Suppressive effect of topoisomerase inhibitors on JC polyomavirus propagation in human neuroblastoma cells

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system, in immunocompromised patients. Because no drugs have been approved for treating PML, many antiviral agents are currently being investigated for this purpose. The inhibitory effects of the topoisomerase I inhibitors topotecan and β‐lapachone were assessed by investigating viral replication, propagation and viral protein 1 (VP1) production in cultured cells. JCPyV replication was assayed using the human neuroblastoma cell line IMR‐32 transfected with the JCPyV plasmid and RT‐ PCR combined with Dpn I treatment. Dpn I digests the input plasmid DNA containing methylated adenosine, but not newly replicated JCPyV DNA, in IMR‐32 cells. It was found that JCPyV replicates less in IMR‐32 cells treated with topotecan or β‐lapachone than in untreated cells. Moreover, drug treatment of JCI cells, which are IMR‐32 cells persistently infected with JCPyV, led to a reduction in the amount of JCPyV DNA and population of VP1‐positive cells. These results demonstrate that topotecan and β‐lapachone affects JCPyV propagation in human neuroblastoma cell lines, suggesting that topotecan and β‐lapachone could potentially be used to treat PML.     

Establishment of COS‐JC cells persistently producing archetype JC polyomavirus

JC polyomavirus (JCPyV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunocompromised patients. Archetype JCPyV circulates in the human population. There have been several reports of archetype JCPyV replication in cultured cells, in which propagation was not enough to produce high titers of archetype JCPyV. In this study, we carried out cultivation of the transfected cells with archetype JCPyV DNA MY for more than 2 months to establish COS‐7 cells (designated COS‐JC cells) persistently producing archetype JCPyV. Moreover, JCPyV derived from COS‐JC cells was characterized by analyzing the viral propagation, size of the viral genome, amount of viral DNA, production of viral protein, and structure of the non‐coding control region (NCCR). Southern blotting using a digoxigenin‐labeled JCPyV probe showed two different sizes of the JCPyV genome in COS‐JC cells. For molecular cloning, four of five clones showed a decrease in the size of complete JCPyV genome. Especially, clone No. 10 was generated the large deletion within the Large T antigen. On the other hand, the archetype structure of the NCCR was maintained in COS‐JC cells, although a few point mutations occurred. Quantitative PCR analysis of viral DNA in COS‐JC cells indicated that a high copy number of archetype JCPyV DNA was replicated in COS‐JC cells. These findings suggest that COS‐JC cells could efficiently propagate archetype JCPyV MY and offer a useful tool to study persistent infection of archetype JCPyV in a kidney‐derived system.

     

Characterization of the VP1 loop mutations widespread among JC polyomavirus isolates associated with progressive multifocal leukoencephalopathy

Recently, we found that JC polyomavirus (JCPyV) associated with progressive multifocal leukoencephalopathy (PML) frequently undergoes amino acid substitutions (designated VP1 loop mutations) in the outer loops of the major capsid protein, VP1. To further characterize the mutations, we analyzed the VP1 region of the JCPyV genome in brain-tissue or cerebrospinal fluid samples from 20 PML patients. VP1 loop mutations occurred far more frequently than silent mutations. Polymorphic residues were essentially restricted to three positions (55, 60, and 66) within the BC loop, one (123) within the DE loop, and three (265, 267, and 269) within the HI loop. The mutations at most polymorphic residues showed a trend toward a change to specific amino acids. Finally, we presented evidence that the VP1 loop mutations were associated with the progression of PML. These findings should form the basis for elucidating the biological significance of the VP1 loop mutations.     

Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning

In both yeast and mammals, the topoisomerase poison camptothecin (CPT) induces fork reversal, which has been proposed to stabilize replication forks, thus providing time for the repair of CPT‐induced lesions and supporting replication restart. We show that Tel1, the Saccharomyces cerevisiae orthologue of human ATM kinase, stabilizes CPT‐induced reversed forks by counteracting their nucleolytic degradation by the MRX complex. Tel1‐lacking cells are hypersensitive to CPT specifically and show less reversed forks in the presence of CPT. The lack of Mre11 nuclease activity restores wild‐type levels of reversed forks in CPT‐treated tel1Δ cells without affecting fork reversal in wild‐type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild‐type, tel1Δ, and mre11 nuclease‐deficient cells and relieves the hypersensitivity of tel1Δ cells to CPT. Altogether, our data indicate that Tel1 counteracts Mre11 nucleolytic activity at replication forks that undergo Mrc1‐mediated reversal in the presence of CPT.     

