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

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 < 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 vacuolated 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.