Purified mouse nerve growth factor (NGF) radiolabeled with 125 was tested for its ability to bind to a variety of different cultured cells. NGF A 36-year-old woman underwent wide excision of an irregular, black, elevated mole at the left scapular area. Pathological examination revealed invasive, malignant melanoma. The patient was re-admitted several months later for excisional biopsy of recurrent melanoma at the left side of the jaw. At that time, left axillary adenopathy was noted; of 20 nodes excised, one was positive for metastatic melanoma. During the following 2' years the patient received radiation therapy to the cranial vault as well as chemotherapy for systemic melanoma. However, a space-occupying lesion developed deep in the right frontal lobe and metastatic melanoma was excised. Part of this tumor was sent to our laboratory for initiation of the A875 line. The pathological report on this specimen indicated a 5 X 3.3 X 0.3 cm mass, containing numerous mitotic figures, giant cells, and pleomorphic cells containing dark brown melanin granules. After a progressively deteriorating course, the patient expired 6 months after surgery.The A875 cells grew rapidly in primary culture with a mass doubling time of approximately 4 days on early passages. The cells grow in soft agar, on monolayers of normal cells, and are tumorigenic in nude mice. In culture they contain abundant intracytoplasmic pigment granules. Frozen samples are available from the fourth through sixty-fifth passages. The experiments presented in this paper were done with cultures at passage 20 to 25. Stock cultures were maintained at 370 in plastic T-75 flasks with Dulbecco's modification of Eagle's medium supplemented with 10% fetal calf serum (Colorado Serum Co.). lodination of NGF. The 2.5S preparation (f3 subunit) of nerve growth factor (NGF) used in these experiments was a generous gift of Dr. Rita Levi-Montalcini. One biological unit (BI.) corresponds to 10 ng of protein, using the conventional bioassay described by . Gel electrophoresis performed in the presence of sodium dodecyl sulfate (5) showed a single band with an apparent molecular weight of 13,000. Twenty micrograms of NGF were mixed with 1 mCi of carrier-free Na'25I (Amersham Searle) in 50 pul of 0.4 M phosphate buffer, pH 7.5. Ten microliters of chloramine T (6) were added (1 mg/ml), and after 30 sec, the iodinated protein was separated from the other reaction products by passing the reaction mixture through a 14 X 1 cm column of Sephadex G-15; the void fractions were pooled. The specific activities of labeled preparations ranged from 5 to 23 ttCi/gg. 125I-labeled NGF was active in binding assays after storage at -60°for at least 2 months.The 6.5S form of NGF was prepared from male mouse submaxillary glands using the method of Varon et al. (7), and was iodinated as above. This preparation was used in some experiments and showed the same specificity for binding, as did the iodinated 2.5S preparations.Binding Assay. Human melanoma cells (A875) (1 to 5 X 105) grown in 60 ...

No abstract

No abstract

No abstract

No abstract