Biomedical subjects

M D Minden

Publications and source records attributed to M D Minden.

At least 109 records · Page 6Linked to original sources

PubMed1988

The effects of combinations of the recombinant growth factors GM-CSF, G-CSF, IL-3, and CSF-1 on leukemic blast cells in suspension culture.

The blast cells of acute myeloblastic leukemia may be considered as a renewal population maintained by stem cells that are capable of both self-renewal and differentiation. Blast stem cells grow in culture usually when stimulated by growth factors normally active on myelopoietic cells. Two culture methods permit an evaluation of the balance between self-renewal and differentiation; previous studies have shown that this balance can be affected by recombinant growth factors. These include interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF), active on early cells in normal myelopoiesis, and G-CSF and CSF-1, restricted in normal hemopoiesis to the granulopoietic and macrophage/monocytic lineages, respectively. In this paper we report the results of evaluating the effects on these recombinant growth factors alone or in mixtures of two at optimal concentrations. The results were obtained either using titrations of colony formation in methylcellulose or growth in suspension. Star diagrams, a technique from exploratory data analysis, were used to provide quantitative and graphic displays of the results of the recombinant factors on the balance between blast self-renewal and differentiation. Blasts from 4 acute myeloblastic leukemia patients and one patient with the blast crisis of chronic myeloblastic leukemia were examined in detail. The great patient-to-patient variation usually observed was seen in both plating efficiency in methylcellulose and growth pattern in suspension. In spite of this variation, a common pattern of response to growth factors emerged. When the early acting factors, IL-3 and GM-CSF, were combined, the effect was quantitatively and qualitatively similar to the largest stimulation seen with either of the factors alone. In contrast, late-acting factors, G-CSF and CSF-1, influenced each other's effects when present together and each affected the activities of GM-CSF and IL-3. Notably, CSF-1, which often led to the accumulation of adherent, terminal cells in suspension, usually maintained or increased this differentiation-like activity in combination. G-CSF also favored differentiation in combination, although the effect was usually to increase the number of colonies in methylcellulose, most of which consist of blast cells incapable of further divisions. The results are discussed as they relate to the postulated structure of the blast population and the normal targets of the recombinant growth factors.

Cell Differentiation↗

PubMed1988

Binding of iodinated recombinant human GM-CSF to the blast cells of acute myeloblastic leukemia.

Granulocyte/macrophage-colony-stimulating factor (GM-CSF) is an effective growth factor for the blasts of acute myeloblastic leukemia (AML). Radioiodinated Chinese hamster ovary (CHO)-cell derived GM-CSF was prepared using Bolton-Hunter reagent to label free amino groups on the protein. Normal human neutrophils and the blast cells from AML patients were examined for binding. We found that there were fewer receptors of higher affinity on blast cells compared with neutrophils. After brief culture in suspension, receptor number increased and affinity decreased. Experiments provided evidence that GM-CSF from Escherichia coli had a higher affinity for neutrophils (kd = 20 pM) than the CHO-cell derived protein (kd = 500 pM-1 nM). This difference was reflected in the increased effectiveness of the E. coli protein over the CHO protein to stimulate colony formation in both normal bone marrow cells and AML blasts.

Colony-Stimulating Factors↗

PubMed1988

Heterogeneity in acute myeloblastic leukemia.

Morphological-identified blast populations are the hallmark of the malignant clones that dominate hemopoiesis in acute myeloblastic leukemia (AML). Marked heterogenity is characteristic of AML blasts. Patient-to-patient variation is seen in their biological properties but is particularly evident in the response to treatment. Intraclonal variation is generated during clonal expansion, particularly as blast stem cells either undergo self-renewal or enter into a series of terminal divisions. These two alternative activities can be monitored in cell culture using a clonogenic assay and a suspension assay. The balance between renewal and differentiation can be altered by exposing blast populations to various growth factors in culture. Further, certain drugs, particularly ara-C, appear to be more toxic for self-renewing divisions than cell-cycle events generally. We suggest that both drugs and growth factors should be assessed for their effects on self-renewal as part of preclinical testing.

