Agar Colony Formation Research Articles

Mutant feline sarcoma proviruses containing the viral oncogene (v-fes) and either feline or murine control elements.

The sequences required for transformation by the Gardner-Arnstein (GA) strain of feline sarcoma virus (GA-FeSV) were defined by site-directed, in vitro mutagenesis of molecularly cloned proviral DNA. Portions of the Ga-FeSV provirus, subcloned in the plasmid pBR322, were mutagenized by deletion or frameshift at XhoI restriction sites flanking the nucleotide sequences presumed to encode the GA-FeSV transforming polyprotein (P108(gag-fes)). The biological activity of subgenomic and reconstructed full-genome-length molecules was assayed by transfection and focus induction in NIH 3T3 cells. Both mutant and wild-type molecules containing the intact P108(gag-fes) coding region induced foci of transformed cells at efficiencies between 10(4) and 10(5) focus-forming units per pmol of DNA; a deletion mutant lacking 3'-terminal v-fes sequences was completely nontransforming in parallel assays. Representative subcloned foci of transformed NIH 3T3 cells synthesized P108(gag-fes) with associated in vitro protein kinase activity. Focus-forming viruses could be rescued from transformed subclones induced by full-length proviral DNA, but not from cells transformed by subgenomic DNA lacking a 3' long terminal repeat (LTR). It was concluded that: (i) nucleotide sequences encoding P108(gag-fes) and its associated kinase activity are responsible for transformation, (ii) the GA-FeSV 3' env and LTR sequences are not required for focus induction, and (iii) the 3' LTR is necessary for rescue of infectious FeSV RNA. A chimeric DNA containing the 5' LTR and P108(gag-fes) coding region of GA-FeSV joined to the 3' LTR of Moloney murine sarcoma virus was both transforming and rescuable at high efficiency. Restriction analysis showed that passaged stocks of rescued transforming virus contained Moloney murine sarcoma virus U3 sequences at both proviral DNA termini, consistent with generally accepted models for LTR formation during reverse transcription. Wild-type GA-FeSV and the chimeric virus (here designated as GAHT), each rescued from NIH 3T3 cells with the same amphotropic murine leukemia virus, yielded approximately equal numbers of foci when titrated on CCL 64 mink cells. By contrast, on mouse NIH 3T3 cells, the focus-forming titer of GAHT was 1 to 2 log higher than that of FeSV. The foci induced on NIH 3T3 cells by GAHT appeared earlier and were reproducibly larger than those induced by GA-FeSV. Differences in transforming activity on NIH 3T3 cells were also found using colony formation in agar, showing that the more rapid appearance and larger size of foci formed in liquid media were not due to virus spread. These data suggest that transcriptional control signals within the viral LTR regulate the levels of the transforming gene product in a species-specific manner.

Autoinduction of differentiation in WEHI-3B leukemia cells.

Cells of the differentiation-responsive mouse myelomonocytic leukemia cell line WEHI-3B D+ form colonies in agar exhibiting a low frequency of spontaneous differentiation mainly in the macrophage pathway. Compared with undifferentiated colonies, spontaneously differentiating colonies have a reduced content of clonogenic cells and surviving clonogenic cells tend themselves to form differentiating colonies, both being characteristics of differentiated colonies induced by the regulator, granulocyte colony-stimulating factor, G-CSF. Colony crowding increased the frequency of spontaneously differentiating colonies and WEHI-3B D+ colony cells were shown to release material able to induce differentiation in WEHI-3B D+ colonies. Cells from spontaneously differentiating D+ colonies were not hyperresponsive to the induction of differentiation by G-CSF and did not release larger amounts of differentiation-inducing material than did cells from undifferentiated colonies. Cells of the differentiation-unresponsive WEHI-3B D-line produced similar amounts of differentiation-inducing material to those produced by D+ cells. Apparently spontaneous differentiation in WEHI-3B D+ colonies seems most likely to be due to exposure of the colony-forming cell or its ancestors to a differentiation-inducing factor of WEHI-3B origin prior to culture in agar, the genetic program initiating differentiation being inherited by the progeny of the exposed cell.

