Abstract: Certain metabolic properties of hormonally responsive osteogenic sarcoma cells derived from a transplantable rat tumor have been compared with those of related normal rat bone cells. All studies were carried out on cells grown in monolayer culture. Normal rat bone cells derived by repeated collagenase/trypsin digestion of newborn rat calvaria. Bone cells selected for comparison were thought to be osteoblast-like, as judged by enrichment of alkaline phosphatase and adenylate cyclase responsiveness to parathyroid hormone and prostaglandin E2. The adenylate cyclases of the two cell strains were similarly stimulated by a range of prostanoids and their metabolites and analogs. Morphology showed the two cell strains to be similar; the only obvious difference was a multilayering of cells in the sarcoma cultures, while the normal cultures showed abundant extracellular fibril formation which was not seen in the tumor cells. Investigation of the cAMP-dependent protein kinase isoenzymes showed the presence of two forms in both cell types, one eluting at a low salt concentration and the other at a high salt concentration. There was approximately twice the amount of the first isoenzyme compared to the second isoenzyme. The results indicate the usefulness of the two cell strains to elucidate further the molecular mechanisms of action of parathyroid hormone and prostaglandins.

Abstract: The mechanism of in vitro cytotoxicity by tumor necrosis serum (TNS) and a purified fraction was examined using sensitive L-M cells. Cell death was assessed by uptake of the dye trypan blue and/or by release of radiolabeled chromium. Cell killing was time and dose dependent. Cell survival was assessed by counting the number of survivors via their ability to internalize the dye neutral red and/or by adherent cell protein. The cytotoxin was not cytostatic. Survival was found to be inversely proportional to cell death. The change in survival was used to estimate the number of cells killed. The number of cells killed was logarithmically related to the amount of toxin. The ability to kill a fixed number of cells was inversely related to the number of cells in the assay well. It was estimated that, at ideal cell seed numbers, 1 µg of TNS protein per 250 µl killed about 50,000 cells in a 20-hr period. Sensitivity was equated with the amount of TNS required to kill 35,000 cells in 20 hr. Inhibitors of RNA and protein synthesis and also elevated temperatures enhanced sensitivity. The combined treatment of 1 µm actinomycin D and 40° enhanced sensitivity by 15-fold. Resistant normal and tumorigenic cell lines (including human) were rendered sensitive by concomitantly treating them with TNS and cycloheximide or actinomycin D. Cytoskeletal-disrupting agents (colchicine, Colcemid, and cytochalasin B), inhibitors of lysosome activity (chloroquine, methylamine, and leupeptin), and 32° all depressed sensitivity. Sensitivity was nearly equivalent in calcium-free medium. Results substituting partially purified cytotoxin were similar. The results suggest that a toxic factor(s) may need to be internalized and that lysosomal activity may be necessary for cell killing. Comparisons with lymphotoxin are discussed.

Abstract: The extent and the specificity of the initial cell attachment induced by various proteins coated on plastic surfaces have been studied with the following results: (a) Cell adhesion on the surfaces coated with sialidase and beta-galactosidase was as strong as on concanavalin A and limulus lectin-coated surfaces and the reactions were strongly inhibited by glycosidase inhibitors or by competitive substrates. The adhesion on sialidase was inhibited by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid and by polysialoganglioside (GT1b) at low concentration (0.05-0.1 mM). The cell adhesion on beta-galactosidase coat was inhibited by 1,4-D-galactonolactone and beta-methylgalactoside but not by alpha-methylgalactoside. Thus, the initiation of cell adhesion on glycosidase surfaces could be mediated through the interactions of the specific binding sites of the enzyme surface with the cell surface substrates under physiological conditions. (b) Cell adhesion on various lectins could be blocked by various competing monosaccharides at the concentrations similar to the inhibitory concentrations for binding of lectins from solution to the cells. (c) Cell adhesion on fibronectin surfaces as well as on gelatin-coated surfaces was equally inhibited by GT1b at relatively high concentrations (0.25-0.5 mM). Lower concentrations of GT1b (0.05-0.1 mM) inhibited the cell adhesion on surfaces of Limulus lectin and sialidase. It is suggested that the cell adhesion mediated by fibronectin is based on yet unknown interactions in contrast to a specific cell adhesion through glycosidases and lectins.

Abstract: In previous studies rat hepatocytes have been shown to adhere to substrates composed of collagen or fibronectin. In the present communication, the basement membrane protein laminin is reported to mediated the attachment and spreading of hepatocytes. The cell attachment-mediating activity of laminin was compared with that of fibronectin. The activity of fibronectin was heat sensitive, whereas laminin retained its activity after boiling. On the other hand, reduction and alkylation or periodate oxidation of the proteins affected only the cell attachment activity of laminin. Preincubation of cells with soluble fibronectin inhibited initial cell attachment to fibronectin but not to laminin substrates, and, reversely, soluble laminin selectively inhibited cell attachment to laminin. These results suggest that attachment of cells to substrates of the two proteins involves different cellular receptors recognizing distinct and nonidentical structures in the proteins.

