Viral oncogene expression during differentiation of Abelson virus-infected murine promonocytic leukemia cells

A cloned, virus-producing, tumorigenic, promonocytic leukemia cell line, AC8, derived from an Abelson murine leukemia virus-infected mouse can differentiate in vitro. Differentiated cells, purified from a population containing undifferentiated cells on the basis of expression of the macrophage differentiation antigens Mac-1 (C3 receptor) and F4/80, were phagocytic, produced lysozyme, were less tumorigenic, and had a reduced replicative capacity compared with undifferentiated cells. Differentiated cells produced less infectious Abelson virus than undifferentiation. Cloning studies indicated that differentiation was the cause rather than the effect of reduced Abelson virus production. However, the intracellular amount and the tyrosine-specific protein kinase activity of the Abelson virus oncogene product, P120v-abl, were not affected by differentiation of the leukemic cells. Thus, these results show that Abelson virus-transformed myeloid lineage cells can differentiate without expression of the viral oncogene product being affected, which implies, in turn, that P120v-abl expression is not sufficient for maintaining transformation by blocking differentiation.     

Clonal analysis of the response of HL60 human myeloid leukemia cells to biological regulators

Human myeloid leukemia (HL60) cells formed colonies in semi-solid agar cultures with a cloning efficiency of 20–90%. Addition of unfractionated human placental conditioned medium (HPCM) or the two semi-purified granulocyte-macrophage colony stimulating factors from HPCM (GM-CSFα and β) increased colony size and the frequency of colonies exhibiting differentiation of colony cells. Differentiation induction using GM-CSFα or β did not reduce the total number of clonogenic cells per colony but did reduce the proportion of clonogenic cells within colonies. Sera from some patients with acute infections exhibited an elevated capacity to induce differentiation both in HL60 colonies and in colonies of the mouse myelomonocytic leukemia cell line, WEHI-3B. A low percentage of HL60 colonies contained maturing eosinophils but the frequency was not influenced by human-active eosinophil colony stimulating factors or by sequential recloning of HL60 colonies.The studies suggest that, as is true for mouse myeloid leukemia cell lines, the granulocyte-macrophage colony stimulating factors are able to induce significant differentiation in human HL60 myeloid leukemia cells.     

Correlation of growth morphology and clonability with malignancy of WEHI-7 T-cell lymphoma sublines

This study examined the development of subclones of different proliferative capacity and malignancy in the WEHI-7 tumor. The mouse T-cell lymphoma WEHI-7 can be cloned in agar with a cloning efficiency of 40 to 50%. On the basis of growth morphology, two types of colonies were distinguished. Most colonies were compact, but a few, no more than 5%, were diffuse. Sublines of the two colony types were established. The cloning efficiency in agar was 40 to 50% for the compact and 15 to 25% for the diffuse sublines. The recloning efficiency did not change for compact colonies. In contrast, the cloning efficiency of diffuse colonies decreased with repeated reclonings. The mice died within 24 to 34 days of i.v. injection of 10(4) WEHI-7 cells. The same number of cells of the individual sublines administered to mice resulted in improved survival. Injection of compact subline cells resulted in death in 24 to 48 days, while diffuse subline cells resulted in death in 30 to 75 days. The sublines were indistinguishable by light and electron microscopy. Both subline types were negative for terminal deoxynucleotidyl transferase and positive for Thy 1.2. Most cells from the two types of sublines were Lyt-1 and Lyt-2 positive. The doubling time was 10 hr for compact and 14 hr for diffuse sublines. Colony morphology was conserved after passage in vivo and after more than 10 transfers in vitro in liquid and agar media. In conclusion, the different growth morphology in vitro distinguished the subclones of different malignancy in the WEHI-7 tumor cell line.     

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.     

Antigens present on human myeloid leukemia cell lines

Two human acute myelogenous leukemia cell lines have recently been established: KG-1 and HL-60. Both lines retain the morphology and cytochemistry of myeloid cells, are not infected with EB virus, and respond to colony-stimulating activity (CSA) with increased colony formation in soft-gel culture. The K 562 cell line is composed of undifferentiated blast cells which are rich in glycophorin and may be induced to produce human fetal and embryonic hemoglobin. We used a variety of antisera to characterize the antigen composition of the leukemic colony-forming cells (CFC) and the general population of leukemic cells. Cells were incubated with antisera and complement, washed, plated in agar, and the colonies counted after 12 days' incubation. Culture data were correlated with the results obtained using direct and indirect immunofluorescence and dye exclusion microcytotoxicity.The majority of KG-1 cells, including CFC, expressed the ‘Ia-like’ antigen. The HL-60 and K562 cells did not express the Ia antigen, although indirect evidence using cytogenetic and isoenzyme studies suggests that the HL-60 gene locus for the antigen was probably intact.We found that the leukemia CFC had the HLA-A and -B antigens as did the general leukemia cell population. The leukemia CFC did not express a T-lymphocyte antigen, certain erythrocyte antigens, nor antigens associated with acute lymphocytic leukemia. HL-60 cells underwent morphological and functional maturation in the presence of DMSO, but no change in surface antigenic composition was noted. All antigens expressed on the leukemia CFC were also present on the general cell population.     

