Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes

1983 ◽  
Vol 3 (4) ◽  
pp. 596-604
Author(s):  
C A Whitlock ◽  
S F Ziegler ◽  
O N Witte
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Lymphoid Cells ◽  
Growth Potential ◽  
Malignant Growth ◽  
Wide Range ◽  
Feeder Layers ◽  
Growth Phenotype ◽  
Abelson Virus

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.

scholarly journals Progression of the transformed phenotype in clonal lines of Abelson virus-infected lymphocytes.

1983 ◽  
Vol 3 (4) ◽  
pp. 596-604 ◽  
Author(s):  
C A Whitlock ◽  
S F Ziegler ◽  
O N Witte
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Lymphoid Cells ◽  
Growth Potential ◽  
Malignant Growth ◽  
Wide Range ◽  
Feeder Layers ◽  
Growth Phenotype ◽  
Abelson Virus

Some molecular changes which correlate with the tumorigenic progression of neoplastic cells can best be studied with in vitro cell lines that represent each stage in the progression. Lymphoid cells infected by Abelson murine leukemia virus exhibit a wide range of growth potential in vitro and in vivo. Uncloned populations that are poorly oncogenic early after infection become progressively more oncogenic with successive passages of the cells in culture. In such mass cultures, it is difficult to evaluate whether a rare subpopulation of highly oncogenic cells becomes dominant in the culture or whether the individual cells progress in oncogenic phenotype. To examine this latter possibility, Abelson virus-infected lymphoid cells were cloned by limiting-dilution culture 10 days postinfection. We isolated two clones that grew poorly in agar, required feeder layers of adherent bone marrow cells for growth in liquid culture, and were extremely slow to form tumors in syngeneic animals. Both clones, after passage in the presence of adherent feeder layers for 3 months, grew well in liquid and agar-containing cultures in the absence of feeder layers and formed tumors in animals at a rapid rate. The progression of these clonal cell lines to a more malignant growth phenotype occurred in the absence of detectable changes in the concentration, half-life, phosphorylation, in vitro kinase activity, or cell localization of the Abelson virus-encoded transforming protein. No change in the concentration or arrangement of integrated Abelson viral DNA sequences was detected in either clone. Thus, perhaps changes in the expression of cellular genes would appear to alter the growth properties of lymphoid cells after their initial transformation by Abelson virus. Such cellular changes could complement the activity of the Abelson virus transforming protein in producing the fully malignant growth phenotype.

Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells

1986 ◽  
Vol 6 (9) ◽  
pp. 3221-3231
Author(s):  
R C Schwartz ◽  
L W Stanton ◽  
S C Riley ◽  
K B Marcu ◽  
O N Witte
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Retroviral Vectors ◽  
Soft Agar ◽  
Lymphoid Cells ◽  
Neoplastic Progression ◽  
Direct Role ◽  
Murine Bone Marrow ◽  
Myc Oncogene

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.

Rearrangement and diversification of immunoglobulin light-chain genes in lymphoid cells transformed by reticuloendotheliosis virus

1989 ◽  
Vol 9 (11) ◽  
pp. 4970-4976
Author(s):  
J Y Zhang ◽  
W Bargmann ◽  
H R Bose
Keyword(s):  
Cell Lines ◽  
Light Chain ◽  
Lymphoid Cells ◽  
Chain Gene ◽  
Gene Rearrangements ◽  
B Cell Differentiation ◽  
Immunoglobulin Light Chain ◽  
Light Chain Gene ◽  
Transformed Cell Lines

Avian lymphoid cells transformed by reticuloendotheliosis virus (REV-T) serve as a model to analyze the mechanism by which B-cell differentiation and antibody diversification occur in birds. Immunoglobulin light-chain gene rearrangements, diversification, and expression were analyzed in 72 independently derived REV-T-transformed cell lines. Lymphoid cells transformed as the result of expression of the v-rel oncogene were divided into two distinct groups based on light-chain gene rearrangements. The status of the light-chain gene loci in these REV-T-transformed cell lines was determined in part by the ages of the chickens whose spleen cells were transformed. In embryonic spleen cell lines transformed by the v-rel oncogene, rearrangements were not detected, even after prolonged culture in vitro, indicating that these cells are arrested in B-cell differentiation. REV-T transformants derived from spleens obtained from chickens 2 weeks old or older, however, had at least one light-chain allele rearranged. All of the cell lines analyzed which exhibited rearranged light-chain genes contained light-chain transcripts, and most of the REV-T-transformed cells which displayed light-chain rearrangements expressed immunoglobulin protein. REV-T, therefore, transforms B-lymphoid cells at phenotypically different stages of development. Many REV-T-transformed cells undergo immunoglobulin chain gene rearrangements during prolonged propagation in vitro. Most of the cell lines which rearrange their light-chain alleles also undergo diversification during cultivation in vitro. Light-chain diversification occurs during or after the rearrangement event.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals Quantitative analysis of tumour spheroid structure

