Clonal Growth of Leukaemic Cells In Vitro

Human leukaemic cell specimens were obtained from patients and directly plated into soft agar (t= 0) or cultured for 1 week in liquid phase and then plated in soft agar. Growth for 1 week in liquid phase allowed the clonal growth in agar of leukaemic specimens which were unable to clone at t= 0. Clonal growth after liquid culture consisted of the usual leukaemic type of cluster-colonies, growth of a new type of ‘syncytial’ cell colony or a mixture of colony types. In addition, marrow from a patient with acute lymphocytic leukaemia produced normal-appearing colonies after 1 week of growth in liquid phase. These studies suggest a similarity in the growth requirements of some leukaemic cells and normal CFUd cells.     

反义RNA对人胃癌细胞生长抑制及恶性表型的阻断作用

ｒａｓ原癌基因的点突变是人胃癌发生发展的重要机理之一。利用能表达ｃ－Ｈ－ｒａｓ癌基因反义ＲＮＡ的质粒,导入人胃癌细胞系ＢＧＣ－８２３,研究了ｒａｓ癌基因反义ＲＮＡ对人胃癌细胞生长及恶性表型的作用,结果表明,ｃ－Ｈ－ｒａｓ反义ＲＮＡ可引起ＢＧＣ－８２３生长速率及形态的变化,在半固体培养基中细胞集落形成能力减弱,部分地抑制了ＢＧＣ－８２３在裸鼠体内的致瘤性。ｃ－Ｈ－ｒａｓ反义ＲＮＡ对其ＲＮＡ的过量表达呈特异性抑制作用。     

Normal mouse serum contains peptides which induce fibroblasts to grow in soft agar

The untransformed mouse fibroblast cells NIH/3T3, C3H/10T1/2, and rat NRK cells do not grow in soft agar in medium supplemented with 10% fetal calf serum. When fetal calf serum in the growth medium was supplemented with less than 1% of sera from mice or other vertebrates, however, these cells responded, forming large colonies. The morphology of soft agar colonies was a function of the treated cell type. In the presence of 10% serum from C57BL/6 mice, NRK cells grew to smooth-surfaced spherical colonies, while NIH/3T3 colonies showed individual round cells on their surface and C3H/10T1/2 cells grew as extended cells forming columns of end to end connected fibroblasts. Mus Musculus Castaneus-Epithelial (MMC- E) cells were not stimulated to grow in soft agar under these conditions. The major fibroblast colony-inducing factor (F-CIF) was partially purified from mouse serum by acid/ethanol-extraction, gel permeation chromatography, and reverse-phase high-pressure liquid chromatography. F-CIF is a polypeptide, which does not compete for binding to epidermal growth factor (EGF) receptors, but stimulates normal fibroblasts to form small colonies in semisolid medium and very large colonies in the presence of added EGF (2 ng/ml). In contrast to unfractionated mouse serum, purified F-CIF did not induce C3H/10T1/2 cells to grow in soft agar, suggesting that serum contains additional cell type-specific agar growth-stimulating activities.     

A T lymphoid cell line responds to a thymic stromal cell line by expression of Thy-1 and CD4

We have cloned both T lymphoid and stromal lines from a single murine thymic tumor that was induced by a retrovirus carrying the v-myc oncogene (M-MuLV(myc]. The T lymphoid line, L4, was cloned by growth in agar. L4 cells were initially negative for Thy-1.2 and CD4 (although they contained rearranged TCR-beta genes), and they remained so if passaged in medium alone. However, cocultivation of these Thy-1.2- CD4- cells with the cloned stromal cell line, St3, resulted in sequential expression of Thy-1.2 and CD4 in subpopulations of cells. Thy-1.2+ CD4- and Thy-1.2+ CD4+ L4 subclones were obtained from the cocultures by subsequent cloning in agar. Derivation of these subclones from the starting Thy-1.2- CD4- clone was verified by Southern blot analyses specific for TCR-beta gene rearrangements and for M-MuLV(myc) proviral integration sites. Continuous cocultivation of Thy-1.2+ CD4+ L4 subclones with the St3 stromal cells was necessary for maintenance of CD4 on the cell surface. Furthermore, CD4 expression which was lost when CD4+ L4 cells were removed from the stroma could be reinduced if they were again cultured on St3 stroma. These cells may provide a model system for studying thymocyte-stromal cell interactions in induction and maintenance of expression of Thy-1 and CD4 molecules.     

