The c-myc oncogene is regulated independently of differentiation in myeloid cell lines

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.

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Deregulation of c-myc gene expression in human colon carcinoma is not accompanied by amplification or rearrangement of the gene

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.1969-1976.1985 ◽

1985 ◽

Vol 5 (8) ◽

pp. 1969-1976

Author(s):

M D Erisman ◽

P G Rothberg ◽

R E Diehl ◽

C C Morse ◽

J M Spandorfer ◽

...

Keyword(s):

Colon Carcinoma ◽

Cell Lines ◽

Blot Hybridization ◽

Human Colon ◽

Normal Colonic Mucosa ◽

Dna Rearrangement ◽

Dot Blot ◽

Myc Oncogene ◽

Elevated Expression ◽

High Level

The structure and expression of the c-myc oncogene were examined in 29 primary human colon adenocarcinomas. Dot blot hybridization of total RNA showed that 21 tumors (72%) had considerably elevated expression of c-myc (5- to 40-fold) relative to normal colonic mucosa. These data were corroborated by Northern blots of polyadenylated RNA, which showed a 2.3-kilobase transcript. Southern analysis of the c-myc locus in these tumors indicated the absence of amplification or DNA rearrangement in a 35-kilobase region encompassing the gene. In a parallel study, elevated expression of c-myc without amplification or DNA rearrangement was also observed in three of six colon carcinoma cell lines examined; in addition, unlike a normal colon cell line control, these three cell lines exhibited constitutive, high-level expression of the gene during their growth in cultures. These results indicate that elevated expression of the c-myc oncogene occurs frequently in primary human colon carcinomas and that the mechanism involved in the regulation of c-myc expression is altered in tumor-derived cell lines.

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Cell membrane-associated proteoglycans mediate extramedullar myeloid proliferation in granulomatous inflammatory reactions to schistosome eggs

Journal of Cell Science ◽

10.1242/jcs.104.2.477 ◽

1993 ◽

Vol 104 (2) ◽

pp. 477-484

Author(s):

M. Alvarez-Silva ◽

L.C. da Silva ◽

R. Borojevic

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Connective Tissue ◽

Endogenous Growth ◽

Cell Lines ◽

Cell Layer ◽

Heparan Sulphate ◽

Myeloid Cells ◽

Myeloid Cell ◽

Gm Csf

In chronic murine schistosomiasis, extramedullar myelopoiesis was observed, with proliferation of myeloid cells in liver parenchyma and in periovular granulomas. We have studied the question of whether cells obtained from granulomatous connective tissue may act as myelopoietic stroma, supporting long-term myeloid proliferation. Primary cell lines (GR) were obtained in vitro from periovular granulomas, induced in mouse livers by Schistosoma mansoni infection. These cells were characterized as myofibroblasts, and represent liver connective tissue cells involved in fibro-granulomatous reactions. They were able to sustain survival and proliferation of the multipotent myeloid cell lines FDC-P1 and DA-1 (dependent on interleukin-3 and/or granulocyte-macrophage colony stimulating factor, GM-CSF) without the addition of exogenous growth factors. This stimulation was dependent upon myeloid cell attachment to the GR cell layer; GR cell-conditioned medium had no activity. Primary murine skin fibroblasts could not sustain myelopoiesis. The endogenous growth-factor was identified as GM-CSF by neutralization assays with monoclonal antibodies. The stimulation of myelopoiesis occurred also when GR cells had been fixed with glutardialdehyde. The observed stimulatory activity was dependent upon heparan sulphate proteoglycans (HSPGs) associated with GR cell membranes. It could be dislodged from the cell layer with heparin or a high salt buffer. Our results indicate a molecular interaction between endogenous growth-factor and HSPGs; this interaction may be responsible for the stabilization and presentation of growth factors in myelopoietic stromas, mediating extramedullar proliferation of myeloid cells in periovular granulomas.

