Liposomes as vehicles for cellular incorporation of biologically active macromolecules

Lipid vesicles (liposomes) have recently been shown to be a useful vehicle for the delivery of a variety of compounds to cultured cells. Using large unilamellar vesicles composed of phosphatidylserine [LUV(PS)] we were able to encapsulate poliovirus and purified poliovirus ribonucleic acid (RNA) and show that it can be delivered efficiently to cells in an infectious form. LUV-entrapped poliovirus RNA produced infectious titers 100-fold higher than comparable RNA preparations delivered to cells by other techniques. We have made a quantitative analysis of the uptake and infectivity of the vesicle-encapsulated RNA by using various ratios of RNA copies per vesicle and by determining the percentage uptake of labelled lipid and RNA by HeLa cells.     

The importance of stroma in morphogenesis and functional activity of urogenital epithelium

Summary Urogenital morphogenesis and cytodifferentiation are presented in the context of the epithelial-stromal interaction. The essential role of stroma in these processes is reviewed. Presented in the formal symposium on Sexual Differentiation in Vitro and in Vivo at the 29th Annual Meeting of the Tissue Culture Association, Denver, Colorado, June 4–8, 1978. The study was supported in part by Grant No. PDT-8 from the American Cancer Society, and Contract Grants N01-CP-55649 and N01-CP-75875 from the National Cancer Institute.     

The analysis of malignancy by cell fusion

Summary The lecture reviews some aspects of the work on the analysis of malignancy that have been, and are now being, pursued in the Dunn School. A brief outline of the early experiments that first demonstrated that the malignancy of mouse tumor cells can be suppressed by the fusion with normal cells is given, and then two areas of current interest in the laboratory are described. The first is an attempt to analyze the clinically important property of tumors to metastasize and the second is the work on the isolation and identification of an abnormal membrane glycoprotein present in tumor cells. In addition the value of cell fusion methods as a general test of hypotheses of malignancy is emphasized. Presented in the symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society.     

Cholera toxin stimulation of human mammary epithelial cells in culture

Summary Addition of cholera toxin to human mammary epithelial cultures derived from reduction mammoplasties and primary carcinomas greatly stimulated cell growth and increased the number of times the cells could be successfully subcultured. Other agents known to increase intracellular cAMP levels were also growth stimulatory. The increased growth potential conferred by cholera toxin enhances the usefulness of this cell culture system. This work was supported by USPHS Grant CA-24844 from the National Cancer Institute and Grant CD-61B from the American Cancer Society.     

Genetic and biochemical studies on the suppression of and a recovery from the tumorous state in higher plants

Summary Four neoplastic diseases of plants: crown gall, which is caused by Ti plasmid DNA; Black's wound tumor disease by an RNA virus; the Kostoff genetic tumors by chromosomal imbalance; and habituation, which results from a spontaneous activation of select biosynthetic systems, have been analyzed and compared. It has been found that both the development of a capacity for autonomous growth and the nature of the heritable cellular change that underlies tumorigenesis are similar in the four instances. All develop a capacity for autonomous growth as a result of the persistent activation of select biosynthetic systems, the products of which are concerned with cell growth and division. That the persistent activation of these biosynthetic systems does not involve heritable changes of an irreversible type is indicated by the finding that a reversal of the neoplastic state occurred in three of the test systems. Since the tumor cells in these instances were found to remain totipotent the results suggest that whether the normal or tumor phenotype is expressed is determined by how the genetic information is regulated in a cell. Regulation appears to be accomplished in part through positive feedback control mechanisms. Foreign genetic information could act either in a regulatory manner to persistently activate normal biosynthetic systems or it could code for one or more essential but normally limiting substance(s) and thus replace a substance(s) that in the case of the Kostoff tumors or habituation is specified by host cell genes, or it could do both. In either case, the foreign genetic information can be regulated in much the same manner as are the host cell genes to give rise to either the normal or tumor phenotype. Presented in the symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society. Certain of the investigations described above were supported in part by Grant Number CA-13808, awarded by the National Cancer Institute, U.S. Department of Health, Education, and Welfare, in which the author is the coprincipal investigator.     

The introduction of poliovirus RNA into cells via lipid vesicles (liposomes).

