Showing papers on "Cell culture published in 1976"

Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.

Abstract: A single cell clonal line which responds reversibly to nerve growth factor (NGF) has been established from a transplantable rat adrenal pheochromocytoma. This line, designated PC12, has a homogeneous and near-diploid chromosome number of 40. By 1 week's exposure to NGF, PC12 cells cease to multiply and begin to extend branching varicose processes similar to those produced by sympathetic neurons in primary cell culture. By several weeks of exposure to NGF, the PC12 processes reach 500-1000 mum in length. Removal of NGF is followed by degeneration of processes within 24 hr and by resumption of cell multiplication within 72 hr. PC12 cells grown with or without NGF contain dense core chromaffin-like granules up to 350 nm in diameter. The NGF-treated cells also contain small vesicles which accumulate in process varicosities and endings. PC12 cells synthesize and store the catecholamine neurotransmitters dopamine and norepinephrine. The levels (per mg of protein) of catecholamines and of the their synthetic enzymes in PC12 cells are comparable to or higher than those found in rat adrenals. NGF-treatment of PC12 cells results in no change in the levels of catecholamines or of their synthetic enzymes when expressed on a per cell basis, but does result in a 4- to 6-fold decrease in levels when expressed on a per mg of protein basis. PC12 cells do not synthesize epinephrine and cannot be induced to do so by treatment with dexamethasone. The PC12 cell line should be a useful model system for neurobiological and neurochemical studies.

Establishment and characterization of a human histiocytic lymphoma cell line (U‐937)

Abstract: A human hematopoietic cell line (U-937) with exceptional characteristics was derived from a patient with generalized histiocytic lymphoma. The morphology of the cell line was identical to that of the tumor cells in the pleural effusion from which the line was derived. Since Epstein-Barr virus (EBV) carrying diploid lymphoblastoid cell lines unrelated to the tumor population often become established in vitro from non-Burkitt lymphoma explants, several parameters were studied to discriminate the U-937 from such lines: morphology in vitro, growth characteristics, cytochemistry, surface receptor pattern, Ig production, lysozyme production, beta2-microglobulin production, presence of EBV genome and karyotype. In all these respects U-937 differed from prototype lymphoblastoid cell lines. The histiocytic origin of the cell line was shown by its capacity for lysozyme production and the strong esterase activity (naphtol AS-D acetate esterase inhibited by NaF) of the cells. It is therefore concluded that the U-937 is a neoplastic, histiocytic cell line.

Fusion between immunoglobulin-secreting and nonsecreting myeloma cell lines.

Abstract: The defects in two nonsecreting variant clones of the mouse plasmacytoma MOPC 21 (P3) were studied by tissue culture methods. The variants (NSI and NSIII) do not synthesize detectable heavy chains. NSI synthesizes, but does not secrete, light chains and NSIII does not synthesize light chain. A screening procedure was used allowing the detection of revertant cells secreting immunoglobulin. The method is based on a hemolytic plaque assay using anti-immunoglobulin-coated red cells. No revertants were detected among 2 x 10(7) cells. Both variant lines were fused to another myeloma line (PI) which secretes a complete immunoglobulin and excess light chains. Analysis of the products by isoelectric focusing showed that in the hybrids there was no reactivation of synthesis of the nonexpressed chains. The defects leading to loss of synthesis cannot therefore be complemented in the hybrid lines. The secretion of light chain in NSI, on the other hand, could be complemented in the hybrid but the light chain was only secreted as part of a new immunoglobulin hybrid molecule.

The origin and characteristics of a pig kidney cell strain, LLC-PK.

Abstract: A stable epithelial-like pig kidney cell strain has been established. This strain has been carried through more than 300 serial passages, has remained free of microbial and viral contaminants, and has retained a near diploid number of chromosomes. Attempts to produce tumors with these cells in immunosuppressed laboratory animals have been uniformly negative. The cells have grown rapidly in monolayer cultures with a split ratio of 1 to 15 at weekly intervals, but have failed to proliferate in suspension cultures. A subline adapted to growth on serum-free medium 199 has been carried through 145 passages on this medium. Several unusual morphologic features have been observed in these cultures including three-dimensional “domelike” structures. These cells have been found susceptible to some viruses and have been especially useful for viruses of domestic animals. LLC-PK1 cells have produced significant levels of plasminogen activator.