Antineoplastic Agents↗

PubMed1988

Structure and expression of genes of GM-CSF and G-CSF in blast cells from patients with acute myeloblastic leukemia.

The hematopoietic growth factors granulocyte/macrophage colony-stimulating factor (GM-CSF) and G-CSF, available as recombinant products, stimulate the growth in culture of blasts from patients with acute myeloblastic leukemia (AML). We used cDNA probes for each gene to study the genomic organization in blast cells of 22 patients and expression in the blast cells of 18 patients. Alteration in the structure of G-CSF (two instances) and GM-CSF (two instances) was found. In two patients in whom it was possible to study DNA from bone marrow obtained at remission, the new bands detected in the leukemic cells were not found. Fifteen of 18 patients showed no RNA expression of either growth factor. Both patients with GM-CSF abnormalities as seen by Southern analysis expressed an abnormally large GM-CSF message but no G-CSF messages. One patient with an abnormal Southern pattern with G-CSF expressed normal-sized G-CSF and GM-CSF messages. The biologic significance of these findings remains to be determined. Nonetheless, the abnormal Southern patterns may prove to be useful clonal markers in the study of AML.

Adolescent↗

PubMed1988

The isolation of the human delta chain gene and its expression in normal T cells and T cell leukemias.

In this paper we describe a gene that lies 85 kb 5' of the constant region of the human alpha chain and some 30-50 kb 3' of the alpha chain V region (H. Griesser, unpublished observation). This gene undergoes somatic recombination and is transcribed in thymocytes, PHA-stimulated peripheral blood T cells, and some T cell leukemic cell lines. Sequence analysis revealed that the gene has a structure similar to that of immunoglobulin and other T cell antigen receptor genes. Comparison of the sequence to a mouse gene found in a similar location revealed 80% homology at the protein level. Recently, M. Davis and A. Weiss (personal communication) have demonstrated that the protein product of the mouse gene is the delta chain gene. Thus, the gene described in this paper represents the human homolog of the delta chain gene of the T cell antigen receptor.

Amino Acid Sequence↗

PubMed1987

Small-airways disease in recipients of allogeneic bone marrow transplants. An analysis of 11 cases and a review of the literature.

In a retrospective review of 116 consecutive allogeneic bone marrow transplants (BMT), severe obstructive airways disease was identified in 11 patients. Lung pathology demonstrated bronchiolitis in 9 patients and physiologic studies showed small-airways disease consistent with bronchiolitis in the other 2. None of the 5 patients with associated infection survived, while 3 of the 6 patients without an identified pathogen stabilized or improved. Analysis of the 11 cases presented and all 25 cases reported in the literature (1982 to 1985) supports the conclusion that graft-versus-host disease is a major risk factor for bronchiolitis in BMT recipients. Among the proposed mechanisms for the development of bronchiolitis after allogeneic BMT, the 2 most likely are graft-versus-host disease directly causing bronchiolitis, and increased immunosuppressive therapy given for graft-versus-host disease predisposing to viral bronchiolitis. The available evidence would suggest that it is prudent to obtain serial pulmonary function tests even in asymptomatic patients post-BMT, and particularly in those with chronic graft-versus-host disease, in the hope that early detection will allow for early intervention that will arrest or reverse the progression of the obstructive airways disease.

Adolescent↗

PubMed1987

Genetically determined regulators acting on the blast cells of acute myeloblastic leukemia.

Regulatory mechanisms affecting the growth of leukemic cells are attractive targets for new treatments. The blast cells of acute myeloblastic leukemia (AML) may be considered as a lineage; a minority are stem cells capable of both self-renewal and determination followed by terminal divisions ending in proliferatively inert cells retaining blast morphology. Two cell culture methods are available for the study of blasts. The first is a clonogenic assay. Blast stem cells form colonies in methylcellulose, containing proliferatively inert blast cells, together with a small number of new progenitors. Growth factor(s) are usually required. These may be supplied by media conditioned by the continuous bladder carcinoma cell line HTB9 (HTB9-CM). The recombinant growth factors GM-CSF and G-CSF are also active, and in many instances are synergistic. Blast progenitors will also grow in suspension, provided the cell density is high and growth factors are provided. In these cultures, blast progenitors increase in number, reflecting their self-renewal capacity. Evidence is also available that specific genes may be involved in the self-renewal process. Thus, three forms of growth regulation, similar to those encoded by proto-oncogenes, can be shown to be operative in AML blast cell cultures.