Large unilamellar vesicles (LUV) composed of phosphatidylserine are capable of encapsulating poliovirus ribonucleic acid (RNA) and delivering it efficiently to cells in an infectious form. The biological activity of vesicle-entrapped poliovirus RNA was 1-2 x 10(4) plaque forming units/nanogram (pfu/ng) and appeared to be enhanced by ribonuclease treatment of the vesicle preparations (infectivity = 1-2 x 10(5) pfu/ng). Vesicle-mediated RNA infection produced equivalent titers in primate and nonprimate cells. Moreover, the data strongly suggest that the ratio of molecules per infectious unit is close to one when the RNA is properly delivered to the cell. A comparative study of LUV and multilamellar vesicles (MLV) indicates that LUV deliver their contents to the cell cytoplasm much more efficiently than MLV. LUV-entrapped poliovirus RNA produced infectious titer 10-100 fold higher than comparable RNA preparations delivered to cells by other techniques.     

Keratinization and the effect of vitamin A in aggregates of a squamous carcinoma cell line NBT II

Summary The terminal differentiation, keratinization, of a rat bladder tumor cell line, NBT II, occurred in multicellular aggregates. After aggregation, these cells did not undergo a round of mitosis before keratinization. 5-Bromodeoxyuridine added to the monolayer cell culture 2 days before aggregation completely prevented this differentiation; it was ineffective when added at the time of cell aggregation. Vitamin A prevented the keratinization of NBT II cells in aggregates but did not inhibit aggregate formation; it enhanced the number of cells engaged in DNA synthesis. This model appears to be very useful for analyzing the mechanisms of terminal differentiation and its modulation by vitamin A in tumor cells. This research was supported by Institutional Research Grant 731-01-E from the American Cancer Society and in part by Research Grant CA 14137 from the National Cancer Institute to Dr. J. Leighton.     

Rat hepatocyte primary cell cultures

Summary The conditions for obtaining representative, adult rat hepatocyte primary cell cultures were improved such that viable yields of 50% of the liver were produced which gave rise to cultures representing 30% of the liver. The survival of the cultures in various media was compared revealing that in complex media, particularly containing galactose, survival was improved. This study was supported by Contract No. N01-CP-55705 from the National Cancer Institute and Research Grant No. BC-133B from the American Cancer Society.     

Differing effects of isoleucine deficiency on the toxicity of MNNG for 10T1/2 and CHO cells

Summary Isoleucine deficiency sensitizes C3H 10T1/2 cells to the cytotoxic effects of MNNG. Synchrony of proliferation is not required for this effect since it occurs prior to full growth arrest and subsequent establishment of synchronous proliferation after refeeding in complete medium. Furthermore, confluence arrest of proliferation of 10T1/2 cells does not affect their cytotoxic response to MNNG, although they proliferate synchronously after replating at low density. In contrast, the toxicity of MNNG for CHO cells is not altered by isoleucine deficiency, even though these cells are readily synchronized by refeeding in complete medium after transient isoleucine deficiency. This research was supported by Grant BC-142 from the American Cancer Society and Grants CA-16086 and CA-17973 from the National Cancer Institute.     

Plant tumor reversal associated with the loss of foreign DNA

Summary Transformation of plant tissues into crown gall tumors has been associated with the transfer of a portion of a tumor-inducing plasmid (Ti-plasmid) into plant DNA. Various laboratories have regenerated normal-appearing plants from a number of crown gall tumors. This study investigates the fate of the foreign DNA in a series of tissues derived from various parts of a plant regenerated from the tumor BT-37 by Braun and his coworkers. It was found that all the foreign DNA sequences were lost from tissues that had lost all their tumorous traits; whereas the plasmid DNA sequences were still present in tissues that appeared normal but still exhibited tumorous traits when returned to tissue culture media. From these studies it would appear that the presence of the Ti-plasmid sequences in the plant DNA is required for the maintenance of the transformed state. Presented in the Symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society.     