The relationship between in vitro cellular aging and in vivo human age.

Abstract: Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging". However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflect human cellular aging in vivo. Studies were therefore undertaken to determine if significant differences would be observed in the in vitro lifespans of skin fibroblast cultures from old and young normal, non-hospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in vivo. Statistically signigificant (P less than 0.05) decreases were found in the rate of fibroblast migration, onset of cell culture senescence, in vitro lifespan, cell population replication rate, and cell number at confluency of fibroblast cultures derived from the old donor group when compared to parallel cultures from young donors. No significant differences were observed in modal cell volumes and cellular macromolecular contents. The differences observed in cell cultures from old and young donors were quantitatively and qualitatively distinct from those cellular alterations observed in early and late passage WI-38 cells (in vitro "aging"). Therefore, although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging.

Production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells

Abstract: We have studied cells dispersed with proteolytic enzymes from rheumatoid arthritic synovectomy specimens to determine the cell type(s) responsible for joint destruction. Initially 10-50% of these cells adhered to culture dishes within 24 hr and were of two main types: small, round cells and larger, stellate cells. During 1-4 days of culture, 5-25% had Fc receptors and 25-50% showed brisk phagocytosis. Daily producition, per 10(6) cells of collagenase (EC 3.4.24.3) (after trypsin pretreatment) was up to 70 mug of collagen fibrils lysed per min at 37 degrees (70 units), of prostaglandin (PGE2), up to about 1200 ng, and of lysozyme, up to about 100 mug. Under identical conditions of assay, fibroblasts grown from explants of synovium produced no detectable collagenase or lysozyme, and PGE2 was only 2-4 ng. With the dispersed cell preparations, macrophage markers (Fc receptors and lysozyme) were undetectable after 4 days and PGE2 decreased rapidly after about 7 more days. However, collagenase production continued for 3-25 weeks, and in some cultures, after cell passage. At these later stages, large, slow-growing stellate cells were predominant and could phagocytose carbon particles if incubated for greater than 6-8 hr. Indomethacin (14 muM) inhibited PGE2 but stimulated collagenase production whereas dexamethasone (10 nM) inhibited both. Production of PGE2 and collagenase in large amounts in vitro by these cells suggests that they may be involved in joint destruction in vivo. The precise origin of these synovial cells and the mechanisms responsible for the sustained production of collagenase at a high rate remain unidentified.

Transepithelial transport in cell culture

Abstract: In cell culture a kidney epithelial cell line MDCK, forms a continuous sheet of identically oriented asymmetrical cells joined by circumferential occluding junctions. The reconstructed epithelial membrane has transport and permeability qualities of in vivo transporting epithelia. The cell layer can be readily manipulated when cultured on a freely permeable membrane filter and, when placed in an Ussing chamber, electrophysiological measurements can be taken. In the absence of a chemical gradient, the cell layer generates an electrical potential of 1.42 mV, the apical surface negative. It is an effective permeability barrier and lacks significant shunting at the clamped edge, as indicated by a resistance of 84 ohms-cm2, which increased when bulk flow from basolateral to apical was induced by an osmotic gradient or electroosmosis. The MDCK cell layer is cation selective with a relative permeability ratio, PNa/PCl, of 1.7. Net water flux, apical to basolateral, was 7.3 mul cm-2 hr-1 in the absence of a chemical gradient. The morphological and functional qualities of a transporting epithelium are stable in cell culture, and the potential use of a homogeneous cell population in cell culture would enhance studies of epithelial transport at the cellular and subcellular levels.

The Effects of Methyl [5-(2-Thienylcarbonyl)-1H-benzimidazol-2-yl]carbamate, (R 17934; NSC 238159), a New Synthetic Antitumoral Drug Interfering with Microtubules, on Mammalian Cells Cultured in Vitro

Abstract: Ultrastructural investigations on mammalian cells cultured in vitro show that R 17934, a new synthetic anticancer drug, interferes with the structure and function of microtubules, both in interphase and mitotic cells. The activity of this compound in a wide range of experimental tumor systems can thus be explained partly as a direct antimitotic effect and partly as the disintegration of the normal subcellular organization of the nondividing cells. Preliminary investigations in experimental animals show that malignant cells are more susceptible to the antimicrotubular effect of R 17934 than are the nonmalignant cells of the host.

