An evaluation of techniques for labelling the surface proteins of cultured mammalian cells.

We have evaluated four techniques for labelling the surface proteins of cultured mammalian cells. The techniques are: (a) the lactoperoxidase system; (b) the pyridoxal phosphate-[3H]borohydride system; (c) the [3H]4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate sysem and (d) the galactose oxidase-[3H]borohydride system. The subcellular distribution of radiolabel produced by these technics has been evaluated by autoradiography at the light microscope level and by cellular fractionation. We find that while all four systems label the surface membranes in the majority of the cell population, they also heavily label internal sites in a small subpopulation of nonviable cells. The contribution of the internally labelled cells to further biochemical analysis may represent a severe problem in investigations which rely solely on surface labels for the study of plasma membrane organization.

Ornithine decarboxylase enzyme activity in human and hamster pancreatic tumor cell lines.

We measured ornithine decarboxylase (ODC) enzyme activity, protein and DNA levels in 4 pancreatic cancer cell lines, 2 derived from human tumors (COLO-357 and PANC-1) and 2 derived from hamster tumors (WDPaCa and PDPaCa). We measured the parameters in confluent stage conditions and in log-growth phase. We report that the enzyme levels in all cell lines are elevated with respect to normal pancreatic tissue. Half-life studies of ODC in the PDPaCa cell line indicate a 3-fold increase in half-life.

Comparative uptake and retention of adriamycin and N-benzyladriamycin-14-valerate in human CEM leukemic lymphocyte cell cultures.

N-Benzyladriamycin-14-valerate (AD 198) is a new lipophilic adriamycin (ADR) analogue that shows marked therapeutic superiority to ADR in murine tumor model systems yet differs mechanistically from ADR in a number of ways. Among its other properties, AD 198 produces a delayed but profound effect on cell-cycle progression and a pattern of continuing DNA damage in cultured cells briefly exposed to the drug. Using radiolabeled drug forms and radioassays combined with HPLC separation and fluorimetric detection techniques, aspects of drug accumulation, biotransformation, and retention in cultured human CEM leukemic lymphocytes were studied, in part to determine a possible pharmacologic basis for the latent effects seen with this drug. In addition, the cellular pharmacology of AD 198 and ADR were comparatively examined under identical experimental conditions. When CEM cells were incubated with drug at equi-growth inhibitory/minimally cytotoxic concentrations (AD 198, 1.0 microM; ADR, 0.1 microM), a number of differences were apparent. Under conditions of continuous 24-h drug exposure, a slow cellular accumulation and equilibration was observed with ADR (cell: medium equilibrium, 1:11 after 4-6 h), whereas the uptake of AD 198 was rapid and extensive (cell: medium equilibrium, 3:1 within 30 min). In drug-retention studies, when cells were pretreated at the same drug concentrations as before (AD 198 for 1 h; ADR for 4 h) and then transferred to drug-free media, both compounds re-equilibrated their intracellular drug content with the fresh media, losing about 50% of their respective anthracycline levels. Liquid chromatographic analysis of ADR-treated cultures under both sets of conditions showed the parent drug to be the only intracellular anthracycline species, whereas analysis of AD 198-treated cultures revealed two fluorescent signals corresponding to the parent drug and its 14-de-esterified biotransformation product, N-benzyladriamycin (AD 288). Levels of AD 288 rose from 2% of the total intracellular anthracycline content immediately on drug admixture to 61% following 24 h continuous drug exposure and to 69% at 24 h in cells exposed to drug for 1 h and then continued in drug-free media for 24 h. At all times, the balance of the intracellular anthracycline fluorescence was attributable to the parent drug; no ADR was detectable in AD 198-treated cells by either fluorescence detection or radioassay.(ABSTRACT TRUNCATED AT 400 WORDS)

Response of human HT-29 colorectal tumor cells to extended exposure to bromodeoxyuridine.