Rat hepatocyte primary cultures

Summary Primary cultures of rat hepatocytes survived well for up to 4 days in defined medium in the presence of dexamethasone but not in its absence. The loss of viability was accompanied by a loss of ultrastructural features characteristic of hepatocytes. The cultures began producing plasminogen activator and a neutral protease after 24 hr in culture. Dexamethasone inhibited the production of both of these substances. The deterioration of the cultures appeared not to be related to plasminogen activator, but prolongation of survival by a variety of protease inhibitors suggested that the neutral protease might contribute to deterioration. Dr. Goldblatt was supported by Grant No. SG-87 from the American Cancer Society as an American Cancer Society Scholar while on sabbatical leave from the Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut. This study was supported by Contracts NO1-CP-55705 from the National Cancer Institute and 68-02-2483 from the Environmental Protection Agency.     

Presence of retrovirus in the B95-8 Epstein-Barr virus-producing cell line from different sources

Summary The B95-8 cell line, a widely used source of highly transforming Epstein-Barr virus (EBV), obtained from the laboratory of origin, harbored an infectious retrovirus. This retrovirus generally resembled the Type D retroviruses structurally and developmentally and like the Type D retroviruses preferred Mg²⁺ to Mn²⁺ in its RNA-directed DNA polymerase reaction. Evidence for the presence of retrovirus was found in B95-8 cultures from two other sources within the United States, either by assay for polymerase or by electron microscopy. Comparison of two B95-8 cell lines showed cytogenetic differences as well as differences in retroviral activities. The results suggest that any B95-8 culture should be tested for the presence of retrovirus before its use as a source of EBV. This research was supported through the National Research and Demonstration Center (HL-17269-07) awarded to Baylor College of Medicine by the National Heart, Lung, and Blood Institute, Bethesda, MD, by RD-125 from the American Cancer Society, by K06 CA14219, CA16781, CA25465, and CA16672 from the National Cancer Institute, Bethesda, MD, and by G-429 from the Robert A. Welch Foundation. G. E. G. was supported by Public Health Service training Grant CA-09299.     

Gene transfer and gene mapping in mammalian cells in culture

Summary The ability to transfer mammalian genes parasexually has opened new possibilities for gene mapping and fine structure mapping and offers great potential for contributing to several aspects of mammalian biology, including gene expression and genetic engineering. The DNA transferred has ranged from whole genomes to single genes and smaller segments of DNA. The transfer of whole genomes by cell fusion forms cell hybrids, which has promoted the extensive mapping of human and mouse genes. Transfer, by cell fusion, of rearranged chromosomes has contributed significantly to determining close linkage and the assignment of genes to specific chromosomal regions. Transfer of single chromosomes has been achieved utilizing microcells fused to recipient cells. Metaphase chromosomes have been isolated and used to transfer single-to-multigenic DNA segments. DNA-mediated gene transfer, simulating bacterial transformation, has achieved transfer of single-copy genes. By utilizing DNA cleaved with restriction endonucleases, gene transfer is being employed as a bioassay for the purification of genes. Gene mapping and the fate of transferred genes can be examined now at the molecular level using sequence-specific probes. Recently, single genes have been clones into eucaryotic and procaryotic vectors for transfer into mammalian cells. Moreover, recombinant libraries in which entire mammalian genomes are represented collectively are a rich new source of transferable genes. Methodology for transferring mammalian genetic information and applications for mapping mammalian genes is presented and prospects for the future discussed. Presented in the symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society. Supported by NIH grants HD 05196 and GM 20454 and by MOD grants 1-485 and 1-692.     

Increased cyclic AMP content directly correlated with morphological transformation of cells infected with a temperature-sensitive mutant of mouse sarcoma virus

Summary Normal rat kidney cells infected with a cold-sensitive mutant of mouse sarcoma virus [NRK(MSV-1b)] morphologically transform when exposed to adenosine 3′∶5′ cyclic monophosphate (cAMP) at the restrictive temperature. The cAMP-induced morphological changes occur rapidly and are reversible. Agents capable of elevating endogenous levels of cAMP [prostaglandin E₁ (PGE₁) and cholera toxin (CT)] induced morphological transformation of NRK(MSV-1b) cells at the restrictive temperature that was concentration dependent, potentiated by cAMP phosphodiesterase inhibitors, and not prevented by inhibitors of DNA, RNA, and protein synthesis. Prostaglandin E₁ stimulated a transient increase in the intracellular level of cAMP with a concomitant morphological transformation and reversion of cells as cAMP levels decline. The maximum increase is reached by 10 min, followed by a decline to near basal level by 80 min. In contrast, incubation of cells with CT resulted in irreversible morphological transformation and increased levels of cAMP first detectable by 1 hr with maximum levels reached by 24 hr. Heated CT (100°C, 20 min) was without effect. Addition of CT to reverted PGE₁-treated cells resulted in morphological transformation suggesting the existence of discrete receptors in NRK (MSV-1b) cells. This research was supported by Grant BC-207 from the American Cancer Society and Cancer Research Emphasis Grant R01 CA 19714 within the Virus Cancer Program of the National Cancer Institute.     

