RESUMEN
Replication-deficient E1-/E3-deleted adenoviral vectors are commonly used to introduce transgenes into cells in vitro and have been used for certain kinds of gene therapy protocols in vivo. We have demonstrated that transduction of cells using these vectors can induce p53 expression in cells containing a wild-type p53 gene. This response is different from p53 induction observed after DNA damage because the time course of induction is slower and because it occurs in cells with an attenuated DNA damage response. However, this vector-induced p53 is transcriptionally active and, therefore, p53 function is not inactivated by viral proteins. The mechanism of induction appears to be an increased rate of protein translation because immunoprecipitation analyses demonstrated increased levels of 35S-labeled p53 protein, even after a short 15-min labeling time. Induction of p53 by adenoviral vectors may have various effects on transduced cells, including apoptosis and altered chemotherapy chemosensitivity. Therefore, the influence of the vector might confound the impact of any particular gene used in a gene therapy application.
Asunto(s)
Adenoviridae/genética , Terapia Genética , Proteínas Nucleares , Proteína p53 Supresora de Tumor/biosíntesis , Replicación Viral , Daño del ADN , Vectores Genéticos , Humanos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Células Tumorales CultivadasRESUMEN
Promising immunotherapies for viral infections and malignancies reflect the successful, rapid translation of laboratory findings into clinical practice. Fletcher et al. [1] present imaging studies of Epstein-Barr virus (EBV)-associated lymphomas before and after immunotherapy. Here, we briefly review the scientific bases of such novel therapies, which have evolved from advances in understanding of immune effector cells, of the cytokines that drive immune responses, and of the mechanisms underlying cell death.
Asunto(s)
Infecciones por Herpesviridae/terapia , Herpesvirus Humano 4 , Inmunoterapia Adoptiva , Trastornos Linfoproliferativos/terapia , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/trasplante , Linfocitos B/inmunología , Trasplante de Médula Ósea/efectos adversos , Trasplante de Médula Ósea/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , HumanosRESUMEN
Although adenovirus is a major source of morbidity for immunocompromised individuals and a popular vector for gene therapy, little is known about the cellular immune responses it evokes in humans. Initial trials using adenovirus vectors have been disappointing, probably owing both to a preexisting immune response to Ad2 and Ad5, the most commonly used vector backbones, and to a response to the transgene. The former problem might be overcome by switching from the common type C adenoviruses, of which Ad2 and Ad5 are members, to other less common serotypes. Evidence for the feasibility of this approach has been provided by a rat model system. However, its success in humans depends on there being no immunological cross-reactivity between groups at the humoral or cellular level. Here, we examine the cross-reactivity of the cellular immune response to adenovirus in a human system, and find that human cytotoxic T lymphocytes (CTLs) prepared in vitro against an adenovirus from two of the six subgroups can lyse cells infected with adenoviruses from the other subgroups. Hence, the proposed use of adenovirus vectors from uncommon subgroups to evade memory immune response to subgroup C adenoviruses may not be successful. However, this same cross-reactivity indicates that adoptive transfer of CTLs generated in vitro against one adenovirus serotype may protect immunocompromised patients from infections by adenoviruses of all serotypes.