Effects of the extended exposure of a human colorectal tumor-cell line (HT-29) to bromodeoxyuridine (BrdUrd) were studied in anticipation of the clinical use of that agent to treat colorectal cancer, particularly as a regionally delivered radiosensitizer. We found that 72-h exposure to a concentration of BrdUrd that is estimated to be locally maintained in the liver (100 microM) was significantly cytotoxic with a 3-log reduction in survival. As measured by GC/MS-SIM method, incorporation of BrdUrd into DNA followed an unexpected time course in that continuous exposure to 10 microM BrdUrd resulted in maximal incorporation at 3 days, after which the extent of incorporated analog fell significantly (despite daily changes of the medium). This finding was apparently due to a greater rate of loss of BrdUrd from the medium at later time points. Flow cytometric analysis using an anti-BrdUrd antibody (IU-4) revealed that antibody binding also peaked and fell off with time. However, at exposure times of greater than 24 h, the timing and extent of this decline were significantly different than had been indicated by the GC/MS method. These results indicate that the quantitative relationship between antibody staining and BrdUrd incorporation changes as drug-exposure time increases and that quantitative studies of anti-BrdUrd antibody binding must be interpreted with caution, especially when extended drug-treatment protocols have been used.

Effect of retinoic acid on human neuroblastoma: correlation between morphological differentiation and changes in plasminogen activator and inhibitor activity.

The relationship between plasminogen activator (PA)/plasminogen activator inhibitor (PAI) activity and morphological differentiation was investigated in human neuroblastoma (NB) cells treated with retinoic acid (RA). Conditioned medium from nine NB cell lines and one closely related neuroepithelioma line was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. All NB cell lines were shown to secrete urokinase (UK)-type PA (mol. wt., 52 kDa), and all except two produced tissue PA (mol. wt., 65 kDa). Identification of the PAs was made based on molecular weight and sensitivity to inhibition by anti-UK and anti-tPA antibodies. Several cell lines expressed PA inhibitory molecules; two molecular-weight forms were observed (35 and 40 kDa) in different cell lines. Complex formation with [125]I-labelled proteases revealed specific binding with UK and trypsin but not thrombin, plasmin, or kallikrein. After treatment for 6 days with 1 microM RA, six of the cell lines exhibited an increase in cell-associated and/or secreted tPA activity, corresponding to morphological differentiation of the cells as manifested by extensive neurite outgrowth. A decrease in UK and UK-complex secretion was observed in several of these cell lines. Three cell lines exhibiting no detectable morphological alterations with RA treatment also showed no dramatic changes in PA/PAI activity. These results suggest that morphological differentiation of NB cells may be associated with alterations in the regulation of PA activity.

The use of restriction fragment polymorphisms to identify the cell line MCF-7.

We have recently documented variations in the level of N-ras gene amplification in independently passaged MCF-7 cells. To establish if these differences are due to a lack of genetic identity between current MCF-7 passages and the original cell line, we have performed karyological and restriction fragment length polymorphism (RFLP) analyses. Documentation of a combination of restriction fragment patterns which are characteristic of MCF-7 has been facilitated by an analysis of DNA from an early-passage of MCF-7 from the Michigan Cancer Foundation (MCF). In addition, such early passage cells and other MCF-7 lines also share a unique amplification of the N-ras oncogene. Using these criteria and karyotypic analysis it can be shown that a sample of MCF-7 obtained from the American Type Culture Collection (ATCC) was derived from a different individual than was the original MCF-7 cell line. It is important that researchers verify the relationship of current cell lines to the original MCF-7. Furthermore, the techniques described here provide a powerful tool which may be used to assess the identity of cell stocks.

Evidence for chemical differences in HLA-DR molecules on autologous acute lymphoblastic leukemia and B-lymphoblastoid cell lines.

HLA-DR molecules on autologous acute lymphoblastic leukemia (ALL) and B-lymphoblastoid cell lines from two individuals were compared by immune precipitation and gel electrophoresis. Cells were surface labeled with 125I and proteins immunoprecipitated with specific monoclonal antibodies (MoAb). Two electrophoretically distinct bands were found in the HLA-DR beta chain region on both ALL cell lines in contrast to only one on each of the autologous B-lymphoblastoid cell lines. Differences in the electrophoretic mobility of the alpha chains were also observed with ALL and B-lymphoblastoid cell lines from one individual. Preclearing of radiolabeled cell lysates with MoAb specific for HLA-DQ and -DP molecules demonstrated that the complexity of the HLA-DR pattern is not the result of antibody cross-reactivity with alpha and beta chains from other class II products. Immunoprecipitation experiments indicated that two beta chain bands are observed with each of the parental HLA-DR molecules on the ALL but not the B-lymphoblastoid cell line from an HLA-DR3,7-positive individual. We conclude that the HLA-DR molecules expressed on ALL and B-lymphoblastoid cell lines from the same individual can differ chemically. Neuraminidase treatment reduced these electrophoretic differences, indicating that these molecules differ in their sialic acid content. Since small changes in class II molecules can profoundly alter cellular interactions, the functional significance of these differences requires further investigation.

