The effect of intracellular ion deregulation, particularly of [Ca2+], on the events following acute cell injury and the progression of change from initiation (reversible) to maintenance (reversible-irreversible) phases and finally to cell death has been the major thrust of experimentation in our laboratory for over 20 years. Cell death, which plays an important role in both normal and pathological phenomena, has been classified into two principal types, accidental and programmed. Recent exploration of programmed cell death (or apoptosis) has revealed extensive data showing it is an important mechanism for the normal maintenance and also differentiation of a variety of cell types and organs. From the results from our laboratory and those of others, we continue to expand and refine our working hypothesis: deregulation of [Ca2+] results in a number of phenomena from activation of signaling mechanisms and alterations in cellular structure to alterations in gene expression, all of which contribute to or play a critical role in cellular toxicity, including carcinogenesis and cell death. Therefore, although much more experimentation is needed to clarify some of these phenomena, the implications of such data for understanding the mechanisms and processes involved in carcinogenesis and the chemotherapeutic killing of cancer cells are extremely exciting. These relationships between [Ca2+], cell injury, and cell death are briefly reviewed here within the framework of our hypothesis.

The pathways and identification of cell injury and cell death are of key importance to the practice of diagnostic and research toxicologic pathology. Following a lethal injury, cellular reactions are initially reversible. Currently, we recognize two patterns, oncosis and apoptosis. Oncosis, derived from the Greek word "swelling," is the common pattern of change in infarcts and in zonal killing following chemical toxicity, e.g., centrilobular hepatic necrosis after CC14 toxicity. In this common reaction, the earliest changes involve cytoplasmic blebbing, dilatation of the endoplasmic reticulum (ER), swelling of the cytosol, normal or condensed mitochondria, and chromatin clumping in the nucleus. In apoptosis, the early changes involve cell shrinkage, cytosolic shrinkage, more marked chromatin clumping, cytoplasmic blebbing, swollen ER on occasion, and mitochondria that are normal or condensed. Following cell death, both types undergo postmortem changes collectively termed "necrosis." In the case of oncosis, this typically involves broad zones of cells while, in the case of apoptosis, the cells and/or the fragments are often phagocytized prior to their death by adjacent macrophages or parenchymal cells. In either case, the changes converge to a pattern that involves mitochondrial swelling, mitochondrial flocculent densities and/or calcification, karyolysis, and disruption of plasmalemmal continuity. The biochemical mechanisms of cell death are currently under intense study, particularly concerning the genes involved in the process. Pro-death genes include p53, the ced-3/ICE proteases, and the Bax family. Anti-death genes include ced-9/Bcl-2 and the adenovirus protein EIB. It is clear that ion deregulation, particularly that of [Ca2+]i plays an important role in cell death following either apoptosis or oncosis. Genetic evidence strongly indicates that activation of proteases is an important step, possibly very near to the point where cell death occurs.

Unique cytoplasmic structures, herein designated as type I cytopathic vacuoles (CPV-I), are found in chick embryo cells early in the logarithmic phase of Semliki Forest virus replication. High resolution autoradiography demonstrated that the CPV-I are loci of 3H-uridine incorporation. This evidence correlates well with previous biochemical data and electron microscopy of the subcellular fractions active in Semliki Forest virus ribonucleic acid synthesis. Origin of the CPV-I within host cell cytoplasm is confirmed by the distribution of electron-dense tracer particles and sequential ultrastructural observations.

