FOXO1 stimulates tip cell-enriched gene expression in endothelial cells.

Forkhead box O (FOXO) family proteins are expressed in various cells, and play crucial roles in cellular metabolism, apoptosis, and aging. FOXO1-null mice exhibit embryonic lethality due to impaired endothelial cell (EC) maturation and vascular remodeling. However, FOXO1-mediated genome-wide regulation in ECs remains unclear. Here, we demonstrate that VEGF dynamically regulates FOXO1 cytosol-nucleus translocation. FOXO1 re-localizes to the nucleus via PP2A phosphatase. RNA-seq combined with FOXO1 overexpression/knockdown in ECs demonstrated that FOXO1 governs the VEGF-responsive tip cell-enriched genes, and further inhibits DLL4-NOTCH signaling. Endogenous FOXO1 ChIP-seq revealed that FOXO1 binds to the EC-unique tip-enriched genes with co-enrichment of EC master regulators, and the condensed chromatin region as a pioneer factor. We identified new promoter/enhancer regions of the VEGF-responsive tip cell genes regulated by FOXO1: ESM1 and ANGPT2. This is the first study to identify cell type-specific FOXO1 functions, including VEGF-mediated tip cell definition in primary cultured ECs.

Factors affecting the number of bacteria in saliva and oral care methods for the recovery of bacteria in contaminated saliva after brushing: a randomized controlled trial.

BACKGROUND: Oral care is important in preventing aspiration pneumonia in older adults. However, it is not clear what kind of oral care can reduce the number of bacteria in saliva. The purposes of this study are to clarify whether there is a relationship between plaque amounts and salivary bacterial counts, and how bacteria dispersed into the oral cavity by brushing can be reduced. METHODS: First, saliva samples were collected from 10 healthy adult volunteers after 30 h of unbrushing and after thorough brushing, and the total bacterial count was determined by real-time PCR. Next, 40 older adults attending an outpatient dental clinic were randomly assigned into two groups: a wiping group (20 patients) and a mouthwashing group (20 patients). Saliva was collected before and after brushing, and after wiping in the wiping group and after mouthwashing in the mouthwashing group, and the total bacterial count was quantified by real-time PCR. RESULTS: In a study of volunteers, there was no association between plaque amounts and salivary bacterial counts. In a study of older adult patients, salivary bacterial counts were significantly higher in patients with higher oral hygiene index and fewer remaining teeth. Brushing increased salivary bacterial counts. Wiping did not significantly reduce the number of bacteria, while mouthwash returned the increased number of bacteria after brushing to the pre-brushing level. CONCLUSIONS: There is no direct relationship between the amount of plaque and the number of bacteria in saliva. Brushing disperses bacteria into the oral cavity, resulting in a marked increase in the number of bacteria in saliva. Wiping does not collect the dispersed bacteria, and it seems essential to rinse the mouth after brushing. TRIAL REGISTRATION: UMIN000045854.

Development and Crosslinking Properties of Psoralen-Conjugated Triplex-Forming Oligonucleotides as Antigene Tools Targeting Genome DNA.

Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been utilized for genome editing and anti-gene experiments for over thirty years. However, the research on Ps-TFOs employing artificial nucleotides is still limited, and their photo-crosslinking properties have not been thoroughly investigated in relation to biological activities. In this study, we extensively examined the photo-crosslinking properties of Ps-TFOs to provide fundamental insights for future Ps-TFO design. We developed novel Ps-TFOs containing 2'-O,4'-C-methylene-bridged nucleic acids (Ps-LNA-mixmer) and investigated their photo-crosslinking properties using stable cell lines that express firefly luciferase constitutively to evaluate the anti-gene activities of Ps-LNA-mixmer. As a result, Ps-LNA-mixmer successfully demonstrated suppression activity, and we presented the first-ever correlation between photo-crosslinking properties and their activities. Our findings also indicate that the photo-crosslinking process is insufficient under cell irradiation conditions (365 nm, 2 mW/cm2 , 60 min). Therefore, our results highlight the need to develop new psoralen derivatives that are more reactive under cell irradiation conditions.

Amphiphilic peptide-tagged N-cadherin forms radial glial-like fibers that enhance neuronal migration in injured brain and promote sensorimotor recovery.