Antimetabolites, Antineoplastic↗

PubMed1987

High-dose cytosine arabinoside in the treatment of acute myelogenous leukemia: contributions to outcome of clinical and laboratory attributes.

High-dose cytosine arabinoside (HDAra-C) has been used for remission induction, and in conventional doses for maintenance in a trial of single-agent therapy in 43 previously untreated patients with acute myelogenous leukemia (AML). Rationale for the trial was provided by the observed decrease in leukemic blast cell self-renewal in culture following exposure to Ara-C. Compared with a previous trial of 57 patients treated with multidrug therapy, single-drug Ara-C was associated with a significantly improved complete remission rate (P = .010), although the survival time was not increased. All patients with low self-renewal responded to HDAra-C in contrast to the previous trial where some patients with this phenotype failed remission induction. The clinical observations are consistent with the view that the antileukemic effect of Ara-C has some specificity for cellular events required for self-renewal of blast cells. Exposure in vivo to Ara-C was associated with an increase in blast stem cell renewal at relapse, indicating that maintenance with other drugs should be tested. The study demonstrates the importance of biological attributes in design and analysis of clinical trials.

Adolescent↗

PubMed1987

Synergism between recombinant growth factors, GM-CSF and G-CSF, acting on the blast cells of acute myeloblastic leukemia.

The genes for the hemopoietic growth factors, GM colony-stimulating factor (CSF) and G-CSF have been cloned, and recombinant material is available for both. We tested these recombinant factors for their effects on the blast cells of acute myeloblastic leukemia (AML). Culture methods are available that support both colony formation by AML blasts and the growth of blast stem cells in suspension. Recombinant GM-CSF is active in both culture systems, although to a varying degree. We found that recombinant G-CSF was also effective; however, the two recombinant factors showed striking synergism for the stimulation of blast growth of cells from five of eight AML patients. In these cases, the combination was equivalent to the stimulating activity of supernatants from the continuous cell line 5637. This conditioned medium (HTB9-CM) is considered the standard for blast growth. Blasts from one of the patients grew without added factor. In another instance, recombinant GM-CSF alone was almost as effective as HTB9-CM. In the third case, both recombinant factors were active, but synergism was not observed and their combined effect was not equivalent to that of HTB9-CM. Both GM-CSF and G-CSF were active on normal bone marrow granulopoietic progenitors, but synergism was not observed. We conclude that the marked heterogeneity observed when AML blasts are examined by other criteria is also observed when their response to growth factors is evaluated.

Acute Disease↗

PubMed1987

The effects of three recombinant growth factors, IL-3, GM-CSF, and G-CSF, on the blast cells of acute myeloblastic leukemia maintained in short-term suspension culture.

The blast stem cells of acute myeloblastic leukemia (AML) respond in cell culture to growth factors by both self-renewal and terminal divisions. Both of these functions have been shown to be stimulated by the recombinant growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). In this paper, recombinant gibbon interleukin-3 (IL-3), homologous to human IL-3, was tested on blast cells and compared with the effects of GM-CSF, G-CSF, and medium conditioned by the bladder cell line 5637 (5637-CM). We found that IL-3 was an effective stimulator of blast renewal and terminal divisions. However, great patient-to-patient variation was found. A graphic method of presenting complex comparisons between growth factors is also included.

Acute Disease↗

PubMed1987

Clonogenic hemopoietic precursors in bone marrow transplantation.

