A critical review of permeabilized cell systems for studying mammalian DNA repair

Permeabilized cell systems have proven valuable for studies of the enzymology of mammalian DNA repair and this review will summarize and contrast the different systems used to this end. Results from permeable cell studies will be reviewed which pertain to 3 questions of DNA repair: the role(s) of ATP, DNA polymerase enzymology, and the isolation of repair factors by in vitro correction of repair-defective cells.     

A tissue culture model for studying ethanol toxicity on embryonic heart cells

A tissue system in which fibroblasts and myocytes from chick embryonic hearts were separately maintained was used to study the toxicity of ethanol. To reproduce the teratogenic effects of acute, high concentrations of ethanol typical of binge drinking, an open tissue culture system was employed. With open cultures, the cells were initially exposed to peak alcohol levels for approximately 6 hr and were exposed to decreasing concentrations of ethanol for the remainder of each 24 hr period. After the first day of ethanol exposure, there was substantial cell loss in both fibroblast and myocyte cultures. Alcohol-induced cell loss was dose-dependent. Despite decreased cell density after the first day of ethanol exposure, the surviving cells differentiated into monolayers of fibroblasts or beating cardiac muscle fibers. However, both ethanol-exposed fibroblasts and myocytes appeared atrophic, that is, smaller and shrunken. Electrophoretic analysis or these ethanol-exposed fibroblast and myocyte cultures revealed specific reduction in the cellular contents of -actinin, myosin, and actin. These decreases in cytoskeletal proteins may be responsible for the morphological abnormalities noted in these cells.Abbreviations FAS, fetal alcohol syndrome - NAD, nicotinamide adenine dinucleotide - PAS, periodic acid Schiff - SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Experimental model for studying the primary cilia in tissue culture cells.

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.     

A permeabilized cell model for studying cell division: a comparison of anaphase chromosome movement and cleavage furrow constriction in lysed PtK1 cells

After lysis in a Brij 58-polyethylene glycol medium, PtK1 cells are permeable to small molecules, such as erythrosin B, and to proteins, such as rhodamine-labeled FAB, myosin subfragment-1, and tubulin. Holes are present in the plasma membrane, and the mitochondria are swollen and distorted, but other membrane-bounded organelles of the lysed cell model are not noticeably altered. After lysis, the mitotic apparatus is functional; chromosomes move poleward and the spindle elongates. Cells lysed while in cytokinesis will continue to divide for several minutes. Addition of crude tubulin extracts, MAP-free tubulin, or taxol to the lysis medium retards anaphase chromosome movements but does not affect cleavage. On the other hand, N-ethylmaleimide-modified myosin subfragment-1, phalloidin, and cytochalasin B inhibit cleavage but have no effect on anaphase chromosome movements under identical lysis conditions. These results suggest that actomyosin plays no functional role in anaphase chromosome movement in mammalian tissue culture cells and that microtubule depolymerization is a rate-limiting step for chromosome-to-pole movements.     

Cytoplasmic inheritance in mammalian tissue culture cells

Summary A series of intraspecific, interspecific and interorder somatic cell cybrids and hybrids have been prepared by fusions in which one of the parents contained the cytoplasmically inherited marker for chloramphenicol (CAP) resistance. A clear relationship has been established between the expression of the CAP-resistant (CAP-R) determinants in the fusion products and the genetic homology of the parents. With increased genetic divergence, the acceptability of the CAP-R mitochondria decreased. Intraspecific cybrids and hybrids of the same strain were stable for the CAP-R marker, while those between strains were stable only in CAP. Intergeneric mouse-hamster cybrids occurred at a high frequency but were unstable in CAP, while CAP suppressed hybrid formation 100-fold. Interorder cybrids (CAP-R human × CAP-S mouse) occurred either at a moderate frequency and were stable or at a low frequency and were unstable in CAP. Interorder hybrids could only be formed by challenging HAT-selected hybrids with CAP or by direct selection in ouabain and CAP. Reciprocal interorder crosses between CAP-R mouse and CAP-S human cells were unsuccessful. Interspecific cybrids contain only the chromosomes of the CAP-S parent. Interspecific hybrids selected directly in CAP segregated the chromosomes of the CAP-S parent, while hybrids selected in HAT and then CAP segregated those of the CAP-R parent. The mitochondrial DNA(mtDNA) of all mouse-human cybrids and most HAT and then CAP-selected hybrids contain only the mtDNA of the CAP-S mouse parent. However, preliminary evidence suggests that one of these hybrids contains both mouse and human mtDNA sequences. 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.P.H.S. research grants GM-18186, GM-1948 and GM-21024 (to J. M. E.), and N.I.H. postdoctoral fellowship No. 1 F22 GM-02655 (to D. C. W.).     