Linkage of the locus for conversion of albumin (Acf-1)in the house mouse,Mus musculus

The linkage of the locus for conversion of albumin (Acf-1) has been established on chromosome 1 with the following gene order and recombination percentages: Id-1 19.3±5.2% Acf-1 4.2±1.7% Dip-1 18.4±4.2% Lp.This work was supported by NIH Postdoctoral Fellowship 1F32 GM0527701, Grant BMS75-03397 from the National Science Foundation, Grant ACS VC-17-R from the American Cancer Society, and Contract NO1-ES42159 from the National Institute of Environmental Health Sciences. The Jackson Laboratory is fully accredited by the American Association for the Accreditation of Laboratory Animal Care.     

Characterization of cells cultured from early lactation milks

Summary Two major types of cells can be cultured from early lactation human milks: a colony-forming epithelial cell and an adherent nondividing cell referred to as a foam cell The epithelial cells show a positive reaction with a specific antiserum reactive against membrane components of the milk fat globule, whereas the foam cells do not. The nondividing foam cells are phagocytic and can be killed by silica particles; they produce lysozyme, are resistant to trypsinization, and have Fc receptors. These properties, together with the lack of reaction with antiserum to the milk fat globule membrane, suggest that the foam cells are not terminally differential epithelial cells, but tissue macrophages. R. L. C. was supported by Grant No. Ca 19455 from the National Cancer Institute, a Yamagiawa-Yoshida Memorial International Cancer Study Grant, and the Imperial Cancer Research Fund. J. A. P. was supported by Grant No. CA 19455 from the National Cancer Institute.     

Methods for the study ofin vitro immunization of mouse spleen cells

Conclusion We have reviewed some of our experiences in developing techniques for studying the functions of the cells of the immune system. It is quite clear that much remains to be done. Improvements in the culture system are needed to permit cells to be grown for longer durations and at lower cell concentrations. The important effects of fetal calf serum should be defined. More sophisticated methods for separating cells into distinct functional populations must be developed. New assays for identifying other functions of the cells, particularly a method for directly assaying the number of precursor cells in a population, are needed. When these techniques are applied to the study of immune cells, further facts should be learned which will permit the development of significant, testable hypotheses on the function and relationships of the cells of the immune system. This is publication No. 298 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California 92037. This work was supported in part by U.S.P.H.S. Grant 7007 and in part by American Cancer Society Grant E-395. Dr. Mishell is supported by American Cancer Society Grant E-395. Dr. Dutton is supported by a Dernham Fellowship of the California Division, American Cancer Society (No. D-100). Dr. Raidt is supported by United States Public Health Service Postdoctoral Fellowship No. 7-F2-A1-31,590.     

Effect of room fluorescent light on the deterioration of tissue culture medium

Summary A major cause of tissue culture medium deterioration is exposure to room fluorescent light. Riboflavin and tryptophan present in Dulbecco's modified Eagle's minimum essential medium, when exposed to light, yield toxic photoproducts responsible for loss of the ability of the medium to support clonal growth of human, mouse and Chinese hamster cell lines. Procedures for minimizing medium deterioration are discussed. This work was supported by American Cancer Society Research Grant No. VC-100B and US PHS Research Career Development Award No. 5 K04 GM70537 from the National Institute of General Medical Sciences.     

Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues

Summary A sensitive radioimmunoassay technique was developed to quantitatite the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by abosorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10⁶ cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10⁶ cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10⁶ cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface. This work was Supported by Grant PTD-99 from the American Cancer Society, Grant CA19455 and CA20286 from the National Cancer Institute, and Biomedical Research Support Grant RR05467 from the National Institutes of Health. Most cells used in the present study were produced with support from National Cancer Institute Contract Y01-CP8-0500, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention, under the auspices of the Office of Naval Research and the Regents of the University of California.