Phenotypic diversity in leukemia cell populations

Olsson, Lennart

doi:10.1007/bf00048967

Cited by 26 publications

(10 citation statements)

References 39 publications

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“…The biological basis for this diversity is unknown, but it can be assumed to be a result of a number of genetic and epigenetic mechanisms. The present results show that the phenotypic diversity, at least in part, can be obtained solely by alteration of the enzymatic DNA methylation pattern and support the idea that the phenotypic features of a tumor cell population are continuously subject to change (42)(43)(44).…”

Section: Discussionsupporting

confidence: 84%

Induction of the metastatic phenotype in a mouse tumor model by 5-azacytidine, and characterization of an antigen associated with metastatic activity.

Olsson¹,

Forchhammer²

1984

Proc. Natl. Acad. Sci. U.S.A.

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The murine Lewis lung carcinoma is a longterm grafted tumor that, after subcutaneous inoculation, forms metastases to the lungs. Forty-two cell lines were established from a primary tumor site and 40 were established from lung metastatic foci. Cloned sublines were established from the original 82 lines, and 2 sublines among 405 were found to be tumorigenic but not metastatic (T+/M-), whereas the remaining 403 sublines were both tumorigenic and metastatic (T+/M+). The Gene expression is influenced by a variety of biological mechanisms that are only partly known (1-6). It has become increasingly evident, however, that enzymatic methylation of cytosine to 5-methylcytosine (m5Cyt) is an important gene silencing factor (7-10) in regard to both cellular (11-14) and viral (15)(16)(17) genes. This has been substantiated by the fact that demethylation of DNA as obtained by replacement of cytosine with 5-azacytidine (5-azaC) may result in expression of otherwise silent genes in a number of biological systems (18)(19)(20)(21)(22), although the biological details still are unknown (5, 6).Metastatic spread of neoplasms is presumably based on multiple genetic and epigenetic features that are expressed by at least some of the tumor cells (23)(24)(25)(26). It is also conceivable that the metastatic phenotype is determined by different gene products in different tumors. It is therefore important to clarify whether metastatic activity can be induced solely by alteration in activity of preexisting genes in nonmetastatic tumor cells.We report here that the metastatic phenotype can be expressed by otherwise stable nonmetastatic mouse tumor cells after demethylation of DNA of proliferating tumor cells with 5-azaC. Metastatic cells could be identified by a monoclonal antibody (mAb) that recognized an epitope specifically expressed on metastatic cells. The same antibody was found to react with a cellular protein of Mr 45,000 and pl 6.7 and two slightly more acidic proteins, which may be modifications of the former. MATERIALS AND METHODS Establishment of Cell Lines and Subclones of the LewisLung Carcinoma (3LL). Cell lines were established from s.c. growing primary 3LL tumor lesions and from metastatic foci as described in detail elsewhere (27). Biopsies from such tumor lesions were seeded into plastic flasks as explant cultures in RPMI 1640 medium with 15% fetal calf serum. After 3-6 weeks in liquid medium, the cultures were cloned in semisolid 0.3% agar RPMI 1640 medium with 10% fetal calf serum. Clones were harvested from the agar cultures and expanded and maintained in liquid RPMI 1640 medium with 10% fetal calf serum. One cloned culture from each biopsy of either primary tumor lesions or metastatic foci was used to establish subclones. All cell lines were maintained in plastic flasks in RPMI 1640 medium with 10% fetal calf serum/0.3% L-glutamine at 370C in a humidified atmosphere with 5%CO2. The cells grew as adherent cultures with cell population doubling times of 20-25 hr in the exponential growth phase. The cells were ...

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Section: Discussionsupporting

confidence: 84%

Induction of the metastatic phenotype in a mouse tumor model by 5-azacytidine, and characterization of an antigen associated with metastatic activity.

Olsson¹,

Forchhammer²

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…Intratumoral phenotypic diversity is known to be expressed in malignant cell populations in respect to a number of biological features, including growth rate and clonability (25)(26)(27)(28)(29)(30) and onc-gene expression (31). The biological basis for this diversity is unknown, but involves probably both genetic and epigenetic factors.…”

Section: Discussionmentioning

confidence: 99%

Treatment of human cell lines with 5-azacytidine may result in profound alterations in clonogenicity and growth rate.