Abstract: Monoclonal antibody against an osteogenic-sarcoma cell line (791T) was prepared by production and cloning of a somatic-cell hybrid between the mouse myeloma P3-NS1 and spleen cells from 791T-immunized mice. Three clones of hybridoma producing antibody against 791T, as detected by 125I-labelled Protein A binding, were tested against a range of normal and tumour cell targets to determine the pattern of expression of the antigen detected. The 3 clones had identical activity. They reacted strongly against 791T cells and another osteogenic sarcoma, 788T, and more weakly against a further 2 from a total panel of 10 osteogenic-sarcoma lines. The antibody was negative for fibroblasts from the donor of 791T, and for other fibroblasts, human red blood cells, human peripheral mononuclear cells and sheep red blood cells. When tested against a panel of unrelated tumours, they reacted against individual cell lines derived from carcinomas of colon, lung, bladder and cervix. These cross-reactions were not observed with other colon or lung carcinomas, and it is suggested that the antibody was reacting with a tumour-associated antigen expressed randomly on different tumour types, rather than specifically on osteogenic sarcomas.

Abstract: The metabolism of low-density lipoprotein (LDL) was studied in neoplastic and non-neoplastic cells of human gynecological origin, in monolayer cultures. The neoplastic cells were derived from epidermoid vaginal carcinoma, epidermoid cervical carcinoma and endometrial adenocarcinoma, in various degrees of differentiation. The non-neoplastic cells were cervical fibroblasts and epithelial cells from proliferative endometrial glands. Both neoplastic and non-neoplastic cells assimilated and degraded LDL in a similar fashion to other human cells (e.g. skin fibroblasts). However, the neoplastic cells metabolized LDL at a higher rate than the non-neoplastic cell (e.g. epidermoid cervical cancer cells metabolized LDL at a 20 times higher rate than did cervical fibroblasts). Such a high rate of LDL metabolism probably enables continuously replicating cancer cells to obtain the large amounts of cholesterol required for cell membrane synthesis. If a high rate of LDL metabolism proves to be a general property of cancer cells, such a property could prove useful for tumor chemotherapy, providing cytotoxic chemicals could be incorporated within the LDL molecule.

Abstract: Twenty micromolar hemin causes no change in the rate of division of K562 cells but results in accumulation of 11 to 14 picograms of embryonic and fetal hemoglobins per cell. This effect is reversible, and hemoglobin induction in response to hemin, and loss of hemoglobin upon removal of hemin, can be cyclically repeated. The cells can be indefinitely subcultured in the presence of the inducer. Thus, the control of hemoglobin levels in K562 cells does not depend on irreversible differentiation.

Abstract: In order to promote cell attachment, fibronectin must first undergo activation by a suitable substrate. In this study, 52 materials have been surveyed for their ability (a) to bind fibronectin, (b) to activate the cell-adhesive property of fibronectin, and (c) to support the growth of cells. Many plastics, polysaccharides, metals, and ceramics were found to support cell growth as well as the fibronectin-dependent attachment of cells. Several other substrates have been identified that were inactive in promoting either cell attachment or growth. Hydrophobic substrates were found to be active in fibronectin activation, whereas hydrophilic substrates were found to be inactive. Since fibronectin binds to substrata of extremely varied chemical composition, it is clear that the binding of fibronectin to such substrata is nonspecific in nature. Since protein pretreatment of all substrata, except collagen and poly(L-lysine), abolished the physical binding of fibronectin, the binding of fibronectin to artificial substrata is probably ascribable to a nonspecific hydrophobic protein-substratum interaction. In contrast, several lines of evidence indicate that the interaction between fibronectin and collagen displays biological specificity. Poly(hydroxyethylmethacrylate)(poly(HEMA)), which has previously been shown to be nonadhesive for cells, is demonstrated here to be unique in its inability to bind fibronectin. Addition of one part per million of an adhesive polymer to poly(HEMA) permits fibronectin binding to occur.