Morphologic and functional heterogeneity of chronic neutropenia of childhood with normal neutrophil colony formation in vitro

In order to obtain further knowledge of chronic neutropenia of childhood, we studied nine neutropenic infants six to ten months of age by in vitro techniques, including bone marrow culture, electron microscopy, and chemotaxis assay. Eight of the nine patients had a benign clinical course and the bone marrow aspirates showed a reduced number of segmented neutrophils. The ninth patient had a moderately severe course and the bone marrow showed maturation arrest at the promyelocyte stage. Bone marrow cultures demonstrated that the in vitro neutrophil colony formation and production of colony-stimulating activity were normal in all of the eight patients studied. Neutrophils from one of the nine patients had ultrastructural abnormalities such as decrease in number of primary and secondary granules and the presence of myelin figures in primary granules. Neutrophil chemotaxis was defective in three of the nine patients. All of the six patients in whom the neutrophil colony formation in agar, the ultrastructure of neutrophils, and neutrophil chemotaxis were normal recovered from the neutropenia between 11 and 30 months of age. These in vitro parameters appear to be useful for evaluating chronic neutropenia of childhood.

Increased eosinophil colony formation in agar by haemopoietic cells from patients with the hypereosinophilic syndrome

Colony formation by granulocyte/macrophage and eosinophilic progenitor cells in the blood and/or bone marrow of four patients with "hypereosinophilic syndrome' (HES) was studied in agar culture. Colony-stimulating activity (CSA) was derived from leucocyte feeder layers (LFL) or from medium conditioned by phytohaemagglutinin-stimulated lymphocytes (LCM). The total numbers of colonies in the patients' blood and marrow were normal. When patients' marrow cells were cultured over LFL, the proportion of colonies that were eosinophilic was greater than normal (mean 21% vs 6%, P less 0.001) and this difference was accentuated when CSA was derived from LCM (mean 53% vs 15%, P less than 0.001). LCM derived from normal subjects and LCM prepared from HES patients had similar effects. In the blood the total number of colonies and the proportion of eosinophilic colonies were similar in patients and controls. The overall pattern of colony formation HES differed distinctly from that observed in a patient with eosinophilic leukaemia reported previously. We conclude that the proportion of eosinophil-committed progenitor cells in the marrow of patients with HES is increased, but we have failed to demonstrate a role for the patient's own lymphocytes in augmenting eosinophil production.

In Vitro Studies of Lactoferrin and Murine Granulopoiesis

Human lactoferrin (LF) has been reported to inhibit in vitro granulopoiesis by means of decreasing colony-stimulating activity production by monocytes. We performed a series of experiments to determine if the reported experimental results could be replicated using highly purified murine LF and murine target cells. Three different types of experiments were performed. (1) Medium was conditioned by lung, femoral shaft, and adherent peritoneal cells in the presence and absence of LF, and the granulopoietic stimulating activity in the conditioned media was assayed by means of a 7-day agar colony assay and a 3-day liquid slide chamber assay, which quantitates 3H-TdR incorporation into DNA. (2) In cultures stimulated by an underlayer of adherent peritoneal cells, marrow cell colony formation in agar was determined after 7 days of culture in the presence or absence of LF. (3) LF was added to 3-day liquid marrow cell cultures that had been stimulated by lung or femoral shaft conditioned media. In all experimental situations, highly purified, iron-saturated LF in concentrations up to 10(-7) M had no effect on in vitro granulopoiesis. These results do not support LF's reputed regulatory role in granulopoiesis.