Replacement of serum by hormones permits growth of cells in a defined medium

Abstract: MOST cell cultures require the addition of serum to synthetic media for their maintenance and growth, and we believe that the primary role of the serum is to provide hormones1. We have been led to this hypothesis by a series of experiments showing that serum depleted of certain hormones no longer supports growth of cells, unless the medium is supplemented with the hormones that were removed2–4. Clear evidence for the validity of this hypothesis has not yet been obtained because it is difficult to grow cells in the absence of serum. Recently, however, we have succeeded in growing an established rat pituitary cell line, GH3, in a defined serum-free medium supplemented with physiological concentrations of four hormones together with the iron transport protein, transferrin. Preliminary investigation shows that serum-free medium supplemented with hormone will also support the growth of several other cell lines.

Properties of the K562 cell line, derived from a patient with chronic myeloid leukemia

Abstract: The K562 cell line derived from a CML patient in blast crisis was examined for properties of B and T lymphocytes and cell lines. K562 lacks the B markers of immunoglobulin, Epstein-Barr virus (EBV) genome and associated nuclear antigen, and receptors for EBV. A low proportion of cells form rosettes with sheep erythrocytes, the frequency of which is considerably increased after neuraminidase treatment. Unlike B lines but like T lines, K562 cells are lysed rapidly by C'/Fc receptor-positive human blood leukocytes and do not stimulate MLC reactions. On the other hand, K562 lacks T antigen, high radiosensitivity and sensitivity to growth inhibition by thymidine. The cells do not contain N-APase, an enzyme found in all lines derived from lymphoid cells and in lymphoproliferative diseases. By scanning electron microscopy, K562 cells were seen to be rounded and relatively smooth, with small numbers of short microvilli resembling undifferentiated leukemic cells. A few cells had narrow ridge-like profiles and small ruffles similar to granulocytic leukemic cells. K562 is strongly positive for immunoglobulin Fc receptors and pinocytosis, but does not phagocytose or mediate antibody-dependent phagocytosis or cytolysis. Among histochemical stains, K562 is positive for esterase, lipid, and acid phosphatase. There seems to be no doubt that K562 is not a B cell line. While it has some T cell properties, these are not exclusive. Some of its characteristics indicate that it is probably not lymphoid. Due to its low level of differentiation, its nature cannot be stated with certainty. On the basis of the possible presence of the cellular marker of chronic myeloid leukemia, the Ph chromosome, it may be regarded as belonging to the granulocytic series of cells. Propretes de la lignee k562, qui provient d'un sujet atteint de leucemie myeǐotde chronique La lignee K562, qui provient d'un sujet atteint de CML en crise blastique, a ete etudee du point de vue des proprietis des lymphocytes B et T et d'autres lignees cellulaires. Elk ne possede ni les marqueurs B de I'immunoglobuline, ni le genome du virus d'Epstein-Barr (EBV), ni l'antigene nucliaire qui h i est assocei, ni les recepteurs de I'EB V. Une faible proportion des cellules forment avec les erythrocytes de mouton des rosettes dont la frequence s'accrǐt considerablement aprds traitement a la neuraminidase. A la difkrence des lignees Bmais comme les ligntes T, les cellules K562 sont lysies rapidement par les leucocytes du sang humain portant des rkcepteurs C'IFc et ne stimulent pas les reactions en MLC. Par contre, elks nepossbdentpas l'antigbne Tet ne sont pas fortement radiosensibles; leur croissance n'est pas inhibke par la thymidine; elles ne contiennent pas de N-APase, une enzyme prisente dans toutes les lignies provenant de cellules lymphoides et dans les maladies Iymphoproliferatives. Au microscope eectronique ´ balayage, on constate que les cellules K562 sont arrondies, relativement lisses, avec quelques courtes microvillosites qui les font ressembler a des cellules leucemiques indifferenciees. Quelques cellules ont des protuberances en forme d'arětes etroites et de petites rides comme les cellules leucemiques granulocytaires. Pour les recepteurs Fc de l'immunoglobuline et la pinocytose, la lignee K562 est fortement positive. Elle ne phagocyte pas et ne declenche pas de phagocytose ou de cytolyse dependant de l'anticorps. Dans les tests de coloration histochimique, on a constate que la lignee K562 est positive pour l'esterase, les lipides et la phosphatase acide. Il ne s'agit incontestablement pas d'une lignee de cellules B. Si elle a quelques-unes des proprietes des cellules T, elle a aussi d'autres caracteristiques, dont certaines indiquent qu'elle n'est probablement pas lymphoide. Du fait de son faible niveau de differenciation, on ne peut pas determiner sa nature avec certitude. En raison de la presence possible du marqueur cellulaire de la leucemie myeloide chronique, le chromosome Ph, on peut considerer qu'elle appartient a la serie granulocytaire.