Asunto(s)
Adenoviridae/inmunología , Linfocitos T Citotóxicos/inmunología , Anticuerpos/inmunología , Reacciones Cruzadas/inmunología , Vectores Genéticos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunidad Celular , Inmunoterapia AdoptivaRESUMEN
We investigated the level of minimal residual disease (MRD) in 26 children with B-lineage acute lymphoblastic leukemia (ALL) after intensive induction therapy. A quantitative semi-nested polymerase chain reaction (PCR) detecting the clone-specific rearranged immunoglobulin heavy chain genes was developed to improve sensitivity and specificity of amplification. In all patients, one leukemic cell could be detected in a background of 10(5) normal blood mononuclear cells. All patients investigated were in complete remission at the end of induction therapy as evaluated by morphologic criteria. Nineteen patients (73%) had no detectable residual leukemic cells using the sensitive semi-nested PCR. Seven patients (27%) were PCR positive. Three had a low level (<2 x 10(-5) leukemic cells per bone marrow cell), while four patients had a high level (>2 x 10(5)) of detectable residual leukemic cells. All patients with low or undetectable levels of residual leukemia remained in complete remission at a median of 63 months from diagnosis (range 40-80 months), while all four patients with a high level of residual leukemia subsequently relapsed at a median of 21 months from diagnosis (range 13-37 months). The patient groups with undetectable or low, and high level of MRD did not differ significantly in other clinical or genetic features with prognostic significance. We conclude that the level of MRD at the end of the intensive induction therapy period is predictive of outcome in childhood B lineage ALL. If confirmed by large prospective studies, the level of MRD might be useful in stratifying patients into high and low risk categories.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Adolescente , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , Niño , Preescolar , Femenino , Humanos , Inmunofenotipificación , Lactante , Masculino , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pronóstico , Inducción de Remisión , Estudios Retrospectivos , Sensibilidad y Especificidad , Resultado del TratamientoRESUMEN
BACKGROUND: Most patients with congenital hypogammaglobulinemia and absent B cells are males with X-linked agammaglobulinemia, which is caused by mutations in the gene for Bruton's tyrosine kinase (Btk); however, there are females with a similar disorder who do not have mutations in this gene. We studied two families with autosomal recessive defects in B-cell development and patients with presumed X-linked agammaglobulinemia who did not have mutations in Btk. METHODS: A series of candidate genes that encode proteins involved in B-cell signal-transduction pathways were analyzed by linkage studies and mutation screening. RESULTS: Four different mutations were identified in the mu heavy-chain gene on chromosome 14. In one family, there was a homozygous 75-to-100-kb deletion that included D-region genes, J-region genes, and the mu constant-region gene. In a second family, there was a homozygous base-pair substitution in the alternative splice site of the mu heavy-chain gene. This mutation would inhibit production of the membrane form of the mu chain and produce an amino acid substitution in the secreted form. In addition, a patient previously thought to have X-linked agammaglobulinemia was found to have an amino acid substitution on one chromosome at an invariant cysteine that is required for the intrachain disulfide bond and, on the other chromosome, a large deletion that included the immunoglobulin locus. CONCLUSIONS: Defects in the mu heavy-chain gene are a cause of agammaglobulinemia in humans. This implies that an intact membrane-bound mu chain is essential for B-cell development.
Asunto(s)
Agammaglobulinemia/genética , Cadenas mu de Inmunoglobulina/genética , Mutación , Agammaglobulinemia/congénito , Linfocitos B , Cromosomas Humanos Par 14/genética , Consanguinidad , Análisis Mutacional de ADN , Femenino , Ligamiento Genético , Humanos , Recuento de Linfocitos , Masculino , LinajeRESUMEN
Adenovirus infections cause significant morbidity and mortality in immunocompromised patients, yet little is known about the immune response to adenovirus infections. We established a system for the generation of a cytotoxic immune response to adenovirus in vitro. Cytotoxic T cells (CTLs) were derived from normal donors by using peripheral blood dendritic cells as antigen-presenting cells. The CTLs were found to contain a mixture of effector cells that recognized virus peptides in the context of both class I and class II antigens. Endogenous viral gene expression was not required to sensitize cells to lysis by adenovirus-specific CTLs. CTLs raised against subgroup C adenovirus type 5 can lyse cells infected with subgroup B adenovirus type 11, indicating that viruses of different subgroups have epitopes in common. This system holds promise for defining the human immune response to adenovirus, including characterization of the viral protein(s) against which the response is generated, and the identity of the effector cells. Such studies are in progress.