5-HT2 Receptors Facilitate JC Polyomavirus Entry

The human JC polyomavirus (JCPyV) causes the rapidly progressing demyelinating disease progressive multifocal leukoencephalopathy (PML). The disease occurs most often in individuals with AIDS but also occurs in individuals receiving immunomodulatory therapies for immune-related diseases such as multiple sclerosis. JCPyV infection of host cells requires the pentasaccharide lactoseries tetrasaccharide c (LSTc) and the serotonin receptor 5-hydroxytryptamine (5-HT) receptor 5-HT_2AR. While LSTc is involved in the initial attachment of virus to cells via interactions with VP1, the mechanism by which 5-HT_2AR contributes to infection is not clear. To further define the roles of serotonin receptors in infection, HEK293A cells, which are poorly permissive to JCPyV, were transfected with 14 different isoforms of serotonin receptor. Only 5-HT₂ receptors were found to support infection by JCPyV. None of the other 11 isoforms of serotonin receptor supported JCPyV infection. Expression of 5-HT₂ receptors did not increase binding of JCPyV to cells, but this was not unexpected, given that the cells uniformly expressed the major attachment receptor, LSTc. Infection of these cells remained sensitive to inhibition with soluble LSTc, confirming that LSTc recognition is required for JCPyV infection. Virus internalization into HEK293A cells was significantly and specifically enhanced when 5HT₂ receptors were expressed. Taken together, these data confirm that the carbohydrate LSTc is the attachment receptor for JCPyV and that the type 2 serotonin receptors contribute to JCPyV infection by facilitating entry.     

Topoisomerase I‐Mediated Antiproliferative Activity of 10‐Substituted and 12‐Substituted Homocamptothecins

Homocamptothecin (hCPT) is an E‐ring modified camptothecin (CPT) analogue, which showed pronounced inhibitory activity of topoisomerase I. In search of novel hCPT‐type anticancer agents, two series of hCPT derivatives were synthesized and evaluated in vitro against three human tumor cell lines. The results indicated that the 10‐substituted hCPT derivatives had a considerably higher cytotoxic activity than the 12‐substituted ones. Among the 10‐substituted compounds, 8a, 8b, 9b , and 9i showed an equivalent or even more potent activity than the positive control drug topotecan against the lung cancer cell line A‐549. Moreover, the hCPT analogues 8a and 8b exhibited a higher topoisomerase I inhibitory activity than CPT at a concentration of 100 μM .     

Replication of IMR‐32‐adapted JC virus clones in human embryonic kidney cells

It has been difficult to study JCV replication because of its restricted host range. In this study, JCV replication was examined using different clones in 293 cells. RT‐PCR assay revealed that large T antigen expression in cells transfected with IMR‐32‐adapted JCVs was significantly greater than in those transfected with Mad‐1 or CY. DNA replication assay and viral load verified that the IMR‐32‐adapted JCVs were replication‐competent in 293 cells, but not Mad‐1 or CY JCVs. These results suggest that a 293 culture system with IMR‐32‐adapted JCVs may be a useful tool for assessing replication of JCV in vitro.     

Efficient propagation of progressive multifocal leukoencephalopathy-type JC virus in COS-7-derived cell lines stably expressing Tat protein of human immunodeficiency virus type 1

The high incidence of progressive multifocal leukoencephalopathy (PML) in AIDS patients compared with many other immunosuppressive diseases suggests that HIV-1 infection is strictly related to the activation of JC virus (JCV) propagation. In this report, propagation of PML-type JCV in COS-7-derived cell lines stably expressing HIV-1 Tat (COS-tat cells) has been examined. In COS-tat cells, production of viral particles and replication of genomic DNA were markedly increased compared to COS-7 cells, as judged by HA and real-time PCR analyses. These results demonstrate that COS-tat cells provide a useful model system for studying HIV-1 Tat-mediated propagation of PML-type JCV.     