A Subclone of HuH-7 with Enhanced Intracellular Hepatitis C Virus Production and Evasion of Virus Related-Cell Cycle Arrest

Hepatitis C virus (HCV) cell culture system with JFH-1 strain and HuH-7 cells enabled us to produce infectious HCV particles in vitro, and such system is useful to explore the anti-HCV compounds and to develop the vaccine against HCV. In the present study, we describe the derivation of a cell line that permits improved production of HCV particles. Specifically, we characterized several subclones that were isolated from the original HuH-7 cell line by limiting dilution. These HuH-7 subclones displayed a notable range of HCV production levels following transfection by full-genome JFH-1 RNA. Among these subclones, HuH-7T1 produced HCV more efficiently than other subclones and Huh-7.5.1 that is known to be highly permissive for HCV replication. Upon transfection with full-genome RNA, HCV production was increased ten-fold in HuH-7T1 compared to Huh-7.5.1. This increase in viral production correlated with increased efficiency of intracellular infectious virus production. Furthermore, HCV replication did not induce cell cycle arrest in HuH-7T1, whereas it did in Huh-7.5.1. Consequently, the use of HuH-7T1 as host cells could provide increased population of HCV-positive cells and elevated viral titer. In conclusion, we isolated a HuH-7 subclone, HuH-7T1, that supports efficient HCV production. High efficiency of intracellular infectious virus production and evasion of cell cycle arrest were important for this phenotype. We expect that the use of this cell line will facilitate analysis of the underlying mechanisms for HCV particle assembly and the cell cycle arrest caused by HCV.     

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen.

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into a retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of psi 2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10(6) cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.     

Proliferation and differentiation of Abelson virus-infected murine myeloid leukemia cell lines does not involve an autocrine mechanism

Culture in agar of cloned promonocytic leukemia cell lines derived from Abelson virus-infected mice produced colonies of both a compact and diffuse morphology. Diffuse colonies contained fewer cells capable of forming colonies when recultured in agar than did compact colonies. Serial subcloning of cells from diffuse, but not compact, colonies ultimately led to the complete loss of colony-forming cells, i.e., to clonal extinction. The production of both compact and diffuse agar colonies was independent of the cell density of either the static liquid culture from which cells were taken for culture in agar, or the number of cells per agar culture. Furthermore, bioassays of culture supernatants indicated the leukemia cells did not secrete hemopoietic growth factors active on normal hemopoietic cells, transforming growth factors active on adherent cell lines, or factors that influenced the growth of the leukemic cells themselves. Collectively, these data suggest neither growth-factor independent replication nor the spontaneous differentiation of Abelson virus-infected myeloid cells involves autocrine secretion of growth regulators.     

Fibronectin Fibrils and Growth Factors Stimulate Anchorage-Independent Growth of a Murine Mammary Carcinoma

Stromal cells are important regulators of mammary carcinoma growth and metastasis. We have previously shown that a 3T3-L1 adipocyte cell line secretes hepatocyte growth factor (HGF), which stimulates proliferation of a murine mammary carcinoma (SP1) in monolayer cultures (DNA Cell Biol.13, 1189–1897, 1994). We now examine the role of growth factors and the extracellular matrix protein fibronectin in stimulation of anchorage-independent growth of SP1 cells. Purified transforming growth factor-β (TGF-β) stimulated significant colony growth in soft agar cultures, whereas HGF had a lesser effect. Analysis by confocal microscopy revealed that carcinoma cell colonies contained extracellular microfibrils composed of fibronectin. Partial depletion of fibronectin from 7% FBS/agar cultures reduced the number of colonies; colony growth could be recovered by adding back exogenous fibronectin. Addition of the 70-kDa N-terminal fragment of fibronectin, which inhibits fibronectin fibril formation, reduced growth of SP1 cell colonies, but an 85-kDa fragment containing the cell binding domain did not inhibit colony growth. These findings indicate that deposition of extracellular fibronectin fibrils is necessary, but not sufficient, for anchorage-independent growth of SP1 mammary carcinoma cells; growth factors are also required. SP1 cells had less fibronectin mRNA and secreted less fibronectin protein under anchorage-independent conditions than under anchorage-dependent conditions, as determined by Northern blotting and immunoprecipitation analysis. Thus, both growth factors (HGF and TGF-β) and fibronectin may be important regulators of paracrine stimulation by stromal cells of anchorage-independent growth of mammary carcinoma cells.     