Asunto(s)
Infecciones por Adenoviridae/inmunología , Adenoviridae/inmunología , Células Dendríticas/virología , Linfocitos T Citotóxicos/inmunología , Presentación de Antígeno , Antígenos Virales/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Humanos , Inmunidad Celular , Linfocitos T Citotóxicos/virologíaRESUMEN
The PCR technique appears to be the most sensitive method for detecting residual disease in ALL and can be applied to a high percentage of cases by amplifying sequences of the antigen-receptor genes. The PCR studies to date suggest that this sensitive technique can detect residual disease in virtually all patients during the first year of treatment. The residual disease becomes undetectable in the majority of patients by the end of treatment; however, a subset of patients remain PCR positive at a time when therapy is electively discontinued. The development of a highly accurate quantitative PCR technique may allow the possibility of distinguishing the patterns of residual disease for patients who will be cured by treatment from those who relapse. If such a pattern can be discerned, then an immediate benefit for PCR monitoring will be that clinicians will have the opportunity to test whether treating patients at the time of 'molecular relapse' will help to improve the cure rate for this disease. The PCR studies of remission marrows at the end of treatment raise a number of questions about the biology of disease persistence in patients who remain in extended 'remission.' A commitment to obtaining and analyzing bone marrow specimens in patients who have completed therapy is necessary to discern whether novel strategies, such as immunomodulatory manipulations, are needed to control or eradicated residual disease in patients who have completed planned chemotherapy. Thus, the long-term benefit of residual disease monitoring by PCR may be a better understanding of the biology and definition of 'cure' in ALL.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Niño , Mapeo Cromosómico , Humanos , Hibridación Fluorescente in Situ , Neoplasia Residual/diagnóstico , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
The polymerase chain reaction (PCR) has been applied to detect occult leukemia cells in children with acute lymphoblastic leukemia who are otherwise considered in complete remission by traditional morphological examination of bone marrow specimens. To determine whether PCR provides unique prognostic information of use for the clinical investigator, we reviewed the 20 clinical studies published to date. From this review, it is evident that discrepancies exist for the detection of residual disease for patients who remain in complete remission and for those who relapse. However, because of the fundamentally different approaches used to apply the PCR method to each of these studies, an entirely different interpretation can be reached when critical technical factors are considered. The combined data from the various studies suggest that a consistent pattern for residual disease disappearance over many months exists for patients who remain in extended complete remission and a pattern of residual disease persistence and reappearance preceding clinical findings exists for the majority of those who ultimately relapse in the bone marrow.
Asunto(s)
Neoplasia Residual/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Médula Ósea/química , Niño , ADN de Neoplasias/genética , Reordenamiento Génico de Linfocito T , Humanos , Pronóstico , ARN Mensajero/genética , ARN Neoplásico/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Estudios RetrospectivosRESUMEN
Adenovirus encodes a 72-kDa single-stranded DNA binding protein (DBP) that is necessary for viral DNA replication and is involved in controlling viral RNA metabolism. Studies of temperature-sensitive (ts) and site-directed mutants of the DBP have identified at least four regions of the protein involved in binding to single-stranded DNA and hence in DNA replication. Two of the ts mutants, Ad2ts111A and Ad2ND1+ts23, are deficient in DNA binding and in supporting in vitro DNA replication. Their effects on viral RNA metabolism are presented here. At early and late times of infection, accumulation of RNAs from the viral E3 region is increased up to sevenfold in Ad2ts111A- and Ad2ND1+ts23-infected cells relative to wild-type virus. This effect is temperature-independent and seems to involve nuclear RNA stability. Steady-state levels of the viral E1B and E4 RNAs increase following the onset of viral DNA replication in cells infected by the wild-type virus, but not in cells infected by ts111A or ts23. The increase in E1B and E4 RNAs at late times of infection is due to a stabilization of the mRNA. The steady-state levels of L3 and L5 RNAs are two- to fourfold higher in cells infected with ts23 and ts111A than with wild-type virus. None of these differences were observed following infection of cells with a temperature-independent revertant of ts111, indicating that the mutations in the DBP were responsible for the phenotypes. However, for the E3 effect, the change brought about by the mutations in the DBP does not seem to be the result of a change in the normal function of the protein, as an essentially DBP-negative virus (Ad5d/802) shows no differences in E3 RNA metabolism compared to wild-type virus at early times of infection. These results demonstrate that mutation of amino acids 280 and 282 of the DBP significantly perturbs the normal regulation of viral RNA metabolism. These effects clearly differ from the phenotypes observed with adenoviruses containing mutations in other early genes and from those ascribed to the ts125 mutation by others. These results are discussed in terms of the mechanisms by which early and late viral RNA metabolism are controlled and the possible effects of the DBP mutations on them.
