The cytostatic and immunosuppressive N'-methyl-N'-beta-chloroethylbenzaldehyde hydrazones B1 and B2 inhibit the colony growth of mouse bone marrow cells and PHA-stimulated human lymphocytes in vitro in a dose-dependent manner. In the presence of B1, however, in contrast to B2, the inhibition of 3H-thymidine (3H-Tdr) uptake by the bone marrow cells and lymphocytes is insignificant. Two further benzaldehydrazones CyB4 and EB4 show little or no influence both on clony growth and nucleoside uptake. On the other hand, CyB4 inhibits the 3H-Tdr uptake by ConA- or LPS-stimulated mouse spleen cells to a gretaer degree than does B1 or B2, although CyB4 unlike B1 or B2 does not display any immunosuppressive effects in the mouse. These findings demonstrate that the 3H-Tdr method is less sensitive than the colony assays and is hence only of limited value as a measure of the vitro proliferation of mammalian cells treated with cytostatics.

Animals↗

PubMed1979

In vitro colony growth of granulocytes, macrophages, T and B lymphocytes in agar capillaries.

Compared with the common agar of hematopoietic colonies in Petri dishes, the use of glass capillary tubes offers a couple of advantages which have been exploited. Among these advantages are less materials, less cells to be seeded, lower costs, easier and quicker colony counting by visual and semi-automated optical scanning, and lower risk of bacterial contamination. In tubes several parameters affecting the colony formation of mouse and human granulocytes and macrophages as well as of human peripheral T and B lymphocyte from mouse spleen were optimized. The applicability of the cultures to assay for inhibiting and stimulating humoral factors and cytostatics was demonstrated.

Agar↗

PubMed1979

Evidence for a rat granulocyte chalone effect on the proliferation of normal human bone marrow and of myeloid leukemias.

Evidence is presented that rat ascites cell extracts, acting as granulocyte chalone, temporarily inhibit the cell doubling of granulocytopoietic precursors in suspension cultures of human bone marrow. The extracts do not affect the proliferation of erythroblasts and do not show any cytotoxicity. In addition, a relative increase of granulocytic precursors capable of proliferation was found, suggesting an increase in the resting population of the granulocytopoietic proliferation pool due to extract treatment. However, the extract capable of depressing the proliferation of the normal granulocytopoiesis did not affect the granulocytopoiesis of 7 chronic myeloid and 2 chronic myelomonocytic leukemias at the same dose level. In contrast to these chronic leukemias, blast proliferation of 4 acute myeloid and monocytic leukemias was greatly depressed not only for 6 h but for the whole culture period of 48 h.

Animals↗

PubMed1979

Screening for specific calf thymus inhibitors (chalones) of T-lymphocyte proliferation.

Using agar colony assays with truly proliferating stimulated human T-lymphocytes and mouse granulocytes, two ultrafiltrate fractions were obtained from calf thymus which preferentially inhibited lymphocyte colony growth: Fraction I in the molecular range 1000-10,000 proved to be stable upon heating, prolonged storage and lyophilization, whereas Fraction II in the molecular range 10,000-30,000, was found to be unstable. Fraction I was also extracted with Tween 80 and cetyltrimethylammonium bromide. Chromatography of Fraction I on Biogel P6 and DEAE-cellulose further increased its specificity of inhibition for lymphocyte colony growth and revealed an estimated molecular weight of below 1400. Its inhibitory activity was found to be reversible and unlikely to result from spermine. Thus the properties of fraction I meet the requirements of a T-lymphocyte chalone as an endogenous non-cytotoxic and reversible inhibitor of T-lymphocyte proliferation.

Animals↗

PubMed1978

[Steroid hormone receptors. Cellular communication systems].

Explore the source record for details and available documents.

Animals↗

PubMed1978

Chalone inhibition of granulocyte colony growth in agar: kinetic quantitation by capillary tube scanning.

