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Biomedical subjects

A Yen

Publications and source records attributed to A Yen.

At least 127 records · Page 7Linked to original sources
PubMed1985

Control of HL-60 myeloid differentiation. Evidence of uncoupled growth and differentiation control, S-phase specificity, and two-step regulation.

Myeloid differentiation of HL-60 human promyelocytic leukemia cells was studied during DMSO-induced differentiation. G 1/0-specific growth arrest could occur without the usual associated subsequent phenotypic differentiation into mature myeloid cells, suggesting that growth arrest and phenotypic differentiation are separately regulated. In the course of differentiating, the cells achieved a semi-stable intermediate state where they had a labile, pre-commitment memory of exposure to inducer, but were not yet committed to differentiation. This state was associated with a nuclear structural change previously found to be associated with the precommitment memory state. The process of differentiation could thus be resolved into two steps, early events up through development of pre-commitment memory and late events subsequents to pre-commitment memory. The kinetics of terminal cell differentiation indicated that the cellular regulatory event initiating a program of differentiation in response to inducer was S phase-specific. A comparison of the present results for DSMO to previous results for retinoic acid (RA)-induced HL-60 myeloid differentiation showed that the two inducers effect different cellular pathways for differentiation of HL-60 cells to mature myeloid cells, but with certain common features including the above S-phase specificity and pre-commitment memory.

Cell Cycle↗
PubMed1985

Control of cell differentiation during proliferation. II. Myeloid differentiation and cell cycle arrest of HL-60 promyelocytes preceded by nuclear structural changes.

The time-dependent dynamics of nuclear structure, cell cycle transit and arrest, and cellular differentiation were studied using the human promyelocytic leukemia cell line HL-60. Myeloid differentiation of HL-60 cells was induced by 10(-6) M beta, all trans, retinoic acid (RA). During exponential growth the cells had G1, S, G2 and M durations of 9, 11, 0.5 and 0.5 h respectively. Significant growth arrest in the G1/0 phase of the cell cycle was apparent after 48 h of RA exposure or after two division cycles. Thereafter, cells arrested in G1/0 with wide dispersion in times of arrest which extended over several cell cycle generation times. The kinetics of phenotypic differentiation, detected by phorbol myristate acetate inducible superoxide production, paralleled those of G1/0 growth arrest with similar lag and dispersion. These kinetics are consistent with a model hypothesizing the existence of an S-phase differentiation control (DC) point regulating both terminal proliferation and differentiation. Before any cell differentiation or termination of cell proliferation occurred, the nuclei of RA-treated cells underwent a structural change detected by narrow-angle light scatter measured with flow cytometry. Narrow-angle light scatter was transiently reduced, reaching a nadir at 24-48 h and returning to control values at 96 h. This change was independent of cell cycle phase or total nuclear protein content. It was associated with a morphological change of the nuclear membrane from a smooth to dimpled or pitted structure. These findings focus attention on the potential significance of nuclear structural reorganization as an early event during cell differentiation.

Cell Cycle↗
PubMed1985

Myeloid differentiation-inducing factors produced by pokeweed mitogen-treated normal G1/0 lymphocytes but not chronic lymphocytic leukemia cells.

HL-60 human promyelocytic leukemia cells undergo myeloid differentiation and G1/0 specific growth arrest in response to an activity produced by pokeweed mitogen stimulated peripheral blood mononuclear cells. Elaboration of this myeloid differentiation inducing activity does not require lymphocytic proliferation or pokeweed mitogen induced B-lymphocyte differentiation. The activity is a product of initially stimulated G1/0 lymphocytes. It is not produced by proliferating lymphocytes. The activity is not elaborated by G1/0 peripheral blood lymphocytes of patients with chronic lymphocytic leukemia, cells which are proliferatively refractory to pokeweed mitogen stimulation.

Cell Differentiation↗
PubMed1984

Dependence of HL-60 myeloid cell differentiation on continuous and split retinoic acid exposures: precommitment memory associated with altered nuclear structure.