Asunto(s)
Adenovirus Humanos/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fosfoproteínas/metabolismo , ARN Viral/metabolismo , Proteínas Virales/metabolismo , Adenovirus Humanos/genética , Línea Celular , Humanos , Células KB , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Proteínas Virales/genética , Dedos de ZincRESUMEN
The polymerase chain reaction (PCR) has been applied to detect occult leukemia (ALL) cells in patients with acute lymphoblastic leukemia who are otherwise considered in complete remission by traditional morphological examination of bone marrow specimens. The combined data from the clinical studies published to date suggest that a consistent pattern for residual disease disappearance over many months exists for patients who remain in complete remission for an extended period of time. Conversely, a pattern of residual disease persistence and reappearance preceding clinical findings exists for the majority of patients who ultimately relapse. The ability to detect residual ALL disease near the end of chemotherapy or after the completion of treatment in some patients who otherwise are deemed likely to be cured of their malignancy raises the possibility that mechanisms other than leukemia cell cytotoxicity are influencing the outcome for this disease.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Niño , Ensayos Clínicos como Asunto , Humanos , Neoplasia Residual , Reacción en Cadena de la Polimerasa/métodos , RecurrenciaRESUMEN
The polymerase chain reaction (PCR) is more sensitive than other methods for detecting residual leukemic cells, and it can be used to analyze the dynamics of the leukemic cell population during and after chemotherapy. Leukemia cells are identified among the normal cells through the use of a leukemic cell-specific signature, such as the junction formed following rearrangement of the immunoglobulin (Ig) or T-cell receptor (TCR) variable (V), diversity (D), and joining (J) regions. We cloned the rearranged Ig or TCR genes from leukemic cells at the time of diagnosis by regular recombinant DNA techniques, or following PCR amplification, and determined the leukemic cell-specific V(D)J junctions. Using oligonucleotides prepared to the unique junctional sequences, we have analyzed Southern blots of PCR-amplified Ig or TCR genes from 11 patients at the time of diagnosis, and in from three to 14 follow-up samples for periods ranging from 17 to 97 months. The level of detection of residual blast cells in these patients was usually on the order of 1 leukemic cell in 10(4) normal ones. Conditions for the hybridization and washing were individually adjusted so that the specificity of the probes was excellent. No residual leukemic cells were found in patients who remained in continuous complete remission at time points more than 6 months post-diagnosis. PCR detected impending relapse in four out of six cases, with PCR-detectable leukemic clones being present 2, 4, 13 and 20 months prior to clinical signs of the disease. Relapse occurred in two patients who lacked PCR evidence of leukemic blasts in marrow samples collected 2 and 6 months before clinical recurrence, respectively. The leukemic cells that persisted for 3 years after a relapse in a patient who remained in clinical remission during this time period eventually became undetectable by PCR.
Asunto(s)
Reordenamiento Génico de Cadena Pesada de Linfocito B , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Médula Ósea/patología , Niño , Sondas de ADN , Estudios de Seguimiento , Humanos , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Recurrencia , Estudios Retrospectivos , Factores de TiempoRESUMEN
The octamer sequence ATGCAAAT is highly conserved in the promoter of immunoglobulin heavy and light chain genes and is one of the sequence motifs involved in the control of transcription of these genes. The promoter region of an human immunoglobulin heavy chain variable gene, the sole member of the VH6 gene family, was found to differ from other VH gene promoters: it contains neither the conserved octamer motif nor a heptamer sequence, and generally bears little resemblance to other VH gene transcriptional control regions. An imperfect octamer sequence with a single nucleotide substitution (AgGCAAAT) is located 108 bp upstream of the ATG translation start site, and 81 bp upstream of the transcription initiation site. We sought to determine which sequence elements within the VH6 promoter were responsible for transcription initiation by creating progressive deletions of a 1 kb fragment from this region and testing their ability to function as promoter elements in B and non-B cells (HeLa). The minimum fragment required for full promoter function was 110 bp, but a fragment with only 65 bp retained 30-50% activity in B cells. Similar levels of transcription were seen when the -146 bp promoter containing two point mutations in the imperfect octamer was tested. Mutation of a possible pyrimidine box sequence located downstream of the TATA box was shown to have only a minor effect (10-30%) on transcription when three nucleotides were changed. Surprisingly, CAT activity was not B cell-specific, as all constructs had virtually the same activity in several B cell lines and in HeLa cells. Removal of the TATA box led to a 50% reduction in CAT activity, and the region upstream of the TATA box functioned as a promoter in both orientations. The transcriptional activity of the VH6 promoter was virtually enhancer independent: only a minor increase was observed when the immunoglobulin or SV40 enhancer was added to the promoter construct. Electrophoretic mobility shift assays of transcription factor binding to the region around the imperfect octamer indicated that binding was weak when nuclear extracts from either B cells or HeLa cells were used. The amount of complex shifted was increased by mutating the imperfect octamer to a perfect one. Chimeras produced between the VH6 promoter and a B cell-specific promoter from a member of the human VH2 gene family demonstrated that the lack of tissue specificity was due to the absence of a repressor of non-B cell transcription in the VH6 promoter. These results indicate that the VH6 promoter is relatively simple, requiring little more than the TATA element and the imperfect octamer, and transcription from this promoter lacks B cell specificity and is not dependent on the enhancer element.