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PubMed1987

Insulin release from a cloned precursor beta cell line.

This paper describes the establishment in long-term tissue culture of a functional, clonal beta (B) cell line UMR 407/3 derived from neonatal rat pancreas. Immunofluorescence demonstrated specific and uniform staining for insulin. Transmission electron microscopy showed the presence of microvilli and cytoplasmic granules. The doubling time in culture was approximately 60 h in 2% (v/v) fetal calf serum with inhibition of growth at confluence. Biochemical studies demonstrated the incorporation of [3H]leucine into proinsulin and insulin, with insulin comprising 43.6% of the total radioactivity incorporated into immune complexes. When incubated at 37 degrees C for 30 min with Krebs-Ringer bicarbonate buffer (pH 7.4), the amount of insulin released on stimulation by 16.7 mmol glucose/l, 20 mmol DL-glyceraldehyde/l or 20 mmol alpha-ketoisocaproate/l was significantly higher compared with 5.6 mmol glucose/l. The mean insulin content was equivalent to 99 +/- 0.4 fmol (S.E.M.)/5 X 10(5) cells. Regulated insulin release was maintained through at least 15 passages in culture. The cells showed morphological evidence of senescence after passage 26 and this was associated with significant reduction in stimulated insulin release as well as insulin content. The ability of the cells of this clonal line to grow in soft agar suggests that it is a precursor cell line. The clonal B cell lines isolated so far may thus represent variably committed rather than fully differentiated B cells in culture. These clonal non-neoplastic cell lines will be useful models with which to study the regulation of maturation/differentiation of B cells and insulin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Achievements in Understanding and Treatment of Myelodysplastic Syndromes.

The myelodysplastic syndromes (MDS) constitute a challenge for the biologist as well as for the treating physician. In Section I, Dr. Willman reviews the current classifications and disease mechanisms involved in this heterogeneous clonal hematopoietic stem cell disorder. A stepwise genetic progression model is proposed in which inherited or acquired genetic lesions promote the acquisition of "secondary" genetic events mainly characterized by gains and losses of specific chromosome regions. The genetic risk to develop MDS is likely multifactorial and dependent on various constellations of risk-producing and -protecting alleles. In Section II Dr. Barrett with Dr. Saunthararajah addresses the immunologic factors that may act as important secondary events in the development of severe pancytopenia. T cells from patients with MDS may suppress autologous erythroid and granulocytic growth in vitro, and T cell suppression by antithymocyte globulin or cyclosporine may significantly improve cytopenia, especially in refractory anemia. Recent studies have also demonstrated an increased vessel density in MDS bone marrow, and a phase II trial of thalidomide showed responses in a subgroup of MDS patients especially in those with low blast counts. In Section III Dr. Hellström-Lindberg presents results of allogeneic and autologous stem cell transplantation (SCT), intensive and low-dose chemotherapy. The results of allogeneic SCT in MDS are slowly improving but are still poor for patients with unfavorable cytogenetics and/or a high score according to the International Prognostic Scoring System. A recently published study of patients between 55-65 years old showed a disease-free survival (DFS) at 3 years of 39%. Consolidation treatment with autologous SCT after intensive chemotherapy may result in long-term DFS in a proportion of patients with high-risk MDS. Low-dose treatment with 5-azacytidine has been shown to significantly prolong the time to leukemic transformation or death in patients with high-risk MSA. Erythropoietin and granulocyte colony-stimulating factor may synergistically improve hemoglobin levels, particularly in sideroblastic anemia. Recent therapeutic advances have made it clear that new biological information may lead to new treatment modalities and, in combination with statistically developed predictive models, help select patients for different therapeutic options.

Journal Article↗

PubMedDate not supplied

[Modulation of voltage-gated sodium channels by recombinant interferon-alpha2b in the human neuroblastoma cell line IMR-32].