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Retroviral insertions in the murine His-1 locus activate the expression of a novel RNA that lacks an extensive open reading frame

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1743-1751.1994 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1743-1751

Author(s):

D S Askew ◽

J Li ◽

J N Ihle

Keyword(s):

Cell Lines ◽

Wild Mouse ◽

Myeloid Cell ◽

Single Copy ◽

Open Reading Frame ◽

Chromosome 2 ◽

Coding Region ◽

Reading Frame ◽

Novel Gene ◽

Myeloid Leukemias

The His-1 locus is a common site of viral insertion in murine myeloid leukemias induced by the wild mouse ecotropic retrovirus, CasBrM. In this report, we describe the cloning of a novel gene at the His-1 locus and show that His-1 expression is associated with the transformed phenotype. Northern (RNA) blot analysis identified His-1 transcripts in four transformed myeloid cell lines but in no normal tissues examined. Two of these cell lines were derived from retrovirus-induced myeloid leukemias that harbor integrated proviruses which drive His-1 gene expression by promoter insertion. The two other cell lines expressed a discrete 3-kb His-1 RNA that is derived from a novel gene consisting of three exons that span 6 kb on mouse chromosome 2. The His-1 gene is conserved as a single-copy sequence in multiple vertebrate species and is expressed as a spliced and polyadenylated RNA. A protein-coding region is not evident from analysis of the His-1 sequence because of the presence of multiple small open reading frames, none of which are greater than 219 bp. This lack of an extensive open reading frame is an unusual feature that is shared by other RNA molecules believed to function in the absence of translation.

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Latent Infection and Reactivation of Human Herpesvirus 6 in Two Novel Myeloid Cell Lines

Blood ◽

10.1182/blood.v93.3.991 ◽

1999 ◽

Vol 93 (3) ◽

pp. 991-999 ◽

Cited By ~ 18

Author(s):

Masaki Yasukawa ◽

Hideki Ohminami ◽

Eiji Sada ◽

Yoshihiro Yakushijin ◽

Masahiko Kaneko ◽

...

Keyword(s):

Cell Lines ◽

Philadelphia Chromosome ◽

Human Herpesvirus ◽

Myeloid Cell ◽

Antigen Expression ◽

Cell Lysis ◽

Myelogenous Leukemia ◽

Human Herpesvirus 6 ◽

Latently Infected ◽

Herpesvirus 6

Abstract It has been reported that reactivation of human herpesvirus-6 (HHV-6) causes a failure of hematopoiesis. To clarify the mechanisms of bone marrow suppression induced by HHV-6 infection, it is necessary to establish an in vitro model of HHV-6 infection in hematopoietic progenitor cells. We have established two novel Philadelphia chromosome–positive myeloid cell lines, SAS413 and SAS527, which possess different hematologic characteristics and show distinct susceptibility to infection by HHV-6, from a patient with blast crisis of chronic myelogenous leukemia (CML). HHV-6 subgroup A (HHV-6A) showed marked replication in SAS413, forming syncytia and inducing cell lysis in short-term culture. On the other hand, HHV-6A–inoculated SAS527 continued to proliferate without cell lysis and only a few cells showed HHV-6 antigen expression. In contrast to HHV-6A infection, inoculation with HHV-6 subgroup B (HHV-6B) did not induce any cytopathic effect (CPE) or viral antigen expression in either of the cell lines. Although HHV-6B replication was undetectable, the presence of the HHV-6 genome in both cell lines was shown by polymerase chain reaction (PCR) during culture for more than 10 months, suggesting that HHV-6B latently infected SAS413 and SAS527. Phorbol ester treatment of SAS527 latently infected with HHV-6B resulted in reactivation of HHV-6, as shown by the appearance of a CPE, positive reactivity for the HHV-6 antigen, and isolation of infectious HHV-6. These novel cell lines should be useful for studying the mechanisms of HHV-6–induced hematopoietic failure and HHV-6 latency and reactivation, as well as differentiation, of the myeloid cell lineage.

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Interleukin 3-specific tyrosine phosphorylation of a membrane glycoprotein of Mr 150,000 in multi-factor-dependent myeloid cell lines.