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Mixed infection with simian virus 40 (SV40) and adenovirus 1 2 can occur in African green monkey kidney (AGMK) cells in vitro, and electron micrographs demonstrating both viruses within the nucleus of a single cell have been presented ( 1 ) . Variable degrees of partial exclusion were produced by altering the time interval between inoculation of SV40 and adenovirus 12, and approximately 40% of the cells contained both viruses when the adenovirus was inoculated 24 hours after SV40(1). Further studies of AGMK cultures infected with both viruses compared with cultures infected with adenovirus 12 only have shown that SV40 enhances the growth of the adenovirus. A similar enhancement of growth of adenovirus 5 has been found when AGMK cultures were infected with SV40 and adenovirus 5.Materials and methods. Cell Cultures: Primary AGMK cell cultures were obtained from Microbiological Associates, Inc., Bethesda, Md., and primary human embryo kidney cell cultures (HEK) from the Virology Research Resources Branch, National Cancer Institute, through the kindness of Drs. Robert E. Stevenson and Theodore Malinin. Roller tube cultures maintained in a medium composed of 2 % fetal bovine serum and 98% mixture 199 and incubated at 36.5"C were used in all experiments.Viruses: SV40 and adenovirus 12 strains were the same as those previously described (1). Adenovirus 5 was obtained from the American Type Culture Collection. SV40 was grown and titrated in AGMK cell cultures, and adenoviruses 12 and 5 were grown and titrated in HEK cultures. Viruses were titrated by serial 10-fold dilutions with 3 to 5 tubes per dilution. Titrations were observed for 2 1 days and titers were calculated by the Reed-Muench method.The amount of adenovirus in the AGMK cultures after infection with both SV40 and adenovirus 12 or adenovirus 5 was determined by titration in HEK cell cultures in which a typical adenovirus-type CPE was produced. Although Shein and Enders have shown that SV40 can produce an incomplete CPE in HEK cell cultures after more than 17 days (2), the cellular degeneration described by them did not resemble the CPE produced by adenovirus 12 and 5, and the inocula they used contained considerably more SV40 than could have been present in the terminal dilutions in our titrations.Experiments and results. In the first experiment, AGMK cell cultures infected with adenovirus 12 only were compared with similar cultures infected simultaneously with both SV40 and adenovirus 12 (Table I). After 48 hours' incubation, clusters of rounded refractile cells were observed in both the singly and doubly infected cultures although the changes were more extensive in the doubly infected cultures. Uninfected control cultures showed no changes. At 72 and 122 hours, the CPE had progressed in all infected tubes; however, it continued to be more severe and extensive in the doubly infected cultures.Electron microscopic studies at 72 and 122 hours showed a significant difference between the singly and doubly infected cultures. In the singly infected cultures, althou...

Production of particles with the ultrastructural appearance of C-type virions persisted for at least 6 h in actinomycin D-treated cells infected with murine leukemia virus. This phenomenon occurred despite severe inhibition of viral RNA synthesis. Virus particles present in a 6-h harvest sedimented in sucrose gradients with the buoyant density characteristic of RNA tumor viruses (1.16 g/cm') and exhibited high levels of reverse transcriptase activity in response to the exogenous template polyriboadenylic acid-oligo deoxythymidylic acid in the range of untreated controls. However, RNase-sensitive endogenous activity was only Y/5 the level found in controls. This observation correlated with a marked

When actinomycin D-treated chick fibroblasts were labeled with 3 H-uridine for varying periods during the log phase of Semliki Forest virus infection, radioactivity was found associated with different cytoplasmic fractions. After a 1-min period of labeling, it appeared in a large cytoplasmic structure which was seen in electron micrographs of infected cells. Sediments of sucrose density gradients of cytoplasmic extracts of these cells also contained these structures. Three forms of viral ribonucleic acid (RNA) were associated with this cytoplasmic structure: a ribonuclease-sensitive 42 S form identical to the RNA of the mature virus, a ribonuclease-sensitive 26 S form, and a ribonuclease-resistant 20 S form. After a 5- to 10-min labeling period, radioactivity was associated with a ribonuclease-sensitive 65 S cytoplasmic fraction which contained only the 26 S RNA form. Finally, after a 1-hr labeling period, a 140 S ribonuclease-resistant particle was the most prominent radioactive structure in the cytoplasm. This particle contained only 42 S viral RNA. Negative-contrast electron micrographs of the 140 S particle and the virion demonstrated structural differences between them. The base compositions of the 42 S and 26 S viral RNA forms were not significantly different. The base composition of the 20 S form differed significantly from that of the other two viral RNA forms, but the values obtained for the mole fractions of the bases present in the 20 S form differed, and depended on the period during the virus growth cycle in which 32 P was present. These results suggested that viral RNA originated in the large cytoplasmic body. The 20 S RNA appeared to be a structure engaged in viral RNA replication and the 140 S particle appeared to be a virus precursor.

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