Abstract: The role of phospholipid methylation and phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) in natural killer (NK) function by human peripheral blood mononuclear cells was studied. Pretreatment of effector cells with a methyltransferase inhibitor, 3-deazaadenosine, in the presence of homocysteine thiolactone, reduced cytotoxicity in a dose-dependent fashion. This effect was closely associated with inhibition of methylation of lipids but not of nucleic acids or proteins. The suggestion for a role of phospholipid methylation was supported by the observation that the interaction between NK-susceptible tumor targets and peripheral blood mononuclear cells caused increased phospholipid methylation only when susceptible target cells were used. Phospholipase A2 was also implicated in human NK activity. Inhibitors of the enzyme such as tetracaine, mepacrine, Rosenthal's inhibitor, and corticosteroids impaired NK function. Rosenthal's inhibitor was also shown to exert an inhibitory effect on a purified NK-cell population obtained by the isolation of large granular lymphocytes on Percoll gradients. Peripheral blood mononuclear cells were also directly shown to display phospholipase A2-like activity, as measured by the decrease in radioactive arachidonate from prelabeled phospholipids, specifically phosphatidylcholine, in effector cells. These data suggest that enhanced phospholipid methylation occurs during the recognition function of NK cells. Consequent activation of phospholipase A2 might be involved in the mechanisms leading to lytic events within the target cell.

Abstract: Rhodamine 123, a fluorescent laser dye that is selectively taken up into mitochondria of living cells, was used to examine mitochondrial morphology in early-passage (young), late-passage (old), and progeric human fibroblasts. Mitochondria were readily visualized in all cell types during growth (mid-log) and confluent stages. In all cell strains at confluence, mitochondria became shorter, more randomly aligned, and developed a higher proportion of bead-like forms. Treatment of cells for six days with Tevenel, a chloramphenicol analog that inhibits mitochondrial protein synthesis, brought about a marked depletion of mitochondria and a diffuse background fluorescence. Cyanide produced a rapid release of preloaded mitochondrial fluorescence followed by detachment and killing of cells. Colcemid caused a random coiling and fragmentation of mitochondria particularly in the confluent stage. No gross differences were discernible in mitochondria of the three cell strains in mid-log and confluent states or after these treatments. Butanol-extractable fluorescence after loading with rhodamine 123 was lower in all cell strains in confluent compared to mid-log stages. At confluence all three cell strains had similar rhodamine contents at zero-time and after washout up to 24 h. At the mid-log stage, young cells contained more rhodamine initially and lost it more rapidly than old or progeria cells, in that order. The data indicate no gross derangement in the morphology or number of mitochondria in old and progeria fibroblasts but there is a reduction of protonmotive force evident in these cells at the mid-log stage that may be growth limiting.

Abstract: Transforming growth factors (TGFs) are growth-promoting polypeptides that cause phenotypic transformation and anchorage-independent growth of normal cells. They have been isolated from several human and animal carcinoma and sarcoma cells. One TGF is sarcoma growth factor (SGF) which is released by murine sarcoma virus-transformed cells. The TGFs interact with epidermal growth factor (EGF) cell membrane receptors. TGFs are not detectable in culture fluids from cells which contain high numbers of free EGF cell membrane receptors. SGF acts as a tumor promoter in cell culture systems and its effect on the transformed phenotype is blocked by retinoids (vitamin A and synthetic analogs). The production of TGFs by transformed cells and the responses of normal cells to the addition of TGFs to the culture medium raise the possibility that cells "autostimulate" their own growth by releasing factors that rebind at the cell surface. The term "autocrine secretion" has been proposed for this type of situation where a cell secretes a hormone-like substance for which it has external cell membrane receptors. The autocrine concept may provide a partial explanation for some aspects of tumor cell progression.

Abstract: In the current study, we examine the mechanism of suppression of cutaneous sensitivity (CS) responses to 4-hydroxy-3-nitrophenyl acetyl succinimide ester. Intravenous administration of haptenated syngeneic spleen cells induces a state of hapten-specific tolerance involving I-J bearing suppressor T cells that function at either the induction phase or the effector phase of the CS response. The effective phase suppressor cells (Tse) are genetically restricted by both Igh and H-2 region genes. However, a third cell population is also required in he immune lymphocyte population for immune suppression. This third cell population, termed Ts3, is an I-J+, cyclophosphamide-sensitive T cell, as shown by reconstitution experiments. Further, the Tse-Ts3 interaction is restricted by genes in he H-2 and Igh gene complexes. The results are discussed with respect to the pathway of cellular interactions leading to immuno suppression.

Abstract: Proliferation of single B cells was observed in response to the combination of LPS and DXS when spleen cells were placed into Terasaki plates at dilutions where the majority of wells containing cells contained one cell. Clones derived from a single cell exhibited varying morphologies and growth characteristics. Neither filler cells nor conditioned medium was required for these responses. Since isolated cells were stimulated, we conclude that B cells can be activated to clonal growth in the absence of accessory cells. Limiting dilution analysis showed that the frequency of cells responding to the combination of LPS + DXS was about 80% of the frequency of input B cells. When only LPS or DXS was present, the frequency of response was less than 25% and less than 5% of the input B cell frequency, respectively. This synergy in mitogenesis is consistent with previous kinetic and cytofluorometric analyses of mitogen-stimulated growth which have indicated that the combination of LPS + DXS removes requirements for cell interaction in growth induction and that B lymphocytes can be stimulated to growth directly.