Metaphase chromosome anomaly: association with drug resistance and cell-specific products

Abstract: Large, homogeneously staining chromosome regions which lack the longitudinal differentiation ordinarily revealed by cytogenetic "banding" methods have been found in antifolate-resistant Chinese hamster cells and also in human neuroblastoma cells established in vitro. The drug-resistant cells are characterized by excessive production of the target enzyme, dihydrofolate reducatase, while the human neuroblastoma cells have phenotypes of normal neuronal cells. The homogeneously staining region appears to represent a novel metaphase chromosome anaomaly which may have functional significance in cells with specialized properties.

Human epidermal growth factor and the proliferation of human fibroblasts.

Abstract: The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of 3H-thymidine incorporation after 20-24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.

Human colonic adenocarcinoma cells. I. Establishment and description of a new line.

Abstract: A series of human colonic epithelial cell lines have been cultured from a single patient: LS-180 the original adenocarcinoma, LS-174T a trypsinized variant, and normal colonic tissue. The malignant cells, 20 to 40, mum in diameter and oval to polygonal, exhibited characteristics of normal colonic mucosal cells, namely, abundant microvilli prominent in secretory cells, and the presence of intracytoplasmic mucin vacuoles. The cultured adenocarcinoma cells, but not normal, demonstrated neoplastic properties by producing high levels of carcinoembryonic antigen (CEA) and by the ability to be propagated in hamster cheek pouches and in immunodeprived mice. The CEA production by the newly established line LS-180 released 900 times more CEA per cell into the culture medium and bore 30 times more cell-associated material than the established line, HT-29. These cell lines may permit detection of distinctive chemical, physiological, pharmacologic, and immunologic characteristics of neoplastic colonic cells.

Establishment of a continuously growing cell line from primary carcinoma of the liver.

Abstract: A continuously growing cell line has been established from autopsy material taken from a primary liver carcinoma, a cancer which has a high incidence in Southern Africa. The cell line was initiated from multifocal areas of outgrowth in the primary culture, and adaptation to in vitro conditions was completed after 18 months. The cells resemble hepatocytes in culture, have a doubling time of 35-40 hours and a plating efficiency of 40-50%. No virus particles have been found in the cells by ultrastructural examination. The isozyme pattern and the karyology of the cells are human. The chromosome pattern is heteroploid (mean number 56) and there are marker chromosomes.

Effect of sodium butyrate on mammalian cells in culture: a review.

Abstract: Sodium butyrate produces reversible changes in morphology, growth rate, and enzyme activities of several mammalian cell types in culture. Some of these changes are similar to those produced by agents which increase the intracellular level of adenosine 3′,5′-cyclic monophosphate (cAMP) or by analogs of cAMP. Sodium butyrate increases the intracellular level of cAMP by about two fold in neuroblastoma cells; therefore, some of the effects of sodium butyrate on these cells may in part be mediated by cAMP. Sodium butyrate appears to have properties of a good chemotherapeutic agent for neuroblastoma tumors because the treatment of neuroblastoma cells in culture causes cell death and “differentiation”; however, it is either innocuous or produces reversible morphological and biochemical alterations in other cell types.

Establishment of a human carcinoembryonic antigen-producing colon adenocarcinoma cell line.

