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.     

Liposomes as vehicles for cellular incorporation of biologically active macromolecules

Summary Lipid vesicles (liposome) 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. 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. The research described here was supported by Grants AI-14042, CA-18527 and GM-18921 from the National Institute of Health and IN-54P-16 from the American Cancer Society.     

Identification of estrogen-inducible growth factors (estromedins) for rat and human mammary tumor cells in culture

Summary The role of polypeptide growth factors (estromedins) as mediators of estrogen-responsive mammary tumor growth is studied in this report. Three possible new mechanisms were investigated that include endocrine, autocrine, and paracrine related growth factors. The first hypothesis being tested is whether estrogens interact with target tissues and cause the biosynthesis and secretion of polypeptide growth factors, which then act as mitogens for normal and neoplastic mammary tissues. Data presented suggest that this mechanism involves estrogen interaction with uterus, kidney, and pituitary gland causing production of growth factors, which then enter the general circulation and promote growth of distant target tissues. This is an endocrine type mechanism. Another type of estromedin control (autocrine control) may be exerted in an autostimulatory way in which the target tissue produces the polypeptide factors for its own growth in response to estrogen stimulation. A variation of the autocrine mechanism may be a paracrine mechanism in which some cells of an estrogen-responsive normal or neoplastic tissue produce growth factors that act on adjacent or neighboring cells. From the data available, all three possible types of growth factors could be functioning synergistically to yield the final result of continuous estrogen responsive tumor growth in vivo. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by American Cancer Society Grant BC-255; D. A. S. is the recipient of an American Cancer Society Faculty Research Award, FRA-212. D. D. is supported by a Rosalie B. Hite predoctoral fellowship from the Rosalie B. Hite Foundation, Houston, Texas. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Differentiation and cancer

Summary Cancer is discussed from a standpoint of a postembryonic differentiation. A differentiation requires the interaction of an exogenous inductive stimulus with competent precursor cell, which then evolve a new tissue with unique, stable heritable properties distinguishable from the progenitor. Evidence is cited pinpointing the normal stem cells of tissues as the competent target precursor cells in carcinogenesis. The resultant phenotype differs from its progenitor and has stable and unique characteristics. All of the characteristics associated with malignancy are expressed during some stage of development, suggesting that the normal genome contains the information necessary for malignant expression, and that the mechanism of malignancy is probably an alteration of control of genomic expression. Malignant tissue, like normal tissue, maintains itself by proliferation and differentiation of its stem cells; at least, that is what was observed in two tumors examined. In each of these tumors the differentiated progeny of the malignant stem cells proved to be benign. A third tumor was adapted to growth in vitro and under the conditions of the experiments could be modulated by altering the in vitro conditions. These data suggest that direction of the naturally occurring differentiation that occurs in tumors may be a suitable therapeutic alternative to cytotoxic chemotherapy. Presented in the Symposium on Regulation in Tumor Cells at the 22nd Annual Meeting of the Tissue Culture Association, Lake Placid, New York. Supported in part by Grant E105 from the American Cancer Society and Grant AM 13112 from the United States Public Health Service. Supported by a Traineeship from National Institutes of Health Training Grant GM 00977.     

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.     

Time sequence and morphological evaluations of cells fused by polyethylene glycol 6000

Summary Mouse L cells (clone 1D) were fused with polyethylene glycol (PEG). The fusion sequence was determined by using sequential light microscopy of the same group of cells, scanning electron microscopy (SEM), transmission electron microscopy, and freeze-etching. The cells were found to fuse only 1 min after PEG had been washed off at small localized areas. Larger fusion images were found after 3 min. Intramembrane particles were observed to have a tendency to aggregate after PEG treatment, but a direct correlation of this activity with the fusion process could not be made. No pathological changes were noted at longer times after PEG removal, except for the extensive widening of the rough-surface endoplasmic reticulum (RER) in some cells. It is proposed that fusion does not occur if apposing cells have many microvilli at the area of apparent contact. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976. This work was supported by U.S. Public Health Service research grants CA 10815 from the National Cancer Institute and GM 21615 from the Institute of General Medical Sciences.     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

T-cell responses to multiple antigens presented by RNA-transfected APCs: a possible immunomonitoring tool

The increasingly deeper understanding of how the immune system recognizes and destroys tumors promises to enable the development of new approaches for gene therapy and immunotherapy. However, a treatment that induces safe and potentially beneficial antitumor responses is expected to require stepwise refinements. As part of this challenge, assays are needed to measure specific antitumor immune responses in patients. This becomes problematic because most tumors express unknown tumor antigens and it is often difficult to obtain sufficient amounts of viable tumor material for in vitro assays. Recently it was demonstrated that RNA derived from tumor cells stimulated T cells in an antigen-specific manner. These studies have formed the basis for the development of dendritic cell vaccines that express tumor antigens following translation of tumor RNA. Therefore, it occurred to us that antigen-presenting cells transfected with total tumor RNA might also be valuable in monitoring the antitumor responses induced in patients who participate in clinical trials. To test this hypothesis, we developed a model in which Epstein-Barr virus (EBV)–transformed lymphoblastoid cell lines were used as a source of RNA. Since this RNA encodes for known EBV antigens, it was possible to determine whether the expected responses were observed. Our results show for the first time that T cells primed to APC transfected with RNA isolated from EBV-infected lymphocytes exhibited a fine specificity that enabled them to recognize individual EBV antigens.This work was supported by a Developmental Research Grant from the Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine.     