Clonal variation in colony morphology and growth of CHO cells cultured on agar

Single Chinese hamster ovary (CHO) cells plated on agar form macroscopic colonies with high efficiency. Colonies produced by cells from the uncloned cell line increase in diameter continuously for 10–12 days after plating to form mounds of cells about 1 mm in diameter. With further incubation, some of these colonies do not increase in diameter (arrested dome), some form an expanding annular monolayer of cells around the central mound (fried egg), and some grow by enlarging the central mound into a low multilayered disc (saucer).These colony types on agar appear to be clonal characteristics of the CHO cell line. Cloning the line gives two kinds of isolates: one forms a mixture of arrested dome and fried egg colonies in an inheritable ratio, and the other forms saucer colonies. Cells from saucer colonies form saucer colonies when replated on agar. Cells from all colony types replate with similar efficiency on plastic or agar, and exhibit the same growth rate and cell size in liquid suspension culture. On plastic substrate, all these CHO cells form colonies which increase continuously in diameter for as long as 21 days, and little clonal difference in the morphology of colonies or of single cells is observed.These observations reveal a previously unsuspected heterogenieity in an established line of cultured mammalian cells and provide a method for studying new classes of In vitro growth control phenomena. These control phenomena may help in the building an in vitro model for tumor growth.     

Antigenic and functional characterization of a rat central nervous system-derived cell line immortalized by a retroviral vector

We have immortalized rat central nervous system (CNS) cells of primary cultures of rat optic nerve with murine leukemia virus psi-2,SV-40-6, which is defective in assembly and contains the SV-40 large T antigen and neomycin resistance genes, to produce a cell line that we named A7. After drug selection, greater than 90% of the growing cells expressed nuclear SV-40 large T cells and a fraction of these contained the astrocyte-specific marker, glial fibrillary acidic protein. The majority of these cells also expressed surface marker A4 (specific for neural tube derivatives), Ran 2, p185 (the 185-kD phosphoprotein product of the neu oncogene), and fibronectin, but did not express the astrocyte enzymes glutamine synthetase and monoamine oxidase B. Surface markers characteristic of glial progenitors (A2B5) and oligodendrocytes (galactocerebroside) were not detected. After two rounds of cell cloning, subclone A7.6-3 expressed Ran 2, fibronectin, and the neural cell adhesion molecule (N-CAM) but not glial fibrillary acidic protein and A4. The A7 cell line and subclones also displayed certain functions of type 1 astrocytes: the conditioned medium of these cells had a potent mitogenic activity for glial progenitor cells which could be neutralized by anti-platelet-derived growth factor antibodies and monolayers of these cells supported the growth of embryonic hypothalamic neurons. We conclude that a retrovirus containing SV-40 large T antigen can immortalize rat CNS cells and that such immortalized glial cells retain at least two important functions of type 1 astrocytes: the ability to secrete platelet-derived growth factor and to support the growth of embryonic CNS neurons. Moreover, such stable immortalized clonal cell lines can be used to study gene regulation in glial cells.     

Intraclonal diversity of fibrosarcoma cells for the production of macrophage colony-stimulating factor and granulocyte colony-stimulating factor

A new cell line was established from fibrosarcoma that had spontaneously developed in a mouse. The cells were maintained growing in culture for two years and constantly produced both macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF). Cloning of the cells by anchorage-independent colony formation gave subclones showing the activity of producing M-CSF and G-CSF in different proportions, whereas no subclone produced G-CSF without producing M-CSF simultaneously. Recloning of the bipotential subclones again gave clonal derivatives producing two types of CSF in various proportions. The observed heterogeneity of the cloned cells seems to be an epigenetic phenomenon, because the cells resumed the G-CSF producing activity in the absence of cell proliferation. After equilibrium was achieved, all of the subclones produced both M-CSF and G-CSF nearly in equal proportions. Tumorigenic and leukocytosis-inducing activity of the cloned cells was nearly comparable with the activity of the original tumor cells.     

