Nucleoside diphosphate kinase at the cell surface of neoplastic human cells in culture

Strong evidence is given that a nucleoside diphosphate kinase is present to some extent on the surface of intact neoplastic cells in culture. Experiments could be performed with cells cultured in a few plates to which an incubation medium was added. The cells were firmly attached to the supporting medium and remained viable during the incubation procedure. Determinations of lactate dehydrogenase were carried out to rule out any possible contamination from the culturing medium as well as from the cell interior. From these analyses, a procedure was developed which easily removed the last traces of the culturing medium and which showed that there was no leakage of intracellular lactate dehydrogenase during the incubation procedure. There was a rather insignificant diffusion of nucleoside diphosphate kinase into the incubation medium. In common with other nucleoside diphosphate kinases, the glioma cell surface enzyme seemed to be nonspecific with regard to nucleotide substrates.     

Formation of extracellular adenosine triphosphate by normal and neoplastic human cells in culture

Normal and neoplastic human cells in culture were suspended under isotonic conditions and incubated for one minute with the substrates, including ³²P-labelled inorganic phosphate, and cofactors of the glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions (complete medium), as well as with an incomplete medium lacking ADP, NAD⁺ and glyceraldehyde-3-phosphoric acid. The neoplastic cell types incubated in the complete medium synthesized three to six times more labelled ATP than the corresponding normal cells. In the incomplete medium only insignificant amounts of labelled ATP were formed during one-minute incubation by all types of cells. From other types of experiments it could be concluded that the labelled ATP, isolated from the cells incubated in the complete medium, was formed at the surface of the cell membranes. Only negligible amounts of enzymes engaged in the synthesis of ATP have leaked out from the cells.     

Recent problems in cell culture systems using normal human cells

Some remarkable studies of human cell culture have contributed to many basic and applied sciences on "normal human cells"; developments of biological products or physiologically activated substances. In this reviews, some problems concerning in vitro culture systems were discussed and recent advances on the researches of normal human cells were described.     

Sensitivity to radiation of human normal, hyperthyroid, and neoplastic thyroid epithelial cells in primary culture

Samples of thyroid tissue removed surgically from 63 patients were cultured in vitro and exposed to X irradiation to investigate the radiosensitivities of various types of thyroid epithelial cells. A total of 76 samples were obtained, including neoplastic cells from patients with papillary carcinoma (PC) or follicular adenoma (FA), cells from hyperthyroidism (HY) patients, and normal cells from the surgical margins of PC and FA patients. Culturing of the cells was performed in a manner which has been shown to yield a predominance of epithelial cells. Results of colony formation assays indicated that cells from HY and FA patients were the least radiosensitive: when adjusted to the overall geometric mean plating efficiency of 5.5%, the average mean lethal dose Do was 97.6 cGy for HY cells and 96.7 and 94.3 cGy, respectively for neoplastic and normal cells from FA patients. Cells from PC patients were more radiosensitive, normal cells having an adjusted average Do of 85.0 cGy and PC cells a significantly (P = 0.05) lower average Do of 74.4 cGy. After allowing for this variation by cell type, in vitro radiosensitivity was not significantly related to age at surgery (P = 0.82) or sex (P = 0.10). These results suggest that malignant thyroid cells may be especially radiosensitive.     

Demonstration of an interferon alpha receptor in human colonic cancer cells in culture

The IFN-alpha 2 receptor present in human cancerous colonic cells in culture has a capacity of 1,800 sites per cell with a kd of 0.5 nM. The molecular mass of the interferon alpha 2 (IFN-alpha 2) receptor complex is 134 kd. The IFN-alpha 2 induces the activity of the 2'5' oligoadenylate (2'5' A) synthetase activity in these cells and partly inhibits their growth.     

Mechanism of thymidylate synthase inhibition by methotrexate in human neoplastic cell lines and normal human myeloid progenitor cells

We have studied the roles of 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2'-deoxyuridine-5'-monophosphate radioenzymatic binding assay, we determined that the 5,10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 micron MTX for up to 21 h. Similar alterations in the 5,10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 micron MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 micron MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 micron MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5,10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.     

Effects of high hydrostatic pressure on normal and neoplastic rat cells in culture

Four types of rat cells in culture were exposed to hydrostatic pressures in the range 1-1,500 bar. Each applied pressure was constant for half an hour. The morphological effects of pressure application were studied by phase contrast microscopy, and mortality was measured by total cell counts and the trypan blue exclusion test. Morphological changes characterized by cell rounding were observed in secondary fetal brain cells and fibroblasts at about 700 bar. In two permanent neoplastic neurogenic cell lines similar changes occurred at 1,000 to 1,100 bar. When approximately 50% of the cells were rounded, mortality began to increase, as compared with controls. This was gradual in secondary cells and comparatively abrupt in the permanent neoplastic lines. Malignant cells in culture may therefore be more resistant to hydrostatic pressure than their normal counterparts.     