Abstract: A human carcinoembryonic antigen-producing colon carcinoma cell line has been established The cells form acinar structures and signet ring cells The lumen of the acini presents microvilli and a glycocalyx Neighboring cells show desmosomes and terminal bars The cells present an aneuploid karyotype with a modal number of 49 No marker chromosomes are found, although a significant proportion of cells show an altered A2 chromosome and an extra B Exponentially growing cultures produce 54 ng of carcinoembryonic antigen/10(6) cells Kinetic parameters are as follows: doubling time, 37 hr; mitotic index, 08%; labeling index, 31%; generation time, 30 hr; G1 phase, 7 hr; S phase, 18 hr; G2 phase, 5 hr; growth fraction 90% This cell line, designated line LoVo, represents an in vitro model for human colon carcinoma

Hepatitis B surface antigen produced by a human hepatoma cell line.

Abstract: The human hepatoma cell line, PLC/PRF/5, was shown to produce hepatitis B surface antigen (HBsAg). Immunologically reactive material was present in the supernatant tissue culture medium in significant amounts, and was associated with spherical particles approximately 20 nm in diameter. The rate of antigen production by the cells was estimated at 500 ng/day/10(6) cells by reference to a purified HBsAg standard. All immunological activity was neutralized by specific antibody and the subtype was ad. The studies reported here broaden the scope of investigations on both the in vitro production of HBsAg and the association between this antigen and primary liver cancer.

Organ Selectivity for Implantation Survival and Growth of B16 Melanoma Variant Tumor Lines

Abstract: The fate of bloodborne malignant melanoma cells selected for their enhanced ability to form lung colonies was examined to determine how specific tumor cells are arrested in certain organs during the experimental metastasis process. After murine B16 melanoma variant tumor cell lines with low (B16-F1) or high (B16-F10) survival and growth potential in vivo were admisistered by iv or intracardiac injections into syngeneic C57BL/6 mice, the quantitative distribution of [125l]5-iodo-2'-deoxyuridine (125IUDR)-labeled cells in the organs and subsequent formation of metastatic lung colonies were assessed. The initial distribution of viable tumor cells was dependent on the route of injection: Soon after iv injection, more 125IUDR-labeled B16 cells were localized in the lungs and fewer in the blood and other organs than after intracardiac injection. However, 1 day after the injection, the number of viable tumor cells in the lungs was independent of the route of injection, and at 14 days the quantity of resulting lung tumor colonies was similar. Variant line B16-F10 cells were better arrested and formed more tumors per input cell than B16-F1, regardless of the injection route. B16-F10 yielded only lung tumor colonies, whereas B16-F1 formed some extrapulmonary tumor growths. The results suggested that the ultimate fate of circulating tumor cells was not determined solely by nonspecific arrest in the capillary bed of the first organ encountered, and that in vivo selection could produce tumor line variants with organs preferences.

An improved technique for obtaining enhanced infectivity with herpes simplex virus type 1 DNA.

Abstract: Summary Cells infected with herpes simplex virus type 1 (HSV-1) DNA by the calcium phosphate precipitation technique produce virus which leads to the formation of plaques (Graham, Veldhuisen & Wilkie, 1973). In the study reported here we show that treatment of cell monolayers with dimethyl sulphoxide (DMSO) solutions after infection with DNA-calcium phosphate complexes leads to a considerable increase in the number of plaques obtained. The conditions for this enhancement of infectivity have been optimized for baby hamster kidney (BHK) cells, and increases in plaque numbers of over 100-fold have been obtained. The treatment appears to increase the proportion of cells which respond to DNA infection by initiating plaque formation, and results in a large increase in the measured specific infectivity of HSV-1 DNA. DMSO causes similar (but quantitatively different) responses in various other cell lines infected with HSV-1 DNA. BHK cells infected with either virus particles, or virus DNA by the DEAE-dextran technique (Laithier & Sheldrick, 1975), do not exhibit this massive enhancement following exposure to DMSO.

Sequential induction of heme pathway enzymes during erythroid differentiation of mouse Friend leukemia virus-infected cells.