Clonal human neuroblastoma cells imr-32 are a suitable model system for studies of neuronal excitability modulation. The ability interferon-alpha 2b "laferon" to modulate the mechanisms of electrical activity was studied in whole-cell patch-clamped undifferentiated human neuroblastoma cells IMR-32. It was shown that 1 h incubation of IMR-32 cells at 37 degrees C in medium with laferon (600 U/ml) exerted changes in voltage-dependent properties of Na(+)-channels. The results of the present study demonstrate that laferon decreased of Na(+)-channels sensitivity to changes of membrane potential leading of IMR-32 cells electrical excitability decrease.

Humans↗

PubMedDate not supplied

Evaluation of cancer chemopreventive agents using clones derived from a human prostate cancer cell line.

BACKGROUND: The successful use of clonal selection through fluctuation analysis of human cancer cells as a means for studying tumor progression has been previously reported. MATERIALS AND METHODS: Three clones derived from a parental population of human prostate cancer (LNCaP) cells were selected based on proliferation, hormone sensitivity and anchorage-independent growth. The effects of five potential cancer preventive agents were evaluated using cell proliferation, anchorage-independent growth and apoptosis as end-points. RESULTS: Clone 21 cells, which represent a presumptive normal phenotype, were generally more sensitive than Clone 17 and Clone 6 cells, which represent a more malignant phenotype, to fluasterone, 7beta-HF, L-selenomethionine and troglitazone in assays for proliferation and/or apoptosis. CONCLUSION: The results confirm the efficacy of the above agents as cancer chemopreventive agents and support our contention that clonal selection of established human cancer cells provides a model to study the efficacy of chemopreventive agents.

Anticarcinogenic Agents↗

PubMed1990

The influence of growth regulation on treatment strategy for acute myeloblastic leukaemia.

Acute myeloblastic leukaemia (AML) is a useful model for generalized malignant disease. Haemopoiesis in AML is clonal; a characteristic cell population (blast cells) has been identified and can be grown in culture. The population is organized as a hierarchy, headed by self-renewing stem cells. Several control mechanisms affect the behaviour of these stem cells. Two classes of regulatory receptors are identified that act by ligand-receptor interaction; in one class the receptors are intracellular, in the other they are in the cell membrane. It is proposed that the many regulatory signals received by blast cells contribute to a genetically determined regulatory milieu that sets the probabilities of blast stem cell renewal and differentiation. Interactions have been identified between regulatory mechanisms and the responses of blast stem cells to chemotherapeutic agents. It is suggested that new therapeutic options are emerging, based on the exploitation of regulatory mechanisms in conjunction with chemotherapy.

Antineoplastic Agents↗

PubMed1996

Antibodies to an autostimulatory growth factor (IL-2) or its receptor induce death of leukemogenic cells.

We show for the first time that Ab-mediated antagonism of growth factor activity can induce death of all cells in clonal populations surviving and growing by an autostimulatory mechanism. Models of autostimulatory leukemia were generated by transfecting a mouse IL-2-dependent cell line (FD.C/2) with vectors directing production of IL-2 by these cells. One series of clones grew in a density-dependent manner in the absence of exogenous IL-2 and produced tumors in syngeneic mice. Although these clones released relatively small amounts of IL-2, their growth and survival was only partially inhibited by Abs to IL-2 or the IL-2R. Another autostimulatory clone was derived, using a different vector, which produced significantly less IL-2. Treatment of cells of this clone with Abs to IL-2 or its receptor resulted in death of all cells. These data demonstrate that Abs that antagonize growth factor action can induce the death of cells transformed by autostimulatory mechanisms. They suggest that while autostimulatory tumors are relatively resistant to Abs that block growth factor action, this is a quantitative phenomenon, and competitive antagonists of growth factor action of sufficiently high affinity may provide effective and specific adjuvants to the treatment of such tumors.

Animals↗

PubMed1998

Alterations in DNA methylation: a fundamental aspect of neoplasia.