In vitro studies of lactoferrin and murine granulopoiesis

Human lactoferrin (LF) has been reported to inhibit in vitro granulopoiesis by means of decreasing colony-stimulating activity production by monocytes. We performed a series of experiments to determine if the reported experimental results could be replicated using highly purified murine LF and murine target cells. Three different types of experiments were performed. (1) Medium was conditioned by lung, femoral shaft, and adherent peritoneal cells in the presence and absence of LF, and the granulopoietic stimulating activity in the conditioned media was assayed by means of a 7-day agar colony assay and a 3-day liquid slide chamber assay, which quantitates 3H-TdR incorporation into DNA. (2) In cultures stimulated by an underlayer of adherent peritoneal cells, marrow cell colony formation in agar was determined after 7 days of culture in the presence or absence of LF. (3) LF was added to 3-day liquid marrow cell cultures that had been stimulated by lung or femoral shaft conditioned media. In all experimental situations, highly purified, iron-saturated LF in concentrations up to 10(-7) M had no effect on in vitro granulopoiesis. These results do not support LF's reputed regulatory role in granulopoiesis.

Severe aplastic anaemia: correlation of in vitro test with clinical response to immunosuppression in 20 patients.

Colony formation in agar (CFU-c) was studied in 20 patients with severe aplastic anaemia by three different assays: (1) cultures of light density untreated marrow cells; (2) cultures of marrow cells manipulated in order to enhance colony formation (pretreatment with antilymphocytic globulin, ALG, or 6-methylprednisolone, 6-MPr, T cell depletion, adherent cell (AC) depletion, depletion of both AC and T cells), and (3) co-culture of putative suppressor T cells with autologous T-depleted marrow cells. By the first assay, all patients showed poor colony formation (1 +/- 1.5 colonies/10(5) cells; normal controls 46 +/- 18 colonies/10(5) cells). By the second assay, ALG and 6-MPr had no significant effect on colony formation. Removal of adherent cells proved equally without effect on colony growth. On the contrary, removal of T cells enhanced significantly (P less than 0.001) colony formation in 10 out of 20 patients. By the third assay, colony formation of marrow cells (deprived of T lymphocytes) was inhibited by the addition of autologous T cells in six patients studied. All patients were given high dose bolus 6-MPr as first treatment on admission: only patients who had detectable suppressor T cells in their marrow achieved a complete autologous haematologic reconstitution after 6-MPr or after 6-MPr and ALG. The results of this study indicate the detection of CFU-c/suppressor T cells correlates with responses to immunosuppressive regimens, and may thus help to identify patients with immune mediated aplastic anaemia.

A murine model system for the study of the human leukemia-associated inhibitory activity

Mice inoculated with the RFV strain of Friend virus develop an erythroleukemia that spontaneously regresses. Bone marrow cells from leukemic mice produce a factor that inhibits colony formation in agar by normal CFU-Cs. CFU-Cs from leukemic animals are resistant to the factor. Bone marrow cells from some mice whose leukemia has regressed continue to produce the factor, and CFU-Cs from some regressed mice retain resistance to the factor. These observations dupllicate findings reported for patients with acute leukemias and those with leukemia in remission. The RFV system thus serves as an accurate experimental model for the study of hematopoietic cell interactions and the proliferative advantage of neoplastic cells in human leukemias.

A Murine Model System for the Study of the Human Leukemia-Associated Inhibitory Activity

AbstractMice inoculated with the RFV strain of Friend virus develop an erythroleukemia that spontaneously regresses. Bone marrow cells from leukemic mice produce a factor that inhibits colony formation in agar by normal CFU-Cs. CFU-Cs from leukemic animals are resistant to the factor. Bone marrow cells from some mice whose leukemia has regressed continue to produce the factor, and CFU-Cs from some regressed mice retain resistance to the factor. These observations duplicate findings reported for patients with acute leukemias and those with leukemia in remission. The RFV system thus serves as an accurate experimental model for the study of hematopoietic cell interactions and the proliferative advantage of neoplastic cells in human leukemias.

Colony formation in agar by adult bone marrow multipotential hemopoietic cells.

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40--50 per 10(5) bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4..5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.