The cell differentiation of HL-60 human leukemic promyelocytes along the myeloid pathway due to various continuous and distributed exposures to retinoic acid was studied. HL-60 myeloid differentiation was a continuously driven process; significant terminal cell differentiation occurred only after a minimum exposure to inducer of two division cycles. Cells so committed to differentiation retained a heritable, finite memory of differentiation commitment over a further division cycle. Prior to becoming committed, cells acquired precommitment memory of exposure to inducer. Precommitment memory abbreviated the subsequent exposure to inducer needed for commitment to differentiation. Precommitment memory was semistable. It was heritable, but was lost after four division cycles. The acquisition and loss of precommitment memory correlated with alterations in nuclear architecture detected by narrow angle light scatter using flow cytometry. The altered nuclear architecture first occurred before any overt cell differentiation or growth arrest. It was thus an early event in the induced program of terminal cell differentiation. Alterations in relative abundances of cytoplasmic proteins also occurred prior to overt cell differentiation or growth arrest. One of these was a 17 kdalton, anionic, probably Ca2+ binding, protein. Retinoic acid thus induced early cellular changes, including cytoplasmic and nuclear alterations, within one cell cycle when cell differentiation was not yet apparent.

Cell Line↗
PubMed1984

Membrane origin for a signal eliciting a program of cell differentiation.

Evidence is presented to indicate that the retiNOic acid (RA)-induced program of myeloid differentiation and growth arrest by HL-60 human promyelocytic leukemia cells was initiated by a signal originating at the cell membrane. Free RA and RA covalently immobilized on a solid substrate elicited similar kinetics of differentiation and G1/0-specific growth arrest. No evidence of cell-induced RA detachment from the solid substrate was found. The data explain why HL-60 cells which are deficient in cellular RA-binding protein (CRABP) nevertheless respond to RA.

Cell Differentiation↗
PubMed1984

Endothelial cell cycle kinetics. Changes in culture and correlation with endothelial properties.

Endothelial cells in culture have emerged as a model system for evaluation of endothelial properties. Culture conditions may affect expression of these endothelial properties or growth characteristics. Therefore, we evaluated cell cycle kinetics, prostacyclin release, and angiotensin-converting enzyme activity in primary and subcultured cells from bovine pulmonary and systemic vessels. Prostacyclin release was highly variable during passage in culture. However, release following stimulation with ionophore A23187 declined with serial passage. Angiotensin-converting enzyme activity also declined with repeated subculturing. Greater than 10% of primary confluent bovine endothelial cells were cycling compared to 2 to 8% of human umbilical vein endothelial cells. With passage in culture, 25% of bovine endothelial cells were cycling at confluence. Occasional strains of subcultured bovine endothelium arrested in G2 but would reenter G1 upon subculturing. Exposure to trypsin did not appear to contribute to this phenomenon. These studies have demonstrated variability in endothelial cell properties and growth characteristics during in vitro propagation. The existence of this variability must be considered when studying properties or responses of subcultured endothelium.

Animals↗
PubMed1984

Evidence for cell cycle phase-specific initiation of a program of HL-60 cell myeloid differentiation mediated by inducer uptake.

The question of whether the initial regulatory event, which directs an uncommitted precursor cell toward terminal differentiation, is cell cycle phase specific was examined using the human promyelocytic leukemia cell line, HL-60. While the HL-60 system does not reflect all of the features of normal hematopoiesis, it does provide a relatively well-defined in vitro experimental system which can be useful for examining aspects of the differentiation process. HL-60 cells were induced to undergo myeloid differentiation by retinoic acid. The subsequent differentiation kinetics of HL-60 populations initially enriched in different cell cycle phases was measured. This was compared to the cellular uptake of retinoic acid as a function of cell cycle position. If the initial differentiation-regulating event were cell cycle phase independent, then the kinetics of differentiation would be independent of the cell cycle status of the initial population. Flow cytometric cell sorting, based on cellular narrow angle and orthogonal light scatter intensity spectra, was used to select G1-enriched and S + G2 + M-enriched cell populations without pharmacological perturbation. These two populations were each induced to undergo myeloid differentiation with 10(-6) M beta-all-trans-retinoic acid. The kinetics of G1/0 arrest associated with terminal cell differentiation, as well as phenotypic differentiation, assayed by development of oxidative metabolism, was measured for both populations. The kinetics of differentiation differed for the two populations, indicating that the initial differentiation-regulating event was cell cycle phase specific. For both of the initial cell populations, significant phenotypic differentiation followed approximately 24 hr after enrichment in the relative number of S-phase cells. When exponentially proliferating HL-60 cells were exposed to a 1-hr pulse of 10(-5) M [3H]retinoic acid and then flow cytometrically sorted by DNA content, cells in late S + G2 + M had an approximately 10-fold higher uptake than cells in G1 or early S. The results indicate that cellular regulation of myeloid differentiation first becomes responsive to the inducer, retinoic acid, in S phase when uptake is enhanced.

Biological Transport↗
PubMed1983

Control of cell differentiation during proliferation. I. Monocytic differentiation of HL-60 promyelocytes.

