The next challenge was to identify the postulated nerve-growth factor being released by the tumor. For this purpose we needed a system much less complex than the developing embryo. Tissue culture (which in the early 1950s had yet to become the universal biological tool it is today) seemed to offer a useful alternative. We reasoned that if sarcoma 180 was releasing a chemical factor with the ability to enhance nerve growth, the same effect should appear when an isolated sympathetic ganglion was incubated with the tumor in laboratory glassware. In 1953 one of us (Levi-Montalcini) and Hertha Meyer did this experiment at the Biophysics Institute in Rio de Janeiro. Sensory and sympathetic ganglia were dissected out of eight-day-old chick embryos and cultured in a semisolid medium in proximity to fragments of mouse sarcoma-180 tumors. Within 10 hours of incubation the isolated ganglion gave rise to a dense halo of nerve fibers, radiating out from the ex-plant like rays from the sun. Control ganglia cultured for the same length of time in the absence of the sarcoma-180 cells displayed only a sparse and irregular outgrowth of nerve fibers.
The discovery that the tumor could exert its growth-enhancing effects on an isolated ganglion in tissue culture was the turning point of the investigation. Whereas our earlier experiments in developing chick embryos had required weeks of painstaking work, we could now in a few hours screen a large number of tissues, organic fluids and chemicals to determine if they were sources of the growth-promoting activity. Furthermore, it now became possible to attempt to isolate the nerve-growth factor from our simplified tissue-culture system.
Stanley Cohen, a biochemist, agreed to join our group at Washington University to undertake the task of identifying the active agent. He soon managed to restrict the growth factor to a fraction of the tumor cells containing both protein and nucleic acid. This finding opened the way to a more precise biochemical analysis, but we would not have made much progress if it had not been for a fortuitous event two years later. In order to determine whether the nerve-growth factor was a protein or a nucleic acid, Cohen and one of us (Levi-Montalcini) treated the tumor-cell extract with snake venom containing a high concentration of the enzyme phosphodiesterase, which degrades nucleic acids. To our surprise we found that adding minute amounts of the venom to the active fraction of the sarcoma-180 cells enhanced the growth-promoting effect of the fraction rather than reduced it. That the snake venom itself was the source of the increased activity was readily confirmed. The addition of a small amount of venom to the culture medium in the absence of the extract of sarcoma 180 elicited the growth of the same dense halo of nerve fibers around an isolated sensory or sympathetic ganglion.
In fact, the nerve-growth factor in the snake venom turned out to be much more abundant and potent than that in the sarcoma-180 cells. Cohen was therefore able to purify the factor from the venom and demonstrate that it was a protein. Injections of the purified snake venom NGF into growing embryos resulted in the same excessive sympathetic innervation of the viscera and the blood vessels as that induced by the sarcoma-180 cells.
The discovery that two unrelated sources—mouse sarcoma and snake venom—harbor NGF suggested the possibility that still other tissues might secrete the factor. The search focused on the submaxillary salivary gland of rodents, which is similar in certain respects to the venom gland of snakes. Indeed, Cohen isolated an NGF from mouse salivary glands that was about 10,000 times more active than that purified from mouse sarcoma 180 and about 10 times more active than that purified from snake venom. Over the next two decades smaller amounts of NGF were found to be secreted by a wide variety of normal and neoplastic (cancer) cells.
INCREASED SIZE AND NUMBER of neurons in the sympathetic ganglia of NGF-treated mice result in an increase in the volume of the ganglia. These micrographs are of sections through two ganglia, one ganglion from a three-week-old rat injected daily with saline solution only (left) and the other from a littermate mouse injected with NGF (right). Neurons in NGF-treated rat are substantially enlarged. Note also that cells in ganglia of animals treated with NGF exhibit a greater affinity for toluidine blue, the dye used to stain the cells.
Image: Scientific American, June 1979