Expression of malignancy traits in the interspecific somatic hybrids of tumor and normal cells

Tumorigenicity and anchorage independence in two types of the interspecies hybrids of the tumor and normal mammalian cells were studied. One hybrid type was derived from fusion of spontaneously transformed Chinese hamster and normal mouse cells; the second type was obtained by fusion of SV40-transformed Djungarian hamster and the same mouse cells. The tumorigenicity in the athymic nude mice was suppressed in the first type of hybrids. The hybrid clones derived from fusion of SV40-transformed and normal cells could form tumor in nude mice. Testing of hybrid clones for their ability to form colonies in soft agar showed that all hybrids grew well in the medium, similar to tumor parental cells. These data suggest that malignancy and anchorage independence are under separate genetic control. The influence of the origin of the tumor parental cells (spontaneous or SV40-virus transformation) on the expression of the malignancy in hybrids of the tumor and normal cells is discussed.     

A human breast tumor cell line (BT-474) that supports mouse mammary tumor virus replication

Summary A human breast tumor cell line BT-474 derived from an invasive ductal carcinoma was experimentally infected in vitro with a mouse mammary tumor virus from the RIII strain (RIII-MuMTV). The virus that replicated in the human cells was characterized as a mouse virus by immunofluorescence, electron microscopy and the presence of a specific RNA-directed DNA polymerase. The cells themselves were human as per the karyotype and isoenzyme migration patterns. It is concluded that human cells are susceptible to the mouse mammary tumor virus and can, eventually, support its replication. This work was supported by USPHS Grant CA-08515 from the National Cancer Institute and by NIH Contract N01-CP-81003.     

2nd PEGS Annual Symposium on Antibodies for Cancer Therapy

The 2nd Annual Antibodies for Cancer Therapy symposium, organized again by Cambridge Healthtech Institute as part of the Protein Engineering Summit, was held in Boston, USA from April 30^th to May 1^st, 2012. Since the approval of the first cancer antibody therapeutic, rituximab, fifteen years ago, eleven have been approved for cancer therapy, although one, gemtuzumab ozogamicin, was withdrawn from the market. The first day of the symposium started with a historical review of early work for lymphomas and leukemias and the evolution from murine to human antibodies. The symposium discussed the current status and future perspectives of therapeutic antibodies in the biology of immunoglobulin, emerging research on biosimilars and biobetters, and engineering bispecific antibodies and antibody-drug conjugates. The tumor penetration session was focused on the understanding of antibody therapy using ex vivo tumor spheroids and the development of novel agents targeting epithelial junctions in solid tumors. The second day of the symposium discussed the development of new generation recombinant immunotoxins with low immunogenicity, construction of chimeric antigen receptors, and the proof-of-concept of ‘photoimmunotherapy’. The preclinical and clinical session presented antibodies targeting Notch signaling and chemokine receptors. Finally, the symposium discussed emerging technologies and platforms for therapeutic antibody discovery.     

The surface membrane as a regulator of animal cell division

Summary The surface membrane of an animal cell is proposed to be the target for regulation of cell division. It undergoes regular periodic changes during the cell division cycle. Interference with these changes by cell-cell surface contacts is proposed to prevent the normal progression of events, and thereby can change the metabolic pattern so as to put the cells into a resting state. Through external influences, cells can escape from this resting state; when this occurs surface changes are the earliest ones observed. Cells that have become malignant, particularly after virus infection, show marked changes in their surface properties. Infection is proposed to prevent the surface changes that lead to the resting state. Recent evidence from in vitro experiments is summarized, and some speculations are made on the connection between the surface and processes of division such as nuclear replication. Presented in the Symposium on Regulation in Tumor Cells at the 22nd Annual Meeting of the Tissue Culture Association. Lake Placid, New York. This work was supported by Public Health Service Grant CA-A1-1195 and Grant E-555 from the American Cancer Society.     

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.     