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alexander P Browning ◽  
Jesse A Sharp ◽  
Ryan J Murphy ◽  
Gency Gunasingh ◽  
Brodie Lawson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tumour Microenvironment ◽  
Culture Conditions ◽  
Experimental Models ◽  
Seeding Density ◽  
Modelling Framework ◽  
Tumour Spheroid ◽  
Tumour Spheroids ◽  
Wide Range

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

scholarly journals IN VITRO CYTOTOXICITY AND ANTIOXIDANT EVALUATION OF BIOGENIC SYNTHESIZED GOLD NANOPARTICLES FROM MARSILEA QUADRIFOLIA ON LUNG AND OVARIAN CANCER CELLS

2018 ◽  
Vol 10 (5) ◽  
pp. 153 ◽  
Author(s):  
Balashanmugam P. ◽  
Mosa Christas K. ◽  
Kowsalya E.
Keyword(s):  
Gold Nanoparticles ◽  
Cell Lines ◽  
Aqueous Extract ◽  
A549 Cell ◽  
In Vitro Cytotoxicity ◽  
Spherical Particles ◽  
X Ray ◽  
Marsilea Quadrifolia ◽  
Wide Range

Objective: The biogenic gold nanoparticles are considered to be extremely impressive for its wide range of applications in pharmaceutics and therapeutics. The present study was aimed at the biogenic synthesis of gold nanoparticles (AuNPs) from Marsilea quadrifolia aqueous extract and to investigate its antioxidant property and cytotoxic effect on human ovarian teratocarcinoma (PA-1) and lung adenocarcinoma (A549) cell lines.Methods: The biogenic AuNPs was synthesized using an aqueous extract of Marsilea quadrifolia. The synthesized biogenic AuNPs were characterized by ultraviolet (UV) visible spectroscopy, transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDX) and X-ray diffraction (XRD). The biogenic AuNPs was assessed for its stability over a period of time and antioxidant activity. The cytotoxicity of biogenic AuNPs against PA-1 and A549 cell lines was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Results: The synthesized biogenic AuNPs showed peculiar ruby red color and a surface plasmon resonance (SPR) peak at 544 nm in the UV-Vis spectrum. The characterization of biogenic AuNPs by TEM, EDX and XRD revealed well dispersed spherical particles ranging from 10-40 nm and the presence of elemental gold and its crystalline nature, respectively. The AuNPs showed good stability and the scavenging activity at 50 μg/ml. The in vitro cytotoxicity of biogenic AuNPs against PA-1 and A549 cell lines recorded half maximal inhibitory concentration (IC50) of 45.88 μg/ml and 52.015 μg/ml, respectively.Conclusion: The biogenic AuNPs demonstrated superior antioxidant and antiproliferative activities against cancer cell lines.

FKLF-2: a novel Krüppel-like transcriptional factor that activates globin and other erythroid lineage genes

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3578-3584 ◽  
Author(s):  
Haruhiko Asano ◽  
Xi Susan Li ◽  
George Stamatoyannopoulos
Keyword(s):  
Cell Lines ◽  
Dna Sequences ◽  
Zinc Finger Protein ◽  
Globin Gene ◽  
Northern Blot Hybridization ◽  
Gene Promoters ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Wide Range ◽  
Carboxyl Terminal

Abstract FKLF-2, a novel Krüppel-type zinc finger protein, was cloned from murine yolk sac. The deduced polypeptide sequence of 289 amino acids has 3 contiguous zinc fingers at the near carboxyl-terminal end, an amino-terminal domain characterized by its high content of alanine and proline residues and a carboxyl-terminal domain rich in serine residues. By Northern blot hybridization, the human homologue of FKLF-2 is expressed in the bone marrow and striated muscles and not in 12 other human tissues analyzed. FKLF-2 is constitutively expressed in established cell lines with an erythroid phenotype, but it is inconsistently expressed in cell lines with myeloid or lymphoid phenotypes. The expression of FKLF-2 messenger RNA (mRNA) is up-regulated after induction of mouse erythroleukemia cells. In luciferase assays, FKLF-2 activates predominantly the γ, and to a lesser degree, the ɛ and β globin gene promoters. The activation of γ gene promoter does not depend on the presence of an HS2 enhancer. FKLF-2 activates the γ promoter predominantly by interacting with the γ CACCC box, and to a lesser degree through interaction with the TATA box or its surrounding DNA sequences. FKLF-2 also activated all the other erythroid specific promoters we tested (GATA-1, glycophorin B, ferrochelatase, porphobilinogen deaminase, and 5-aminolevulinate synthase). These results suggest that in addition to globin, FKLF-2 may be involved in activation of transcription of a wide range of genes in the cells of the erythroid lineage.