Multilineage and single-lineage hemopoietic precursors were studied in 102 bone marrow transplant recipients and their respective donors to determine their contribution to clinical outcome as measured by time to engraftment and survival. The patient population was heterogenous with respect to diagnosis and disease status. They included individuals with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), aplastic anemia, and a few other hematopoietic malignancies. The frequency of various clonogenic precursors in the normal donor population varied considerably. The data yielded a symmetrical distribution. In contrast, most bone marrow transplant recipients presented with significantly reduced numbers of clonogenic cells before transplantation, resulting in skewed distribution profiles. Serial studies of recipients demonstrated a significantly lower than normal level of clonogenic precursors even 3 and 4 years after transplantation. The median values and distribution profiles approximated those observed before transplantation but did not return to measurements obtained for normal donors. Patients with ALL deviated from this pattern. The median values and distribution profiles of clonogenic precursors before transplantation approximated the pattern of normal donors. The frequency of clonogenic progenitors after transplantation, however, remained significantly lower than that of their respective donor or pretransplant values. Cell cycle studies performed after normalization of peripheral blood hematopoietic parameters demonstrated for most recipients that a higher than normal proportion of multipotent cells was in S-phase (P = .011). By univariate and multivariate approaches, clonogenic precursors and clinical parameters were assessed for their contributions to clinical outcome as measured by time to engraftment and survival time. The number of nucleated cells in the transplant inoculum contributed to survival independent of other risk factors. Patients with a higher cell load had a higher probability of surviving than did patients with a lower cell concentration in the transplant inoculum (P = .042). The frequency of clonogenic precursors in the transplant inoculum altered neither survival nor time to engraftment. The time to engraftment was significantly influenced by the frequency of clonogenic megakaryocyte precursors (CFU-M) observed in recipients prior to transplantation (P = .003). Patients with high values engrafted faster than did patients with a low frequency of CFU-M. This was independent of both diagnosis and disease status of the patients at time of transplantation.

Adolescent↗

PubMed1987

The presence of clonogenic cells in high-grade malignant lymphoma: a prognostic factor.

A culture system has been developed that promotes growth of clonogenic lymphoma cells of some patients with intermediate and high-grade malignant lymphoma. The formation of colonies in bone marrow, lymph nodes, and peripheral blood samples is best supported by human plasma. Colony formation of some patients was dependent upon growth factors, which in this study were added in the form of medium conditioned by phytohemagglutinin (PHA)-stimulated leukocytes (PHA-LCM). Some gave rise to lymphoma colonies without PHA-LCM but improved their frequency with PHA-LCM; others were completely independent of PHA-LCM. Colonies grown in primary cultures were routinely recloned and propagated as Epstein-Barr virus (EBV)-negative cell lines with stable B cell phenotype. The cell lines showed the same immunoglobulin rearrangement pattern as that observed in the primary lymphoma sample. In addition, a significant clinical correlation was observed between culture data and clinical outcome. Survival of patients who formed lymphoma colonies at any time during their clinical course was significantly shorter than survival of patients who did not give rise to colonies (P = 0.0009). The same observation was made when the survival assessment was performed for the subset of patients studied at diagnosis (P = 0.0014).

Adult↗

PubMed1986

Sequences and diversity of human T cell receptor beta chain variable region genes.

The nucleotide sequences of 22 human T cell antigen receptor (TcR) beta chain variable region genes isolated from various T lymphocytes have been analyzed. Of the 19 variable gene segment (V beta)-containing sequences, 17 were unique. The V beta gene segments were grouped into 11 families. Comparisons were made with the data of Concannon et al. to unify the nomenclature. The data is consistent with a total V beta gene segment repertoire with a most probable value of 38 members and an upper bound of 104 members at the 95% confidence level. Southern blot data of germline DNA using selected TcR V beta cDNAs as probes support this estimate. The human repertoire is approximately three to four times greater than that reported for the mouse. Explanations for this discrepancy are proposed.

Base Sequence↗

PubMed1986

T cell receptor and immunoglobulin gene rearrangements in acute myeloblastic leukemia.

The organization and expression of the beta chain of T cell antigen receptor gene (beta-TCR) and Ig H and L chain genes were analyzed by Southern blot technique in 24 patients with a diagnosis of acute myeloblastic leukemia (AML). Rearrangements of the beta-TCR genes were seen in DNA samples from 3 of the 24 patients. One of these three patients also showed rearrangement of the Ig H chain gene. RNA samples from all three patients expressed a beta-TCR gene transcript on dot blot analysis. However, on Northern blot analysis, one patient expressed an incomplete 1.0 kb transcript and no Ig H chain mRNA, despite a rearranged configuration. The karyotypes of two of these patients showed abnormalities involving chromosome 7. Rearrangements of T cell antigen receptor genes may occur in nonlymphoid malignancy, and is consistent with the concept of lineage infidelity in AML.