Do cell lines in vitro reflect the properties of the tumours of origin? A study of lines derived from human melanoma xenografts.

The characteristics of 7 human melanoma cell lines were compared with those of the xenografts from which they were established. The ultrastructure, melanin content, isozyme pattern and chromosome numbers of the cell lines were closely similar to those of the corresponding xenografts. The different cell lines gave rise to colonies in soft agar of size and morphology similar to the parent xenografts, and the plating efficiencies were clearly correlated. However, no correlation was found between the growth rates in vivo and either the doubling times and saturation densities in monolayer cultures or the plating efficiencies in soft agar. Moreover, one of the cell lines lost its tumorigenic ability upon establishment in culture. Thus, although the properties of the cell lines by and large reflected those of the parent xenografts, important inconsistencies were seen. The data emphasize that extrapolations from continuous cell lines in vitro to tumour cells in vivo are not necessarily valid. A high content of cellular fibronectin was correlated with a compact colony morphology in soft agar and rapid attachment and spreading on plastic. The growth rates and cellular morphology of the cell lines were strongly influenced by TPA, DMSO, retinoic acid and theophylline, but not by alpha-melanocyte-stimulating hormone. A murine cell line established from one of the xenografts grew in soft agar and produced sarcoma in mice. The malignant murine cells had arisen by transformation of murine stromal cells during the first subcultures in vitro, possibly caused by a factor produced by the human melanoma cells.     

裸小鼠高致瘤性K562-n细胞系的生物学特性研究

目的：探讨高致瘤性K562－n细胞系在胸腺缺陷裸小鼠体内致瘤的细胞生物学机理。方法：通过极限稀释法建立K562－n细胞系致瘤率不同的克隆株，并以K562细胞为对照，对各克隆株进行裸小鼠体内外生物学特性的比较研究，结果：高致瘤性的T562－B、K562－C、K562－E克隆株（致瘤率≥5／6），其软琼脂培养集落形成率，特别是大集落形成率，较对照K562细胞显著增高，（P＜0．01），而无致瘤性或低致瘤性的K562－A、K562－D克隆株（致瘤率≤2／6），其集落形成率与对照无显著差别（P＞0．05）；超微结构观察提示高致瘤性克隆株，常染色质成分增多，异染色质少见，胞浆内微丝增多且排列紊乱；流式细胞仪细胞周期分布分析显示高致瘤性的克隆株S期细胞比例增加。实验结果还表明裸小鼠NK细胞对高致瘤性克隆株的杀伤活性显著低于对照的K562细胞（P＜0．01），而低致瘤性克隆株与对照无显著差别（P＞0．05）；组织病理观察显示低瘤性的克隆株及对照K562细胞，所成裸小鼠肿瘤组织内有大量的炎性细胞浸润，大量肿细胞被杀伤，血供丰富区尤甚，而高致瘤性克隆株所致肿瘤组织内炎性细胞浸润少见，肿瘤组织增殖旺盛，结论：K562－n细胞系的裸小鼠高致瘤机理在于：1．软琼脂集落形成率增高，2．细胞增殖和DNA合成活跃，涉及细胞周期调节因素和细胞骨架成分的改变，3．对裸小鼠NK细胞杀伤的耐受性增强，所接种宿主的免疫力受到抑制，形成肿瘤组织内炎性细胞浸润减少。     

In myelodysplastic syndromes progression to leukemia is directly related to PHA dependency for colony formation and independent of in vitro maturation capacity