Mouse bone marrow cells were seeded into capillary tubes containing agar with colony stimulating factor. The development of myelomonocytic clusters and colonies was followed by daily tube scanning using their light scattering properties. Three kinetic scanning parameters were determined and the significance of different threshold settings was evaluated; viz. the number of signals, the mean signal height and the signal integrals. The inhibitory effect of two extracts with known granulocyte chalone activity which had been prepared from human peripheral leukocytes and rat bone marrow cells, was followed with the scanning method. A continuous reduction of clusters and colony formation and their growth throughout the incubation period was observed which suggested a sustained retardation of proliferation of both the stem cells committed for myelomonopoiesis and their progeny.

Agar↗

PubMed1978

Screening for granulopoiesis inhibitors (chalones) by different assays.

The suitability of various granulocyte chalone sources was examined; for this purpose rat ascites fluid and the conditioned media of ascites and bone marrow cells were fractionated by ultrafiltration and Sephadex gel filtration. To evaluate different assay systems, the ability of the fractions to inhibit the growth of granulocytic and T-lymphocytic colonies in agar capillaries, as well as to inhibit the uptake of [3H]thymidine in bone-marrow cells, T- and B-lymphocytes, was tested and compared. Three granulocyte colony inhibiting fractions were obtained that contained apparent chalone activities, but showed different elution parameters with molecular weights well below 10 000. Comparison of the test systems revealed that the granulocyte colony assay may detect inhibitors different from those found by the [3H]thymidine bone-marrow assay; the validity of the latter test is seriously questioned, however. The need for precisely defined assays to screen for the apparently various inhibitors is emphasized by these studies.

Animals↗

PubMed1978

The formation of B-lymphocyte colonies in agar contained in glass capillaries.

Optimal conditions were established for a micro method for the production of colonies of B lymphocytes from mouse spleen cells cultured in agar in glass capillaries, in the presence of bacterial lipopolysaccharide (LPS). Besides LPS the cultures require 5 X 10(-5) M mercaptoethanol and 20% horse serum for optimal colony growth. Foetal calf serum and heat-inactivated horse or foetal calf serum were found to be inferior. An agar gel strength of 0.3% was best for colony counting. A sigmoid curve was obtained when the number of colonies formed was related to the seeded cell density suggesting that some kind of cell to cell co-operation is essential for colony formation. The daily kinetics of colony growth were followed by microscopic colony counting and photometric capillary scanning with integration of the signal areas. Both methods indicated that colony growth had ceased by day 6. The combination of both methods gave the most realistic picture of B-lymphocyte colony development.

Agar↗

PubMed1978

Drug evaluation on haemopoietic cells in vitro. I. A micro agar colony assay with semi-automatic optical monitoring.

A micro method was developed to culture myelomonocytic colonies from mouse bone marrow cells in semi-solid agar contained in glass capillary tubes. Parameters affecting colony formation in capillaries were studied. An optical, semi-automatic scanning system using the light scattering properties of the colonies was developed; the system was mostly composed of commercially available equipment. The daily growth of single colonies within the tubes was followed. Combined with the capillary technique the scanning system provides an easy, accurate and sensitive method to quantitate the formation of haemopoietic colonies. This is the basis of a micro assay for the evaluation (screening) of drug effects on mammalian cell colonies in vitro offering several advantageous features.

Agar↗

PubMed1977

Temperature-dependence of the proliferation of mouse bone marrow cells cultured in H2O- and D2O-media.

In an attempto to optimize and standardize the in vitro culture conditions of mouse bone marrow cells for assaying growth regulating factors, we studied the effects of incubation at low temperatures and of a nutrient medium containing deuteriumoxide instead of water. It was found that (1) the proliferative capacity of the cells is significantly increased by pre-incubation for 1-2 h at 0 degrees C rather than at 37 degrees C, measured by both a colony-forming and a 3H-thymidine (3H-tdr) uptake assay. A similar temperature effect on the colony-forming and 3H-tdr uptake ability is apparent after pre-incubation in D2O-medium, yet significantly lower than in H2O-medium. It was concluded that the previously observed protective effects of D2O on ascites tumor cell proliferation and viability and for hemolysis of human erythrocytes is not apparent in proliferating and colony-forming mouse bone marrow cells in vitro.

Animals↗

PubMed1977

A lymphocyte chalone assay using lymphocyte colony growth in agar capillaries.