Olsson¹,

Due²,

Diamant³

1985

The Journal of Cell Biology

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Liquid medium cultures of three human cell lines (B-lymphoma, myeloma, and squamous lung carcinoma) with population-doubling times (PDT) and cloning efficiencies (CE) in the range of 32-43 h and 0.01-5.6%, respectively, were exposed to 5-azacytidine (5-azaC) for 3 d. The doses used (1-3/zM) were found to be nontoxic as measured by cell growth in liquid and semisolid agar medium and to be nonmutagenic as measured by the rate of generation of ouabain-and 6-thioguanine-resistant cell variants. After 5-azaC treatment, cell samples were subsequently harvested every day and assayed for their CE in semisolid agar medium. For each cell line, 30 to 42 individual clones were harvested at the day of maximal CE and expanded in liquid culture medium. PDT and CE were determined for each subclone about every 6 wk for 12 too. The majority of the subclones had unaltered PDT and CE compared to the original lines. However, several clones had profoundly changed proliferative activity with PDT on ~12-14 h and/or CE 5 to >50%. Some of the clones with altered growth properties reverted to PDT and/or CE values of untreated clones. However, a few clones of each line had stable alterations with PDT on 12-14 h and CE 5 to >50%; these clones were all significantly hypomethylated. It is concluded that the human gene repertoire does contain genes that appropriately activated can result in growth properties with very short PDT and high CE (and comparable to animal cell lines), and that this activation may be obtained by 5-azaC treatment. It is conceivable that the procedure here described to alter growth properties of human cell lines may be applied to experimental situations, where alterations of cell growth properties are desired.Many reports in recent years on regulation of both cellular 0-6) and viral~ genes (7-11) have provided experimental support to the hypothesis (12-15) that 5-methylcytosine (mSCyt) t may be important in the regulation of gene expression. Moreover, a number of experiments have shown that exposure of cells to 5-azacytidine (5-azaC) may result in hypomethylation of DNA and cause expression of otherwise silent genes of both cellular and viral origin (for review, cf. references 16, 17) to the extent that it mimics mutation induced genetic alterations, e.g., reversion frequency of thyAbbreviations used in this paper." 5-azaC, 5-azacytidine; CE, cloning efficiency; mSCyt, 5-methylcytosine; Oua r, ouabin resistant; PDT, population-doubling time; RPMI/FCS, RPMI-1640 medium SUlSplemerited with 0.3% L-glutamine and 10-15% fetal calf serum; 6-TG ~, 6-thioguanine resistant. midine kinase negative cells to thymidine kinase positive cells (18).The growth properties of malignant tumor cell lines are at least in part reflected in the growth rate and CE. However, it has hitherto not been possible by exogenous factors to alter profoundly these biological features, despite numerous attempts that mainly have been based on alterations in the growth medium conditions such as addition of growth factors like insulin and transferr...

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“…it has been suggested that genomic methylation patterns are important in the generation of intratumoral phenotypic diversity (IPD) (Frost and Kerbel, 1983), a pertinent feature of malignant cell populations (Fidler and Hart, 1982;Olsson, 1983;Heppner, 1984). However, it is unclear whether the methylation pattern in malignant cells has an impact on the progression of malignant cells along differentiative pathways and on activation.…”

Section: -50%mentioning

confidence: 99%

“…5-AzaC has also been shown to result in profound alterations in cellular phenotypes in numerous other experimental systems (for review, see Riggs and Jones, 1983). The effects of 5-azaC are probably a rimary result of gene gene-silencing factor, and when 5-azaC is incorporated into the genome instead of m'Cyt, DNA methylase is inhibited, and a number of genes become demethylated as shown experimentally in several gene systems (for reviews, see Doerfler, 1983;Riggs and Jones, 1983).it has been suggested that genomic methylation patterns are important in the generation of intratumoral phenotypic diversity (IPD) (Frost and Kerbel, 1983), a pertinent feature of malignant cell populations (Fidler and Hart, 1982;Olsson, 1983;Heppner, 1984). However, it is unclear whether the methylation pattern in malignant cells has an impact on the progression of malignant cells along differentiative pathways and on activation.…”

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confidence: 98%

Modulatory effects of 5‐azacytidine, phorbol ester, and retinoic acid on the malignant phenotype of human lung cancer cells

Olsson

Behnke²,

Sørensen

1985

Intl Journal of Cancer

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Cloned human cell lines of squamous-cell lung carcinoma and small-cell lung carcinoma were treated with 5-azacytidine (5-azaC), 12-0-tetradecanoyl-phorbol-13 acetate (TPA), retinoic acid (RA), or a combination of these drugs, and the effects on cellular morphology, in vitro growth properties, antigenicity, tumorigenicity and metastatic activity in nude mice were studied. Antigenicity was measured by the expression of major histocompatibility antigens (MHC) and of lung-tumor-associated antigens. 5-AzaC treatment resulted in subclones with shorter population doubling times (from 40-50 hr down to 14-20 hr) and increased cloning efficiencies (from less than 1% to 5-50%). TPA and RA induced loss of proliferative activity in vitro and tumorigenicity in vivo of both lines. This was morphologically associated with the appearance of an abundance of large vaculated cells which by DNA-analysis were all found to be in G0-G1 phase. However, 2 of the 5-azaC-treated subclones were insensitive to both TPA and RA, whereas the remaining subclones (20) all responded like untreated lines to TPA and RA. One of the sublines insensitive to TPA and RA formed metastases in nude mice in contrast to all the other lines used. The density of lung-tumor-associated antigens was significantly reduced by TPA-RA treatment, whereas the expression of MHC antigens was unaffected. In contrast, 5-azaC resulted in some cases in increased density of MHC antigens. The effects of 5-azaC on cellular phenotypes were not directly correlated to the total genomic content of 5-methylcytosine. The data suggest that this experimental system is suitable for studies of phenotypic features of malignant cells as related to cellular differentiation.

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Phenotypic diversity in leukemia cell populations

Cited by 26 publications

References 39 publications

Induction of the metastatic phenotype in a mouse tumor model by 5-azacytidine, and characterization of an antigen associated with metastatic activity.

Induction of the metastatic phenotype in a mouse tumor model by 5-azacytidine, and characterization of an antigen associated with metastatic activity.

Treatment of human cell lines with 5-azacytidine may result in profound alterations in clonogenicity and growth rate.

Modulatory effects of 5‐azacytidine, phorbol ester, and retinoic acid on the malignant phenotype of human lung cancer cells

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