Beta‐lapachone inhibits pathological retinal neovascularization in oxygen‐induced retinopathy via regulation of HIF‐1α

Retinal neovascularization in retinopathy of prematurity (ROP) is the most common cause of blindness for children. Despite evidence that hypoxia inducible factor (HIF)‐1α ‐VEGF axis is associated with the pathogenesis of ROP, the inhibitors of HIF‐1α have not been established as a therapeutic target in the control of ROP pathophysiology. We investigated the hypothesis that degradation of HIF‐1α as a master regulator of angiogenesis in hypoxic condition, using β‐lapachone, would confer protection against hypoxia‐induced retinopathy without affecting physiological vascular development in mice with oxygen‐induced retinopathy (OIR), an animal model of ROP. The effects of β‐lapachone were examined after intraocular injection in mice with OIR. Intraocular administration of β‐lapachone resulted in significant reduction in hypoxia‐induced retinal neovascularization without retinal toxicity or perturbation of developmental retinal angiogenesis. Our results demonstrate that HIF‐1α–mediated VEGF expression in OIR is associated with pathological neovascularization, not physiological angiogenesis. Thus, strategies blocking HIF‐1α in the developing eye in the pathological hypoxia could serve as a novel therapeutic target for ROP.     

Detection of JC virus-specific cytotoxic T lymphocytes in healthy individuals

The polyomavirus JC (JCV) infects 85% of healthy individuals, and its reactivation in a limited number of immunosuppressed people causes progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease of the central nervous system. We hypothesized that JCV-specific cytotoxic T lymphocytes (CTLs) might control JCV replication in healthy individuals, blocking the evolution of PML. Using 51Cr release and tetramer staining assays, we show that 8 of 11 HLA-A*0201+ healthy subjects (73%) harbor detectable JCV-specific CD8+ CTLs that recognize one or two epitopes of JCV VP1 protein, the HLA-A*0201-restricted VP1p36 and VPp1100 epitopes. We determined that the frequency of JCV VP1 epitope-specific CTLs varied from less than 1/100,000 to 1/2,494 peripheral blood mononuclear cells. More individuals had JCV VP1-specific than cytomegalovirus-specific CTLs (8 of 11 subjects [73%] versus 2 of 10 subjects [20%], respectively). These results show that a CD8+-T-cell response against JCV is commonly found in immunocompetent people and suggest that these cells might protect against the development of PML.     

Enhanced killing of cancer cells by poly(ADP-ribose) polymerase inhibitors and topoisomerase I inhibitors reflects poisoning of both enzymes

Poly(ADP-ribose) polymerase-1 (PARP1) plays critical roles in the regulation of DNA repair. Accordingly, small molecule inhibitors of PARP are being developed as agents that could modulate the activity of genotoxic chemotherapy, such as topoisomerase I poisons. In this study we evaluated the ability of the PARP inhibitor veliparib to enhance the cytotoxicity of the topoisomerase I poisons topotecan and camptothecin (CPT). Veliparib increased the cell cycle and cytotoxic effects of topotecan in multiple cell line models. Importantly, this sensitization occurred at veliparib concentrations far below those required to substantially inhibit poly(ADP-ribose) polymer synthesis and at least an order of magnitude lower than those involved in selective killing of homologous recombination-deficient cells. Further studies demonstrated that veliparib enhanced the effects of CPT in wild-type mouse embryonic fibroblasts (MEFs) but not Parp1(-/-) MEFs, confirming that PARP1 is the critical target for this sensitization. Importantly, parental and Parp1(-/-) MEFs had indistinguishable CPT sensitivities, ruling out models in which PARP1 catalytic activity plays a role in protecting cells from topoisomerase I poisons. To the contrary, cells were sensitized to CPT in a veliparib-independent manner upon transfection with PARP1 E988K, which lacks catalytic activity, or the isolated PARP1 DNA binding domain. These results are consistent with a model in which small molecule inhibitors convert PARP1 into a protein that potentiates the effects of topoisomerase I poisons by binding to damaged DNA and preventing its normal repair.     