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Regiones Promotoras Genéticas , Linfocitos B , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , ADN , Células HeLa , Humanos , Datos de Secuencia Molecular , TransfecciónRESUMEN
The human immunoglobulin (Ig) heavy chain VH6 gene promoter contains an imperfect octamer (AgGCAAAT) and is not dependent on the Ig heavy chain enhancer for activity; reporter constructs containing this promoter are very active in non-B cells. In experiments designed to characterize regions upstream of the transcriptional start site that are important for promoter function, we produced a series of deletion constructs, including one containing sequences between -74 and -146. Surprisingly, this fragment had promoter activity in both orientations. Inspection of the VH6 promoter sequence indicated that there was a possible TATA box in the proper orientation upstream of the imperfect octamer. The -74 to -146 fragment functioned as a promoter in the reverse orientation in three B cell lines and in non-B (HeLa) cells, with a much higher level of activity seen in the HeLa cells. To determine if the promoter could work in both directions simultaneously, reporter genes were positioned up- and downstream of a VH6 promoter fragment. Reporter gene activity was found for both genes in B cells and HeLa cells. Using a reverse transcriptase-polymerase chain reaction procedure (RT-PCR), we found a transcript corresponding to sequences upstream of the VH6 promoter in RNA from both the lymphoblastoid cell line ML-1, which actively transcribes the VH6 promoter, and the REH cell line, which does not. No transcripts were found in the KB epithelial cell line. Two or three mRNA 5' ends were found that mapped between -137 to -143 from the authentic VH6 transcription site, 31-37 nucleotides upstream of the putative TATA box. Inspection of the sequence upstream of the VH6 promoter demonstrated the presence of an open reading frame capable of coding for 96 amino acids. The VH6 promoter represents the second Ig promoter with bidirectional activity.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Regiones Promotoras Genéticas , Transcripción Genética , Secuencia de Aminoácidos , Linfocitos B/metabolismo , Secuencia de Bases , Línea Celular Transformada , ADN , Células HeLa , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
Acute lymphoblastic leukemia (ALL) represents the clonal outgrowth of transformed hematopoietic progenitor cells. We have found that blast cells in some cases of B-precursor cell ALL contain Ig heavy chain gene rearrangements with considerable diversity at the junctions of the variable (VH), diversity (D), and joining (JH) regions. This diversity consists of heterogeneous nucleotide sequences at the VH-D and, less frequently, the D-JH junctions. In two cases, different VH segments were attached to the same D-JH rearrangement. In all cases studied there was a much higher than expected frequency of nucleotide sequence changes in the VH segment. At least three mechanisms may produce these changes in different cases: (1) continuing rearrangement of the heavy chain gene, in some cases by VH addition to a preexisting D-JH; (2) VH replacement; and (3) an open-and-shut mechanism. These findings suggest that an active VDJ recombinase system is present at the time of transformation in a high percentage of ALLs. An active recombinase in the rapidly growing leukemic cell population could lead to genomic instability.
Asunto(s)
Reordenamiento Génico , Genes de Inmunoglobulinas , Variación Genética , Cadenas Pesadas de Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Secuencia de Bases , Médula Ósea/inmunología , Médula Ósea/patología , Clonación Molecular/métodos , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
A monoclonal antibody (MoAb) recognizes a novel 52-Kd cell protein (MKW) that is expressed on cells of the normal myelocytic and monocytic lineage, a subset of B cells, and the U937 cell line. Using the U937 cell line as a model, the MoAb (anti-MKW) was examined for its ability to inhibit the effects of differentiation-inducing factors. In the U937 cell line, recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) inhibits cell proliferation, 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits proliferation and induces the early differentiation antigen CD11b, and vitamin D3 inhibits proliferation and induces both CD11b and the late differentiation antigen CD14. The antiproliferative and differentiation effects of rhGM-CSF and vitamin D3 on U937 cells were inhibited by the anti-MKW MoAb. Similar effects were seen when anti-MKW antibody was added 30 minutes before or 2 hours after rhGM-CSF or vitamin D3, suggesting that its effects are not mediated by blocking or binding to the receptors for these growth factors. The anti-MKW MoAb had no effect on TPA-induced differentiation in U937 cells, indicating that TPA exerts its effects through a pathway different from rhGM-CSF and vitamin D3. These results suggest that the MKW antigen is important in controlling the proliferation and differentiation of monocytic cells.