In an agar-liquid double-layer colony assay in which myeloid leukemia colony-forming cells require the presence of both the lectin PHA and CSF for in vitro proliferation, colony formation of bone marrow cells derived from patients with a myelodysplastic syndrome (MDS) was studied. In five of 14 MDS and all five leukemic transformed MDS cases, colony formation was found to require both PHA and CSF. Three of these five PHA-dependent MDS cases progressed to overt leukemia within 1 year, one progressed from RA to RAEB, and one patient received AML chemotherapy. PHA-dependent colony formation was associated with higher bone marrow blast counts, but not directly to FAB type or cytogenetic abnormalities. In nine other MDS cases only CSF was required for colony formation. In these PHA-independent cases the course of the disease was stable during the observation time (5-17 months). Two types of colonies were observed in this in vitro system: colonies adherent and colonies nonadherent to the agar underlayer. The former consisted of terminally differentiated myeloid cells, and the latter comprised immature cells. This suggests that the percentage of adherent colonies formed in vitro may be used as a measure for the maturation defect in MDS. However, no correlation was found between the percentage of adherent colonies and progression to leukemia of the MDS cases. Our findings suggest that the dependency on PHA for in vitro colony formation of colony-forming cells in MDS is predictive for the progression to leukemia. However, the in vitro differentiation capacity has no apparent prognostic significance.     

Characterization of clones of tumorigenic feline cells transformed by a chemical carcinogen

Tumorigenic clonal lines derived from soft agar colonies induced by DMBA-transformed feline embryo cells were isolated and characterized. The morphologically altered clonal cells formed large aggregates, growing in this aggregate form when suspended in liquid growth medium above an agar base and forming colonies in soft agar with high efficiency. When inoculated into athymic nude mice, chemically altered clonal cells produced progressively growing sarcomas. Cells established from the tumors morphologically resembled the DMBA-transformed feline embryo cells and were characterized as cat cells by karyological analysis. The tumorigenic lines were negative for feline oncornavirus-associated cell membrane antigen (FOCMA), and for "gag-X" the transformation-related polyprotein which is encoded by the replication defective feline sarcoma virus.     

Confluent BALB/c 3T3 cells monolayer provides selective and efficient growth of neoplastic cells

In an attempt to further characterize non-irradiated contact-inhibited confluent monolayer of BALB/c 3T3 cells (= Contact-Sensitive Plates; CSP) as substrata for in vitro drug sensitivity testing, we compared the efficiency of colony formation with panels of cell lines on CSP with that on plastic dishes or in agar. Tumorigenicity in athymic nude mice was also examined. We found that: (1) HeLa cells, 2 esophageal cancer lines, rat 3Y1 fibroblasts transformed by either adenovirus type 12, mouse polyoma virus, Rous avian sarcoma virus, or plasmid DNA carrying v-Ha-ras oncogene all formed colonies on CSP and in agar and at the same time was tumorigenic. The efficiency of colony formation on CSP proved always to be higher than that in agar. (2) None of the 4 "normal" fibroplastic cell lines formed colonies on CSP or in agar and were tumorigenic. (3) Simian virus 40 and adenovirus E1A gene transformed rat 3Y1 fibroblasts formed colonies on CSP but not in agar, and were not tumorigenic. Therefore, CSP was found to provide selective and efficient growth of neoplastic cells when compared to other substrata and is also helpful in detecting incompletely transformed cells.     

Multistage progression of Abelson virus-infected murine pre-B-cells to the tumorigenic state

The tumorigenic potential of pre-B-cells at different stages of Abelson murine leukemia virus-induced transformation was determined. Cell lines with low growth potential in liquid culture were found (a) to have a dose-dependent growth requirement for conditioned medium obtained from bone marrow cultures, (b) to have low colony-forming ability in semisolid medium in the absence of conditioned medium, and (c) to be nontumorigenic when inoculated into syngeneic mice. Culture of the factor-dependent cells in vitro leads to the emergence of factor-independent variants, which eventually dominate the population by overgrowth. Cell lines that acquired a factor-independent phenotype were able to form colonies in semisolid medium and form tumors when inoculated into syngeneic mice. These results suggest that Abelson murine leukemia virus is sufficient to initiate transformation in the infected cell but that an additional genetic alteration is needed to confer tumorigenicity.     

Potentiation of erythroid colony formation in fetal liver cultures containing serum from patients with hemochromatosis or acute myeloid leukemia.

Studies were made on the capacity of 726 serum or plasma samples from normal donors or patients with various diseases to support erythroid colony formation in agar cultures of mouse fetal liver cells stimulated by spleen conditioned medium. Overall, 36% of samples supported the growth of well hemoglobinized erythroid colonies. Sera from patients with hemochromatosis or acute myeloid leukemia in relapse exhibited an exceptional capacity to support erythroid colony formation. Serum activity did not correlate with hemoglobin levels, blood group or mouse red cell hemagglutinin titers but did correlate with a capacity to support neutrophil-macrophage colony formation in the same cultures.     

