Considerable evidence exists that lung cancer cell lines produce large amounts of insulin-like growth factor-binding proteins (IGFBPs). In addition, these cells are subject to an autocrine or paracrine growth control by insulin-like growth factors (IGFs). We now demonstrate by immunocytochemistry with IGFBP-3 antibodies that nuclei of a lung cancer cell line distinctly immunostain for IGFBP-3. This finding led us to investigate in more detail the localization of this protein that, to date, had only been known to occur extracellularly. Ligand blotting revealed that purified nuclear extracts contain a 43,000-Da IGFBP which can bind [I125]IGF-I. By Western blot this protein was identified as IGFBP-3. Thus, our data are consistent with the results of a previous structural study predicting a nuclear localization for IGFBP-3. Moreover, our findings raise the possibility that nuclear IGFBP-3 is functional and involved in the pathogenesis of lung cancer.

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The growth of human lung cancer cells is regulated positively and negatively by a variety of growth factors through autocrine as well as paracrine mechanisms. In the present report, we studied the differential role and expression of a neuropolypeptide growth factor in 26 lung cancer cell lines. Expression of the heparin-binding growth-associated molecule (HB-GAM) in 12 small cell lung cancer (SCLC) cell lines was compared to that in 14 non-small cell lung cancer (NSCLC) cell lines. HB-GAM mRNA was expressed in 9 of 12 SCLC and 3 of 14 NSCLC cell lines as determined by RT-PCR analyses. Normal human bronchial epithelial cells were used as negative controls. All cell lines which expressed HB-GAM mRNA produced HB-GAM protein as well. Western blot analysis showed that the tumor cells secreted HB-GAM into the media. HB-GAM, purified from lung cancer cell lines, exerted biological activity on fibroblasts, endothelial cells and SW13 cells as determined by thymidine incorporation and soft agar cloning assays. In addition, the biological activity of HB-GAM was blocked by a specific antibody in a dosedependent way. Our findings suggest that HB-GAM may serve as a marker for SCLC cell lines and that it may function as a paracrine growth factor in human lung cancer. HB-GAM may be a further member of the network of growth factors involved in proliferation, angiogenesis and metastasis of lung tumors. Int.

Insulin-like growth factors (IGFs) are potent mitogens for lung cancer cells. Their proliferative activity is influenced by their binding proteins (IGFBPs). We report here on the regulatory effects of IGF-I and IGF-II on the production and release of IGFBPs by nonsmall cell lung cancer cell lines (NSCLC). The nine NSCLC cell lines used in this study showed messenger ribonucleic acid (mRNA) expression of all six IGFBPs known, as determined by PCR, and protein secretion of IGFBP-1, -2, -3, -4, and -5, as analyzed by Western immunoblots. The addition of IGFs to a serum-free medium showed divergent effects on IGFBP-3 and IGFBP-4 levels in a conditioned medium (CM). IGF-I and IGF-II, but not insulin, led to a much higher concentration of IGFBP-3 in the CM of all tested NSCLC cell lines, whereas the level of immunologically detected membrane-associated IGFBP-3 was decreased. Furthermore, Northern analysis of mRNA isolated from A549 revealed that IGFBP-3 specific mRNA was not changed by IGF-I or IGF-II, suggesting that the IGF-induced effects on IGFBP-3 depend on the release of cell-associated IGFBP-3. In contrast, IGFBP-4 levels were diminished by increasing concentrations of IGFs in the CM of the NSCLCs A549, NCI-H157, and U1752, with no response to insulin or the IGF-I analog, whereas IGFBP-4-specific mRNA was not changed by IGF-I or IGF-II, as determined by Northern analysis. The same effects were seen in a cell-free system after incubation of the CM with IGFs. The decrease in IGFBP-4 concentrations was prevented by coincubation of the CM with the IGFs and either ethylenediamine tetraacetate or 1,10-phenanthrolene, but not with other protease inhibitors. We suggest that IGFs may either activate an IGFBP-4-specific metalloprotease present in NSCLC CM or that the binding of IGFs to IGFBP-4 may enhance the susceptibility of IGFBP-4 to proteolytic degradation. Based on these data, we present evidence that IGFs may regulate their own availability both by releasing IGFBP-3 from cell membrances and through proteolytic degradation of IGFBP-4.

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