2nd PEGS Annual Symposium on Antibodies for Cancer Therapy: April 30–May 1, 2012, Boston,USA

The 2nd Annual Antibodies for Cancer Therapy symposium, organized again by Cambridge Healthtech Institute as part of the Protein Engineering Summit, was held in Boston, USA from April 30^th to May 1^st, 2012. Since the approval of the first cancer antibody therapeutic, rituximab, fifteen years ago, eleven have been approved for cancer therapy, although one, gemtuzumab ozogamicin, was withdrawn from the market.  The first day of the symposium started with a historical review of early work for lymphomas and leukemias and the evolution from murine to human antibodies. The symposium discussed the current status and future perspectives of therapeutic antibodies in the biology of immunoglobulin, emerging research on biosimilars and biobetters, and engineering bispecific antibodies and antibody-drug conjugates. The tumor penetration session was focused on the understanding of antibody therapy using ex vivo tumor spheroids and the development of novel agents targeting epithelial junctions in solid tumors. The second day of the symposium discussed the development of new generation recombinant immunotoxins with low immunogenicity, construction of chimeric antigen receptors, and the proof-of-concept of ‘photoimmunotherapy’. The preclinical and clinical session presented antibodies targeting Notch signaling and chemokine receptors.  Finally, the symposium discussed emerging technologies and platforms for therapeutic antibody discovery.     

Factors involved in the modulation of cell proliferation in vivo and in vitro: The role of fibroblast and epidermal growth factors in the proliferative response of mammalian cells

Summary The control of proliferation of mesoderm-derived cells by EGF and FGF has been examined taking, as an example, the vascular endothelium. The mechanisms by which cell proliferation can be brought to a stop in vivo and in vitro have been reviewed. Presented in the formal symposium on Mechanisms of Cellular Control at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by Grants HL20-197 and HD 11082 from the National Institutes of Health, and VC-194 from the American Cancer Society. B. R. Zetter was supported by Fellowship 5-F32-CA05149-02 from the U.S. Public Health Service.     

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.     

De novo construction of cell-to-cell channels

Summary Nexus (gap junctions), which are considered to contain cell-to-cell channels, are newly formed in uterine smooth muscle during parturition or in response to estrogen treatment of virginal animals. A mRNA preparation was isolated from estrogen-dominated rat myometria and was encapsulated into liposomes. Subsequently the liposomes were fused with cultured cells of a mouse cell line CL-1D. It is established that these tumor cells normally are neither electrically coupled nor do they contain nexus. The cells, however, become electrically coupled a few hours after being loaded with the mRNA preparation. This de novo expression of cell coupling persisted for a little more than 24 hr after a single loading procedure. Freeze-fracture electron microscopy revealed small nexus-like particle aggregates at the time coupling was present. In control experiments the cells remained noncoupling when the RNA preparation was pretreated with ribonuclease, when cycloheximide was applied to the cells, or when liposomes filled with buffer solution only were used. These data suggest that the de novo expression of cell-to-cell coupling is accomplished by mRNA-induced protein biosynthesis resulting in the formation of cell-to-cell channels. Presented in the symposium on Molecular Morphological Aspects of Cell-Cell Communication at the 31 st Annual Meeting of the Tissue Culture Association, St. Louis, Missouri, June 1–5, 1980. This symposium was supported in part by Contract 263-MD-025754 from the National Cancer Institute and the Fogarty International Center.     

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.     

Adoptive T cell therapy of solid cancers

The development of immune-based approaches for the treatment of cancer has been actively investigated for many years. One strategy that has emerged as a potentially effective strategy for the treatment of aggressive established malignancies is adoptive T cell therapy. The power of this approach has been repeatedly observed in preclinical animal models. However, moving from homogeneous animal models to the heterogeneous human clinical setting has been very difficult. It is only in recent times that we have been able to pinpoint the problems of the clinical translation of adoptive T cell therapy. Some of the major problems are sources of tumor-specific T cells, ex vivo expansion, persistence, and anti-tumor activity. This review overviews the nature of these problems and some of the emerging solutions. This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2004 (PIVAC 4)”, held in Freudenstadt-Lauterbad, Black Forest, Germany, on 22–25 September 2004 Grant support: This grant was supported by K01-CA100764 (KLK) and R01-CA85374 (MLD)     

Ovarian substance induces steroid production in cultured granulosa cells

Summary The rat ovary produces an apparently low molecular weight substance that mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induces temporal cell rounding and, later on, intensive progestin production. However, unlike FSH, OS does not induce accumulation of cyclic AMP (cAMP) in the granulosa cells. The ovarian factor cannot be cAMP as its action is not abolished by phosphodiesterase (PDE) treatment. Neither is it a possible PDE inhibitor, as it does not augment cAMP accumulation in granulosa cells or Friend erythroleukemic cells induced by FSH or PGE₁, respectively. The factor is still active after heating for 20 min at 90° C but is rapidly inactivated by alkali treatment. In addition, treatment with various proteases did not abolish the steroidogenic activity. These findings suggest a possible novel intraovarian regulator of the granulosa cell function. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by the United States-Israel Binational Science Foundation, Grant 2656/81. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.