Cloned human polyomavirus JC DNA can transform human amnion cells.

The genome of the human polyomavirus JC (Mad-1 strain) was molecularly cloned in Escherichia coli by using the plasmid vector pBR322. Recombinant DNA molecules were constructed with the entire JC genome inserted either at its unique EcoRI site at 0.0 map units or at its unique BamHI site at 0.51 map units. Viral DNA from each of these recombinant plasmids was capable of transforming human amnion cells, and cell lines established from transformed foci were positive for JC tumor antigen as assayed by indirect immunofluorescence.     

The investigation and diagnosis of pathogenic mitochondrial DNA mutations in human urothelial cells

Patients with mitochondrial DNA disease are amongst the most challenging to diagnose and manage given the striking phenotypic and genetic heterogeneity, which characterise these conditions. Recently, we and others have demonstrated the m.3243A>G mutation, one of the most common mitochondrial DNA pathogenic mutations, is present at clinically relevant levels in urinary epithelium, thus providing a practical, non-invasive test for diagnosis and mutation screening. In this study we further evaluate the use of these cells in detecting the m.3243A>G mutation, other mtDNA tRNA gene point mutations including the m.8344A>G mutation and single large-scale mtDNA deletions. We observe a robust relationship between m.3243A>G levels in urothelial cells and clinically affected tissues that does not change with time. Conversely, single large-scale mtDNA deletions can be detected in urothelial cells, with higher levels present in younger patients with more severe disease, but generally mtDNA deletion levels are not representative of those seen in a clinically affected tissue. Our results have implications for the diagnosis, management and counselling of families with mtDNA disease.     

CPT11 prevents virus replication in JCI cells persistently infected with JC polyomavirus

JC polyomavirus (JCPyV) is the causative agent of the demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML), which occurs in immunocompromised patients. Moreover, patients treated with natalizumab for multiple sclerosis or Crohn disease can develop PML, which is then termed natalizumab‐related PML. Because few drugs are currently available for treating PML, many antiviral agents are being investigated. It has been demonstrated that the topoisomerase I inhibitors topotecan and β‐lapachone have inhibitory effects on JCPyV replication in IMR‐32 cells. However, both of these drugs have marginal inhibitory effects on virus propagation in JC1 cells according to RT‐PCR analysis. In the present study, the inhibitory effect of another topoisomerase I inhibitor, 7‐ethy‐10‐[4‐(1‐piperidino)‐1‐piperidino] carbonyloxy camptothecin (CPT11), was assessed by investigating viral replication, propagation, and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using real‐time PCR combined with Dpn I treatment in IMR‐32 cells transfected with JCPyV DNA. It was found that JCPyV replicates less in IMR‐32 cells treated with CPT11 than in untreated cells. Moreover, CPT11 treatment of JCI cells persistently infected with JCPyV led to a dose‐dependent reduction in JCPyV DNA and VP1 production. Additionally, the inhibitory effect of CPT11 was found to be stronger than those of topotecan and β‐lapachone. These findings suggest that CPT11 may be a potential anti‐JCPyV agent that could be used to treat PML.

     

Image cytometry detection of breast cancer cells with > 5C DNA content and minor DNA stemlines

OBJECTIVE: To determine if the presence of cells having a DNA content > 5c and occurring at very low frequency is related to breast cancer outcome. STUDY DESIGN: Feulgen-stained imprints of fresh tumors used for routine standard DNA image cytometry were reanalyzed, with the aim of detecting hyperploid (> 5c) cells or minor stemlines. Specially adapted software was used. RESULTS: The new DNA analysis showed discordance of 47.3% with standard DNA cytometry. Minor stemline or rarely occurring 5c exceeding cells were found. These were not detected by the first DNA analysis. The presence of both DNA hyperploid cells occurring as rare events and a DNA hyperploid stemline was related to outcome. CONCLUSION: The detection of DNA hyperploid cells, even in very small numbers, appears essential to outcome, particularly in diploid or single DNA aneuploid breast cancers.     

Plasmodium berghei - infected red cells sorted according to DNA content.

A cell-sorting method is described for the analysis and separation of red blood cells in Plasmodium berghei-infected mouse blood based on their DNA content. This method involves a selective uptake of the bis-benzimidazole dye 33258 Hoechst, a DNA-binding dye, by red blood cells containing parasites. Infected blood is incubated at 37 degrees C with the dye then washed at 4 degrees C to remove unbound dye. Uninfected cells are then non-fluorescent at the characteristic wavelengths for 33258 Hoechst excitation and emission, whereas parasitized cells display fluorescence intensities in approximately direct proportion tothe number of parasite nuclei (i.e. amount of parasite DNA) within the cell and can be sorted accordingly. Providing cells were incubated in a complex nutrient medium during dye uptake at 37 degrees C, the sorted parasite-infected cells produced lethal P. berghei infections when injected into BALB/c mice. The dye-labelling technique is simple and sufficient red blood cells at various stages of infection can be collected for biochemical or immunochemical studies by cell sorting.     

