntos
Vírus da Leucemia Murina de Friend/patogenicidade , Interleucina-3/farmacologia , Lactoferrina/farmacologia , Lactoglobulinas/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Feminino , Vírus da Leucemia Murina de Friend/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos DBA , Policitemia/microbiologia , Vírus Formadores de Foco no Baço/efeitos dos fármacos , Vírus Formadores de Foco no Baço/patogenicidade , Replicação Viral/efeitos dos fármacosRESUMO
Combinations of low dosages of purified murine hematopoietic colony-stimulating factors (CSFs)--L-cell CSF type 1 (CSF-1), recombinant interleukin 3 (IL-3), and recombinant granulocyte/macrophage CSF (GM-CSF)--were compared with single CSFs for their influence on the cycling rates and numbers of bone marrow granulocyte/macrophage, erythroid, and multipotential progenitor cells in vivo in mice pretreated with human lactoferrin. Lactoferrin was used to enhance detection of the stimulating effects of exogenously administered CSFs. Concentrations of CSFs that were not active in vivo when given alone were active when administered together with other types of CSF. The concentrations of CSF-1, IL-3, and GM-CSF needed to increase progenitor cell cycling rates were reduced by factors of 40-200, 10-50, and 40- greater than 400, respectively; the concentrations needed to increase progenitor cell numbers were reduced by factors of 40-500 (CSF-1), 20-80 (IL-3), and greater than 40- greater than 200 (GM-CSF) when these forms of CSFs were administered in combination with low dosages of one of the other forms of CSFs. The results demonstrate that different CSFs can synergize when administered in vivo to increase the cycling rates and numbers of marrow hematopoietic progenitor cells. These findings may be of relevance physiologically to the regulation of myeloid blood cell production by CSFs.