Neoplastic cells simultaneously harbor widespread genomic hypomethylation, more regional areas of hypermethylation, and increased DNA-methyltransferase (DNA-MTase) activity. Each component of this "methylation imbalance" may fundamentally contribute to tumor progression. The precise role of the hypomethylation is unclear, but this change may well be involved in the widespread chromosomal alterations in tumor cells. A main target of the regional hypermethylation are normally unmethylated CpG islands located in gene promoter regions. This hypermethylation correlates with transcriptional repression that can serve as an alternative to coding region mutations for inactivation of tumor suppressor genes, including p16, p15, VHL, and E-cad. Each gene can be partially reactivated by demethylation, and the selective advantage for loss of gene function is identical to that seen for loss by classic mutations. How abnormal methylation, in general, and hypermethylation, in particular, evolve during tumorigenesis are just beginning to be defined. Normally, unmethylated CpG islands appear protected from dense methylation affecting immediate flanking regions. In neoplastic cells, this protection is lost, possibly by chronic exposure to increased DNA-MTase activity and/or disruption of local protective mechanisms. Hypermethylation of some genes appears to occur only after onset of neoplastic evolution, whereas others, including the estrogen receptor, become hypermethylated in normal cells during aging. This latter change may predispose to neoplasia because tumors frequently are hypermethylated for these same genes. A model is proposed wherein tumor progression results from episodic clonal expansion of heterogeneous cell populations driven by continuous interaction between these methylation abnormalities and classic genetic changes.

Aging↗

PubMed1982

Differentiation of cultured pre-adipose cells: a probability model.

Some cells of the established preadipose cell line, 3T3-L1, synthesize triglyceride after becoming confluent and quiescent. An analysis of the distribution of clusters of lipid-containing cells was consistent with a commitment event during exponential growth followed by clonal growth of committed cells. Experiments were designed to determine if the final clonal pattern of fat among nonfat cells could be described by a probability model. Undifferentiated cells (fibroblastic cells with no detectable accumulation of triglycerides) were plated at various cell numbers so that the total number of cell divisions to confluence could be controlled. Cells were passaged by trypsinization and replating, or trypsinization followed by passage through a narrow-bore needle before replating. Passing cells through a 22G needle seems to eliminate already committed cells from the population. We determined the percentage of fat cells and the range of clone sizes in cultures in which clone sizes depended upon the number of allowed cell divisions. Patterns of clone sizes in experimental cultures were compared to expected patterns obtained by computer simulations of several programmed and stochastic commitment models. Both the observed range of clone sizes and pattern of clones can be approximated by a simple stochastic model, suggesting that commitment to fat production in 3T3-L1 cells is a random process occurring with a fixed probability in single cells in exponential growth, followed by division of both committed and uncommitted cells. The probability of commitment was essentially constant at each cell division. The number of cells committed during each passage is just large enough to replace "terminally differentiated" lipid-containing cells that have been lost, thereby maintaining a constant percentage of fat cells in any given culture of 3T3-L1.

Adipose Tissue↗

PubMed2000

Characterization of superantigen-induced clonal deletion with a novel clan III-restricted avian monoclonal antibody: exploiting evolutionary distance to create antibodies specific for a conserved VH region surface.

Evolution of the Ab system has yielded three clans of VH region genes that are represented in almost every known higher species with an adaptive immune system. These clans are defined by sequence homologies primarily in highly conserved framework (FR) subdomains, which serve a scaffolding function maintaining the conformation of loops responsible for Ag binding. Structural analyses indicate that the VH FR1 and FR3 form a conserved composite exposed surface, which has been implicated in interactions with B cell superantigens. To directly investigate the expression of clan-defined supraclonal sets, we exploited the evolutionary distance of the chicken immune system and the selection power of phage display, to derive Abs diagnostic for clan III Ig. Using a specially tailored immunization and selection strategy, we created recombinant avian single chain Fv Abs specific for the clan III products, including those from the human VH3 family, and the analogous murine 7183, S107, J606, X24, and DNA4 families, and binding was competitive with natural B cell superantigens. The archetype, LJ-26, was demonstrated to recognize a clan-specific surface expressed in diverse mammalian, and also the Xenopus and chicken, immune systems. In flow-cytometric studies with LJ-26, we found that treatment of heterozygous T15i transgenic mice with a model B cell superantigen induced a clan III-restricted clonal deletion. These studies demonstrate the utility of a novel recombinant serologic reagent to study the composition of the B cell compartment and also the consequences of B cell superantigen exposure.