The cell proliferation relating an uncommitted precursor cell to a differentiated terminal cell has been quantitated. HL-60 promyelocytes, a bipotent precursor cell capable of differentiating along either the myeloid or monocytic pathway, were induced by a human lymphocyte-conditioned medium (CM) to differentiate into macrophage-like cells. The promyelocytes had a generation time of approx. 42 h. Most promyelocytes which differentiated became macrophage-like cells after only one cell division. Some, a minority, underwent more than one division. The time between induction of differentiation and expression of differentiated characteristics could thus be very short. Labelled S-phase promyelocytes could differentiate after traversing S. G2 and undergoing mitosis. Some, approx. 21%, required a subsequent complete cell cycle before differentiating. The data suggest a model in which cells must undergo a S-phase-specific differentiation control event in the presence of CM in order to differentiate in the subsequent G1 phase. This model proposes that a discrete time in S phase exists when cells are susceptible to exogenous regulation directing them to yield differentiated daughter cells.

Cell Differentiation↗
PubMed1983

Serum-free media for a human lymphocyte cell line and for pwm-stimulated peripheral blood lymphocytes: requirements for insulin, transferrin and albumin.

Two simple chemically defined media are reported to support cell proliferation of a human lymphocyte cell line and of mitogenically (PWM) stimulated peripheral blood lymphocytes. The medium in the former instance is Ham's F12 supplemented with insulin, transferrin, albumin and cholesterol. In the latter case, RPMI 1640 supplemented with insulin, transferrin and albumin was found to support cell proliferation as well as fetal calf serum. There was no apparent cell cycle specificity observed for the mode of action of insulin and transferrin. A comparison of the two defined media showed that the one for the cell line was more enriched for lipids and lipid precursors.

Adult↗
PubMed1982

Interferon inhibits PWM induced B cell differentiation but not onset of proliferation.

The dependence of B lymphocyte differentiation into plasmacytes on anteceding B and T cell proliferation was studied using interferon as a probe. Possible correlations of the effect of interferon on PWM induced T and B cell proliferation and B cell differentiation into either kappa or lambda light chain immunoglobulin synthesizing plasmacytes have been investigated. The hypothesis that the observed inhibition of the PWM induced formation of plasmacytes by interferon is due to putative enhanced suppressor cell activity resulting from increased T cell proliferation is tested. Human, peripheral blood lymphocytes were exposed to PWM in the presence or absence of human leukocyte interferon. Proliferation was assayed by pulse cytophotometric analysis of cell kinetics, as well as [3H]TdR labelling of S-phase cells. Incidence of plasmacytes was detected by immunofluorescence using kappa or lambda light chain specific antibody. During continuous [3H]TdR labelling of stimulated cells, interferon inhibited incorporation of precipitable label by 40% at 96 and 144 h, indicating reduced net DNA synthesis by interferon treated cells. The relative fraction of cells in S-phase as well as G1- and G2 + M- was similar for treated and untreated cells. The fraction of cells rosetting SRBC remained stable for both treated and untreated cells. The size of the interferon treated population was persistently smaller once proliferation began. The time of initiation of proliferation was comparable for treated and untreated cells. Consistent with the findings of others using cell lines, interferon apparently induces a dilation of all cell cycle phases, thereby reducing the rate of proliferation. The same reduction occurred for both T and B cells. Time of initiation of DNA synthesis was, in contrast, not delayed by interferon, suggesting it is specific for events during the proliferative cell cycle. The occurrence of both kappa and lambda light chain immunoglobulin secreting plasmacytes was inhibited by interferon. The degree of inhibition was comparable for both kinds of plasmacytes detected. While not delaying the onset of DNA synthesis, interferon apparently retards subsequent cell proliferation and inhibits the differentiation of B cells to plasmacytes. The data indicate that active cellular proliferation and B cell differentiation require interferon sensitive events which cells initially recruited from quiescence by PWM do not. The inhibition of the incidence of plasmacytes cannot be attributed to an imbalance of T cell proliferation relative to non-T cells.

B-Lymphocytes↗
PubMed1981

Cloning of fresh lymphoma cells.