The mammalian brain has very limited ability to regenerate lost neurons and recover function after injury. Promoting the migration of young neurons (neuroblasts) derived from endogenous neural stem cells using biomaterials is a new and promising approach to aid recovery of the brain after injury. However, the delivery of sufficient neuroblasts to distant injured sites is a major challenge because of the limited number of scaffold cells that are available to guide neuroblast migration. To address this issue, we have developed an amphiphilic peptide [(RADA)3-(RADG)] (mRADA)-tagged N-cadherin extracellular domain (Ncad-mRADA), which can remain in mRADA hydrogels and be injected into deep brain tissue to facilitate neuroblast migration. Migrating neuroblasts directly contacted the fiber-like Ncad-mRADA hydrogel and efficiently migrated toward an injured site in the striatum, a deep brain area. Furthermore, application of Ncad-mRADA to neonatal cortical brain injury efficiently promoted neuronal regeneration and functional recovery. These results demonstrate that self-assembling Ncad-mRADA peptides mimic both the function and structure of endogenous scaffold cells and provide a novel strategy for regenerative therapy.

Clone Dynamics and Its Application for the Diagnosis of Enzootic Bovine Leukosis.

Bovine leukemia virus (BLV) infection results in polyclonal expansion of infected B lymphocytes, and ~5% of infected cattle develop enzootic bovine leukosis (EBL). Since BLV is a retrovirus, each individual clone can be identified by using viral integration sites. To investigate the distribution of tumor cells in EBL cattle, we performed viral integration site analysis by using a viral DNA capture-sequencing method. We found that the same tumor clones existed in peripheral blood, with a dominance similar to that in lymphoma tissue. Additionally, we observed that multiple tumor tissues from different sites harbored the identical clones, indicating that tumor cells can circulate and distribute systematically in EBL cattle. To investigate clonal expansion of BLV-infected cells during a long latent period, we collected peripheral blood samples from asymptomatic cattle every 2 years, among which several cattle developed EBL. We found that no detectable EBL clone existed before the diagnosis of EBL in some cases; in the other cases, clones that were later detected as malignant clones at the EBL stage were present several months or even years before the disease onset. To establish a feasible clonality-based method for the diagnosis of EBL, we simplified a quick and cost-effective method, namely, rapid amplification of integration sites for BLV infection (BLV-RAIS). We found that the clonality values (Cvs) were well correlated between the BLV-RAIS and viral DNA capture-sequencing methods. Furthermore, receiver operating characteristic (ROC) curve analysis identified an optimal Cv cutoff value of 0.4 for EBL diagnosis, with excellent diagnostic sensitivity (94%) and specificity (100%). These results indicated that the RAIS method efficiently and reliably detected expanded clones not only in lymphoma tissue but also in peripheral blood. Overall, our findings elucidated the clonal dynamics of BLV- infected cells during EBL development. In addition, Cvs of BLV-infected cells in blood can be used to establish a valid and noninvasive diagnostic test for potential EBL onset. IMPORTANCE Although BLV has been eradicated in some European countries, BLV is still endemic in other countries, including Japan and the United States. EBL causes huge economic damage to the cattle industry. However, there are no effective drugs or vaccines to control BLV infection and related diseases. The strategy of eradication of infected cattle is not practical due to the high endemicity of BLV. Furthermore, how BLV-infected B cell clones proliferate during oncogenesis and their distribution in EBL cattle have yet to be elucidated. Here, we provided evidence that tumor cells are circulating in the blood of diseased cattle. Thus, the Cv of virus-infected cells in blood is useful information for the evaluation of the disease status. The BLV-RAIS method provides quantitative and accurate clonality information and therefore is a promising method for the diagnosis of EBL.

Posterior reversible encephalopathy syndrome following spine surgery: A case report and review of the literature.

Posterior reversible encephalopathy syndrome (PRES) following spine surgery was first documented in 2011. Reports have been rare, and sufficient consensus has not been established for clinical application. We presented a case of PRES following spine surgery. The patient was a 35-year-old woman with a history of hypertension who successfully received microendoscopic L5-S1 lumbar discectomy for lumbar disc herniation at L5-S1 under general anesthesia. Six hours after surgery, she suffered from headache, nausea, visual disturbance, and seizures. Magnetic resonance imaging revealed vasogenic edema in the occipital lobe, and she was diagnosed with PRES. Prompt symptomatic treatment resulted in a full recovery at 3 days after surgery. Subsequently, we reviewed the literature pertaining to PRES following spine surgery. The review of the relevant literature on PRES following spine surgery identified 12 cases (male, n = 2; female, n = 10; average age, 59.5 years). Approximately 92% patients received multi-level decompressive laminectomy and/or fusion. This case and the review of the relevant literature suggest that even minimally invasive spine surgery in a young woman with specific characteristics (eg, hypertension) can cause PRES.