A novel cell culture model for studying ischemia-reperfusion injury in lung transplantation.

Many cell culture models have been developed to study ischemia-reperfusion injury; however, none is specific to the conditions of lung preservation and transplantation. The objective of this study was to design a cell culture model that mimics clinical lung transplantation, in which preservation is aerobic and hypothermic. A549 cells, a human pulmonary epithelial cell line, were preserved in 100% O(2) at 4 degrees C for varying periods in low-potassium dextran glucose solution, simulating ischemia, followed by the introduction of warm (37 degrees C) DMEM plus 10% fetal bovine serum to simulate reperfusion. Cultures were assayed for cell attachment and viability. Sequential extension of ischemic times to 24 h showed a time-dependent loss of cells. There was a further decrease in cell number after simulated reperfusion. Cell detachment was due mainly to cell death, as determined by cell viability. The effects of chemical components such as dextran 40 and calcium in the preservation solution and various preservation gas mixtures were examined by use of this model system. With its design and validation, this model could be used to study mechanisms related to ischemia-reperfusion injury at the cellular and molecular level.     

A mammalian cell culture collection for biotechnology

Conclusion Culture collections are key to the success of biotechnology companies. Protection of patent strains and manufacturing inoculum; standardization of biological materials for research, development and manufacturing; and documentation of organism transfers are essential functions provided by a culture collection in a biotechnology company. Certified, stable mammalian cell cultures will continue to be key in research advances and in manufacturing of biotechnology products in the future.     

Cytoplasmic inheritance in mammalian tissue culture cells.

A series of intraspecific, interspecific and interorder somatic cell cybrids and hybrids have been prepared by fusions in which one of the parents contained the cytoplasmically inherited marker for chloramphenicol (CAP) resistance. A clear relationship has been established between the expression of the CAP-resistant (CAP-R) determinants in the fusion products and the genetic homology of the parents. With increased genetic divergence, the acceptability of the CAP-R mitochondria decreased. Intraspecific cybrids and hybrids of the same strain were stable for the CAP-R marker, while those between strains were stable only in CAP. Intergeneric mouse-hamster cybrids occurred at a high frequency but were unstable in CAP, while CAP suppressed hybrid formation 100-fold. Interorder cybrids (CAP-R human X CAP-S mouse) occurred either at a moderate frequency and were stable at a low frequency and were unstable in CAP. Interorder hybrids could only be formed by challenging HAT-selected hybrids with CAP or by direct selection in ouabain and CAP. Reciprocal interorder crosses between CAP-R mouse and CAP-S human cells were unsuccessful. Interspecific cybrids contain only the chromosomes of the CAP-S parent. Interspecific hybrids selected directly in CAP segregated the chromosomes of the CAP-S parent, while hybrids selected in HAT and then CAP segregated those of the CAP-R parent. The mitochondrial DNA(mtDNA) of all mouse-human cybrids and most HAT and then CAP-selected hybrids contain only the mtDNA of the CAP-S mouse parent. However, preliminary evidence suggests that one of these hybrids contains both mouse and human mtDNA sequences.     

Extraction of RNA from mammalian tissue culture cells

A rapid method is described for the effective fractionation and extraction of undergraded nuclear and cytoplasmic RNA species from mammalian tissue culture cells. It is compared in detail with several existing procedures.     