DNA content in the Purkinje cells of the rat cerebellum spontaneously infected with Kilham virus]

The DNA content in the cerebellum Purkinje cells of rats naturally contaminated with the Kilham virus and in the animals, in which no infection was observed, was determined cytospectrophotometrically on the Feulgen-stained preparations. The incidence of the cells with hyperdiploid nuclei was shown to be equal in the groups compared. It was concluded that the nonmultiple increase of the DNA content in the Purkinje cells of rat cerebellum was apparently unrelated to the virus infection.     

Flow cytometry analysis of early DNA content changes in human and monkey cells following infection with simian virus 40

Simian virus 40 (SV₄₀) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfects (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI-38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (～60%) of the cells were blocked in the G₂ + M phase of the cell cycle. At later time intervals, an increase in the G₁ population was demonstrated. The two monkey cell lines responded to SV₄₀ virus with an accumulation of cells in the G₂ + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV₄₀ virus. The one strain (WI-38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transformin) cells infected with SV₄₀ virus.     

Increased DNA content of HSR-marker chromosomes of human neuroblastoma cells

Scanning cytophotometry demonstrates that the DNA content of human neuroblastoma chromosomes containing a specific abnormal segment—the Homogeneously Staining Region (HSR)—is significantly greater than that of homologous normal chromosomes without this segment. The potential significance of this finding and its relationship to another chromosome abnormality, the double minutes, is discussed.     

DNA damage and oxidative stress in human cells infected by Trypanosoma cruzi

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase-1 (PARP1) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2–related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells treated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.     

Anion homeostasis is important for non-lytic release of BK polyomavirus from infected cells

BK polyomavirus (BKPyV) is a member of a family of potentially oncogenic viruses, whose reactivation can cause severe pathological conditions in transplant patients, leading to graft rejection. As with many non-enveloped viruses, it is assumed that virus release occurs through lysis of the host cell. We now show the first evidence for a non-lytic release pathway for BKPyV and that this pathway can be blocked by the anion channel inhibitor DIDS. Our data show a dose-dependent effect of DIDS on the release of BKPyV virions. We also observed an accumulation of viral capsids in large LAMP-1-positive acidic organelles within the cytoplasm of cells upon DIDS treatment, suggesting potential late endosome or lysosome-related compartments are involved in non-lytic BKPyV release. These data highlight a novel mechanism by which polyomaviruses can be released from infected cells in an active and non-lytic manner, and that anion homeostasis regulation is important in this pathway.     

Image analysis of DNA content and nuclear morphometry for predicting radiosensitivity of nasopharyngeal carcinoma

OBJECTIVE: To investigate the relationship among nuclear DNA content, nuclear morphology, clinical response, and radiosensitivity in nasopharyngeal carcinoma (NPC) and the suitability of image cytometric analysis of DNA content and nuclear morphology for predicting radiosensitivity of NPC prior to radiotherapy. STUDY DESIGN: Nuclear DNA content and morphology features were detected by image cytometric analysis in 51 biopsy specimens of NPC prior to radiotherapy. The radiotherapeutic effect experienced by the NPC patients was classified as CR (complete response [i.e., complete tumor disappearance]) and PR (partial response [i.e., residual tumor]) according to pathologic analysis of tumor specimens after completion of the scheduled treatment. RESULTS: The mean DNA index; the percentage of cells with the DNA pattern of 2C, 5C, aneuploidy respectively; the mean nuclear area; the mean nuclear perimeter and the mean nuclear diameter in the CR group were significantly higher than they were in the PR group. CONCLUSION: DNA content and nuclear morphometry by image cytometric analysis were significantly correlated with patient outcome and radiosensitivity of NPC. Other measurements of more biomarkers for predicting the radiosensitivity of NPC await further study.     

Proliferative capacity and DNA content of aging human diploid cells in culture: A cytophotometric and autoradiographic analysis

Using a combination of DNA-cytophotometry and tritiated thymidine-autoradiography, we have shown that the majority of nondividing cells in serially propagated human diploid cell populations have the 2C DNA content consistent with their being arrested in the G₁ phase of the diploid cell cycle. Unlabeled 4C cells appear increasingly with time in culture. These may be arrested G₂ diploids or they may be G₁ tetraploids, since there is an associated increase in polyploidy in older cultures as evidenced by the appearance of labeled 8C cells.     