Abstract: PHA-mediated mitogenesis of human peripheral blood T lymphocytes was studied by using highly purified cell populations. The kinetics of human, mature T cell [3H]-Tdr incorporation were examined with respect to those elements necessary and sufficient for the progression of the activated T cell into the S-phase of the cell cycle. These experiments indicated that although a lectin may independently initiate morphologic T cell blastogenesis, this event is not associated with significant progression through the G1 phase of the cell cycle. This blastogenic response is associated with the subsequent T cell receptivity to monocyte-initiated cell cycle progression, and the effect of monocytes can be substituted by partially purified Interleukin 1 (IL-1). Progression of a lectin exposed T cell into the S-phase of the cell cycle could also be achieved by exposing the activated T cell to partially purified Interleukin 2 (IL-2). Given the prior demonstrations that IL-1 functions to induce the T cell-dependent production of the IL-2, it appears that IL-2 is the requisite signal necessary for the activated human lymphocyte to actually progress through the prereplicative phase of the cycle into the S-phase.

Abstract: Two epithelial cell lines have been established from the duodenum (IEC-17) and the ileum (IEC-18) of outbred germfree Crl:CD(SD)GN rats. They have a very similar morphology and ultrastruture, a normal rat diploid karyotype, comparable growth rates, and a similar set of surface antigens as detected with monoclonal antibodies specific for intestinal epithelial cell surface proteins in vivo. Both cell lines do not grow in soft agar and when injected into syngeneic animals do not form tumors. The toxic effects of benzo(a)pyrene (BP) and 7,12-dimethylbenz(a)anthracene (DMBA) were studied in these two cell lines. The IEC-18 cells were found to be much more sensitive to both chemicals than the IEC-17 cells. In paticular, at concentrations greater than 1 ..mu..g/ml, DMBA completely and irreversibly inhibited cell proliferaion in the IEC-18 cells, whereas it produced a less marked and reversible inhibition in the IEC-17 cells. The metabolism of BP and DMBA to water-soluble products was studied; BP was apparently metabolized at similar rates in the two cell lines, whereas a striking difference was observed with DMBA, which was metabolized at a rate 10-15 times greater in the IEC-18 cells than that in the IEC-17 cells. These results suggest that cultured cell lines derived frommore » different portions of the intestinal tract have similar propertes in vitro, but they may have very different responses to chemical carcinogens.« less

Abstract: Membrane rigidity has been widely accepted as the dominant cause of reduced deformability both of ATP-depleted erythrocytes and erythrocytes containing excess calcium (Ca). However, recent studies have shown normal membrane deformability in ATP-depleted erythrocytes. In addition, Ca accumulation causes massive ion and water loss, and it has been shown that extensive dehydration causes an increase in intracellular viscosity with attendant loss of whole cell deformability. To obtain a detailed understanding of the processes accompanying ATP depletion and/or Ca accumulation that limit cell deformability, we have used a viscodiffractometric method to identify the cellular factors contributing to reduced whole cell deformability. Analysis of the influence of the suspending medium osmolality on deformability showed the presence of two independent processes. One was a Ca-independent reduction in cell surface area/volume ratio, resulting from the spheroechinocyte formation that follows total ATP consumption. The other was a Ca-dependent increase in intracellular viscosity resulting from a Ca-induced loss of intracellular potassium and water. This deformability loss due to increased intracellular viscosity was found for cells depleted of ATP in the presence of Ca and in cells treated with Ca and A23187 without prior depletion. Ionophore-treated cells at high Ca concentration (>500 μM) formed spheroechinocytes with reduced surface area and a further loss of whole cell deformability. The rate of deformability loss associated with Ca-induced spheroechinocytosis was much more rapid than that associated with ATP-depletion-induced spheroechinocytosis, suggesting different mechanisms for the morphologic changes. No major effects of altered membrane elasticity on the reduced deformability of either ATP-depleted or Ca-loaded cells were observed.

Abstract: Eleven cases of primary pancreatic adenocarcinomas have been investigated histochemically, immunohistochemically and with electron-microscopy. Endocrine-paracrine (EP) cells were present in six of these tumours. In one case numerous 5HT-enterochromaffin cells (EC) of the intestinal type and a few somatostatin immunoreactive D cells were found. Two cases contained insulin-immunoreactive cells and another case displayed glucagon-IR elements. In the remaining two cases argyrophilic cells were present. These findings demonstrate that polypeptide hormone or amine production is not restricted to islet cell tumours. It is suggested that both endocrine and exocrine components of the tumours studied might have derived from a common precursor.