Study of some stages of poliovirus morphogenesis in MiO cells.

Five to seven minutes after addition of (14)C-amino acids to poliovirus-infected MiO cells, radioactivity is found only in 5S virus-specific structures. With increased time of labeling, radioactivity appears in considerable amounts also in the 14S area. In the presence of guanidine blocking the transition of 14S particles in subsequent structures in MiO cells, radioactivity is first detected in the 5S zone and then accumulates predominantly in 14S particles. These results indicate that formation of 5S particles and conversion of a part of their protein into 14S particles reflect the earliest stages of the morphogenesis of poliovirus. In contrast to poliovirus-infected HeLa cells, no 73S particles could be detected in MiO cells, nor could 73S structures be detected in poliovirus-infected MiO cells after a short incubation period (20 to 30 min) with radioactive amino acids, although formation of 150S virions was observed. Addition of guanidine to infected MiO cells leads to accumulation of 14S particles; however, 73S particles are not detected. After removal of guanidine, radioactivity increases only in the 150S area. Incubation of 14S particles isolated from the sucrose gradient of the cytoplasmic extract of infected MiO cells resulted in formation of 73S particles. The results obtained show the modification of the late stages of poliovirus morphogenesis in MiO cells.

Demonstration of infectious deoxyribonucleic acid in transformed cells. I. Recovery of simian virus 40 from yielder and nonyielder transformed cells.

Deoxyribonucleic acid (DNA) was extracted from virus-free simian virus 40 (SV40)-transformed hamster, mouse, and monkey cells and was inoculated into simian cells in the presence of diethylaminoethyl (DEAE)-dextran; infectious SV40 was recovered by using DNA from cell lines which fail to yield virus by the fusion technique as well as from cell lines which readily yield virus by fusion. The rescued virus was identified as SV40 by three methods: (i) neutralization of plaque formation by specific antiserum; (ii) induction of synthesis of viral-specific antigens detected by immunofluorescence; and (iii) presence of papovavirus particles seen by the electron microscope. Treatment of the transformed cell DNA with deoxyribonuclease or omission of the DEAE-dextran prevented the rescue of virus. Large amounts of transformed cell DNA were required (>10 mug/culture of 10(6) cells) to effect rescue of SV40 by passage through monkey cells. A linear response was obtained between the input of DNA with inocula between 10 and 45 mug of DNA/culture and the yield of SV40 recovered. Biological activity was demonstrable irregularly when the transformed cell DNA was assayed directly in the presence of DEAE-dextran. The DNA induced plaque formation in about 50% of the trials as well as the synthesis of SV40 tumor and viral antigens in rare simian cells. The infectious DNA appeared to be associated with cellular DNA. The infectivity was found in the pellet of precipitated DNA obtained by the Hirt technique and was inactivated by boiling for 15 min. These properties are characteristic of linear cellular DNA and not of free, circular SV40 DNA.

Fatty chains of different lipid classes of Semliki forest virus and host cell membranes.

Semliki Forest virus was grown in BHK-21 cells. The major classes of phospho-and glycolipids of the virus were analyzed for the compositions of fatty acids, aldehydes, and sphingosine bases, and the major glycerophospholipids were analyzed for the relative proportions of alkenyl-acyl, alkyl-acyl, and diacyl forms. All viral lipid classes proved to be mixtures of several molecular species. Each class contained a characteristic mixture of fatty chains, which was different in all other classes. All viral lipid classes resembled their counterparts of the host plasma membrane and also those of the endoplasmic reticulum. The gangliosides of the virus and the plasma membrane proved to be similar even at the level of individual molecular species. The number of certain lipid molecules in an average virion was less than the number of the protein molecules.

Immunological studies on viral polypeptide synthesis in cells replicating murine sarcoma-leukemia virus.

