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The murine Lewis lung carcinoma is a longterm grafted tumor that, after subcutaneous inoculation, forms metastases to the lungs. Forty-two cell lines were established from a primary tumor site and 40 were established from lung metastatic foci. Cloned sublines were established from the original 82 lines, and 2 sublines among 405 were found to be tumorigenic but not metastatic (T+/M-), whereas the remaining 403 sublines were both tumorigenic and metastatic (T+/M+). The Gene expression is influenced by a variety of biological mechanisms that are only partly known (1-6). It has become increasingly evident, however, that enzymatic methylation of cytosine to 5-methylcytosine (m5Cyt) is an important gene silencing factor (7-10) in regard to both cellular (11-14) and viral (15)(16)(17) genes. This has been substantiated by the fact that demethylation of DNA as obtained by replacement of cytosine with 5-azacytidine (5-azaC) may result in expression of otherwise silent genes in a number of biological systems (18)(19)(20)(21)(22), although the biological details still are unknown (5, 6).Metastatic spread of neoplasms is presumably based on multiple genetic and epigenetic features that are expressed by at least some of the tumor cells (23)(24)(25)(26). It is also conceivable that the metastatic phenotype is determined by different gene products in different tumors. It is therefore important to clarify whether metastatic activity can be induced solely by alteration in activity of preexisting genes in nonmetastatic tumor cells.We report here that the metastatic phenotype can be expressed by otherwise stable nonmetastatic mouse tumor cells after demethylation of DNA of proliferating tumor cells with 5-azaC. Metastatic cells could be identified by a monoclonal antibody (mAb) that recognized an epitope specifically expressed on metastatic cells. The same antibody was found to react with a cellular protein of Mr 45,000 and pl 6.7 and two slightly more acidic proteins, which may be modifications of the former. MATERIALS AND METHODS Establishment of Cell Lines and Subclones of the LewisLung Carcinoma (3LL). Cell lines were established from s.c. growing primary 3LL tumor lesions and from metastatic foci as described in detail elsewhere (27). Biopsies from such tumor lesions were seeded into plastic flasks as explant cultures in RPMI 1640 medium with 15% fetal calf serum. After 3-6 weeks in liquid medium, the cultures were cloned in semisolid 0.3% agar RPMI 1640 medium with 10% fetal calf serum. Clones were harvested from the agar cultures and expanded and maintained in liquid RPMI 1640 medium with 10% fetal calf serum. One cloned culture from each biopsy of either primary tumor lesions or metastatic foci was used to establish subclones. All cell lines were maintained in plastic flasks in RPMI 1640 medium with 10% fetal calf serum/0.3% L-glutamine at 370C in a humidified atmosphere with 5%CO2. The cells grew as adherent cultures with cell population doubling times of 20-25 hr in the exponential growth phase. The cells were ...

In HeLa cells deprived of valine, histidine, or methionine initiation of protein synthesis decreases rapidly and disaggregation of polyribosomes also occurs. The mechanism of inhibition does not seem to involve the supply of RNA in the cell, and thus it differs from the initiation of inhibition at elevated temperatures. Polyribosomes rapidly form again if the missing amino acid is restored, even in the presence of actinomycin D.Studies of HeLa cells deprived of single essential amino acids -have shown that protein synthesis continues in this situation at a reduced rate determined by the rate of endogenous protein turnover (1). Such cells are in a condition of suspended growth and offer a unique opportunity for the study of metabolic regulation. It has previously been established that the rate of synthesis of ribosomal precursor RNA is reduced in these cells (2, 3), and maturation into complete ribosomal subunits is restricted (3), though synthesis of ribosomal proteins is uninterrupted (4).In several recent studies (5, 6), it was found that ascites tumor cells deprived of a complete mixture of essential -amino acids had extensive disaggregation of polyribosomes into inactive monoribosomes. The disaggregation was reversed if the set of essential amino acids was restored. However, since all of the essential amino acids were withheld or supplied at the same time, it is uncertain whether the apparent -regulation of protein synthesis that was observed involved the availability of amino acids per se, or amino acids in the role of donors of amide or methyl groups.We have found that HeLa cells, deprived of any one of several essential amino acids, promptly exhibit a marked inhibition of translational initiation. This inhibition and its -reversal have been studied and compared to previous demonstrations of control of translational initiation in cells undergoing mitosis (7) and in cells exposed to elevated temperatures (8). MATERIALS AND METHODS Cell cultureSuspension cultures of HeLa cells were maintained in Eagle's medium with 5% calf serum, as described (9, 10), at a con-* To whom reprint requests should be sent. Reprints will be available only outside of North America. Amino-acid Depriation, Cell Labeling, and Fractionation. Cells were collected by centrifugation and rinsed in Eagle's medium without valine, histidine, or methionine, but with leucine at a concentration of 0.02 mM (one-twentieth of the normal concentration), and 5% dialyzed calf serum. They were then resuspended in a medium of the same composition at a concentration of 3 X 106 cells/ml. Cells acting as growing controls were resuspended in medium to which the missing amino acid had been added to its normal concentration. All cultures were incubated at 370C, except where noted Suspension cultures deprived of an amino acid for specified times, or growing control cultures, were incubated for 45 min, and then labeled for various times with ['4C]leucine (0.25 pCi/ml, 12.5 Ci/mol final specific activity). 8-ml samples were then rapidly cooled and fract...

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