Anti-sense trefoil factor family-3 (intestinal trefoil factor) inhibits cell growth and induces chemosensitivity to adriamycin in human gastric cancer cells

Intestinal trefoil factor (ITF), which is normally absent in gastric mucosa, is over-expressed in gastric cancer. However, the functional significance of ITF in gastric cancer is unknown. We examined the effects of blocking ITF expression on the growth of gastric cancer cells and their responses to chemotherapeutic agents. Anti-sense ITF cDNA was cloned into mammalian expression vector pcDNA3 and was transfected into an ITF-expressing gastric cancer cell line SNU-1. We assessed the doubling time and anchorage dependent growth of the transfected cells using growth curve and soft agar assay respectively. Cell cycle analysis and apoptosis were determined by flow cytometry and cell death ELISA. The response to chemotherapeutic agents after transfecting anti-sense ITF was also examined. Anti-sense ITF transfectant (3A-5) had a significantly longer doubling time as compared to control cells which were transfected with empty vector (32.4 hr vs 26.9 hr, p < 0.05). In the soft agar assay, 3A-5 formed fewer colonies than control (3.5 colonies vs 23.5 colonies, p < 0.05). Although there was no significant difference in the cell cycle distribution between 3A-5 and control, anti-sense ITF resulted in marked increase in adriamycin-induced apoptosis. Our results demonstrated that blocking the expression of ITF inhibits growth of gastric cancer cells and enhances the response to chemotherapy.     

The purification of an acid- and heat-labile transforming growth factor from an avian sarcoma virus-transformed rat cell line

A new class of transforming growth factor (TGF), with chemical characteristics differing from previously reported TGFs, was isolated and purified from an avian sarcoma virus-transformed rat cell line, 77N1 . Purification steps were simple and consisted of ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, ammonium sulfate precipitation, Chromatofocusing, and DEAE-Sephadex A-25 chromatography. The purified TGF is a heat- and acid-labile protein with a molecular mass of 12,000 daltons and isoelectric point of 5.2-5.4. Because of the acid lability of this TGF, purification was carried out at neutral pH. The TGF induced DNA synthesis in growth-arrested BALB 3T3 cells and promoted anchorage-independent growth of nontransformed BALB 3T3 cells in soft agar; the latter activity is specific for the peptide growth factors, called TGFs, but it did not compete with epidermal growth factor (EGF) for binding to the EGF membrane receptors. The TGF activity was not potentiated by EGF. The purified preparation of the TGF stimulated BALB 3T3 cells to grow progressively in soft agar at a dose of 20 ng/ml.     

Sialylation and sulfation of lactosylceramide distinctly regulate anchorage-independent growth,apoptosis, and gene expression in 3LL Lewis lung carcinoma cells

To investigate the significance of sialylation and sulfation of lactosylceramide in transformed cells, we established ganglioside GM3- and lactosylsulfatide (SM3)-reconstituted cells by transfecting cDNAs of GM3 synthase and cerebroside sulfotransferase into the J5 subclone of 3LL Lewis lung carcinoma cells. The J5 clone was selected for the transfection of these genes because it lacks GM3 and SM3 but accumulates lactosylceramide. The anchorage-dependent growth of both GM3- and SM3-reconstituted cells was similar. However, anchorage-independent growth (as measured by colony-forming ability in soft agar) of the SM3- reconstituted cells was almost completely lost, which supports our previous observation showing the suppression of tumorigenic potential in vivo and beta1 integrin gene expression induced by the introduction of cerebroside sulfotransferase gene (Kabayama et al. [2001] J. Biol. Chem., 276, 26777-26783). The GM3-reconstituted cells formed a significantly higher number of colonies in soft agar compared to mock-transfected cells and began to proliferate and become resistant to apoptosis when serum was depleted, indicating that endogenous GM3 is essential for maintaining these fundamental properties of malignant cells. We also found that serum-induced ERK1/2 activation was suppressed in the GM3-reconstituted cells, suggesting that anchorage-independent cell cycle initiation by endogenous GM3 is elicited through pathway(s) independent of ERK1/2 activation. The selective down-regulation of platelet-derived growth factor (PDGF)-dependent ERK1/2 activation in the GM3-reconstituted cells was due to the substantial decreases of PDGF alpha receptor mRNA and protein, but in the SM3-reconstituted cells PDGF alpha receptor expression was similar to mock cells. Thus, endogenously produced GM3 and SM3 differentially and distinctly regulate tumor-progression ability, that is, GM3 leads the transformed phenotype of J5 cells to promotion and SM3 to abrogation.     

Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells

The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF)β-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGFβ-like activities at an apparent molecular weight of 6–10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal expiants. The relation between this activity and a recently described TGFβ-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.     

Tumor promoters and epidermal growth factor stimulate anchorage-independent growth of adenovirus-tranformed rat embryo cells.