Abstract: The process of erythroid differentiation in mouse Friend leukemia virus transformed cells (T3-C1-2) was examined by following changes in several enzyme activities of the heme biosynthetic pathway and in heme concentration while the cells were undergoing erythroid differentiation after treatment with dimethylsulfoxide. Untreated cells on the one hand, have a limited capacity for spontaneous differentiation. On the other hand, dimethylsulfoxide(DMSO)-treated cells showed an increase in the activities of delta-aminolevulinic acid (ALA) synthetase, ALA dehydratase, uroporphyrinogen-I synthetase, ferrochelatase, and heme concentration by days 1, 1.5, 2, and 4, respectively. The increase of the heme pathway enzymes and heme concentration followed the order of these enzymes or products as they are arranged in the heme biosynthetic pathway. These changes induced by DMSO were effectively inhibited by treatment with actinomycin D, suggesting that continued RNA synthesis is required for the differentiation process. 5-bromo-2'-deoxyuridine (BrdU) (10(-5) M) inhibited the DMSO-induced changes of the heme pathway enzymes. BrdU was most effective when it was present during the first 2 days of cell culture. It gradually lost its inhibitory effect when added after the 3rd day or later. The BrdU-mediated inhibition was completely overcome by the addition of thymidine (7 x 10(-5) M), but not by uridine (7 x 10(-5) M). All these data suggest that a sequential induction of the heme pathway enzyme takes place during erythroid differentiation of Friend leukemia cells, and that the sequential induction of the enzymes may be due to a sequential activation of genes coding for these enzyme activities.

Cytological and enzymological characterization of adult human adipocyte precursors in culture.

Abstract: Cell strains were derived from the stromal-vascular fraction of human omental adipose tissue and grown in culture. Since the purpose of this study was to isolate adipocyte precursors from adults, the cells were obtained from nonobese patients 40-60 yr of age. After treatment of adipose tissue with collagenase, mature adipocytes were separated from stromal-vascular fraction cells, and cell strains of the latter replicated in culture with a doubling time of 40-60 h. They were initially fusiform; upon reaching monolayer confluency, they accumulated lipid and became rounder. Skin fibroblasts from the same patients and grown under the same culture conditions remained fusiform and did not accumulate lipid. The stromal-vascular fraction cells of adipose tissue may be fibroblasts with the potential to become adipocyte precursors. Subcellular preparations of the cells grown from the stromal-vascular fraction revealed lipoprotein lipase activity (characterized by such properties as inhibition by 1 M NaCl) that was not detectable in skin fibroblasts. The overall specific activity of the enzymes that catalyze triglyceride synthesis was 15 times higher and that of fatty acid synthetase was 2 times higher in the cells cultured from the stromal-vascular fraction. The difference was significant in each case. Conversely, when isolated mature adipocytes were cultured, they lost considerable lipid and acquired morphological characteristics similar to those of stromal-vascular fraction cells. Thus, adipose tissue stromal-vascular fraction cells acquire in culture many of the morphological and enzymological characteristics of mature fat cells.

Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers

Abstract: Improved media have reduced the amount of serum protein required for clonal growth of normal human and chicken fibroblast-like cells. In the presence of limiting amounts of serum protein, attachment of colonies to tissue culture plastic surfaces is weak. Treatment of the culture surface with polylysine or other basic polymers causes the cells to adhere much more tightly. Growth is also improved on the surfaces treated with basic polymers, and further reductions in the concentration of serum as possible. At sufficiently low protein concentrations, growth of some types of cells is totally dependent on the use of a treated surface. Several different types of normal human and chicken fibroblast-like cells show improved growth on polylysine-coated surfaces, but no improvement was obtained in growth of a line of SV-40 transformed WI-38 cells. Acidic and neutral polymers are generally inactive. Collagen and gelatin improve growth slightly, but the effect is much less than that obtained with basic polymers. Both natural and synthetic polymers with an excess of basic groups are active, including histone, polyarginine, polyhistidine, polylysine, polyornithine, and protamine. The only critical requirement appears to be a polymer that carries a positive charge at a physiological pH.

