Epithelial cell cultures from normal and cancerous human tissues.

Thirty epithelial cell strains were isolated from human carcinomas and normal epithelial tissues by collagenase digestion and selective removal of fibroblasts with trypsin-Versene. Most strains were obtained from metastatic carcinomas or epithelia of the urinary and intestinal tracts. The success rate for growth of both neoplastic and normal tissues (excluding skin) was 38%. Six of these strains showed gross morphologic and chromosome changes typical of malignant cells. Nine resembled normal epithelium. The other 15 exhibited some degree of morphologic change from normal.

Two syngeneic cell lines from human breast tissue: the aneuploid mammary epithelial (Hs578T) and the diploid myoepithelial (Hs578Bst) cell lines.

We characterized two human cell lines (Hs578T and Hs578Bst), which provide several unique features that should be useful in the study of breast disease. Hs578T, derived from a carcinosarcoma, is epithelial in origin. Hs578Bst, established from normal tissue peripheral to the tumor, is myoepithelial in origin. This is the first report of companion cell lines, one malignant and one normal, established from the same organ.

Comparative study of the cytoplasmic organelles of epithelial cell lines derived from human carcinomas and nonmalignant tissues.

The cytoplasmic organelles of 16 human epithelial cell lines have been characterized by electron microscopy. The cell lines were derived from normal, nonmalignant tissues of cancerous organs and from primary and metastatic carcinomas. Every cell section on a grid which contained a clearly defined nucleus, nucleolus, and cytoplasm was scored blindly utilizing a checklist of markers. Mitochondrial pleomorphism was expressed slightly by normal, to variable degrees by lines derived from nonmalignant tissues of cancerous organs, and to a much greater extent by all lines derived from malignant tissues. Hypertrophied mitochondria and longitudinal cristal arrangement were found in almost all the malignant lines, but not in any lines derived from nonmalignant tissues of cancerous organs or from normal tissues. Although malignant and nonmalignant cell lines could not be distinguished using microfilament bundles (stress fibers) and microtubules as markers, this may reflect the effect of insufficient cell spread or the preparative procedures used. All the lines appeared differentiated and showed slightly to moderately developed Golgi and smooth and rough endoplasmic reticula. There were no significant ultrastructural differences in cells at different passage levels or subconfluent and confluent tumor cells; however, more tight junctions were observed in confluent than in subconfluent normal cells.

Nuclear ultrastructure of epithelial cell lines derived from human carcinomas and nonmalignant tissues.

The nuclear ultrastructure of sixteen human epithelial cell lines has been characterized in detail by transmission electron microscopy. The cell lines were derived from normal tissues, nonmalignant tissues of cancerous organs, primary carcinomas, and metastatic carcinomas. Every cell section on a grid containing a clearly defined nucleus and nucleolus was scored blindly utilizing a checklist of markers. The goal of these studies was to determine whether any ultrastructural markers consistently distinguished the different stages of malignant progression represented among the lines. Nuclear bodies and perichromatin granules were found in all lines derived from cancer and were not observed in any nonmalignant lines. Nuclear envelope dilation was seen in all lines derived from cancerours organs as well as from malignant tissues but not in any lines derived from normal tissue. Margination of chromatin, irregularity of nuclear outline, redistribution of nucleolar components, and margination were expressed slightly by the normal lines, to variable degrees by the lines derived from cancerous organs, and to a much greater extent by all lines derived from malignant tissues. No differences were found between lines derived from primary carcinomas and those derived from metastatic specimens. There were no ultrastructural differences comparing subconfluent and confluent cells or cells at different passage levels. In addition, the nuclear ultrastructure of a malignant line in culture was similar to that of a tumor induced by those cells in an immunosuppressed mouse.

Expression of antigenic crossreactivity to RD114 p 30 protein in a human fibrosarcoma cell line.

An antigen crossreacting with the 30,000-molecular-weight protein (p30) of the feline endogenous oncornavirus (RD114) was detected in a well-characterized human fibrosarcoma cell line, HT1080, by indirect immunofluorescence. Three antisera against RD114 p30 gave similar positive results, while two antisera prepared against simian sarcoma virus p30, one antiserum prepared against murine leukemia virus p30, and one antiserum prepared against feline leukemia virus p30 gave no immunofluorescence. The reactivity observed with the antiserum against RD114 p30 was detected in 10-40% of the cells at early passages and was no longer expressed by the forty-first subculture. The reactivity could be removed by adsorption of the antiserum with RD114-infected dog or human cells, but not by uninfected cells or by cells infected with an antigenically unrelated oncornavirus, feline leukemia virus. Neither complete virus particles nor reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity was detected in the culture. These experiments suggest that the fibrosarcoma cell line is expressing an antigen related to the p30 protein of RD114 baboon endogenous virus group of oncornaviruses without producing complete virions.