Adolescent↗

PubMed1986

Characterization of reticulofibroblastoid colonies (CFU-RF) derived from bone marrow and long-term marrow culture monolayers.

The maintenance of hemopoietic precursors in long-term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose-clotted plasma in the presence of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU-RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid-laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU-F). The relationship of CFU-RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU-RF-derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU-RF-derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU-RF is not part of the repertoire of hemopoietic stem cells.

Antigens↗

PubMed1986

The use of restriction fragment polymorphisms to identify the cell line MCF-7.

We have recently documented variations in the level of N-ras gene amplification in independently passaged MCF-7 cells. To establish if these differences are due to a lack of genetic identity between current MCF-7 passages and the original cell line, we have performed karyological and restriction fragment length polymorphism (RFLP) analyses. Documentation of a combination of restriction fragment patterns which are characteristic of MCF-7 has been facilitated by an analysis of DNA from an early-passage of MCF-7 from the Michigan Cancer Foundation (MCF). In addition, such early passage cells and other MCF-7 lines also share a unique amplification of the N-ras oncogene. Using these criteria and karyotypic analysis it can be shown that a sample of MCF-7 obtained from the American Type Culture Collection (ATCC) was derived from a different individual than was the original MCF-7 cell line. It is important that researchers verify the relationship of current cell lines to the original MCF-7. Furthermore, the techniques described here provide a powerful tool which may be used to assess the identity of cell stocks.

Breast Neoplasms↗

PubMed1986

Molecular evidence for the clonal origin of blast crisis in chronic myeloid leukaemia.

Cytogenetic and enzymatic studies have shown that chronic myeloid leukemia (CML) represents the clonal proliferation of a pluripotent stem cell. The Philadelphia chromosome (Ph') is the characteristic karyotypic abnormality seen in this disease, although the exact role of this clonal marker in the pathogenesis of CML is uncertain. At a molecular level, the Ph' has recently been shown to represent the translocation of c-abl to a limited (breakpoint cluster region, bcr) on chromosome 22. We have used probes for the bcr gene to obtain molecular evidence for the clonal origin of blast crisis in 2 patient with CML. In both cases, the first with myeloid and the second with lymphoid blast crisis, there was rearrangement of the bcr gene. The patterns of rearrangement varied between patients but were identical when comparing acute and chronic phases within the same individual. As the Ph' translocation is thought to represent a random recombination event these data not only provide further evidence for the clonal origin of blast crisis in CML, but also suggest that in the second patient this translocation event had already occurred at the pluripotent stem cell.

Adult↗

PubMed1986

Gene encoding the alpha chain of the T-cell receptor is moved immediately downstream of c-myc in a chromosomal 8;14 translocation in a cell line from a human T-cell leukemia.

The SKW-3 cell line, which was established from the malignant cells of a patient with T-cell chronic lymphocytic leukemia, is characterized by a translocation involving chromosome 8 (band q24) and chromosome 14 (band q11) [t(8;14)(q24;q11)]. To determine the position of the gene encoding the alpha chain of the T-cell receptor and of the human protooncogene myc (c-myc) in relation to the breakpoint junctions and to evaluate their possible role in the pathogenesis of T-cell neoplasia, we applied the techniques of in situ chromosomal hybridization and Southern blot analysis to SKW-3 cells. Our results indicate that the breakpoint on chromosome 14 at band q11 occurs close to a joining sequence of the gene encoding the alpha chain of the T-cell receptor. Additional rearrangements within the alpha-chain locus appear to split the variable region cluster. As a result of the rearrangements, the constant region of this gene, as well as some variable region segments, are translocated to chromosome 8, to the 3' side of the c-myc-coding exons. The identification of a breakpoint to the 3' side of c-myc suggests that this translocation is analogous to the variant (2;8) and t(8;22) translocations observed in the B-cell malignancies.

Chromosome Mapping↗