Previous studies indicated that the potent tumor promoter 12--0--tetradecanoyl-phorbol-13-acetate (TPA) enhances transformation of rat embryo cells (2 degrees RE) by a mutant of human Ad5 (H5ts125). This study examines the effect of TPA, its structural analogs and epidermal growth factor (EGF) on anchorage-independent growth of a cloned population of H5ts125-transformed 2 degrees RE cells (clone E11). Both TPA and EGF (approximately 10(-8) M) induced a 3--5 fold increase in agar cloning efficiency of E11 cells. In addition, macroscopic colonies appeared earlier and were larger and more diffuse. The TPA analogs phorbol--12,13--didecanoate (PDD) and ingenol--3,20--dibenzoate also enhanced growth in agar of E11 cells, whereas phorbol, 4 alpha PDD and 4--0--meTPA, which are inactive as tumor promoters, failed to enhance agar growth. In contrast to the results obtained with E11 cells, TPA, PDD or ingenol--3,20--bidenzoate failed to induce growth in agar of normal 2 degrees RE cells. Dexamethasone (10(-5)--10(-6) M), trans retinoic acid (10(-5)--10(-6) M) and the protease inhibitors leupeptin, antipain and elastatinol did not inhibit the ability of TPA to enhance the growth of E11 cells in agar. The TPA-enhanced anchorage independence was a stable property, since subclones of E11 cells isolated from TPA-agar plates had a higher agar cloning efficiency than the parental E11 cells when retested in the absence of TPA. This effect of TPA does not appear to reflect simple selection of a subpopulation of cells. When the parental E11 cells were first cloned in monolayer culture in the absence of TPA, all ten randomly picked clones showed enhanced growth in agar in the presence of TPA. In addition, prior growth of E11 cells in monolayer culture in the presence of TPA did not enhance their subsequent growth in agar. This system therefore provides an example in which TPA appears to enhance the acquisition of a stable cell property, and thus may be a useful model for studying mechanisms of tumor promotion and progression.     

Stability of the cytokinin requirement in Paul's scarlet rose cells in culture

Summary Cells ofRosa sp. cv Paul's scarlet have been reported to require cytokinin for growth in suspension culture. This report was verified in the present study. However, a rose cell line that was maintained for 2 yr in suspension culture by routine subculturing developed the capacity to grow without exogenous cytokinin. The stability of the cytokinin requirement and the basis for this altered response to cytokinin was investigated. The parental cell line, which has been maintained independently on agar-solidified medium, was subcloned and the cytokinin dependence of the subclones was determined. The subclones were found to exhibit a continuous spectrum of responses, ranging from a high degree of cytokinin dependence for growth to rapid growth upon the initial transfer to cytokinin-deficient medium. The average growth constant (K=1n W/Wo; Wo=initial fresh weight,W=fresh weight after growth for the stated time interval) of 30 subclones grown on medium containing 0.5 μM zeatin was 3.1, with a range of 1.1 to 4.0. The average growth constant of the same subclones when grown on medium lacking a cytokinin was 1.5, with a range of 0 to 3.9. By comparison, the parental cell line exhibited growth constants of 3.5 in the presence of 0.5 μM zeatin and 1.6 in the absence of exogenous cytokinin. Although the growth of some of the subclones after transfer to cytokinin-deficient medium suggested that they were cytokinin autotrophs, this was not the case because none of them grew after a second transfer to medium lacking cytokinin. Culture in medium containing cytokinin conferred upon the cells the capacity for a limited amount of growth after subsequent transfer to medium lacking cytokinin. The extent of this cytokinin-induced growth potential varied from subclone to subclone. Efforts to determine the frequency with which cytokinin autotrophs appeared in a subclone that required cytokinin suggested that it is a rare event and that the cytokinin requirement is a fairly stable phenotypic characteristic of these cells. This research was supported by Grant PCM 7722398 from the National Science Foundation.     

Possible activity of acidic fibroblast growth factor as a progression factor rather than a transforming factor.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are mitogens for mesoderm- and neuroectoderm-derived cells. The facts that FGF-related proteins are oncogenic and that FGFs are expressed in a variety of tumor cell lines or tumor tissues suggest the transforming activities of FGFs. To examine such an activity of aFGF, we introduced a human aFGF expression vector into two populations of Rat-1 cells; one was non-transformed (nRat-1), the other was partially-transformed (tRat-1). tRat-1 cells transfected with aFGF cDNA formed larger colonies in soft agar and produced larger and more malignant tumors in nude mice than those of parental cells. In contrast, nRat-1 cells transfected with aFGF cDNA neither formed colonies in soft agar nor produced tumors in nude mice. Our results suggest that high expression of aFGF may enhance a tumorigenic potential of Rat-1 cells rather than confer such a potential de novo.