Asunto(s)
Anticuerpos Monoclonales , Antígenos de Neoplasias/fisiología , Antígenos de Superficie/fisiología , Diferenciación Celular/fisiología , División Celular/fisiología , Colecalciferol/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Antígenos CD/fisiología , Antígenos de Neoplasias/inmunología , Antígenos de Superficie/inmunología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Interacciones Farmacológicas , Humanos , Interleucina-3/farmacología , Interleucina-6/farmacología , Cinética , Factor Estimulante de Colonias de Macrófagos/farmacología , Antígeno de Macrófago-1/análisis , Proteínas Recombinantes/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Adenovirus requires the virus-encoded single-stranded DNA-binding protein (DBP) to replicate its DNA. We have previously shown (M. Tsuji, P. C. van der Vliet, and G. R. Kitchingman, J. Biol. Chem. 266:16178-16187, 1991) that the inability of three temperature-sensitive (ts) mutant DBPs (Ad2+ ND1ts23, Ad2ts111A, and Ad5ts125) to support DNA replication at the nonpermissive temperature was associated with impaired ability to bind to DNA. In this study, we examined these mutant proteins for structural alterations that might be linked to the functional changes. All three ts mutants, but not the wild-type protein, showed different proteolytic cleavage patterns before and after heating at 40 degrees C (the nonpermissive temperature), suggesting a possible conformational change during heating. The Ad2+ND1ts23 and Ad2ts111A DBPs have single amino acid changes located in a putative zinc finger subdomain (positions 282 and 280). In the presence of zinc ions, these ts mutants showed significantly increased resistance to inactivation at 40 degrees C. Surprisingly, however, the stabilizing effect of zinc was also observed with the Ad5ts125DBP, which contains a mutation located more than 100 amino acids from the zinc finger. Other related metal ions, such as cobalt, cadmium, and mercury, did not protect the ts DBPs from inactivation at 40 degrees C. These results indicate that functional changes of the ts DBPs in DNA replication and DNA binding are accompanied by structural alterations in the protein and that zinc and the metal-binding subdomain may play an important role in the structure and/or function of the DBP.
Asunto(s)
Adenoviridae/genética , Proteínas de Unión al ADN/genética , Mutación , Proteínas Virales/genética , Proteínas E2 de Adenovirus , Anticuerpos Monoclonales , Línea Celular , ADN de Cadena Simple/metabolismo , ADN Viral/metabolismo , ADN Viral/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Endopeptidasas , Microscopía Electrónica , Temperatura , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteínas Virales/ultraestructura , Zinc/metabolismoRESUMEN
After achieving remission, approximately one-third of patients with T-cell acute lymphoblastic leukemia (T-ALL) relapse due to the resurgence of residual leukemic cells that cannot be detected in remission by morphologic methods. Thus, the early detection of residual disease is highly desirable to monitor the efficacy of therapy, or to institute an alternative mode of therapy. Toward this aim, we have examined the applicability of polymerase chain reaction (PCR) amplification in the detection of minimal residual disease (MRD) in bone marrow samples from patients with T-ALL in morphologic remission. Two different approaches were taken to identify leukemic clone-specific sequences that could be used as targets for PCR amplification. The first technique used T-cell receptor-delta (TCR-delta) gene rearrangements that were sequenced directly after PCR amplification of leukemic DNA. This method was successful in generating clone-specific probes for 76% of T-ALL patients screened. An alternative method was used to clone and sequence a TCR-beta chain gene from leukemic cells to generate a specific probe. The PCR assays that we used were specific for each patient's leukemic clone, and were capable of routinely detecting one leukemic cell in 10(4) normal cells. Using these sensitive PCR-based assays, we found no evidence for persistence of the leukemic clone in any of the bone marrow samples from four T-ALL patients who are in long-term (3.9 + to 8.1 + years) remission. In contrast, we detected residual disease in clinical remission samples from two patients who subsequently relapsed. In one patient, where we had appropriate samples, we observed a dramatic expansion of the leukemic clone 3 months before clinical relapse. These results suggest that PCR-based assays for detection of MRD in T-ALL patients have great potential in predicting impending relapse, and in determining the efficacy of the anti-leukemic therapy. These methods may also allow the identification of long-term survivors.