Amino Acid Sequence↗

PubMed1996

Some drug-resistant models for cancer chemotherapy. Part 1: Cycle-nonspecific drugs.

The pioneering mathematical analyses for describing the phenomenon of clonal selection, whereby cancer cells mutate and become resistant to treatment, were carried out by Goldie & Coldman (1979, 1983). The present analysis yields more generalized drug-resistant models arising in chemotherapy treatment when cycle-nonspecific (CNS) drugs (i.e. drugs which are toxic to cells irrespective of their position in the cell cycle) are deployed. This is accomplished by modifying models developed by Wheldon (1988) and Usher (1994) for investigating the effect of chemotherapy drugs on homogeneous cell populations (i.e. in the absence of drug resistance); both types of migration, of drug-sensitive cells to drug-resistant cells, and the possibility of a migration of drug-resistant cells to drug-sensitive cells, are incorporated into the models. Subsequently, drug concentrations are sought such that both the drug-resistant cell subpopulation and the drug-sensitive cell subpopulation either simultaneously decay or remain of constant size. The analysis reveals that the role played by the migration of drug-resistant cells to drug-sensitive cells is significant, if it occurs, when seeking appropriate drug concentrations. The qualitative results from these models provide further insight into the effective treatment of cancer by chemotherapy in the presence of drug resistance.

Antineoplastic Agents↗

PubMed2025

Colchicine and Longitudinal Dynamics of Clonal Hematopoiesis: An Exploratory Substudy of the LoDoCo2 Trial.

BACKGROUND: Clonal hematopoiesis (CH) is an aging-related hematologic condition associated with increased risk for cardiovascular events. Larger CH clones associate more strongly with cardiovascular risk. Preclinical data indicate that inflammatory signaling drives expansion of CH clones and CH-associated cardiovascular disease. However, the effect of anti-inflammatory therapies on CH clonal dynamics in humans is unclear. OBJECTIVES: The goal of this study was to test the association of randomization to colchicine vs placebo with CH growth in participants with chronic coronary artery disease. It also assessed the association of colchicine use with change in inflammatory biomarkers over time according to CH status. METHODS: In this exploratory substudy of the LoDoCo2 (Low-Dose Colchicine 2) trial, high-coverage targeted sequencing was used to detect CH driver mutations and to quantify variant allele frequency at 4 timepoints: baseline, after a 30-day open-label colchicine run-in phase (0.5 mg daily), 1 year postrandomization to colchicine or placebo, and at end of study (median follow-up of 25.0 months). Clonal dynamics were assessed by using a generalized linear mixed model. High-sensitivity C-reactive protein and interleukin-6 were additionally measured at baseline, randomization, and 1 year postrandomization. RESULTS: In total, 854 participants contributed 2,047 observations across 4 timepoints, including before and after the prerandomization colchicine run-in period. Randomization to placebo was associated with a 14.9% annual increase in CH clone size (βtime = 0.14; 95% CI: 0.08 to 0.21) vs a nonsignificant 6.3% increase with colchicine (βtime on colchicine: 0.06; 95% CI: -0.01 to 0.14), although this difference between treatment arms was not statistically significant (Pinteraction = 0.13). Compared with placebo, colchicine was associated with attenuated clonal growth in TET2 CH (βtime on colchicine: 0.09 [95% CI: -0.04 to 0.22]; βtime placebo: 0.27 [95% CI: 0.16 to 0.37]; Pinteraction= 0.04). Among individuals with non-DNMT3A CH, interleukin-6 levels increased to a lesser extent in those receiving colchicine vs placebo over 1 year (30.0% vs 98.1% increase, respectively; Pinteraction = 0.01). CONCLUSIONS: In this exploratory analysis, treatment with low-dose colchicine was associated with attenuated clonal expansion in TET2 CH. These findings suggest the potential for colchicine to curb the proliferative advantage of key CH driver mutations and to mitigate their associated risk of cardiovascular disease. Further validation in prospective studies is warranted.

Humans↗

PubMed2003

Transiently beneficial insertions could maintain mobile DNA sequences in variable environments.