Identification and characterization of a novel enhancer in the HTLV-1 proviral genome.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), a cancer of infected CD4+ T-cells. There is both sense and antisense transcription from the integrated provirus. Sense transcription tends to be suppressed, but antisense transcription is constitutively active. Various efforts have been made to elucidate the regulatory mechanism of HTLV-1 provirus for several decades; however, it remains unknown how HTLV-1 antisense transcription is maintained. Here, using proviral DNA-capture sequencing, we found a previously unidentified viral enhancer in the middle of the HTLV-1 provirus. The transcription factors, SRF and ELK-1, play a pivotal role in the activity of this enhancer. Aberrant transcription of genes in the proximity of integration sites was observed in freshly isolated ATL cells. This finding resolves certain long-standing questions concerning HTLV-1 persistence and pathogenesis. We anticipate that the DNA-capture-seq approach can be applied to analyze the regulatory mechanisms of other oncogenic viruses integrated into the host cellular genome.

Clonality of HIV-1- and HTLV-1-Infected Cells in Naturally Coinfected Individuals.

BACKGROUND: Coinfection with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) diminishes the value of the CD4+ T-cell count in diagnosing AIDS, and increases the rate of HTLV-1-associated myelopathy. It remains elusive how HIV-1/HTLV-1 coinfection is related to such characteristics. We investigated the mutual effect of HIV-1/HTLV-1 coinfection on their integration sites (ISs) and clonal expansion. METHODS: We extracted DNA from longitudinal peripheral blood samples from 7 HIV-1/HTLV-1 coinfected, and 12 HIV-1 and 13 HTLV-1 monoinfected individuals. Proviral loads (PVL) were quantified using real-time polymerase chain reaction (PCR). Viral ISs and clonality were quantified by ligation-mediated PCR followed by high-throughput sequencing. RESULTS: PVL of both HIV-1 and HTLV-1 in coinfected individuals was significantly higher than that of the respective virus in monoinfected individuals. The degree of oligoclonality of both HIV-1- and HTLV-1-infected cells in coinfected individuals was also greater than in monoinfected subjects. ISs of HIV-1 in cases of coinfection were more frequently located in intergenic regions and transcriptionally silent regions, compared with HIV-1 monoinfected individuals. CONCLUSIONS: HIV-1/HTLV-1 coinfection makes an impact on the distribution of viral ISs and clonality of virus-infected cells and thus may alter the risks of both HTLV-1- and HIV-1-associated disease.

HTLV-1 infection promotes excessive T cell activation and transformation into adult T cell leukemia/lymphoma.

Human T cell leukemia virus type 1 (HTLV-1) mainly infects CD4+ T cells and induces chronic, persistent infection in infected individuals, with some developing adult T cell leukemia/lymphoma (ATL). HTLV-1 alters cellular differentiation, activation, and survival; however, it is unknown whether and how these changes contribute to the malignant transformation of infected cells. In this study, we used single-cell RNA-sequencing and T cell receptor-sequencing to investigate the differentiation and HTLV-1-mediated transformation of T cells. We analyzed 87,742 PBMCs from 12 infected and 3 uninfected individuals. Using multiple independent bioinformatics methods, we demonstrated the seamless transition of naive T cells into activated T cells, whereby HTLV-1-infected cells in an activated state further transformed into ATL cells, which are characterized as clonally expanded, highly activated T cells. Notably, the greater the activation state of ATL cells, the more they acquire Treg signatures. Intriguingly, the expression of HLA class II genes in HTLV-1-infected cells was uniquely induced by the viral protein Tax and further upregulated in ATL cells. Functional assays revealed that HTLV-1-infected cells upregulated HLA class II molecules and acted as tolerogenic antigen-presenting cells to induce anergy of antigen-specific T cells. In conclusion, our study revealed the in vivo mechanisms of HTLV-1-mediated transformation and immune escape at the single-cell level.