Expression of cell surface sialic acid and galactose by normal adult human melanocytes in culture

Normal adult human melanocytes grown either in the presence of phorbol ester or dialyzed hypothalamic extract were analyzed for their cell surface sialic acid and galactose content. In both cases, cells expressed large amounts of sialic acid, whereas they differed in their terminal nonreducing beta-D-galactosyl residues linked to N-acetyl galactosamine; such residues were accessible to peanut agglutinin and Bauhinia purpurea lectin on cells grown in phorbol ester and inaccessible on cells grown with dialyzed hypothalamic extract. In addition, striking differences in morphology and growth characteristics were observed between adult melanocytes grown with phorbol ester or with dialyzed hypothalamic extract. Thus, pure cultures of normal adult human melanocytes grown in the presence of dialyzed hypothalamic extract displayed cell surface properties different from those of melanocytes grown with phorbol ester. Cultures of melanocytes with dialyzed hypothalamic extract are likely to reflect known cell surface characteristics of human melanocytes in the skin. Such cultures could represent a useful model to study normal behavior and tumor progression of pigmented cells.     

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.     

Fibrinolytic activity associated with cultured human neoplastic and normal cells

Summary Fibrinolytic activity was studied in a number of different established as well as secondary human cell cultures derived from both malignant and normal tissues. The ability to degrade [¹²⁵I]-labeled fibrin was found to be characteristic of some malignant cultures as well as some normal cultures, and to be dependent upon the presence of serum. For the most part, this activity was detected in cultures with a relatively shortin vitro passage history (<30 passages). Low passaged colon and rectal carcinoma cells, HCT-8 and HRT-18, as well as normal rectal, colon and foreskin fibroblasts were positive for fibrinolytic activity, while long established (>100 passages) cultures of malignant cells (colon carcinoma, HeLa, Hep-2, KB) as well as normal cells (HEI, AV₃) were negative. It is proposed that although some normal cells synthesize plasminogen activators, the fibrinolytic capability of both malignant and normal cells may be lost on prolongedin vitro cultivation.     

Ultraviolet radiation-induced neoplastic transformation of normal human cells,in vitro

Human foreskin cell cultures in scheduled DNA synthesis (S phase) of the cell cycle were exposed to UV irradiation at a dose of 10 J · m^?2 in the presence of insulin. These treated cell populations, when selectively passaged in a high amino acid supplemented complete growth medium (CM) after 20 Dulbecco's phosphate buffered saline (pH 6.8) (PDL), were able to be grown in soft agar. These treated cell populations were also grown in 1% serum supplemented growth medium and at 41°C in 10% serum supplemented growth medium. Cell populations 4–5 PDL after treatment exhibited altered colony morphology and altered lectin agglutination profiles but would not grow in soft agar. These events appeared to be associated with the early stages in the expression phase of the transformed phenotype. After 20 PDL, we observed that these cells would grow in soft agar at a frequency of 20 colonies/10⁵ cells seeded in soft agar. The cell populations derived from these colonies, when propagated and injected into the nude mice, formed myxofibromas at the injection sites rather than the type of tumor (fibrosarcoma) previously described for chemical carcinogen-induced neoplasms.     

Radiosensitivity of normal human epidermal cells in culture

Using an in vitro culture system we have derived radiation survival curves for the clonogenic cells of normal human epidermis. The culture system used allows the epidermal cells to stratify and form a multi-layered sheet of keratinizing cells. The cultures appear to be a very good model for epidermis in vivo. The survival curves show a population which is apparently more sensitive than murine epidermis in vivo. It remains unclear whether this is an intrinsic difference between the species or is a consequence of the in vitro cultivation of the human cells.     

Growth of normal human mammary cells in culture

Summary Reduction mammoplasty tissue was used to obtain short-term cultures of human epithelial cell populations. Digestion of tissue with collagenase and hyaluronidase resulted in cell clusters (organoids) resembling ductal and alveolar structures; these could be separated by filtration from the stromal components. Epithelial outgrowth from these organoids was greatly enhanced by the addition of conditioned medium from other human epithelial and myoepithelial cell lines. Additionally, the mammary epithelial growth was stimulated by insulin, hydrocortisone, epidermal growth factor, and steroid hormones. With this enriched nutritional environment, active cell division could be maintained for 1 to 3 months and cells could be serially subcultured 1 to 4 times. This research was supported by Grant PDT-72 from the American Cancer Society and Grant CP-70510 from the National Institutes of Health.