PHA-induced colony formation in acute non-lymphocytic and chronic myeloid leukemia

A method which has previously been introduced for growing colonies in vitro from T-lymphocytic cells in normal subjects, has been applied to studying the growth of colonies from the bone marrow and blood of patients with acute non-lymphocytic leukemia and chronic myeloid leukemia. The technique consists of an underlayer of agar containing irradiated leukocytes and a liquid overlayer containing PHA. In leukemic patients, the assay permitted the formation of T-lymphocytic as well as leukemic colonies. Removal of the E-rosette forming cells before culture resulted in the appearance of mainly leukemic colonies. Cytogenetic analysis demonstrated the presence of the acquired karyotypic changes characteristic of the leukemic cells of these patients in the colony cells. Study of eight cases of acute non-lymphocytic leukemia, four cases of blast crisis of CML, and seven cases of CML chronic phase revealed the assay to be efficient in growing large numbers of leukemic blast colonies, as compared to the Robinson culture assay where large colony formation is only found in chronic leukemia [10]. Removal of the progenitors of lymphocytic colonies was sometimes incomplete in acute leukemia but not in CML.     

Characterization of hemopoietic precursor cells in juvenile-type chronic myelocytic leukemia

In order to study the pathogenesis of juvenile-type chronic myelocytic leukemia (CML), we examined the colony-forming capacity and colony composition in the bone marrow (BM) and peripheral blood (PB) of three children with juvenile-type CML. Large numbers of granulocytes and macrophage colonies were formed by BM and PB cells. Whole agar culture staining revealed that especially macrophage colonies increased in comparison with normal controls.After removal of carbonyl iron-laden cells with a magnet or deprivation of cells adherent to glass from BM cells, the number of macrophage colonies markedly reduced in comparison with the number of colonies formed by untreated BM cells, suggesting that some of the macrophage colony-forming cells (M-CFC) may have phagocytic and/or adherent activity. Radiation sensitivity and thymidine suicide rate of these M-CFC were not different from those of granulocyte colony-forming cells (G-CFC).The predominance of M-CFC in juvenile-type CML may be one of the reflections of fetal-type myelopoiesis since M-CFC are predominant in cord blood and PB in the neonatal period. Moreover, considerable numbers of erythroid-colony-forming units (CFU-E) were present in PB of all patients. It may be concluded that juvenile-type CML is a panmyelopathy with the predominance of M-CFC.     

Qualitative changes in the biologic characteristics of cultured fetal rat keratinizing epidermal cells during the process of malignant transformation after benzo[a]pyrene treatment.

In vitro malignant transformation of fetal rat keratinizing epidermal cells from inbred SD rats after benzo[a]pyrene (BP) treatment was analyzed from various biologic viewpoints. BP treatment directly and indirectly effected changes in cell growth characteristics, i.e., temperature dependence for growth, in vitro keratinization, chromosome structure, and the ability to form colonies on plastic substrate, on 0.57% agar medium layer, and in 0.33% soft agar medium. BP-treated cells at 30 degrees C remained in the premalignant stages and showed shifts in chromosome structure toward the hypodiploid range and parakeratotic changes in their keratinization process. However, the cells failed to form colonies even on a plastic substrate. BP-treated cell lines that adapted to temperatures of 35 and 37.5 degrees C remained in the premalignant stages; however, they acquired the ability to form colonies on plastic substrates during subcultivation. Malignantly transformed colonies appeared in these cell lines. In vitro keratinization processes were classified into nearly normal (diffuse lamellar, focal lamellar, and parakeratotic), intermediate, and atypical subtypes (columnar, spherical, and single-cell type). Cells of atypical keratinization subtypes and some of the intermediate subtypes formed squamous cell carcinomas in syngeneic hosts. Malignantly transformed cells showed shifts in chromosome structure toward the hypotetraploid range and colony formation on the 0.57% agar medium layer. However, they failed to form colonies in 0.33% soft agar medium. With the use of changes in biologic characteristics of the cells as indicators, fetal rat keratinizing epidermal cells in culture were classified into five stages. The appearance of stage III cells seemed to be the first key step in their malignant transformation.     

The E5 gene from human papillomavirus type 16 is an oncogene which enhances growth factor-mediated signal transduction to the nucleus.

