The growth of a panel of 22 different human
tumor,
leukemia, and
lymphoma cell lines was examined in a human
tumor cloning assay in
agar or
methylcellulose and a tritiated
thymidine uptake assay. The cultures were performed in the absence or presence of increasing concentrations (0.5-500 ng/ml) of
nerve growth factor (
NGF). The growth of 17 of the 22 cell lines was not significantly and reproducibly affected by
NGF. There was minor (1.2-fold) but reproducible stimulation of clonal growth in one
glioblastoma cell line (86-HG-39) by
NGF, but in this cell line
NGF induced no growth modulation in a tritiated
thymidine uptake assay. However, clonal growth of another
glioblastoma cell line (87-HG-31) and all three
lung cancer cell lines tested (
HTB 119,
HTB 120, CCL 185) could be stimulated up to 3-fold by
NGF with a dose-response relationship for the
growth factor. Growth stimulation by
NGF could be completely reversed by neutralizing anti-
NGF antibody and by the
tyrosine kinase inhibitor genistein. Evaluation of secondary plating efficiency revealed the stimulation of colony formation as representing self-renewal and not terminal differentiation.
Reverse transcriptase-PCR experiments in the five responding cell lines showed expression of both low-affinity
NGF receptor (
glycoprotein 75) and c-trk transcripts on the
mRNA level. Of the five responding cell lines, only 86-HG-39, the cell line with the lowest responsiveness, revealed low-affinity
NGF receptor on the
protein level; the other four cell lines with high responsiveness, including the three
lung cancer cell lines, expressed no low-affinity
NGF receptor as shown by fluorescence-activated cell sorter analysis and immunoprecipitation using the ME 20.4 antibody. Immunoprecipitation using anti-trk
antibodies was negative in all five responding cell lines. However, binding studies with iodinated
NGF showed only low-affinity binding on the 86-HG-39 cell line and only high-affinity binding on the high-responder cell lines CCL 185 and 87-HG-31. In summary, our data suggest that
NGF can be operative in stimulation of clonal growth of malignant
tumor cells. High-affinity but not low-affinity binding sites mediate signal transduction for clonal growth and signaling involves
tyrosine kinase activity.