Viral identification by scanning electron microscopy of preparations stained with fluorescein-labeled antibody.

A new technique combines the specificity of fluorescent-antibody labeling and the resolution of the scanning electron microscope to identify and distinguish between viruses. Hemagglutination of chicken erythrocytes by influenza virus was used as a model system to demonstrate the technique.

Studies on morphological transformation of BALB/3T3-derived clones by murine leukemia virus.

We have described a cell line, UC1-B, derived spontaneously from BALB/3T3 mouse embryo cells, which, unlike the standard BALB/3T3, are morphologically transformed and produce bizarre viral forms in response to murine leukemia virus. Although UC1-B and BALB/3T3 are morphologically similar, and both form contact-inhibited monolayers at confluence, the UC1-B cells are partially transformed because: they grow to a slightly higher saturation density than 3T3 cells, they grow in medium lacking serum growth factors, and they produce tumors in mice. Another clone, 12A-3, derived from BALB/3T3, also transforms and produces bizarre viral forms after infection with murine leukemia virus. Unlike UC1-B cells, the 12A3-8 cells are identical in growth properties to BALB/3T3; therefore, a partially altered morphology is not required for the induction of transformation by murine leukemia virus.

Alteration of the cell membrane architecture during suspension and monolayer culturing.

Using suspension-growth-adapted HeLa (S3) cells, we followed the alterations in cell morphology during attachment and spreading on a glass surface in vitro by scanning electron microscopy. Suspension cultivation obviated the need for trypsinization or EDTA treatment of the cells before seeding, thus the cell surface was not chemically altered. The suspension-grown cells were sperical and covered with microvilli. Three hours after seeding into stationary culture, attached cells had surface blebs and large processes, but no microvilli. During the following 9 h, the surface topography altered stepwise producing a complex lamelloplasm. The paper discusses how these membrane events are related to the cell cycle and metastasis.

Liposome-mediated delivery of deoxyribonucleic acid to cells: enhanced efficiency of delivery related to lipid composition and incubation conditions.

Delivery of liposome-encapsulated simian virus 40 (SV40) DNA to African green monkey Related to been used as a probe to study liposome--cell interactions and to determine conditions which favor the intracellular delivery of liposome contents to cells. The efficiency of DNA delivery by various liposome preparations (monitored by infectivity assays) was found to be dependent both on the magnitude of vesicle binding to cells and on the resistance of liposomes to cell-induced leakage of contents. Acidic phospholipids were much more effective in both binding and delivery, and phosphatidylserine (PS) was the best in both aspects. The inclusion of 50 mol % cholesterol in liposomes reduces the cell-induced leakage of vesicle contents (2--5-fold) and substantially enhances the delivery of DNA to cells (2--10-fold). Following incubation of cells with negatively charged liposomes containing SV40 DNA, infectivity can be enhanced greatly by brief exposure of the cells to glycerol solutions. In contrast, only slight enhancement by glycerol was observed for SV40 DNA encapsulated in neutral or positively charged liposomes. The results of competition experiments between empty phosphatidylcholine liposomes and DNA-containing PS liposomes also suggest possible differences in the interaction of neutral and negatively charged liposome preparations with cells. Morphological studies indicate that the glycerol treatment stimulates membrane ruffling and vacuolization and suggest that the enhanced uptake of liposomes occurs by an endocytosis-like process. Results obtained with metabolic inhibitors are also consistent with the interpretation that the enhancement of liposome delivery in glycerol-treated cells occurs via an energy-dependent endocytotic pathway. Pretreatment of cells with chloroquine, a drug which alters lysosomal activity, further enhanced infectivity in glycerol-treated cells (4-fold). This observation suggests the involvement of a lysosomal processing step at some point in the expression of liposome-encapsulated DNA and, more importantly, illustrates the possibility of altering cellular mechanism to engineer more efficient delivery by liposomes. Under optimal conditions determined in this study, the efficiency of liposome-mediated SV40 DNA delivery was increased more than 1000-fold over that obtained by simply incubating cells with liposomes. It is also demonstrated that these conditions enhance delivery of other molecules, besides DNA, which are encapsulated in liposomes.