A target enrichment high throughput sequencing system for characterization of BLV whole genome sequence, integration sites, clonality and host SNP.

Bovine leukemia virus (BLV) is an oncogenic retrovirus which induces malignant lymphoma termed enzootic bovine leukosis (EBL) after a long incubation period. Insertion sites of the BLV proviral genome as well as the associations between disease progression and polymorphisms of the virus and host genome are not fully understood. To characterize the biological coherence between virus and host, we developed a DNA-capture-seq approach, in which DNA probes were used to efficiently enrich target sequence reads from the next-generation sequencing (NGS) library. In addition, enriched reads can also be analyzed for detection of proviral integration sites and clonal expansion of infected cells since the reads include chimeric reads of the host and proviral genomes. To validate this DNA-capture-seq approach, a persistently BLV-infected fetal lamb kidney cell line (FLK-BLV), four EBL tumor samples and four non-EBL blood samples were analyzed to identify BLV integration sites. The results showed efficient enrichment of target sequence reads and oligoclonal integrations of the BLV proviral genome in the FLK-BLV cell line. Moreover, three out of four EBL tumor samples displayed multiple integration sites of the BLV proviral genome, while one sample displayed a single integration site. In this study, we found the evidence for the first time that the integrated provirus defective at the 5' end was present in the persistent lymphocytosis cattle. The efficient and sensitive identification of BLV variability, integration sites and clonal expansion described in this study provide support for use of this innovative tool for understanding the detailed mechanisms of BLV infection during the course of disease progression.

A widely distributed HIV-1 provirus elimination assay to evaluate latency-reversing agents in vitro.

Persistence of HIV-1 latent reservoir cells during antiretroviral therapy (ART) is a major obstacle for curing HIV-1. Even though latency-reversing agents (LRAs) are under development to reactivate and eradicate latently infected cells, there are few useful models for evaluating LRA activity in vitro. Here, we establish a long-term cell culture system called the "widely distributed intact provirus elimination" (WIPE) assay. It harbors thousands of different HIV-1-infected cell clones with a wide distribution of HIV-1 provirus similar to that observed in vivo. Mathematical modeling and experimental results from this in vitro infection model demonstrates that the addition of an LRA to ART shows a latency-reversing effect and contributes to the eradication of replication-competent HIV-1. The WIPE assay can be used to optimize therapeutics against HIV-1 latency and investigate mechanistic insights into the clonal selection of heterogeneous HIV-1-infected cells.

HTLV-1 contains a high CG dinucleotide content and is susceptible to the host antiviral protein ZAP.

BACKGROUND: Human T cell leukaemia virus type 1 (HTLV-1) is a retrovirus associated with human diseases such as adult T-cell leukaemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis. In contrast to another human retrovirus, human immunodeficiency virus type 1 (HIV-1), HTLV-1 persists in the host not via vigorous virus production but mainly via proliferation and/or long-term survival in the form of silent proviruses in infected host cells. As a result, HTLV-1-infected cells rarely produce virus particles in vivo even without anti-retroviral treatment. That should be an advantage for the virus to escape from the host immune surveillance by minimizing the expression of viral antigens in host cells. However, why HIV-1 and HTLV-1 behave so differently during natural infection is not fully understood. RESULTS: We performed cap analysis of gene expression (CAGE) using total RNAs and nascent, chromatin-associated, RNAs in the nucleus and found that HTLV-1 RNAs were processed post-transcriptionally in infected cells. RNA processing was evident for the sense viral transcripts but not the anti-sense ones. We also found a higher proportion of CG di-nucleotides in proviral sequences of HTLV-1-infected cells, when compared to the HIV-1 genomic sequence. It has been reported recently that CG dinucleotide content of viral sequence is associated with susceptibility to the antiviral ZC3HAV1 (ZAP), suggesting the involvement of this protein in the regulation of HTLV-1 transcripts. To analyse the effect of ZAP on HTLV-1 transcripts, we over-expressed it in HTLV-1-infected cells. We found there was a dose-dependent reduction in virus production with ZAP expression. We further knocked down endogenous ZAP with two independent targeting siRNAs and observed a significant increase in virus production in the culture supernatant. Other delta-type retroviruses such as simian T-cell leukaemia virus and bovine leukaemia virus, also contain high CG-dinucleotide contents in their viral genomes, suggesting that ZAP-mediated suppression of viral transcripts might be a common feature of delta-type retroviruses, which cause minimal viremia in their natural hosts. CONCLUSIONS: The post-transcriptional regulatory mechanism involving ZAP might allow HTLV-1 to maintain a delicate balance required for prolonged survival in infected individuals.