Colony formation in agar by multipotential hemopoietic cells

Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen-stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7-day mixed colonies was 1,800-7,300 cells. When 7-day mixed colonies were recloned in agar, low levels of colony-forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophil and/or macrophage colonies. Injection of pooled 7-day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony-forming cells per 7-day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed. The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.

Marrow adherent cell colony-stimulating activity production in acute myeloid leukemia

Provision of colony-stimulating activity (CSA) by human marrow cells was determined utilizing culture techniques in vitro to assess intramedullary cellular interactions on human granulopoiesis. CSA production from marrow cells of 21 patients with acute myeloid leukemia (AML) was evaluated and compared to normal marrow by testing the capacity of conditioned media from adherent marrow cells to promote granulocytic colony formation in agar of relatively-light-density nonadherent human marrow target cells. Morphologic, cytochemical, density, and phagocytic characteristics of normal marrow cells suggested that marrow CSA production was provided by middensity adherent cells including those of the monocyte-macrophage series. Decreased marrow cell CSA provision was found in 13 of 21 studies of patients with AML at diagnosis or relapse (p

Relationship between somatic mutation and neoplastic transformation.

Somatic mutation and neoplastic transformation of diploid Syrian hamster embryo cells were examined concomitantly. Mutations induced by benzo[a]pyrene and N-methyl-N'-nitro-N-nitrosoguanidine were quantitated at the hypoxanthine phosphoribosyltransferase and Na(+)/K(+) ATPase loci and compared to phenotypic transformations measured by changes in cellular morphology and colony formation in agar. Both cellular transformations had characteristics distinct from the somatic mutations observed at the two loci. Morphological transformation was observed after a time comparable to that of somatic mutation but at a frequency that was 25- to 540-fold higher. Transformants capable of colony formation in agar were detected at a frequency of 10(-5)-10(-6), but not until 32-75 population doublings after carcinogen treatment. Although this frequency of transformation is comparable to that of somatic mutation, the detection time required is much longer than the optimal expression time of conventionally studied somatic mutations. Neoplastic transformation of hamster embryo cells has been described as a multistep, progressive process. Various phenotypic transformations of cells after carcinogen treatment may represent different stages in this progressive transformation. The results are discussed in this context and the role of mutagenesis in the transition between various stages is considered. Neoplastic transformation may be initiated by a mutational change, but it cannot be described completely by a single gene mutational event involving a dominant, codominant, or X-linked recessive locus. Neoplastic transformation induced by chemical carcinogens is more complex than a single gene mutational process. Thus, this comparative study does not give experimental support to predictions of the carcinogenic potential of chemicals based on a simple extrapolation of the results obtained from conventional somatic mutation assays.

Molecular weight and some chemical properties of the granulocytic chalone.

Granulocytic Chalone (GCh) has been highly purified from culture medium conditioned by human peripheral leukocytes. Purification was performed by column chromatography on Sephadex G-25 and G-10, rechromatography on G-10, preparative paper chromatography and thin-layer-chromatography. The inhibitory activity and specificity of GCh was monitored by agar colony formation and 3H-thymidine incorporation into bone marrow and thymic cells. The gelchromatographic behavior of GCh is discussed in detail with regard to its molecular weight. The results obtained indicate that GCh may be a small (MW 500--600), acidic and highly polar peptide containing aspartic and glutamic acid among others. The N-terminal aminogroup seems to be blocked.

Human bone marrow colony formation in diffusion chambers: Normal values

Two-hundred and twentyfive marrow samples, aspirated from the iliac crests of hematologically normal individuals, have been assessed for colony formation in agar in diffusion chambers. These assays were made over a period of 30 months and the results show that the mean, range and distribution of normal values has remained the same during this time.

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.