scholarly journals Expression of human adenosine deaminase in mice after transplantation of genetically-modified bone marrow

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1733-1741 ◽  
Author(s):  
M Kaleko ◽  
JV Garcia ◽  
WR Osborne ◽  
AD Miller
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
Dna Sequences ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Helper Virus ◽  
Lymphoid Tissues ◽  
Hematopoietic Stem ◽  
Wide Range

Abstract A high titer retroviral vector was used to transfer a human adenosine deaminase (h-ADA) cDNA into murine bone marrow cells in vitro. The h- ADA cDNA was linked to the retroviral promoter, and the vector also contained a neomycin phosphotransferase gene as a selectable marker. Infected marrow was transplanted into syngeneic W/Wv recipients, and h- ADA expression was monitored for 5.5 months. Several weeks after transplantation, h-ADA was detected in the erythrocytes of all nine recipients, eight of which expressed levels equal to the endogenous enzyme. This level of expression persisted in two of six surviving mice, while expression in three others stabilized at lower, but readily detectable, levels. Only one mouse had no detectable h-ADA after 5.5 months. Vector DNA sequences with common integration sites were found in hematopoietic and lymphoid tissues of the mice at 5.5 months, providing evidence that hematopoietic stem cells had been infected. Furthermore, all mice transplanted with marrow that had been selected in G418 before infusion had multiple vector copies per genome. While this category included the two highest h-ADA expressors, it also included the negative mouse. Thus, multiple copies of the vector were not sufficient to guarantee long-term h-ADA expression. Mice were monitored for “helper virus” infections with an assay designed to detect a wide range of replication-competent retroviruses, including those endogenous to the mouse genome. No helper virus was detected in the two highest h-ADA expressors, ruling out helper-assisted vector spread as a cause of the high h-ADA expression. These results help provide a foundation for the development of somatic gene therapy techniques to be used in the treatment of human disease.

The Cell Polarity PTK7 Receptor Is Expressed in Myeloid Cells and Acts as a Modulator of the Chemotherapeutic Response in AML Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1571-1571 ◽  
Author(s):  
Thomas Prebet ◽  
Anne Catherine Lhoumeau ◽  
Christine Arnoulet ◽  
Anais Aulas ◽  
Sylvie Marchetto ◽  
...  
Keyword(s):  
Cell Migration ◽  
Tyrosine Kinase ◽  
Cell Polarity ◽  
Cell Lines ◽  
Tyrosine Kinase Receptor ◽  
Epithelial Tissue ◽  
Relapse Free Survival ◽  
Free Survival ◽  
Wide Range

Abstract Abstract 1571 Poster Board I-596 The pseudo tyrosine kinase receptor 7 (PTK7) is an orphean tyrosine kinase receptor assigned to the planar cell polarity pathway (PCP). It has been recently described and plays a major role during embryogenesis and epithelial tissue organisation. To date there is no report in the litterature considering a potential implication in hematopoiesis. In silico and in vitro analysis found that PTK7 was also expressed in normal myeloid progenitors and CD34+ CD38- bone marrow cells in humans. Preliminary results from our team showed that PTK7 was also expressed in various leukemic cell lines such Jurkat, TF-1 or KG-1a. We decided to perform a wide range multicolour immunophenotyping screen on patients with acute myeloid leukemia (AML) at diagnosis and to investigate the role of PTK7 in AML in vitro. More than 250 patient samples were evaluated and we demonstrated that PTK7 was largely expressed in AML as 72% of the samples were PTK7 positive. Its expression mostly correlates with granulocytic lineage differentiation. PTK7 expression was associated with a lower WBC count at diagnosis and a lower frequency of extramedullary disease whatever was FAB subtype. Interestingly, PTK7 expression was associated with some cytogenetic subgroups including CBF-AML and APL. There was no correlation with molecular subgroups (i.e. FLT3-ITD/NPM1/CEBPA status). Overall Survival and Relapse Free Survival were evaluated in non-APL patients treated with induction chemo (n=182). Patients with PTK7 positive AML are more resistant to anthracycline-based frontline therapy with a significantly reduced Relapse Free Survival in a multivariate analysis model integrating all pre treatment variables (2 year probability of RFS= 29% vs 66% for PTK7 negative patients, p= 0.003). Forrest plot analysis showed that the negative impact of PTK7 expression was the most significant in intermediate cytogenetic risk subgroup and when PTK7 was aberrantly expressed in M4-M5 FAB subtypes. There was no demonstrated impact on CR. In cultured cells, expression of PTK7 promotes leukemia cell migration, cell survival and resistance to anthracyclin-induced apoptosis. There was no effect of PTK7 expression on cell proliferation in tritiated thymidine assay. In the absence of known inhibitor of PTK7, we produced a soluble recombinant PTK7-Fc protein that efficiently competes for PTK7 functions in cell migration and survival assays in cell lines and primary AML samples. These data were confirmed using a shRNA strategy. We conclude that PTK7 is a PCP component expressed in the myeloid progenitor compartment that conveys promigratory and anti-apoptotic signal to leukemia cells. Its use as a potential biomarker or therapeutical target should be investigated. Disclosures No relevant conflicts of interest to declare.

