Murine sarcoma virus-transformed mouse fibroblasts produce polypeptide growth factors and release them into serum-free medium. These factors stimulate cells to divide in monolayer cultures and also to form colonies that grow progressively in soft agar. Three major peaks of activity are seen, with apparent molecular weights of 25,000, 12,000, and 7000.

In the last decade, several groups have shown a direct correlation between the inappropriate or ectopic release of interleukin (IL)-8 by tumor cells in vitro and their growth and metastatic potential using in vivo models of tumor growth. IL-8 is a potent neutrophil chemoattractant. Neutrophils, as "early responders" to wounds and infections, release enzymes to remodel the extracellular matrix of the tissues through which they migrate to reach the site of the wound or infection. It is proposed that the host's cellular response to IL-8 released by tumor cells enhances angiogenesis and contributes to tumor growth and progression. The activities released by the responding neutrophils could serve as enablers of tumor cell migration through the extracellular matrix, helping them enter the vasculature and journey to new, metastatic sites. The reactive oxygen species produced by neutrophilic oxidases to kill invading organisms have the potential to interact with tumor cells to attenuate their apoptotic cascade and increase their mutational rate. It is proposed that the increase in metastatic potential of tumors ectopically releasing IL-8 is, in part, attributable to their ability to attract neutrophils. Discussed here are possible mechanisms by which the neutrophils responding to ectopic IL-8 contribute to the in vivo growth, progression, and metastatic potential of tumor cells. Possible targets are also presented for the development of therapies to attenuate the effects of the ectopic IL-8 release by tumor cells. DISCOVERY OF INTERLEUKIN-8The cytokine interleukin (IL)-8 is a small basic protein first purified on the basis of its neutrophil chemoattractant properties (1, 2). Soon after it was purified, a cDNA clone was obtained, and its gene was characterized (3, 4). IL-8 is a member of the ␣-chemokine family and a very potent neutrophil chemoattractant. It and its homologs are induced in wounds by the G 0 to G 1 transition, and its expression is greatly enhanced by the inflammatory mediators IL-1␤ and tumor necrosis factor (TNF) ␣ (5, 6). IL-8 recruits neutrophils to the site of wounds to protect the tissue from invading microorganisms and enhance the healing process (7). However, the release of IL-8 by the wrong cells, at the wrong time, or at too high a concentration can lead to undesired pathologies, such as rheumatoid arthritis, inflammatory bowel disease, idiopathic pulmonary fibrosis, and cerebral and myocardial ischemia (8). ECTOPIC INTERLEUKIN-8 EXPRESSIONIn the last decade, several groups have observed a direct correlation between the level of ectopic IL-8 expression by individual clones of tumor cell lines and their in vivo growth rate and metastatic potential. This correlation has been shown for many tumor cell types (9 -13) as well as for fresh breast tumor samples (14). Highly metastatic tumor cells produce more IL-8 constitutively than their poorly metastatic counterparts, and the amounts of IL-8 they release in response to the pro-inflammatory cytokines IL-1␤ and TNF-␣ are much greater (13, 15). ...

Three different human tumor lines in culture, a rhabdomyosarcoma, a bronchogenic carcinoma and a metastatic melanoma, release proteins (transforming growth factors, TGFs) into the medium that confer the transformed phenotype on untransformed fibroblasts. These proteins are acid and heat-stable; produce profound morphologic changes in rat and human fibroblasts; and enable normal anchorage-dependent cells to grow in agar. Removal of the transforming protein results in a reversion of cell phenotype. The major activity interacts with epidermal growth factor (EGF) cell membrane receptors. The peptides from these tumor cells are similar in their action to the sarcoma growth factor (SGF) released by murine sarcoma virus-transformed rodent cells. The most anchorageindependent tumor cells released the most TGFs. EGF-related TGFs were not detectable in fluids from cultures of cells with high numbers of free EGF membrane receptors (normal human fibroblasts and human carcinomas). Mouse sarcoma virus (MSV)-and rat sarcoma virus-transformed cells release a potent growth-stimulating peptide that interacts with epidermal growth factor (EGF) receptors (1, 2). This ability has been utilized to purify sarcoma growth factor (SGF) produced by MSV-transformed cells (3). It has been noted that certain human sarcoma and carcinoma cells (4) and most melanomas (5) lack EGF receptors and, therefore, may produce endogenous factors related to EGF and SGF. To test this possibility, serum-free media were collected from four human tumors and normal human fibroblasts and partially purified. Cells that lack EGF receptors released a potent growth-stimulating activity that enabled normal fibroblasts and epithelial cells to proliferate in soft agar. Supernates from normal human fibroblasts possessed <2% as much activity.A human epidermoid carcinoma cell (A431) with an exceptionally high number of EGF receptors (4, 6, 7) released little growth-stimulating activity when compared to the other tumor cells. That activity did not compete with EGF. Tumor cells that lack EGF receptors and form colonies in soft agar released a greater quantity of the transforming growth factors (TGFs) than did normal or tumor cells that grow poorly in agar.We conclude that certain human tumor cells release potent transforming protein(s) that transform normal indicator cells in a manner similar to SGF. MATERIALS AND METHODSCell Cultures. Cell cultures were maintained at 37°C in 75-cm2 plastic tissue culture flasks (Falcon no. 3024) with Dulbecco's modification of Eagle's medium (DME medium) with 10% calf serum (Colorado Serum). Five human tumor cell cultures were used. The human rhabdomyosarcoma line, A673, and the bronchogenic carcinoma line, 9812, produce progressively growing tumors in immunologically depressed mice and grow readily in soft agar (8). Neither has detectable EGF receptors (4). The human epidermoid carcinoma A431, from a primary vulvar carcinoma in an 85-year-old woman, has an exceptionally high number of EGF receptors (4, 6). The human metastatic m...

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 ...

TPA (12-O-tetradecanoyl-phorbol-13-acetate) reversibly inhibits the binding of (125)I-labelled epidermal growth factor (EGF) to treated mouse and human cells, but does not affect the binding of various other ligands to their membrane receptors. It alters the affinity of the receptors for EGF without changing the total number of available receptors per cell. Those phorbol esters which stimulate cell growth in culture and have tumour-promoting activity in vivo alter the EGF-receptor affinity, while the biologically inactive derivatives fail to change the affinity of EGF for its receptors.