The Nature of the HTLV-1 Provirus in Naturally Infected Individuals Analyzed by the Viral DNA-Capture-Seq Approach.

The retrovirus human T-cell leukemia virus type 1 (HTLV-1) integrates into the host DNA, achieves persistent infection, and induces human diseases. Here, we demonstrate that viral DNA-capture sequencing (DNA-capture-seq) is useful to characterize HTLV-1 proviruses in naturally virus-infected individuals, providing comprehensive information about the proviral structure and the viral integration site. We analyzed peripheral blood from 98 naturally HTLV-1-infected individuals and found that defective proviruses were present not only in patients with leukemia, but also in those with other clinical entities. We further demonstrated that clones with defective-type proviruses exhibited a higher degree of clonal abundance than those with full-length proviruses. The frequency of defective-type proviruses in HTLV-1-infected humanized mice was lower than that in infected individuals, indicating that defective proviruses were rare at the initial phase of infection but preferentially selected during persistent infection. These results demonstrate the robustness of viral DNA-capture-seq for HTLV-1 infection and suggest potential applications for other virus-associated cancers in humans.

HIV-1 DNA-capture-seq is a useful tool for the comprehensive characterization of HIV-1 provirus.

Regardless of recent advances in the development of anti-retroviral drugs, it is still extremely difficult to eradicate HIV-1 from infected individuals. The characterization of the HIV-1 provirus, a type of viral reservoir, with a high resolution is key to HIV-1 cure research. Here, we demonstrate that DNA-capture-seq is a powerful tool to obtain comprehensive information on the HIV-1 provirus. We use biotinylated DNA probes targeting the entire HIV-1 sequence to capture fragments containing HIV-1 sequences from DNA-seq libraries prepared for high throughput sequencing. We demonstrate that the protocol provided the entire proviral sequence from the beginning of the 5' LTR to the end of the 3' LTR. Since HIV-1 DNA-probes can hybridize not only viral fragments but also virus-host chimeric ones, the viral integration site information can also be obtained. We verify the efficiency of the protocol by using latently infected cell lines, such as ACH-2 and J1.1, and newly generated ones. The results reveal that the 2 new clones that we analyse harbour one copy of replication-competent provirus, suggesting that latency is not caused by genetic mutations or deletions of the provirus. In conclusion, HIV-1 DNA-capture-seq is a powerful tool to characterize the HIV-1 provirus at a single nucleotide resolution and therefore might be useful for various experiments aiming for an HIV-1 cure.

[The role of HTLV-1 provirus in clonal selection of the infected cells].

HTLV-1 inserts its viral genome into the host cellular DNA in the form of a provirus. The proviral DNA is a key to understand the persistence and pathogenesis of HTLV-1 infection. There has been a significant progress in proviral research due to technological advances on DNA sequencing.Next generation sequencing technology revolutionized our understanding of the human genome,showing how it is organized and regulated, not only by the nucleotide sequence itself but also by epigenetic features and higher-order chromatin structure. We will review recent findings regarding the role of HTLV-1 provirus in HTLV-1 infection.

Triplex- and Duplex-Forming Abilities of Oligonucleotides Containing 2'-Deoxy-5-trifluoromethyluridine and 2'-Deoxy-5-trifluoromethylcytidine.

A facile synthesis of 2'-deoxy-5-trifluoromethyluridine and 2'-deoxy-5-trifluoromethylcytidine phosphoramidites from commercially available 2'-deoxyuridine and 2'-deoxycytidine was achieved, respectively. The obtained phosphoramidites were incorporated into oligonucleotides, and their binding affinity to double-stranded DNA (dsDNA) and single-stranded RNA (ssRNA) was evaluated by UV-melting experiments. The triplex-forming abilities of oligonucleotides including 5-trifluoromethylpyrimidine nucleobases with dsDNA were decreased. Especially, the stability of the triplex containing a trifluoromethylcytosine (CF3C)-GC base triplet was low, likely due to the low pKa of protonated CF3C by the electron-withdrawing trifluoromethyl group. A slight decrease in stability of the duplex formed with ssRNA by oligonucleotides including 5-trifluoromethylpyrimidine nucleobases was only observed, suggesting that they might be applicable to various ssRNA-targeted technologies using features of fluorine atoms.