The maintenance of mobile DNA sequences in clonal organisms has been seen as a paradox. If selfish mobile sequences spread through genomes only by overreplication in transposition, then sexuality is necessary for their spread through populations. The persistence of bacterial transposable elements without obvious dominant selectable markers has previously been explained by horizontal transfer. However, advantageous insertions of mobile DNAs are known in bacteria. Here we model maintenance of an otherwise selfish mobile DNA element in a clonal species in which selection for null mutations occurs during one of two temporally alternating environments. Large areas of parameter space permit maintenance of mobile DNAs where, without selection, they would have gone extinct. Horizontal transfer diminishes, rather than enhances, mean copy number. In finite populations, effective population sizes are greatly reduced by selective sweeps, and mean copy number can be increased as the reduced variance in copy number results in reduced selection.

Clone Cells↗

PubMed2002

Leukemic B cells clonally identical to myeloma plasma cells are myelomagenic in NOD/SCID mice.

OBJECTIVE: In multiple myeloma (MM), the immunoglobulin gene rearrangement characterizing malignant plasma cells is unique. For a patient with multiple myeloma who underwent a B-cell leukemic blast transformation, using the immunoglobulin molecular signature, we characterized the clonal relationship to autologous plasma cells and the impact on normal polyclonal B-lymphocyte populations. METHODS: Single-cell reverse transcriptase polymerase chain reaction (RT-PCR)/PCR was used to determine the clonal relationship between autologous MM plasma cells and leukemic B cells. A murine xenograft model was used to determine the myelomagenic potential of the leukemic B cells. RESULTS: Single-cell analysis showed that circulating leukemic-phase cells were clonotypic, with an IgH VDJ sequence identical to that of diagnosis plasma cells. Analysis of IgH transcripts indicates MM clonal dominance over normal B-cell components of the immune system at diagnosis and during leukemic disease. Leukemic B cells were xenografted to irradiated NOD/SCID mice, leading to lytic bone lesions and clonotypic cells in murine BM. Although human cells in murine BM expressed CD138, a marker largely absent from ex vivo leukemic cells, the expression of CD45, CD19, and CD20 confirmed that engrafting cells were mature, probably late-stage B cells rather than plasma cells. CONCLUSIONS: Leukemic B cells are able to exert strong clonal dominance over normal components of the immune system, colonize the murine BM in a xenograft model, and disrupt normal bone metabolism leading to lytic bone lesions. This supports the hypothesis that clonotypic MM B cells are reservoirs of disease that persist throughout therapy and give rise to relapse.

Animals↗

PubMedDate not supplied

An interregional validation of ECOPHYS, a growth process model of juvenile poplar clones.

Field data from poplar plantations in Michigan, Washington, and Wisconsin were used to validate ECOPHYS, a whole-tree growth process model for juvenile poplar. Five clones representing a range of morphological, phenological, and physiological characteristics were planted on the same date at the three sites. Height and diameter measurements were made monthly on 20 trees per clone, and intensive morphological measurements were made every two weeks on two trees per clone. Hourly solar radiation and temperature data were recorded at each site over the growing season. The model was run for each clone x site combination with the weather data and clonal parameters as inputs. A repeated measures ANOVA indicated that there were significant differences in height growth patterns among both clones and sites, as well as significant clone x site interactions. The model generally predicted height growth within a standard deviation of the field plantation means; three of the 15 clone x site simulations were significantly different from the plantation means. The median error between predicted and observed values was 5%. Evaluation of the clonal parameters showed that differences in photosynthetic rates, morphological attributes such as specific leaf area, and timing of budset are primary factors leading to differences in growth.

Journal Article↗

PubMed2000

Human T lymphopoiesis. In vitro and in vivo study models.