The EMBO Journal ◽

10.1002/j.1460-2075.1987.tb02740.x ◽

1987 ◽

Vol 6 (13) ◽

pp. 3979-3984 ◽

Cited By ~ 81

Author(s):

S. Koyasu ◽

A. Tojo ◽

A. Miyajima ◽

T. Akiyama ◽

M. Kasuga ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Cell Lines ◽

Myeloid Cell ◽

Membrane Glycoprotein ◽

Interleukin 3

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Nonhistone protein antigen profiles of five leukemic cell lines reflect the extent of myeloid differentiation

Blood ◽

10.1182/blood.v63.3.701.701 ◽

1984 ◽

Vol 63 (3) ◽

pp. 701-710 ◽

Cited By ~ 28

Author(s):

A Goldberger ◽

G Brewer ◽

LS Hnilica ◽

RC Briggs

Keyword(s):

Cell Lines ◽

Myeloid Cell ◽

Leukemic Cell ◽

Nuclear Extract ◽

Myeloid Differentiation ◽

Chromosomal Protein ◽

Protein Antigens ◽

Nonhistone Protein ◽

Leukemic Cell Lines ◽

Chromatin Proteins

Abstract The human leukemic cell lines, K562, KG-1, and HL-60, and the blast subclones, KG-1a and HL-60 blast, were utilized to relate differences in nonhistone protein antigens to stages of myeloid cell differentiation. Chromatin proteins were separated on SDS- polyacrylamide gels, transferred electrophoretically to nitrocellulose sheets, and visualized by the peroxidase-antiperoxidase method of Sternberger. Screening with antisera raised against total and dehistonized chromatin and a nuclear extract from these cells revealed quantitative as well as qualitative differences between the cell lines. A decrease in antigen content seemed to parallel progressive stages of myeloid cell development. The results indicate that a number of chromosomal protein antigens are lost or modified during differentiation. An antigen(s) of approximately 55,000 molecular weight was found in HL-60 chromatin, but was not present in its less differentiated subclone or in the other lines representative of earlier stage cells. Upon the induction of HL-60 cells to mature to end stages with 4 microM retinoic acid, a significant increase in the mol wt 55,000 activity was seen. This antigen was detected only with antisera against HL-60 total chromatin and granulocyte nuclei, and it was found only in normal mature granulocytes and in the later stage cells of the HL-60 culture. Thus, the antigen appears to be associated with a differentiated myeloid function.

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Biologic significance of GATA-1 activities in Ras-mediated megakaryocytic differentiation of hematopoietic cell lines

Blood ◽

10.1182/blood.v96.7.2440 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2440-2450 ◽

Cited By ~ 38

Author(s):

Itaru Matsumura ◽

Akira Kawasaki ◽

Hirokazu Tanaka ◽

Junko Sonoyama ◽

Sachiko Ezoe ◽

...

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Cell Lines ◽

Direct Interaction ◽

Myeloid Cell ◽

Megakaryocytic Differentiation ◽

Dna Binding Domains ◽

Binding Domains ◽

Endogenous Expression ◽

Ras Activation

Abstract Lineage-specific transcription factors play crucial roles in the development of hematopoietic cells. In a previous study, it was demonstrated that Ras activation was involved in thrombopoietin-induced megakaryocytic differentiation. In this study, constitutive Ras activation by H-rasG12V evoked megakaryocytic maturation of erythroleukemia cell lines F-36P and K562, but not of myeloid cell line 32D cl3 that lacks GATA-1. However, the introduction of GATA-1 led to reprogramming of 32D cl3 toward erythrocytic/megakaryocytic lineage and enabled it to undergo megakaryocytic differentiation in response to H-rasG12V. In contrast, the overexpression of PU.1 and c-Myb changed the phenotype of K562 from erythroid to myeloid/monocytic lineage and rendered K562 to differentiate into granulocytes and macrophages in response to H-rasG12V, respectively. In GATA-1–transfected 32D cl3, the endogenous expression of PU.1 and c-Myb was easily detectable, but their activities were reduced severely. Endogenous GATA-1 activities were markedly suppressed in PU.1-transfected and c-myb–transfected K562. As for the mechanisms of these reciprocal inhibitions, GATA-1 and PU.1 were found to associate through their DNA-binding domains and to inhibit the respective DNA-binding activities of each other. In addition, c-Myb bound to GATA-1 and inhibited its DNA-binding activities. Mutant GATA-1 and PU.1 that retained their own transcriptional activities but could not inhibit the reciprocal partner were less effective in changing the lineage phenotype of 32D cl3 and K562. These results suggested that GATA-1 activities may be crucial for Ras-mediated megakaryocytic differentiation and that its activities may be regulated by the direct interaction with other lineage-specific transcription factors such as PU.1 and c-Myb.

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