Low frequency of cytotoxic T lymphocytes against the novel HLA-A*0201-restricted JC virus epitope VP1(p36) in patients with proven or possible progressive multifocal leukoencephalopathy

JC virus (JCV)-specific cytotoxic T lymphocytes (CTL) in peripheral blood are associated with a favorable outcome in patients with progressive multifocal leukoencephalopathy (PML). However, the frequency of these cells in the peripheral blood mononuclear cells (PBMC) of PML patients is unknown. To develop a highly sensitive assay for detecting the cellular immune response against this virus, we performed a CTL epitope mapping study of JCV VP1 major capsid protein by using overlapping peptides. A novel HLA-A*0201-restricted epitope, the VP1(p36) peptide SITEVECFL, was characterized. The cellular immune response against JCV was assessed in 32 study subjects. By combining the results of the (51)Cr release assay on pooled peptides and staining with the HLA-A*0201/JCV VP1(p36) tetramer, VP1-specific CTL were detected in 10 of 11 PML survivors (91%) versus only 1 of 11 PML progressors (9%, P = 0.0003). VP1-specific CTL were also detected in two of two patients recently diagnosed with PML and in four of four human immunodeficiency virus-positive patients with possible PML. The frequency of CTL specific for the novel VP1(p36) and the previously described VP1(p100) epitopes was determined. In two patients, the frequency of CTL specific for the VP1(p36) or VP1(p100) epitopes, as determined by fresh blood tetramer staining (FBTS), ranged from 1/6,000 to 1/24,000 PBMC. A CTL sorting technique combining tetramer staining and selection with immunomagnetic beads allowed the detection of epitope-specific CTL in two cases that were determined to be negative by FBTS. The phenotype of these CTL in vivo was consistent with activated memory cells. These data suggest that, although present in low numbers, JCV-specific CTL may be of central importance in the containment of JCV spread in immunosuppressed individuals.     

Inhibition of JC polyomavirus infectivity by the retrograde transport inhibitor Retro-2.1

JC polyomavirus (JCPyV) is a common human pathogen that results in a chronic asymptomatic infection in healthy adults. Under conditions of immunosuppression, JCPyV spreads to the central nervous system and can cause the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML), a disease for which there are no vaccines or antiviral therapies. Retro-2 is a previously identified small molecule inhibitor that was originally shown to block retrograde transport of toxins such as ricin toxin from endosomes to the Golgi apparatus and endoplasmic reticulum (ER), and Retro-2.1 is a chemical analog of Retro-2 that has been shown to inhibit ricin intoxication of cells at low nanomolar concentrations. Retro-2 has previously been shown to prevent retrograde transport of JCPyV virions to the ER, but the effect of Retro-2.1 on JCPyV infectivity is unknown. Here it is shown that Retro-2.1 inhibits JCPyV with an EC₅₀ of 3.9 μM. This molecule inhibits JCPyV infection at dosages that are not toxic to human tissue culture cells. Retro-2.1 was also tested against two other polyomaviruses, the human BK polyomavirus and simian virus 40, and was also shown to inhibit infection at similar concentrations. Viral uncoating studies demonstrate that Retro-2.1 inhibits BKPyV infectivity in a manner similar to Retro-2. These studies demonstrate that improved analogs of Retro-2 can inhibit infection at lower dosages than Retro-2 and further optimization of these compounds may lead to effective treatment options for those suffering from JCPyV infection and PML.     

Altered phosphorylation of topoisomerase I following overexpression in an ovarian cancer cell line.

