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Human soluble galactose-binding lectin (galectin-1) has been expressed as an Escherichia coli fusion protein, following the amplification by polymerase chain reaction of cDNA prepared from a human osteosarcoma cell line. The fusion protein is a functional beta-galactoside-binding lectin, as is the recombinant galectin when purified from the cleaved fusion protein. The recombinant galectin has a biphasic effect on cell proliferation. Unlike the fusion protein, it functions as a human cell growth inhibitor, confirming earlier findings with natural human galectin-1, though it is less effective than the natural galectin. This reaction is not significantly inhibited by lactose, and is thus largely independent of the beta-galactoside-binding site. At lower concentrations, recombinant galectin-1 is mitogenic, this activity being susceptible to inhibition by lactose, and thus attributable to the beta-galactoside-binding ability of the protein. Some tumour cells are susceptible to the growth-inhibitory effect, and the galectin-1 gene is expressed in both normal and tumour cells.

We have previously shown that thrombin reverses the growth inhibition caused in human fibroblasts when a cell-surface proteinase is inhibited. A similar result was obtained with a synthetic thrombin receptor agonist peptide, which mimics the conformational change resulting from receptor cleavage by thrombin. Consideration of the effects of the growth-related proteinase on intracellular second messengers indicates that cleavage of the thrombin receptor by this endogenous proteinase is not a significant event for normal fibroblast growth in culture. Inhibition of the growth-related proteinase also fails to block the mitogenic action of bombesin. In conjunction with earlier results, this suggests that the intracellular point of action of GRP inhibition may be at, or closely connected with, the receptor-linked tyrosine kinases, and evidence for inhibition of protein phosphorylation following from GRP inhibition is presented.

Several reports have indicated that proteinase inhibitors can act as both stimulators and inhibitors of the growth of cultured cells. To confirm and extend these reports, we have purified the trypsin inhibitor from human urine (UTI). We have demonstrated a biphasic effect of this protein on the proliferation of human fibroblasts, and have identified two types of cellular binding site which may be responsible for the stimulatory and inhibitory aspects of this effect. Both aspects of UTI action have also been observed in human tumour cell cultures, but they are not consistently associated in these cells.

Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors.

1. Three distinct lines of evidence indicate that proteinases are involved in the growth of cultured animal cells. 2. Endogenous growth-related proteinases have been identified, and exogenous proteinases can also stimulate cell proliferation, probably by different mechanisms. In some cases, higher concentrations of proteinases are cytotoxic. 3. Proteinase inhibitors, not surprisingly, inhibit cell growth, but can also be mitogenic at sub-inhibitory concentrations. 4. There must, therefore, be at least three major cellular processes in which proteinases or proteinase inhibitors can operate to exert a direct effect on cell proliferation. 5. Details of one action of an exogenous proteinase, typified by thrombin and the thrombin receptor, are becoming clear at the molecular level, but thrombin probably activates at least two intracellular signalling systems, as well as acting as a growth inhibitor in some situations. 6. Much remains to be investigated in other examples.

Human alpha 1-proteinase inhibitor (alpha 1-antitrypsin) and bovine pancreatic trypsin inhibitor both inhibit cell-surface proteolytic activity in human fibroblasts. At the inhibitory concentrations, these proteins also inhibit cellular proliferation, as assayed by a microassay based on spectrophotometry of fixed and stained cells. Despite testing a wide range of proteinase inhibitor concentrations, no evidence was found for mitogenic or growth-stimulatory activity. ovomucoid was inactive against cell-surface proteinase activity and did not inhibit cellular proliferation.

Specific antibodies and protein proteinase inhibitors will inhibit cell-surface proteinase activity on human fibroblasts and cause a concomitant inhibition of DNA synthesis and of cell multiplication. An insolubilized proteinase inhibitor also inhibits cell multiplication. The same reagents partially inhibit the multiplication of mouse L cells, both in monolayer and suspension culture, and inhibit the mitogenic effect of epidermal growth factor (EGF) on both types of cell.