Using a recently developed method of culturing T-lymphocyte colonies in agar-containing capillary tubes, the capacity of three different lymphoid extracts with lymphocyte chalone (LC) activity to inhibit colony growth was demonstrated. Sephadex fractions from a calf spleen extract were tested on the colony growth of granulocytic cells and PHA-stimulated T-lymphocytes as well as on the in vitro uptake of 3H-thymidine by bone marrow and ConA- and LPS-stimulated mouse spleen cells. The data strongly suggest that it is only the combination of several different assay systems applied to the same fractions that permits a clear-cut determination of a specific lymphocyte proliferation inhibitor like LC.

Agar↗

PubMed1977

Inhibition of agar colony formation by partially purified granulocyte extracts (chalone).

Granulocytic extracts (GE) of different sources, presumably containing the granulocytic chalone, were prepared in different laboratories and purified to some extent. They specifically inhibited the formation of granulocyte and macrophage colonies in agar. The effect was however most pronounced on granulocyte and mixed granulocyte-macrophage colonies, and less on macrophage types. Addition of GE to bone marrow cells at the time of plating in agar, as well as short incubation of the cells together with GE prior to plating, inhibited subsequent colony formation. The inhibitory effect could easily be reversed by washing the cells with an excess of medium prior to plating during the first hour of preincubation, but not after five hours. Increasing the doses of colony stimulating activity (CSA) (at low doses of GE) released the inhibitory effect, but not at high doses of GE. The inhibitory effect of GE on colony formation was dose dependent down to almost 100% inhibition. No apparent cytotoxic effect of GE on bone marrow cells could be found and lymphoblastic cells were not inhibited. Extracts containing a specific inhibitor of erythropoiesis (EIF) stimulated myelopoietic colony formation in agar.

Agar↗

PubMed1977

In vitro culture of lymphocyte colonies in agar capillary tubes after PHA-stimulation.

Human peripheral lymphocytes were stimulated with phytohemagglutinin (PHA) in liquid culture, suspended in agar and incubated in glass capillary tubes. Compact colonies of lymphocytes were found growing along the tube bottom in a buffer film, while compact clusters and rare diffuse colonies were observed inside the agar. Several parameters affecting the clonal growth were studied and optimized: PHA-dose, agar concentration, gel length (volume), quantity and density of seeded cells per capillary and gel length. Colony yield mainly depends on the seeded-cell density with a sharp optimum at 2 X 10(5) cells/ml irrespective of gel length; higher cell densities reduce the colony yield, suggesting that colony growth is the result of both stimulatory and inhibitory factors produced by cooperating cells. Following the daily clonal growth was only possible with undisturbed tubes; the number of colonies steadily increased from day 2 until day 7. Densitometric colony scanning is possible, yet problematic. Colony yield (plating efficiency is 10--50-fold higher in agar capillaries than in the usual Petri dishes. An additional advantage is that the capillaries provide a basis for a simple and reliable assay system for determining regulatory factors of lymphocyte proliferation (including chalones).

Agar↗

PubMed1976

Evaluation of a short term in vitro test for granulocyte chalone activity.

A short term in vitro test for granulocyte chalone activity eas examined for its specificity and reliability. The test used the inhibition, by granulocyte extracts, of 3H-thymidine (3H-Tdr) uptake in to the acid-insoluble material by rat bone marrow cells in vitro to measure possible chalone activity. Among the many possible 3H-Tdr artifacts pool size dilution by Tdr contained in the extracts was excluded using an E. coli mutant requiring thymine. Several amino acids and biogenic amines do not affect the test. However, continuous and pulse labelling of bone marrow cells with 3H-Tdr, viability tests and micro flow fluorometric measurements of the cell cycle distribution following colcemid treatment strongly suggests that the cells do not proliferate in vitro during short term incubation, since practically no cells enter the S-phase, cells in the S-phase die and few if any cells proceed through G2 and mitosis. Moreover, the test cannot exclude cytotoxicity. Thus, the in vitro test may only sceem for an unspecific S-phase inhibitor and must hence be supplemented by another assay to prove the chalone nature of an extract or fraction. The test per se fails to meet most of the requirements of a valid granulocyte chalone assay.

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