There is a growing interest regarding the use of camptothecins (CPTs) for the management of ovarian cancer. Since topoisomerase I has been established as a prime target of these drugs in other experimental models, it was important to determine whether sensitivity to CPTs in ovarian cancer cells is also correlated with the cellular level of this enzyme. Despite the 7-fold increase in topoisomerase expression achieved by adenovirus-mediated expression, the sensitivity to a CPT derivative (topotecan), was not improved compared with control cells harboring an endogenous level of the enzyme. This observation is in accordance with the similar level of topoisomerase I activity found in control and overexpressing cells and suggests that these cells may efficiently regulate the enzyme activity. Indeed, topoisomerase I overexpressing cells are characterized by a lack of alkaline phosphatase sensitivity and elimination of the hyperphosphorylated form of the protein. Taken together, these observations strongly suggest that an alteration in the phosphorylation state of topoisomerase I could limit its activity and prevent improvement of CPT response in ovarian cancer cells. In addition, a limited extent of topoisomerase I phosphorylating activity was found in nuclear extract of OVCAR-3 cells. Hence, providing enhancement in topoisomerase I expression may not result in improvement of CPT response in ovarian cancer cells because of an efficient control of the phosphorylation state of the enzyme.     

Synthesis and Biological Evaluation of 7‐Alkenyl Homocamptothecins as Potent Topoisomerase I Inhibitors

Homocamptothecin (hCPT) is a camptothecin (CPT) derivative with a seven‐membered β‐hydroxylactone E ring, which shows higher lactone stability and improves topoisomerase I (Topo I) inhibition activity. In an attempt to improve the antitumor activity of homocamptothecins, a series of 7‐alkenyl‐homocamptothecin derivatives was designed and synthesized based on a semisynthetic route starting from CPT. Most of the synthesized compounds exhibit higher cytotoxic activities on the A‐549 tumor cell line than topotecan (TPT). Some compounds such as 2a and 2o show a broad in vitro antitumor spectrum and exhibit superior Topo I‐inhibition activity.     

WRN protects against topo I but not topo II inhibitors by preventing DNA break formation

The Werner syndrome helicase/3′-exonuclease (WRN) is a major component of the DNA repair and replication machinery. To analyze whether WRN is involved in the repair of topoisomerase-induced DNA damage we utilized U2-OS cells, in which WRN is stably down-regulated (wrn-kd), and the corresponding wild-type cells (wrn-wt). We show that cells not expressing WRN are hypersensitive to the toxic effect of the topoisomerase I inhibitor topotecan, but not to the topoisomerase II inhibitor etoposide. This was shown by mass survival assays, colony formation and induction of apoptosis. Upon topotecan treatment WRN deficient cells showed enhanced DNA replication inhibition and S-phase arrest, whereas after treatment with etoposide they showed the same cell cycle response as the wild-type. A considerable difference between WRN and wild-type cells was observed for DNA single- and double-strand break formation in response to topotecan. Topotecan induced DNA single-strand breaks 6 h after treatment. In both wrn-wt and wrn-kd cells these breaks were repaired at similar kinetics. However, in wrn-kd but not wrn-wt cells they were converted into DNA double-strand breaks (DSBs) at high frequency, as shown by neutral comet assay and phosphorylation of H2AX. Our data provide evidence that WRN is involved in the repair of topoisomerase I, but not topoisomerase II-induced DNA damage, most likely via preventing the conversion of DNA single-strand breaks into DSBs during the resolution of stalled replication forks at topo I–DNA complexes. We suggest that the WRN status of tumor cells impacts anticancer therapy with topoisomerase I, but not topoisomerase II inhibitors.     

Tipin Functions in the Protection against Topoisomerase I Inhibitor

The replication fork temporarily stalls when encountering an obstacle on the DNA, and replication resumes after the barrier is removed. Simultaneously, activation of the replication checkpoint delays the progression of S phase and inhibits late origin firing. Camptothecin (CPT), a topoisomerase I (Top1) inhibitor, acts as a DNA replication barrier by inducing the covalent retention of Top1 on DNA. The Timeless-Tipin complex, a component of the replication fork machinery, plays a role in replication checkpoint activation and stabilization of the replication fork. However, the role of the Timeless-Tipin complex in overcoming the CPT-induced replication block remains elusive. Here, we generated viable TIPIN gene knock-out (KO) DT40 cells showing delayed S phase progression and increased cell death. TIPIN KO cells were hypersensitive to CPT. However, homologous recombination and replication checkpoint were activated normally, whereas DNA synthesis activity was markedly decreased in CPT-treated TIPIN KO cells. Proteasome-dependent degradation of chromatin-bound Top1 was induced in TIPIN KO cells upon CPT treatment, and pretreatment with aphidicolin, a DNA polymerase inhibitor, suppressed both CPT sensitivity and Top1 degradation. Taken together, our data indicate that replication forks formed without Tipin may collide at a high rate with Top1 retained on DNA by CPT treatment, leading to CPT hypersensitivity and Top1 degradation in TIPIN KO cells.     

