nm23 has properties of a metastasis suppressor gene and also has been implicated in the control of response to transforming growth factor beta 1 (TGF beta 1) by studies in melanoma cells. In this report, we have examined the role of nm23 in two HT29 colon carcinoma sublines at different stages in tumor progression with different responses to TGF beta 1: the HD3 subline, which shows TGF beta 1-induced growth arrest and differentiation; and the more invasive and tumorigenic U9 subline, which induces tumors 7-fold as large as those induced by HD3 cells with one-half the latency. Analysis by semiquantitative reverse transcription-polymerase chain reaction showed that antisense phosphorothiolated oligonucleotides to the nm23 initiation site (nm23 AS oligos) decreased nm23 mRNA levels 2-8-fold in HD3 and U9 cells when normalized to beta-actin mRNA levels. However, a role for nm23 in TGF beta 1-mediated responses could only be found in HD3 cells. nm23 AS oligos inhibited the differentiation property of cell adherence over 90% in HD3 cells, and this loss of adherence could be partially blocked by concurrent treatment with TGF beta 1. In contrast, U9 cell adherence was not detectably altered by nm23 AS oligos, whether added in the presence or absence of TGF beta 1. The TGF beta 1-induced inhibition of HD3 cell proliferation was blocked by nm23 AS oligos, whereas the TGF beta 1-induced proliferation of U9 cells was unaffected by nm23 AS oligos. TGF beta 1 increases nm23 mRNA and protein levels in HD3 cells, which may explain the opposing effects of nm23 AS oligos and TGF beta 1 in these cells. No mutation in the coding sequence of nm23 was detected in U9 cells to explain the lack of effects of nm23 AS oligos. nm23 mRNA and protein levels were similar in the two lines. We have recently shown that TGF beta 1 induces two distinct signal transduction pathways in epithelial cells (Z. Yan et al, J. Biol. Chem., 269:13231-13237, 1994), one leading to increased cell adherence and growth arrest in HD3 cells, with the other leading to increased invasion and tumorigenicity in U9 cells. The data in the current study indicate that nm23 functions only in the TGF beta 1 signaling pathway leading to growth arrest and differentiation. After colon carcinoma cells have progressed to a more aggressive phenotype and use TGF beta 1 to stimulate growth and invasion (S. Hsu, et al, Cell Growth & Duff., 5: 267-275, 1994), the second TGF beta 1 signaling pathway is used which does not include nm23. Loss of nm23 function occurs in melanoma cells because of a decrease in expression level, whereas by this model in more aggressive colon carcinoma cells, loss of nm23 function occurs by use of an alternative TGF beta 1 signaling pathway.