A pilot plant for mammalian cell culture

Studies of the possible viral etiology of human leukemia have required large quantities of cultured cells derived from human hematopoietic tissues. Since cultures sufficiently large and free from contamination could not readily be produced according to existing methods, a pilot, cell culture plant has been constructed for the production of mammalian cells in mass quantity. 500-ml to 20-liter trophocell units have already proved to be scientifically and economically practical, as they provide good reliability, excellent growth rates, and sustained yield of human cells. 200-liter stainless steel culture units have now been added to the trophocell system. Five complete 200 liter units are now in operation. The design of the original stainless steel unit was based on that of a stainless steel, jacketed soup kettle. There are no openings in the vessel other than those in the lid, which provide convenient access points for sampling, sensor probes, etc. Environmental parameters, e.g., liquid level, temperature, and pH, are monitored and controlled with commercially available apparatus. Many initial problems connected with the new 200 liter units have been resolved, but operational and design problems remain in the areas of stable instrumentation, cell harvesting, salvaging and reuse of unspent media components, establishment of physiologic steady stale, recovery of virus-containing cells with reculture of the remaining unaffected cells, and the recovery and separation of cell components and special products such as immunoglobulins, interferons, and hormones. A definitive cell plant with culture units of 20, 50, 250, and 1250 liters is now being constructed.     

A new method of cloning mammalian tissue culture cells in suspension

Chinese hamster cells (line CHO) were cloned in suspension with a carboxymethylcellulose-containing medium, and the colonies were counted with an electronic particle counter after enzymatically degrading the carboxymethylcellulose with cellulase. Survival of x-irradiated cells was determined by both the standard plating technique and the suspension technique, and comparable results were obtained.     

Focal adhesion components are essential for mammalian cell cytokinesis

The final stages in mammalian cytokinesis are poorly understood. Previously, we reported that the ADP-ribosyltransferase activity of Pseudomonas aeruginosa type III secreted toxin ExoT inhibits late stages of cytokinesis. Given that Crk adaptor proteins are the major substrates of ExoT ADP-ribosyltransferase activity, we tested the involvement of Crk in cytokinesis. We report that the focal adhesion-associated proteins, Crk and paxillin are essential for completion of cytokinesis. When their function is absent, the cytoplasmic bridge fails to resolve and the daughter cells fuse to form a binucleated cell. During cytokinesis, Crk is required for syntaxin-2 recruitment to the midbody, while paxillin is required for both Crk and syntaxin-2 localization to this compartment. Our data demonstrate that the subcellular localization and the activity of RhoA and citron K, which are essential for early stages of cytokinesis, are not dependent on paxillin, Crk, or syntaxin-2. These studies reveal a novel role for Crk and paxillin in cytokinesis and suggest that focal adhesion complex, as a unit, may partake in this fundamental cellular process.     

A high-throughput,homogeneous microplate assay for agents that kill mammalian tissue culture cells

Screens for cytostasis/cytoxicity have considerable value for the discovery of therapeutic agents and the investigation of the biology of apoptosis. For instance, genetic screens for proteins, protein fragments, peptides, RNAs, or chemicals that kill tissue culture cells may aid in identifying new cancer therapeutic targets. A microplate assay for cell death is needed to achieve throughputs sufficient to sift through thousands of agents from expression or chemical libraries. The authors describe a homogeneous assay for cell death in tissue culture cells compatible with 96- or 384-well plates. In combination with a previously described system for retroviral packaging and transduction, nearly 6000 expression library clones could be screened per week in a 96-well plate format. The screening system may also prove useful for chemical screens.     

The ultrastructure of centriole in mammalian tissue culture cells

Structural polarity of centriole has been shown by analyzing serial sections of centrioles in the tissue culture cells of mouse, man, pig and Chinese hamster. Its major component is nine microtubule triplets. The inclination of the triplets towards the radius at the proximal end of the centriole is smaller than at the distal one. The internal tubule of the triplet has a smaller diameter than the middle and external ones; The triplets are bound by links of various nature all over their length. In the middle part, in the centriole lumen there is an amorphous hub; in the distal part, a thin fibre that is helically wound. In the proximal part, there are bases along the triplets, and handles stretch from the internal tubules. In the middle and distal parts, there are accumulations of an electron dense substance along the middle tubules. At the distal end, the centriole lumen is filled with an amorphous substance, whereas the proximal end is free from it. From outside, appendages are attached to the triplets at the distal end. The centriole structure is identical in all the cell types studied, except for those of Chinese hamster.