Transcriptional and Epigenetic Regulatory Mechanisms Affecting HTLV-1 Provirus.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with human diseases, such as adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/Tropic spastic paraparesis (HAM/TSP). As a retrovirus, its life cycle includes a step where HTLV-1 is integrated into the host genomic DNA and forms proviral DNA. In the chronic phase of the infection, HTLV­1 is known to proliferate as a provirus via the mitotic division of the infected host cells. There are generally tens of thousands of infected clones within an infected individual. They exist not only in peripheral blood, but also in various lymphoid organs. Viral proteins encoded in HTLV-1 genome play a role in the proliferation and survival of the infected cells. As is the case with other chronic viral infections, HTLV-1 gene expression induces the activation of the host immunity against the virus. Thus, the transcription from HTLV-1 provirus needs to be controlled in order to evade the host immune surveillance. There should be a dynamic and complex regulation in vivo, where an equilibrium between viral antigen expression and host immune surveillance is achieved. The mechanisms regulating viral gene expression from the provirus are a key to understanding the persistent/latent infection with HTLV-1 and its pathogenesis. In this article, we would like to review our current understanding on this topic.

Application of targeted enrichment to next-generation sequencing of retroviruses integrated into the host human genome.

The recent development and advancement of next-generation sequencing (NGS) technologies have enabled the characterization of the human genome at extremely high resolution. In the retrovirology field, NGS technologies have been applied to integration-site analysis and deep sequencing of viral genomes in combination with PCR amplification using virus-specific primers. However, virus-specific primers are not available for some epigenetic analyses, like chromatin immunoprecipitation sequencing (ChIP-seq) assays. Viral sequences are poorly detected without specific PCR amplification because proviral DNA is very scarce compared to human genomic DNA. Here, we have developed and evaluated the use of biotinylated DNA probes for the capture of viral genetic fragments from a library prepared for NGS. Our results demonstrated that viral sequence detection was hundreds or thousands of times more sensitive after enrichment, enabling us to reduce the economic burden that arises when attempting to analyze the epigenetic landscape of proviruses by NGS. In addition, the method is versatile enough to analyze proviruses that have mismatches compared to the DNA probes. Taken together, we propose that this approach is a powerful tool to clarify the mechanisms of transcriptional and epigenetic regulation of retroviral proviruses that have, until now, remained elusive.

Involvement of both tumor necrosis factor-alpha-induced necrosis and p53-mediated caspase-dependent apoptosis in nephrotoxicity of cisplatin.

We previously reported that necrosis occurs predominantly in porcine renal tubular LLC-PK1 cells, when the cells were exposed transiently to a high concentration of cisplatin. Moreover, we demonstrated that generation of reactive oxygen species and subsequent production of tumor necrosis factor-alpha (TNF-alpha) through phosphorylation of p38 MAPK are implicated in the pathogenesis of cisplatin-induced renal cell injury. However, some TUNEL-positive cells appeared in renal proximal tubules of rats after systemic injection of cisplatin, suggesting an involvement of apoptosis. In the present study, we found in LLC-PK1 cells that both apoptosis and necrosis were elicited when the cells were exposed to 200 microM cisplatin for 1 h followed by incubation for 24 h in the presence of 20 microM cisplatin. The cisplatin-induced necrosis was largely attenuated by the antioxidant N-acetylcysteine, while apoptosis was prevented by the specific inhibitors for caspases-2, -8, and -3 and a p53 inhibitor pifithrin-alpha but not by the p38 MAPK inhibitor SB203580. On the other hand, SB203580 attenuated the cisplatin-induced increase in TNF-alpha production. These findings suggest that p53-mediated activations of caspases-2, -8 and -3 play a key role in cisplatin-induced renal cell apoptosis, while oxidative stress-induced TNF-alpha synthesis via p38 MAPK phosphorylation contributed to the necrosis.