Disaggrcgatcd mouse embryo cells, grown in monolaycrs, undcrwcnt a progrcssivc dcclinc in growth ratc upon succcssivc transfer, the rapidity of the decline dcpcnding, among othcr things, on the inoculation density. Ncvcrthclcss, ncarly all culturcs dcvclopcd into cstablishcd lincs within 3 months of culture. Thc first sign of thc emcrgcncc of an established line was the ability of thc cells to maintain a constant or rising potential growth ratc. This occurred while thc cultures wcrc morphologically unchangcd. Thc growth rate continued to incrcasc until it cqualcd or cxcccdcd that of the original culturc. The carly cstablishcd cclls showed an increasing mctabolic autonomy, as indicated by dccrcasing dependence on ccll-to-ccll fccding. It is suggcstcd that the process of cstablishmcnt involvcs an altcration in ccll pcrmcability propcrtics. Chromosome studics indicatcd that thc cells rcsponsiblc for thc upturn in growth rate wcrc diploid, but latcr the population shifted to the tctraploid range, often very rapidly. Still later, marker chromosomes appcarcd. Different lines acquired diffcrcnt propcrtics, dcpcnding on thc culture conditions cmploycd; one line dcvclopcd which is cxtrcmcly scnsitivc to contact inhibition.

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The A549 tumor-cell line, initiated from a human alveolar cell carcinoma, has been continuously propagated in vitro for more than 3 years (more than 1,000 cell generations). These cells have a human karyotype and appear to have been derived from a single parent cell. All A549 cells examined by electron microscopy at both early and late passage levels contain multilamellar cytoplasmic inclusion bodies typical of those found in type II alveolar epithelial cells of the lung. At early and late passage levels, the cells synthesize lecithin with a high percentage of disaturated fatty acids utilizing the cytidine diphosphocholine pathway; such a pattern of phospholipid synthesis is expected for cells believed to be responsible for pulmonary surfactant synthesis. The A549 cell line should permit in vitro analysis of human surfactant synthesis and secretion and possibly provide a source of human surfactant for therapeutic intervention in pulmonary disease states characterized by surfactant deficiency.

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.

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The cells of an established mouse fibroblast line, 3T3, have a high plating efficiency and grow rapidly in sparse culture, but stop growing at a very low saturation density in comparison with other lines, because 3T3 is extremely sensitive to contact inhibition of cell division. After each medium change, however, there occurs in a small fraction of the cells in a saturation density culture a series of changes that results in a single rather synchronized division 30 hours later. This is due to a macromolecular substance in the serum which appears to act by reducing the sensitivity o f the cells to contact inhibition. The first recognizable event following the addition of serum to a stationary phase culture is a ten fold increase in the rate of RNA synthesis, occurring within 30 minutes. An increase in the rate of protein synthesis follows several hours later. DNA synthesis does not begin before 12 hours, but by two hours after niedium change an appreciable fraction of the cells become committed to eventuaI DNA synthesis and cell division. The sequence of event suggests that regulation of RNA synthesis is the means by which contact inhibition controls cell division.

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...

An epithelioid cell line, started from a human pancreatic carcinoma of ductal cell origin, has been maintained in culture for over 2 years and has been subcultured more than 40 times. The PANC-1 cell line has a doubling time of 52 h and G6PD activity of the slow mobility of B type. Chromosome studies show a modal number of 63 with three distinct marker chromosomes and a small ring chromosome. The malignant nature of the PANC-1 cell line was verified by: (1) the ready growth of PANC-1 cells in soft agar and on top of a fibroblast monolayer; and (2) the formation of a progressively growing anaplastic carcinoma after injection of a nude-athymic mouse with PANC-1 cells.