CD74 Expression by AML Cell Lines and Bone Marrow Specimens, and Augmented In Vitro Cytotoxicity of Anti-CD74 Antibody After Interferon-Gamma (IFN-γ) Treatment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2888-2888
Author(s):  
Abhinav B. Chandra ◽  
Jack Burton ◽  
Rhona Stein ◽  
Susan Chen ◽  
Nidhi Mishra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cellular Signaling ◽  
In Vitro Cytotoxicity ◽  
Cancer Type ◽  
Phosphorylated Akt ◽  
Wide Range ◽  
Ifn Γ

Abstract Abstract 2888 Background: CD74 (HLA-DR-associated invariant chain) is expressed alone or along with DR in a wide range of hematologic cancers and solid tumors. Humanized anti-CD74 mAb, milatuzumab (Immunomedics, Morris Plains, NJ), exhibits direct cytotoxicity for NHL, CLL and MM cell lines, and is undergoing clinical evaluation for treatment of these malignancies. CD74 is upregulated by interferons in hematologic and epithelial cancer cell lines. Here we present the results of our analysis of CD74 expression and function in AML, and the effect of CD74 upregulation by treatment with IFN-γ on the cytotoxicity of milatuzumab for AML cell lines. Methods: CD74 expression in bone marrow biopsy (BMB) specimens from non-M3 AML patients was evaluated by immunohistochemistry and, for the 3 human AML cell lines, by flow cytometry, with/without permeabilization and with/without IFN-γ (40 and 200 U/mL). These cell lines were also tested in proliferation assays for responses to milatuzumab, with/without IFN-γ. Also, assessment of apoptosis and cellular signaling was performed. Results: In the initial group of AML cases, 13/14 BMB specimens showed moderate to strong CD74 expression by leukemic blasts, which was mostly intracellular, usually with a perinuclear distribution. Three AML cell lines also showed moderate to strong expression of CD74, which was mostly intracellular. Without IFN-γ, surface expression of CD74 was present, but IFN-γ treatment of these 3 lines resulted in upregulation of surface CD74 by 69–117%. Much higher levels of intracellular CD74 were observed in all 3 lines (with and without IFN-γ), with IFN-γ-induced upregulation of intracellular CD74 in all 3 lines (from 85%-868%; P<0.001). In 2/3 lines, IFN-γ increased milatuzumab-mediated growth inhibition (23.7 to 44.8% and -3.9 to 30.9%, P=0.01 and P<0.05, respectively). Cytotoxicity was in part due to apoptosis, as significant increases in Annexin V binding (P=0.01) were observed after treatment with IFN-γ plus milatuzumab. Initial experiments addressing cellular signaling suggest a role for AKT, because phosphorylated AKT levels increased (P=0.06) in response to IFN-γ + milatuzumab. Conclusions: CD74 is expressed in AML patient specimens and in AML cell lines, with the majority of CD74 expression found intracellularly. Cell surface and cytoplasmic expression of CD74 were upregulated in AML lines after IFN-γ exposure. This increased expression resulted in increased cytotoxicity of the anti-CD74 mAb, milatuzumab, in 2/3 AML lines. This effect was through apoptosis and involved the AKT pathway. Thus, AML is another cancer type where combined IFN-γ and milatuzumab treatment may be useful. Supported in part by NIH grant PO1-CA103985 (DMG). Disclosures: No relevant conflicts of interest to declare.

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