Antibodies to disrupted murine sarcoma-leukemia virus (MSV[MLV]) were used to study the synthesis of viral polypeptides in the transformed, virus-producing rat cell line 78A1. When cultures were labeled for 10 min with radioactive amino acids, about 9% of the total labeled proteins were precipitated with antiserum against purified MSV(MLV), and 3 to 4% were precipitated with the same antiserum after it had been absorbed with an extract from uninfected rat cells. The difference is due to the presence in the unabsorbed antiserum of antibodies to cellular proteins that are present in purified virus preparations. Intracellular viral proteins labeled with radioactive amino acids were isolated by immunoprecipitation and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The mobilities of intracellular viral polypeptides were identical to those of the purified virion. However, labeled polypeptides having electrophoretic mobilities lower than that of the major virion polypeptide, the group-specific antigen of molecular weight 31,000, were present in higher proportion in the total cell extract and in the membrane fraction than in the virion. These polypeptides appear to be of cellular origin for they were present only in minute amounts in the immunoprecipitates obtained with the absorbed serum. After a 10-min labeling period, radioactive proteins were assembled into extracellular virions rapidly for the first 4 hr followed by a slower rate. More than 2% of the total proteins of the cell labeled in a 10-min pulse were assembled into virions at the completion of a 24-hr chase. The high-molecular-weight polypeptides with the same mobilities as those detected in the immunoprecipitate of intracellular proteins were found in virions released from cells after a 10-min pulse. A larger proportion of these high-molecular-weight proteins was detected in virions released after short chase periods (30-120 min) than after longer chase periods (6-24 hr). Two possible interpretations of these data are that the high-molecular-weight cell-derived polypeptides (i) have a turnover rate higher than that of the major virion polypeptides or (ii) are cleaved proteolytically from the virions during long incubation in the culture media.

Contact-inhibited revertant cell lines isolated from simian virus 40-transformed cells. 3. Concanavalin A-selected revertant cells.

Contact-inhibited variants have been isolated by treatment of simian virus 40 (SV40)-transformed Balb/c 3T3 cells (SVT2) with the plant lectin concanavalin A. These con A revertant cells exhibit the following properties: (i) they resemble 3T3 cells morphologically and grow to saturation densities which are similar to that of 3T3 cells; (ii) they synthesize the SV40-specific T antigen and yield infectious virus after fusion with permissive monkey cells; (iii) they contain a high sialic acid content similar to that of 3T3 cells and not to that of SVT2 cells; sialic acid composition was found to be independent of serum concentration; (iv) they contain more chromosomes with the average number in the tetraploid range than the SVT2 cells from which they were derived; and (v) SVT2 and revertant cells, confluent or subconfluent, produce more collagen than Balb/3T3 cells. The relationship of surface membrane properties to contact inhibition of growth and the mechanisms for generating revertant cells are discussed.

Establishment and characterization of cell line SFCC from clear cell adenocarcinoma of the uterine cervix.

A human cervical clear cell adenocarcinoma cell line was established and named SFCC. The tumor cells were obtained from ascites of a 39-year-old woman who underwent a radical hysterectomy. She had no history of exposure to diethylstilbestrol (DES). The histologic feature of the tumor cells at autopsy showed abundant clear cytoplasm with diastase digested glycogen granule growing in solid nest and tubular pattern. SFCC cells were continuously propagated in vitro during the past 29 months and grown in a monolayered sheet with a doubling time of about 67 hours. SFCC cells resembled the structure of the original tumor and had abundant glycogen granules, lipid droplets in the cytoplasm, and numerous microvilli. Immunohistochemically, SFCC cells had carcinoembryonic antigen (CEA) immunoreactivity in some parts of the cell population. Moreover, they had a relatively high amount of progesterone receptor (20.0 fmol/mg protein; kd, 6.6 nM) but did not have either an estrogen receptor or EGF receptor. The SFCC cell line secreted a high content of tissue peptide antigen (TPA) into the medium, indicating that the SFCC cell line is useful for analyzing the progesterone receptor and TPA production in clear cell adenocarcinoma of uterine cervix.

Tumour cells which develop resistance to cytolysis by tumour necrosis factor have a different glycoform of a 105-kDa glycoprotein and lose the capacity to invade and metastasize.