JC virus-specific cytotoxic T lymphocytes in individuals with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by a reactivation of the polyomavirus JC (JCV) within a setting of immunosuppression. The nature of the immune response that contains replication of this virus is unknown. We have explored JCV-specific cellular immune responses in patients with PML and control subjects. JCV antigen-stimulated peripheral blood mononuclear cells (PBMC) of four human immunodeficiency virus (HIV)-infected patients who were survivors of PML and one HIV-uninfected patient recently diagnosed with PML lysed autologous B-lymphoblastoid cell lines expressing either the JCV T regulatory protein or the VP1 major capsid protein. This lysis was mediated by CD8(+) T lymphocytes and was major histocompatibility complex class I restricted. These cells were therefore cytotoxic T lymphocytes (CTL). JCV-specific CTL could not be detected in PBMC of three HIV-infected PML patients who had progressive neurologic disease and an eventual fatal outcome. These data suggest that the JCV-specific cellular immune response may play a crucial role in the containment of PML. This finding may also prove useful as a favorable prognostic marker in the clinical management of these patients.     

Specific antibodies reacting to JC polyomavirus capsid protein mimotopes in sera from multiple sclerosis and other neurological diseases-affected patients

Published data support the hypothesis that viruses could be trigger agents of multiple sclerosis onset. This link is based on evidence of early exposure to viral agents in patients affected by this neurologic disease. JC (JC polyomavirus [JCPyV]), BK (BKPyV), and simian virus 40 (SV40) neurotropic polyomavirus footprints have been detected in brain tissue specimens and samples from patients affected by different neurological diseases. In this investigation, serum samples from patients affected by multiple sclerosis and other inflammatory and noninflammatory neurologic diseases, as well as healthy subjects representing the control, were investigated for immunoglobulin G (IgG) antibodies against JCPyV. To this end, an immunologic approach was employed, which consists of employing indirect enzyme-linked immunosorbent assay testing with synthetic peptides mimicking viral capsid protein 1 antigens. A significantly lower prevalence of IgG antibodies against JCPyV VP1 epitopes, with a low titer, was detected in serum samples from patients with multiple sclerosis (MS) and other neurologic diseases than in healthy subjects. Our study indicates that the prevalence of JCPyV antibodies from patients with multiple sclerosis is 50% lower than in healthy subjects, suggesting specific immune impairments. These results indicate that patients affected by neurological diseases, including MS, respond poorly to JCPyV VP1 antigens, suggesting specific immunologic dysfunctions.     

Early years of biological agents therapy in Crohn's disease and risk of the human polyomavirus JC reactivation

Although the remarkable efficacy of biological therapy has resulted in significant success in inflammatory bowel disease (IBD) management, susceptibility to infections remains a concern. The biological agents include the tumor necrosis factor‐α (TNF‐α) inhibitors, for instance infliximab, and other immunomodulating agents, such as natalizumab. Progressive multifocal leukoencephalopathy (PML), a rare but mostly fatal opportunistic brain infection caused by reactivation of the human polyomavirus JC virus (JCV), has been found in two patients with multiple sclerosis and one patient with Crohn's disease (CD), linked to treatment with natalizumab. After these cases of PML, the commercial and investigational use of natalizumab was suspended in February 2005 but was subsequently resumed for multiple sclerosis and for CD, only through a special restricted distribution program. This review, starting from an extensive literature search by the PubMed database, resumes the clinical aspects and pathophysiology of CD and focuses on the biologics in current use in CD (infliximab, adalimumab, and natalizumab), in order to provide a reference and gateway to prevention, recognition, and management of JCV, in the early years of biological agents therapy. It also proposed to provide an overview on the hypothetical mechanism of reactivation of JC virus related to the use of these drugs. J. Cell. Physiol. 224: 316–326, 2010. © 2010 Wiley‐Liss, Inc.