We attempted to induce in vivo clonal growth of neoplastic lymphoid cells from fresh specimens of involved tissue from 22 patients with no-Hodgkin lymphoma (NHL). Conditioned media derived from two human B-lymphocyte tissue culture lines were tested for their ability to promote colony growth. In addition, we compared the incidence of colony induction to the flash-3H-thymidine labeling index (LI) of the cells in the tissues cultured. Successful colony induction occurred in three-tenths of the cases of diffuse histiocytic lymphoma and one-half of the cases of nodular lymphoma. Cloning efficiencies were low, ranging from 0.0003 to 0.04%. There was no apparent relationship between successful instances of cloning and LI. We have confirmed the observations of Jones et al that lymphoid tumor cells can be cloned from tissues involved by NHL using a soft agar system. The possibility of using such systems for clinical predictive assays of chemotherapeutic drug toxicity toward tumor cells of NHL is discussed. We conclude that further refinements of the assays are desirable before they can be applied to widespread clinical use.

Clone Cells↗
PubMed1981

Inaccuracy of estimations of s phase fraction by reduction in cloning efficiency with hydroxyurea or tritiated thymidine.

The current hypothesis, that the fractional reduction of cloning efficiency in semi-solid culture systems induced by pretreatment of the cells with hydroxyurea (HU) or [3H]TdR equals the fraction of cells initially in S phase, is tested. A lymphoblastoid cell line, SK-L7, with known cell cycle kinetics was exposed to cytotoxic concentrations of HU or suicidal doses of [3H]TdR and then initiated in semi-solid and liquid culture. Although approximately 0.6 of the initial population was in S, 1-hr exposures of HU at concentrations of up to 10(-2) M failed to reduce subsequent cloning efficiency. The 1-hr exposure to HU did not reduce either the immediate cell number or the gross population doubling rate over 24 hr. A 24-hr exposure to 10(-3) M HU reduced the cloning efficiency by approximately 98%, confirming the drug's cytotoxic capability. [3H]TdR at doses of 100 microCi/ml for 20--40 min reduced the cloning efficiency by approximately 60 and 70%, respectively. Although no cytotoxicity immediately after exposure was observed in either case, gross population doubling rate in liquid culture was reduced. While HU failed to reduce subsequent cloning efficiency, [3H]TdR reduced cloning efficiency by approximately the fraction of initial cells in S. The above hypothesis, therefore, cannot be applied naïvely as a technique for quantitating the fraction of a clonogenic cell population in S phase.

B-Lymphocytes↗
PubMed1981

Human megakaryocyte stimulation of proliferation of bone marrow fibroblasts.

Human marrow cells were processed sequentially by density centrifugation and by velocity sedimentation in serum-free Percoll gradients in order to purify megakaryocytes and to determine if these cells are the source of the growth factor derived from platelets. Cell homogenates were made from the resulting fractions and tested for growth-promoting activity(ies) in 3T3 cells and in well characterized human marrow fibroblasts. Growth was evaluated by 3H-TdR incorporation and changes in DNA cell content, as measured by flow microfluorometry. The highest mitogenic activity was derived from homogenates of low density (less than 1.050 g/cu cm), rapidly sedimenting cells. This fraction contained the highest percentage of megakaryocytes. The assessment of growth-promoting activity(ies) derived from various megakaryocyte-enriched marrow cell homogenates containing different proportions of megakaryocytes demonstrated a positive correlation between the number of megakaryocytes and their stimulatory capacity as determined by 3H-TdR uptake. The growth-promoting activities elicited from homogenates of platelets and marrow fractions enriched for megakaryocytes were similar. The dose--response curves for both were parallel, and they were both temperature resistant and trypsin sensitive. These findings implicate megakaryocytes as a source of the growth factor derived from platelets and suggest that megakaryocytes may play a role in the pathogenesis of the marrow fibrosis observed in myeloproliferative disorders by stimulating fibroblast proliferation and collagen secretion.

Blood Platelets↗
PubMed1979

Role of nuclear size in cell growth initiation.

Swiss 3T3 cells arrested in B0 (quiescent state) by reducing serum content of the medium all contain the same amount of DNA but vary in nuclear volume over approximately a twofold range. By use of flow microfluorimetry, scatterplots of nuclear volume versus DNA content were obtained in intervals after serum stimulation. The earliest cells to enter DNA synthesis were those with the largest nuclei, whereas cells with the smallest nuclei were among the latest. Regulation of cellular transit from G0 to the S phase was therefore, at least in part, deterministic, since all G0 cells did not have equal probabilities of entry into S at a given moment. All cells having the same nuclear volume did not initiate DNA synthesis at the same moment; therefore, factors other than nuclear volume must also influence this timing. Nuclear volume correlated with the maximum rate at which cells could enter S. The kinetic model of the cell cycle postulating a probabilistic event as solely responsible for entry into S thus appears too simple.

Animals↗