Cell death induced by cisplatin was studied in Chinese hamster ovary cell lines, one proficient and the other deficient (100-fold sensitive) in DNA excision repair. Previous experiments demonstrated that cells progressed to and arrested in the G2 phase of the cell cycle before dying. DNA double-strand breaks were detected following G2 arrest and prior to loss of membrane integrity. These DNA breaks have been studied in more detail. DNA fragments were observed consisting of multimers of approximately 180 base pairs. These fragments are consistent with internucleosomal cleavage of chromatin by an endonuclease. At LC90 concentrations, DNA digestion began 48 hr cisplatin treatment followed by loss of membrane integrity and cell shrinkage 24 hr later. High concentrations of cisplatin (170 logs of kill) induced DNA digestion 12 hr after drug treatment but loss of membrane integrity occurred 12 hr later. Both cell death and DNA fragmentation were inhibited by cycloheximide, suggesting the requirement for new protein synthesis. Cells incubated with many other agents demonstrated the same characteristic pattern of DNA degradation. At 90% lethal conditions, DNA digestion was induced within 30 min by hyperthermia, 18 hr by methotrexate, and 48-72 hr by all other agents tested. DNA digestion always preceded loss of membrane integrity and cell shrinkage. These observations are consistent with cell death occurring by the process of apoptosis, or prorammed cell death, and demonstrate the importance of DNA digestion as an early and presumably essential step in cell death. The results suggest that, irrespective of the primary site of action of a drug, cell death by most pharmacologic agents is mediated by activation of the signal transduction pathway for apoptosis. The results also suggest two signal pathways for apoptosis, one directly associated with drug action and a second that requires cell cycle-related events.

DNA is the accepted target for cisplatin, but recent evidence has shed doubt on DNA synthesis as the critical process. L1210/0 cells incubated for 2 hours with cisplatin progress to the G ₂ phase of the cell cycle and are arrested there for several days. They then either progress in the cell cycle or die. In cells that eventually die, total transcription, polyadenylated [poly(A) ⁺ ] RNA synthesis, and protein synthesis were markedly inhibited only after 48 hours. Nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) levels decreased after 3 days. Cell membrane integrity was lost after 4 days. These results demonstrate that cells can be lethally damaged, yet continue to undergo apparently normal metabolic activities for several days. In a previous study, DNA double-strand breaks were detected after 1 day. We now show that by 2 days, breaks are visible as fragmentation in the nucleosome spacer regions of chromatin. This type of damage is consistent with cell death occurring by the process of apoptosis. Cell shrinkage and morphology were also consistent with this type of cell death. The slow cell death reported here appears to occur at the G ₂ /M transition and may involve events that normally occur at this stage of the cell cycle. These results demonstrate the importance of DNA degradation as an early and possibly essential step in cell death. [J Natl Cancer Inst 82: 749–755, 1990]

Cell death occurs by apoptosis during programmed deletion of cells and following exposure to cytotoxic agents. Central to the mechanism of apoptosis is internucleosomal DNA digestion by an endogenous endonuclease which is thought to mediate cell death. An axiom of apoptosis is that the endonuclease involved is a Ca²⁺/Mg²⁺-dependent endonuclease. During purification of endonucleases from Chinese hamster ovary cells, we found little Ca²⁺/Mg²⁺-dependent endonuclease activity, but large amounts of an endonuclease active below pH 7. This acidic endonuclease was activated in intact cells by reducing intracellular pH values below 7 with a proton ionophore. This activity generated internucleosomal digestion of DNA characteristic of apoptosis. Nuclear extracts contained a cation-independent endonuclease with identical pH-dependent activity. We have compared the acidic endonuclease to bovine deoxyribonuclease II (DNase II) and have found them nearly identical by all tests, including sensitivity to various inhibitors, purification by the same chromatographic steps, and recognition by antibody raised against the bovine enzyme. Addition of either the acidic endonuclease or bovine DNase II to isolated nuclei induced internucleosomal DNA digestion up through pH 6.5. These data demonstrate that DNase II can mediate internucleosomal DNA digestion characteristic of apoptosis following intracellular acidification. Furthermore, these data question the premise that the Ca²⁺/Mg²⁺-dependent endonuclease is the only endonuclease involved in apoptosis.

Viruses are reprogrammed into vectors for cancer treatment based on three types of modification: targeting, arming and shielding. Viruses that are turned into therapeutics are beginning to find their place in cancer clinical practice, in combination with chemotherapy and radiation. The principles of virus reprogramming are illustrated in this article using adenovirus, a DNA virus with a naked icosahedral capsid, and measles virus, an enveloped RNA virus with a helical capsid. Targeting introduces multiple layers of cancer specificity, thereby improving safety and efficacy. The four basic layers of specificity are: particle activation through cancer-specific proteases; cell entry through cancer-specific cell-surface proteins; control of viral transcription and replication by tissue-specific promoters; and preferential spread of viruses that exploit cancer-specific molecular defects. Arming occurs through genes that express prodrug convertases, pro-apoptotic proteins or immuno-activating proteins. Coating with polymers and sequential usage of different envelopes or capsids provides shielding from the host immune response. The window of therapeutic opportunity can be extended by temporary immunosuppression. A five-step plan to turn a virus of choice into a potential oncolytic is discussed.

Human species C adenovirus serotype 5 (Ad5) is the most common viral vector used in clinical studies worldwide. Ad5 vectors infect liver cells in vivo with high efficiency via a poorly defined mechanism, which involves virus binding to vitamin K-dependent blood coagulation factors. Here, we report that the major Ad5 capsid protein, hexon, binds human coagulation factor X (FX) with an affinity of 229 pM. This affinity is 40-fold stronger than the reported affinity of Ad5 fiber for the cellular receptor coxsackievirus and adenovirus receptor, CAR. Cryoelectron microscopy and single-particle image reconstruction revealed that the FX attachment site is localized to the central depression at the top of the hexon trimer. Hexon-mutated virus bearing a large insertion in hexon showed markedly reduced FX binding in vitro and failed to deliver a transgene to hepatocytes in vivo . This study describes the mechanism of FX binding to Ad5 and demonstrates the critical role of hexon for virus infection of hepatocytes in vivo .

More than two decades have passed since genetically modified HIV was used for gene delivery. Through continuous improvements these early marker gene-carrying HIVs have evolved into safer and more effective lentiviral vectors. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production. Accordingly, lentivector technologies now have widespread use in basic biology and translational studies for stable transgene overexpression, persistent gene silencing, immunization, in vivo imaging, generating transgenic animals, induction of pluripotent cells, stem cell modification and lineage tracking, or site-directed gene editing. Moreover, in the present high-throughput ‘-omics’ era, the commercial availability of premade lentiviral vectors, which are engineered to express or silence genome-wide genes, accelerates the rapid expansion of this vector technology. In the present review, we assess the advances in lentiviral vector technology, including basic lentivirology, vector designs for improved efficiency and biosafety, protocols for vector production and infection, targeted gene delivery, advanced lentiviral applications and issues associated with the vector system.