Presence of diadenosine 5',5''' -P1, P4-tetraphosphate (Ap4A) in mamalian cells in levels varying widely with proliferative activity of the tissue: a possible positive "pleiotypic activator"

Abstract: An accurate assay of diadenosine 5',5'''- P1,P4-tetraphosphate [A(5') pppp(5')A], which was shown to be formed in vitro in the backreaction of the amino acid activation step, has been developed in various cell lines in culture and in normal mouse liver or hepatoma in vivo. Use of radioactive labeling of acid-soluble nucleotides to high specific activity followed by chromatographic separation techniques yielded levels of Ap4A varying from 5 to 0.05 muM (from 30 pmol/mg of protein to 0.15 pmol), depending on the doubling time of the cell line or the proliferative state of the cells. The levels of Ap4A incells is inversely related to their doubling time, varying from 0.1 X 10(-4) of the cellular ATP levels in slowly growing cells to 20 X 10(-4) of the ATP levels of cells with rapid doubling times. The steady-state levels of ATP of different cell lines, although showing some fluctuations, are not related to the doubling time of the cells. Arrest of cellular proliferation by serum deprivation or amino acid starvation, which does not alter the cellular ATP levels more than 2-fold, does nevertheless cause a decrease of 30 to 50-fold in the Ap4A levels. Inhibition of protein synthesis by pactamycin or puromycin, or inhibition of DNA synthesis by hydroxyurea, leads to a more dramatic decrease of 50 to 100-fold in intracellular Ap4A levels. The metabolic lability of Ap4A is also demonstrated by its rapid depletion after decreases in the ATP/ADP ratio. The possibility of Ap4A being a metabolic "signal nucleotide" that is formed at the onset of protein synthesis and is active in positive growth regulation (positive pleiotypic activation) is discussed.

Hormone responsive human breast cancer in long-term tissue culture: effect of insulin.

Abstract: The mechanisms of steroid and peptide hormone action in human breast cancer are poorly understood. We have previously characterized a cell line of human breast cancer in long-term tissue culture that possesses various steroid hormone receptors and responses, providing a model for the study of steroid hormone action. The present studies describe a human breast cancer in vitro that responds to physiologie concentrations of insulin with an increased rate of macromolecular synthesis and growth. Thymidine and uridine incorporation in cells in serum-free medium are stimulated by 10(-11) M insulin and are maximal with 10(-8) M. Leucine incorporation is stimulated by 5 X 10(-11) M insulin and is maximal with 10(-9) M. Significant stimulation of uridine and leucine incorporation is evident by 3 hr and maximal by 10 hr. A 10-hr lag period exists for insulin stimulation of thymidine incorporation, which is maximal form 14 to 24 hr. The effect of 10(-8) M insulin on macromolecular synthesis is accompanied by a 69% increase above controls in the number of cells after 24 hr. The effect on macromolecular synthesis is observed in glucose-free medium. Insulin's effect on protein synthesis is not blocked by inhibition of RNA synthesis with actinomycin D. Glucocorticoids partially inhibit the action of insulin in these cells. This system provides a model for studying insulin action, and suggests that some human breast cancer may show growth regulation by insulin.

Isolation and partial characterization of three methotrexate-resistant phenotypes from Chinese hamster ovary cells.

Abstract: Three mechanisms for resistance to methotrexate (Mtx) have been identified in Chinese hamster ovary (CHO) cells selected from resistance to this drug. First-step selections produce cells with either an apparent structural alteration in the enzyme dihydrofolate reductase (class I), or a decreased permeability to the drug (class II). Mutagenesis with ethyl methanesulfonate increases the proportion of Mtx-resistant cells 5-10-fold. Second-step selections to higher resistance using class I resistant cells as parents results in cells with an increased activity of the reductase enzyme (class III) with no apparent further qualitative alterations in the enzyme. All three classes of resistant cells retain their Mtx-resistant phenotype when cultured under nonselectivve conditions.

Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts

Abstract: The trace element selenium is essential for clonal growth of diploid fibroblasts from human fetal lung (WI-38) in media containing small amounts of serum protein. Maximum growth stimulation is obtained when 30 nM neutralized selenious acid is added to a synthetic medium containing 1.5 mg/ml of dialyzed fetal bovine serum protein (equivalent to a 3% serum concentration). Serum appears to be a source of selenium in most culture media, since higher concentrations of serum protein or whole serum mask the selenium requirement of WI-38 cells. Selenium is also required by a Chinese hamster cell line that can be grown in a protein-free synthetic culture medium.