Although human papillomavirus type 16 (HPV-16) is believed to be a major etiological agent in the development of cervical cancer, the biological function of several of its early genes remains to be established. In the present study, we have defined some of the biological properties of the E5 gene from HPV-16. Expression of the HPV-16 E5 gene in 3T3-A31 cells induced transformation to anchorage-independent growth (colony formation in soft agar). Addition of epidermal growth factor (EGF) to the soft-agar medium caused the E5-expressing cells to form larger colonies than those formed in the absence of EGF. Parental 3T3-A31 cells did not form colonies in soft agar either in the presence or in the absence of EGF. Analysis of clones expressing high levels of E5 mRNA revealed that these cells also expressed higher levels of c-fos mRNA in response to serum, EGF and platelet-derived growth factor (PDGF) than did the parental 3T3-A31 cells. Cells expressing the E5 gene were also capable of accelerated growth in low serum and were more tumorigenic in nude mice than were control cells. We conclude that the E5 gene from HPV-16 is an oncogene which transforms cells in part through enhancing signal transduction from growth factors to the nucleus.     

Colony stimulating factor-1 induced growth stimulation of v-fms transformed fibroblasts

The v-fms oncogene, which is capable of transforming fibroblasts, was derived by recombination of a feline leukemia virus with a cellular gene (c-fms) that encodes the receptor for colony stimulating factor 1 (CSF-1). We examined the capacity of recombinant human CSF-1 (produced in a yeast expression system) to stimulate the growth of v-fms transformed rat fibroblasts. Recombinant human CSF-1 bound to v-fms transformed fibroblasts with high affinity (apparent Kd = 6.0 x 10(-10) M); only non-specific binding was observed on control cells. The number of colonies formed in soft agar by v-fms transformed cells was increased by CSF-1 treatment in a dose-dependent manner; a nine fold increase in the number of colonies was seen in the presence of 10(-8) M CSF-1. CSF-1 did not stimulate the growth of either non-transformed cell lines, a non-transformed cell line that expresses a mutated v-fms protein on the cell surface, or cells transformed by the v-fgr oncogene. The growth stimulating effect of CSF-1 on v-fms transformed cells was also seen in monolayer culture. The v-fms transformed cells treated with CSF-1 had a more refractile, rounded morphology than non-treated cells; no morphology change was observed in CSF-1 treated control cells. CSF-1 treatment also increased both the number and size of foci that arose from fibroblasts following transfection with the v-fms oncogene. These data show that the altered CSF-1 receptor encoded by the v-fms oncogene retains a capacity to bind, and be stimulated by, human CSF-1.     

Clonal suppression of HL60 and U937 cells by recombinant human leukemia inhibitory factor in combination with GM-CSF or G-CSF

The in vitro actions of recombinant human leukemia inhibitory factor (LIF) were studied on the human leukemia cell lines HL60 and U937. Parameters analyzed were the suppression of stem cell generation using sequential clonal cultures, alterations of surface antigen expression, and morphological changes. When acting alone, LIF had no observable effects on the number, size, or morphology of colonies formed by HL60 or U937 cells, surface phenotype expression, or recloning capacity of cells of either line. In combination with GM-CSF and G-CSF, however, LIF significantly reduced the number of colonies formed in agar respectively by HL60 and U937 cells. GM-CSF alone greatly reduced the clonogenicity of U937 cells. Using sequential recloning, marked suppression of clonogenicity was observed using combinations of LIF with GM-CSF in HL60 cultures and with G-CSF in U937 cultures. These results suggest that human LIF may have some capacity to suppress human leukemia cells with loss of clonogenicity, at least in combination with G-CSF or GM-CSF.     

The transformation of primary and established mouse mammary epithelial cells by p21-ras is concentration dependent

We constructed a retroviral vector (pZSR) which is capable of simultaneously expressing the neomycin resistance gene and the viral ras oncogene. Primary mammary gland epithelial cells were prepared from mid-pregnant mice and infected with this virus. Cell lines with epithelial cell characteristics could be established with a low frequency. High expression of p21 v-ras was observed in these cells. They are tumorigenic and form soft agar colonies dependent on the presence of EGF and insulin in the growth medium but progress to hormone independent growth at higher passage numbers. A cloned cell line of non-tumorigenic, established mammary epithelial cells (NOG8) was also infected with the v-ras expressing virus. Individual cell clones expressing increasing amounts of p21 v-ras were selected. The level of p21 v-ras expression directly influences the morphology of the epithelial cells in culture, determines their cloning efficiency in soft agar and their tumorigenicity.     

Survival of human cells in the aggregate form: potential index of in vitro cell transformation.

The ability of cell populations to survive in the aggregate form was compared to colony formation in soft agar and tumorigenicity in nude mice. Nontumorigenic human osteogenic sarcoma (HOS) cells, which formed colonies in soft agar, could not survive in the aggregate form. Tumorigenic HOS cell lines, which also formed colonies in soft agar, survived and proliferated in the aggregate form. Other cell types were tested with the same results. This approach, based on cell survival in the aggregate form, may provide an additional, reliable method for predicting the tumorigenic status of a cell population.