A plastic-adherent variant of human myelomonocytic leukaemia cells (U937) is highly susceptible to direct TNF cytolysis in vitro. Previously, we found that a subline selected for resistance to TNF cytolysis (U937/R) was much less motile and more plastic-adherent than the parental line. In the present study we show that U937 and U937/R cells have different glycoforms of a 105-kDa cell-surface glycoprotein. This protein is predominantly N-glycosylated and has the physicochemical properties of the LAMP-I glycoprotein. In nude mice, U937 cells are highly malignant whereas U937/R cells form a benign, encapsulated tumour. Therefore, possession of a different glycoform of the 105-kDa glycoprotein by U937/R cells correlates not only with loss of TNF susceptibility but also with reduced invasiveness and metastasis.

Effect of VIP on the glycogen metabolism of human colon adenocarcinoma cells studied by 13C nuclear magnetic resonance spectroscopy.

Metabolic pathways of glucose utilization have been investigated in a human colon adenocarcinoma cell line (HT29) using carbon-13 Nuclear Magnetic Resonance spectroscopy. HT29 cells were adapted to grow on a polystyrene beaded microcarrier and were perfused when attached to the beads in a specially designed NMR cell. Abnormalities in carbohydrate metabolism already observed in several cancer cells were studied in HT29 cells fed with (1-13C)-enriched glucose. The cells were first perfused with a glucose-free medium for 2 h in order to deplete the intracellular store of glycogen, and they were subsequently perfused with a medium containing enriched glucose at an initial concentration of 5.5 mM. Sequential 13C-NMR spectra, recorded at 100.5 MHz (5 min accumulation), show that HT29 cells were able to utilize glucose through the glycolytic pathway while storing glucose as glycogen (glucose was utilized at a rate of 3.9 mumol/mg protein/hr). The glycolytic activity determined by the amount of lactic acid produced was 4.6 microns/mg protein/hr, corresponding to the formation of 1.2 lactic acid per glucose molecule. Glycogen accumulation corresponded to 16 micrograms/mg of protein. Treatment of HT29 with 10 nM vasoactive intestinal peptide (VIP) induced a transient decrease in the level of labelled glycogen to 50% of the initial value. Control level was recovered 12 min after VIP loading.

Inhibition of growth and squamous-cell differentiation markers in cultured human head and neck squamous carcinoma cells by beta-all-trans retinoic acid.

Vitamin A and some of its metabolites such as beta-all-trans retinoic acid (RA) have been implicated in the regulation of differentiation of normal and malignant epithelial cells in vivo and in vitro. In the present study the effects of RA on the growth and differentiation of 7 cell lines derived from human head and neck squamous-cell carcinomas (HNSCCs) were examined. RA (greater than 0.01 microM) inhibited the proliferation in monolayer culture of 6 of 7 HNSCC cell lines. One cell line (UMSCC-35) was very sensitive, 5 (UMSCC-10A, -19, -30, -22B and HNSCC 1483) were moderately sensitive, and 1 (HNSCC 183) was insensitive. Three of the cell lines (UMSCC-22B, -30, and HNSCC 1483) were capable of forming colonies in semisolid medium--a capability that was suppressed by RA. The HNSCC cell lines expressed various levels of the squamous-cell differentiation markers type I (particulate, epidermal) transglutaminase (TGase) and cholesterol sulfate (CS). RA treatment (I microM, 6 days) decreased TGase activity by more than 50% in 3 (UMSCC-10A, -22B and 1483) of the 7 cell lines, and the effect on UMSCC-22B was dose-dependent. Type II TGase (soluble, tissue type) activity was detected in 3 cell lines, and after RA treatment its activity increased in HNSCC 1483 and 183 cells and decreased in UMSCC-19. Following RA treatment, CS levels decreased by 20, 25, 70, 76, 89 and 91% in cell lines UMSCC-30, -10A, 183, UMSCC-35, -22B, and HNSCC 1483, respectively. The suppression by RA of CS accumulation in the 1483 cells was dose-dependent. Cholesterol sulfotransferase activity, which is responsible for CS synthesis, was suppressed by 40-97% after RA treatment of UMSCC-19, -22B, and HNSCC 1483. Our results demonstrate that RA inhibits the growth and decreases the level of 2 squamous differentiation markers in HNSCC cells.