Successive steps in T lymphocyte differentiation and T potential of human stem cells (HSC) can be tested in the following models: (a) the infusion of cells in NOD-SCID mice, (b) the injection of cells in renconstituted SCID/hu mice, (c) the differentiation of cells in fetal thymus organ culture (FTOC), and (d) on thymic stromal layers. Using mixed human-murine FTOC, we showed (a) TCR alpha beta, TCR gamma delta lymphocytes, NK cells, and dendritic cells complete their differentiation, (b) IL-7R alpha signaling and IL-7 are essential, (c) a detailed phenotypic and functional analysis of discrete successive steps of positively selected thymocytes, (d) an efficient transduction of genes in HSC with persistent gene expression throughout the T-lymphocyte differentiation, and (e) adaptation to submerging high oxygen culture increases the test sensitivity to a clonal assay. Other approaches are the in vivo SCID/hu reconstitution model. With this method small fragments of human fetal liver and thymus are implanted under the kidney capsule of an adult SCID mouse with result in an impressive human thymus organ, six months after transplantation. We use this model to study thymus T-cell developmental kinetics, development of gene-marked precursor cells and thymic homing of precursor cells.

Adult↗

PubMed2006

TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a clonal disease that evolves through the accrual of genetic rearrangements and/or mutations within the dominant clone. The TEL-AML1 (ETV6-RUNX1) fusion in precursor-B (pre-B) ALL is the most common genetic rearrangement in childhood cancer; however, the cellular origin and the molecular pathogenesis of TEL-AML1-induced leukemia have not been identified. To study the origin of TEL-AML1-induced ALL, we generated transgenic zebrafish expressing TEL-AML1 either ubiquitously or in lymphoid progenitors. TEL-AML1 expression in all lineages, but not lymphoid-restricted expression, led to progenitor cell expansion that evolved into oligoclonal B-lineage ALL in 3% of the transgenic zebrafish. This leukemia was transplantable to conditioned wild-type recipients. We demonstrate that TEL-AML1 induces a B cell differentiation arrest, and that leukemia development is associated with loss of TEL expression and elevated Bcl2/Bax ratio. The TEL-AML1 transgenic zebrafish models human pre-B ALL, identifies the molecular pathways associated with leukemia development, and serves as the foundation for subsequent genetic screens to identify modifiers and leukemia therapeutic targets.

Animals↗

PubMed2002

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell.

Many applications of hematopoietic gene therapy require selection for clones with active transgene expression. However, it was unclear whether the clonal progeny of a retrovirally transduced hematopoietic stem cell would be capable of maintaining transgene expression through serial repopulation and multilineage differentiation. Such investigations require simultaneous analyses of clonality, multilineage activity and transgene copy numbers. Using a mouse model, the present study demonstrates that a single hematopoietic stem cell expressing a marker gene from one or two insertions of a simple retroviral vector actively maintains multilineage transgene expression in the vast majority (80-99%) of bone marrow and peripheral blood cells. Gene expression persisted through serial transplantations for at least 97 weeks post gene transfer and was observed in the lymphoid (B, T and NK cells), myeloid (CD11b(+), Gr-1(+)), erythroid (Ter119(+), mature red blood cells) and megakaryocytic (as indicated by platelets) progeny. Therefore, a single immunoselection for hematopoietic stem cells expressing the transgene in vivo was sufficient to establish a completely chimeric hematopoiesis. These observations imply that the retroviral vectors used in this study contain cis-elements that mediate expression through massive clonal expansion and multilineage differentiation, provided the insertion occurred in genetic loci permissive for expression in hematopoietic stem cells.

Animals↗

PubMed1987

Facilitation of emergence of multidrug-resistant state by alteration of tumor environment: implications from competitive ecology models.

The presence of multidrug-resistant (MDR) cells in a solid tumor constitutes a major problem in cancer therapy. Current thinking suggests that the resistant phenotype arises de novo during the tumor's evolution via somatic mutation mechanisms. The proportion of MDR cells, once established, may be enriched during therapy as a consequence of differential cell kill. Michelson et al have developed mathematical models of these phenomena to gain an insight into the dynamics of clonal subpopulation emergence in general and MDR emergence in particular, and I now show that one unexpected consequence of therapy may be the facilitation of MDR emergence due to damage inflicted on the host. The therapeutic damage to the host is modeled as a decreased ability to carry a specific tumor burden.

Antineoplastic Agents↗