Asunto(s)
Leucemia-Linfoma de Células T del Adulto/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Southern Blotting , Médula Ósea/inmunología , Médula Ósea/patología , ADN de Neoplasias/genética , Reordenamiento Génico , Reordenamiento Génico de Linfocito T , Humanos , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/inmunología , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , PronósticoRESUMEN
The adenovirus single-stranded DNA-binding protein (DBP) is an essential factor in viral DNA replication. Three temperature-sensitive (ts) adenoviruses (Ad2+ND1ts23, Ad2ts111A, and Ad5ts125) are known to have single amino acid substitutions in their DBPs that result in defective DNA replication at the nonpermissive temperature. To elucidate the mechanism(s) involved in the ts phenotype, we purified the three mutant DBPs and studied their DNA-binding properties and their ability to support DNA replication in an in vitro system. The results confirm that the three ts DBPs were incapable of supporting DNA replication at the nonpermissive temperature (40 degrees C). The defect was found at both the initiation and elongation steps of DNA replication. The 2-fold stimulation of pTP.dCMP formation by the DBP was lost by prior heating of the ts DBPs. The pronounced effect of the DBP on the early elongation process was severely diminished, but not abolished, by prior heating to 40 degrees C. The functional change at 40 degrees C was irreversible, as the ts DBPs preincubated at 40 degrees C were no longer active when assayed at 30 degrees C. Upon heating to 40 degrees C, all three ts DBPs lost their ability to bind to oligonucleotides, although they still retained some binding activity for large single-stranded DNAs such as M13 DNA. Thus, the inability of these three ts DBPs to support DNA replication is attributable to their altered DNA-binding properties.
Asunto(s)
Adenoviridae/genética , Replicación del ADN , ADN de Cadena Simple/metabolismo , ADN Viral/biosíntesis , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/metabolismo , Proteínas E2 de Adenovirus , Western Blotting , Cromatografía Liquida , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Poliacrilamida , Genes Virales , Cinética , Mutación , Temperatura , Proteínas Virales/genéticaRESUMEN
Approximately 25% of acute leukemias of the B-cell lineage demonstrate more than two rearranged immunoglobulin heavy chain genes when examined by Southern blot analysis. The origin of the extra bands was investigated by molecular cloning and sequencing of four rearranged genes from one patient's leukemic cells. All four rearrangements were apparently derived independently. Two of the rearrangements used the VH6 variable region, attached to different diversity and joining regions. One of the two rearrangements contained a mutation in the coding sequence leading to the generation of a nonsense codon. This rearranged gene also differed from the other VH6 containing gene starting at about 330 bp upstream of the ATG initiation codon. The third rearranged gene used a member of the VH2 variable gene family. A DH-JH rearrangement was found in the fourth rearranged gene. The data indicate that the leukemia probably arose as a result of the transformation of an early B-cell progenitor that lacked rearranged immunoglobulin genes but retained some differentiation potential.
Asunto(s)
Linfoma de Burkitt/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Southern Blotting , Clonación Molecular , ADN de Neoplasias/genética , Humanos , Mapeo RestrictivoRESUMEN
Transcription events are thought to precede gene rearrangement in the immunoglobulin (Ig) loci and may be the mechanism by which the various gene regions are made accessible for recombination. If this is the case, identification and characterization of transcripts from the Ig loci should permit a better understanding of the gene rearrangement process. We have isolated a 2.3 kb cDNA clone from the human pre-B cell line Nalm-1 that contains enhancer-specific sequences from the Ig heavy (H) chain gene locus. The 2.3 kb transcript initiated within the enhancer region and showed extreme 5' heterogeneity, with more than 50 initiation sites mapping near the Ig-specific octamer ATTTGCGT. Sequencing of the cDNA clone demonstrated that 644 nucleotides from the Ig enhancer region were incorporated as a leader exon spliced to the mu constant (Cmu) region. This leader exon contained many translation termination codons and may function to inhibit the translation of sterile Cmu polypeptides. Using an enhancer-derived probe, we detected two low-abundancy mRNA transcripts with sizes of 2.3 and 12 kb. Northern blot analysis suggested that the 12 kb transcript was the unspliced precursor mRNA of a VDJ rearrangement. The potential role of these enhancer-containing transcripts in the opening of the IgH chain gene for rearrangement and for class switching is discussed.