Properties of mammalian cells transformed by temperature-sensitive mutants of avian sarcoma virus

Fibroblasts from European field vole (Microtus agrestis) and from normal rat kidney (NRK) have been infected by avian sarcoma virus mutants which are temperature-sensitive for the maintenance of transformation. These cells are transformed at 33°C, but show normal cell characteristics in morphology, colony formation in agar, saturation density, sugar uptake and membrane proteins at 39°C and 40°C, the nonpermissive temperatures. Ts mutant virus was rescued from most of the ts transformed cell lines. NRK cells infected by avian sarcoma virus ts mutants and kept at the nonpermissive temperature can be transformed by wild-type avian sarcoma virus. The susceptibility of the temperature-sensitive NRK lines to this transformation is higher than the susceptibility of uninfected NRK at either permissive or nonpermissive temperature.

Evaluation of Congenital Neutropenic Disorders by in Vitro Bone Marrow Culture

The congenital neutropenias are a heterogenous group of diseases whose etiology and pathogenesis are largely unknown. We studied nine neutropenic patients from seven families. Evaluation included peripheral blood cell and differential cell counts, epinephrine and typhoid vaccine stimulation studies. Rebuck skin windows, and bone marrow aspirations for morphological assessment and for in vitro culture in liquid suspension and in agar plates. Parallel cultures were set up with and without colony-stimulating activity (CSA), and peripheral leukocytes were assayed for cellular production of CSA. Patients were initially classified on the basis of their clinical course: benign, mild, moderately severe, or severe disease. One patient in the moderately severe group had an immunoglobulin disorder. Morphologically normal mature granulocytes were seen in bone marrow aspirates of two patients, and maturational defects of varying degree were seen in the remaining seven. Colony formation in agar was markedly reduced below normal in three of seven, moderately reduced in two of seven, and greater than normal in two patients. Colonies in six of seven patients consisted exclusively of macrophages. Marrow from all but one of the nine patients demonstrated poor neutrophil development in suspension culture, and addition of CSA did not result in augmented granulocytic proliferation or maturation. A scheme of normal neutrophil maturation is proposed, and the nine patients were categorized according to this scheme. Four patterns of congenital neutropenia emerged: type 1 was the most benign form of disease with essentially normal clinical and in vitro parameters, and a defect considered to be due to a small committed stem cell pool, abnormal release, or excessive utilization peripherally; type 2 had mild disease with presumed defective committed stem cell differentiation along the granulocyte line; type 3 included benign to severe clinical expression with an apparent defect at the level of the committed granulocyte precursor more severe than in type 2; type 4 disease had varied clinical expression but evidence for a defect at the level of the pluripotent stem cell.

Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail

Several continuous tissue culture cell lines were established from methylcholanthrene-induced fibrosarcomas of Japanese quail. The lines consist either of fibroblastic elements, round refractile cells or polygonal cells. They show transformed characteristics in agar colony formation and hexose uptake, and most are tumorigenic. Their cloning efficiency in plastic dishes is not increased over that of normal quail embryo fibroblasts. The quail tumor cell lines do not produce endogenous avian oncoviruses and fail to complement the Bryan high titer strain of Rous sarcoma virus; those tested lack the p27 protein of avian oncoviruses. Most of the cell lines are susceptible to subgroup A avian sarcoma viruses, but are relatively resistant to viruses of subgroups C, E and F as compared to normal quail embryo fibroblasts.

Stimulation of B-lymphocyte colony formation in vitro by sera from patients with leukaemia or lymphoma.

Studies were made on the effects of 665 sera, from normal donors or patients with various diseases, on B-lymphocyte colony formation in agar by mouse spleen cells. Undiluted serum from most normal donors inhibited colony formation, but 43-53% of sera from patients with histiocytic lymphoma, lymphocytic lymphoma or Hodgkin's disease stimulated colony formation, serum activity correlating with the stage of the disease. Moderate colony-stimulating activity was observed with serum taken from patients with acute lymphoid or myeloid leukaemia following, but not prior to, chemotherapy. Colony stimulating activity was not correlated with the blood group of serum donors and could not be ascribed to the presence of endotoxin, red cells or mouse red cell haemagglutinins in the active sera. Elevated colony stimulating activity was not observed in sera from patients with non-neoplastic disorders ot haemopoiesis or with diseases of other organ systems.