A cell-free replication system for human polyomavirus JC DNA.

The human polyomavirus JC virus (JCV) establishes persistent infections in most individuals and is the etiologic agent of progressive multifocal leukoencephalopathy. In this report, we describe the establishment of a soluble cell-free system that is capable of replicating exogenous plasmid DNA containing the JCV origin of replication. Replication in this system is completely dependent on the addition of JCV large T antigen (TAg). To prepare JCV TAg for replication analysis, a recombinant baculovirus containing the JCV TAg-coding sequence was generated. TAg expressed in insect cells was purified by metal chelate chromatography. JCV TAg supported initiation of JCV DNA replication in the presence of DNA polymerase alpha-primase, replication protein A, and topoisomerase I in a dose-dependent manner and was also capable of supporting DNA replication in crude human cell extracts. Point mutation of TAg-binding site I strongly diminished TAg binding and concomitantly reduced JCV DNA replication in vivo and in vitro by approximately 50%. Point mutation of TAg-binding site II or deletion of the early palindrome completely abolished replication of JCV origin-containing plasmid DNA in vivo and in vitro, marking these sequences as essential components of the JCV core origin. A comparison of several TAgs showed that simian virus 40 TAg, but not mouse polyomavirus (PyV) TAg, supported replication of a plasmid containing a JCV origin. These findings provide evidence that replication in the cell-free system faithfully mimics JCV DNA replication in vivo. Therefore, it may be a useful tool for future analysis of interactions between JCV and its host cell.     

Reversible tenfod reduction in mitochondrial DNA content of human cells treated with ethidium bromide

Summary Cells of the human line VA₂-B in suspension culture have been treated with very low concentrations of ethidium bromide for the purpose of reducing the amount of mitochondrial DNA (mit-DNA) per cell. Cells maintained in the presence of 5 ng/ml ethidium bromide grew at a normal rate for three days; thereafter, their doubling time gradually increased to a stable value of about 60 h. In these cells, the rate of ³H thymidine incorporation into mit-DNA decreased very rapidly to 60% of the normal, and remained thereafter at this level, while the amount of mit-DNA per cell stabilized around a level of 70–80% of the control. In cells long-term treated with 5 ng/ml ethidium bromide, the rate of mitochondrial protein synthesis was about 35% of the normal, and the cytochrome c oxidase activity about 50% of the control. Cells treated with 20 ng/ml of the drug underwent 3–4 cell doublings at control rates, then gradually stopped growing, and eventually died. In these cells, the rate of incorporation of ³H thymidine into mit-DNA was reduced to 50% of the control value after 10 min treatment with ethidium bromide, and became barely detectable after three cell doublings. At this time, the cells had on the average less than 10% of the control amount of mit-DNA, the rate of mitochondrial protein synthesis was reduced to 3% of the normal, and the specific activities of cytochrome c oxidase and rutamycin-sensitive ATPase were less than 20% of the control values. In spite of these marked changes, the cells exhibited only a 20–30% loss in cell viability, as estimated by cloning efficiency, after three days of exposure to the drug. Cells treated with ethidium bromide at 20 ng/ml for three days, and then transferred to drug-free medium, recovered a near-to-normal growth rate and cloning efficiency and a near-to-normal rate of synthesis and amount of mit-DNA in about five days.     

Zinc-finger-nucleases mediate specific and efficient excision of HIV-1 proviral DNA from infected and latently infected human T cells

HIV-infected individuals currently cannot be completely cured because existing antiviral therapy regimens do not address HIV provirus DNA, flanked by long terminal repeats (LTRs), already integrated into host genome. Here, we present a possible alternative therapeutic approach to specifically and directly mediate deletion of the integrated full-length HIV provirus from infected and latently infected human T cell genomes by using specially designed zinc-finger nucleases (ZFNs) to target a sequence within the LTR that is well conserved across all clades. We designed and screened one pair of ZFN to target the highly conserved HIV-1 5′-LTR and 3′-LTR DNA sequences, named ZFN-LTR. We found that ZFN-LTR can specifically target and cleave the full-length HIV-1 proviral DNA in several infected and latently infected cell types and also HIV-1 infected human primary cells in vitro. We observed that the frequency of excision was 45.9% in infected human cell lines after treatment with ZFN-LTR, without significant host-cell genotoxicity. Taken together, our data demonstrate that a single ZFN-LTR pair can specifically and effectively cleave integrated full-length HIV-1 proviral DNA and mediate antiretroviral activity in infected and latently infected cells, suggesting that this strategy could offer a novel approach to eradicate the HIV-1 virus from the infected host in the future.