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

Abstract

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...

Can cancer chemotherapy enhance the malignant behaviour of tumours?

“…We have obtained increased metastasis in two tumour lines, a murine [48] and a human [107] melanoma, following brief exposure to low concentrations (3-5/zM) of aza-C. Similar results have been obtained in other murine tumours [108][109][110], while the in vitro clonogenicity and growth rates of human tumour lines, presumed indicators of malignant capacity, have been increased by comparable protocols [117]. While initial treatments with aza-C brought about significant decreases (about 50%) in the percentage of methylated cytosine residues, this extensive hypomethylation was not maintained in lung turnout nodule-derived cell lines [107].…”

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

“…During studies on the cellular phenotypes of 3 human malignant cell lines, it has been recently observed that 5-azaC treatment may induce profound changes in the proliferative activity as expressed by significantly shorter PDT and higher CE (Olsson et al, 1985). The PDT and CE values obtained by 5-azaC treatment were comparable to the fastest growing rodent cell lines, and since the 5-azaC doses were non-mutagenic and non-toxic, the experiments indicate that the human genome does contain a gene repertoire that upon appropriate activation can result in cell populations with PDT and CE values comparable to rodent cell systems.…”

“…The schedule for treatment with 5-azaC (Sigma, St. Louis, MO) has been described in detail elsewhere (Olsson et al, 1985). Briefly, cells were exposed for 3 days to 3 pM 5-azaC dissolved in RPMUFCS, then washed and resuspended in RPMUFCS, and aliquots of cells were cloned in semi-solid agar medium for 16 days.…”

On the Advent and Necessity of Molecular Biology in the Clinical Management of Lung Cancer*

“…Intratumoral phen otyp ic diversity is well established for a numb er of phenot yp ic featu res and experiment ally confirmed in many experimen tal as well as huma n tumo r type s (14, 2B). Antigen ic heterogene ity in human lung cancer cell lines was recentl y de scribed and the impact on analysis with Mabs discussed (31)(32)(33). Of significant impo rtance, and only analyzed to a very limited extent , is the intratumoral phen otypic diversity in the stem cell populati on that is the crucial cellular sou rce for maintenance and expansion o f the cell population.…”