EBV infection mediated BDNF expression is associated with bladder inflammation in interstitial cystitis/bladder pain syndrome with Hunner's lesion.

Interstitial cystitis/bladder pain syndrome with Hunner's lesion (HIC) is characterized by chronic inflammation and nerve hyperplasia; however, the pathogenesis of HIC remains a mystery. In this study, we detected both Epstein-Barr virus (EBV) latency infection genes EBNA-1 and LMP-1 and EBV lytic infection BZLF-1 and BRLF-1 expression in the HIC bladders, indicating the coexistence of EBV persistence and reactivation in the B cells in HIC bladders. Upregulation of EBV-associated inflammatory genes in HIC bladders, such as TNF-α and IL-6, suggests EBV infection is implicated in the pathogenesis of bladder inflammation. Nerve hyperplasia and upregulation of brain-derived neurotrophic factor (BDNF) were noted in the HIC bladders. Double immunochemical staining and flow cytometry revealed the origin of BDNF to be EBV-infected B cells. Inducible BDNF expression was noted in B cells upon EBV infection, but not in the T cells. A chromatin immunoprecipitation study revealed BDNF transcription could be promoted by cooperation between EBV nuclear antigens, chromatin modifiers, and B-cell-specific transcription. Knockdown of BDNF in EBV-infected B cells resulted in the inhibition of cell proliferation and viability. Downregulation of phosphorylated SMAD2 and STAT3 after BDNF knockdown may play a role in the mechanism. Implantation of latent EBV-infected B cells into rat bladder walls resulted in a higher expression level of CD45 and PGP9.5, suggesting tissue inflammation and nerve hyperplasia. In contrast, implantation of BDNF depleted EBV-infected B cells abrogated these effects. This is the first study to provide insights into the mechanisms underlying the involvement of EBV-infected B cells in HIC pathogenesis. © 2022 The Pathological Society of Great Britain and Ireland.

DNA topoisomerase III alpha regulates p53-mediated tumor suppression.

PURPOSE: Human DNA topoisomerase III alpha (hTOP3α) is involved in DNA repair surveillance and cell-cycle checkpoints possibly through formatting complex with tumor suppressors. However, its role in cancer development remained unsolved. EXPERIMENTAL DESIGN: Coimmunoprecipitation, sucrose gradient, chromatin immunoprecipitation (ChIP), real time PCR, and immunoblotting analyses were performed to determine interactions of hTOP3α with p53. Paired cell lines with different hTOP3α levels were generated via ectopic expression and short hairpin RNA (shRNA)-mediated knockdown approaches. Cellular tumorigenic properties were analyzed using cell counting, colony formation, senescence, soft agar assays, and mouse xenograft models. RESULTS: The hTOP3α isozyme binds to p53 and cofractionizes with p53 in gradients differing from fractions containing hTOP3α and BLM. Knockdown of hTOP3α expression (sh-hTOP3α) caused a higher anchorage-independent growth of nontumorigenic RHEK-1 cells. Similarly, sh-hTOP3α and ectopic expression of hTOP3α in cancer cell lines caused increased and reduced tumorigenic abilities, respectively. Genetic and mutation experiments revealed that functional hTOP3α, p53, and p21 are required for this tumor-suppressive activity. Mechanism-wise, ChIP data revealed that hTOP3α binds to the p53 and p21 promoters and positively regulates their expression. Two proteins affect promoter recruitments of each other and collaborate in p21 expression. Moreover, sh-hTOP3α and sh-p53 in AGS cells caused a similar reduction in senescence and hTOP3α mRNA levels were lower in gastric and renal tumor samples. CONCLUSION: We concluded that hTOP3α interacts with p53, regulates p53 and p21 expression, and contributes to the p53-mediated tumor suppression.

Anthracenedione-methionine conjugates are novel topoisomerase II-targeting anticancer agents with favorable drug resistance profiles.

Structure-associated drug resistance and DNA-unwinding abilities have greatly limited the clinical usage of anthracenediones, including mitoxantrone (MX) and ametantrone (AT), which intercalate into DNA and induce topoisomerase II (TOP2)-mediated DNA break. We studied a series of 1,4-bis(2-amino-ethylamino) MX- and AT-amino acid conjugates (M/AACs) and showed that abilities in cancer cell killing correlate with the amounts of chromosomal DNA breaks induced by M/AACs. Notably, the 1,4-bis-L/l-methionine-conjugated MAC (L/LMet-MAC) exhibits DNA-breaking, cancer cell-killing and anti-tumor activities rivaling those of MX. Interestingly, l- and d-form Met-M/AACs unwind DNA poorly compared to MX and AT. The roles of the two human TOP2 isozymes (hTOP2α and 2ß) in the L/LMet-MAC-induced DNA breakage and cancer cell-killing were suggested by the following observations: (i) M/AAC-induced DNA breakage, cytotoxicity and apoptosis are greatly reduced in various TOP2-deficient conditions; (ii) DNA breaks induced by MACs are highly reversible and effectively antagonized by the TOP2 catalytic inhibitors; (iii) MACs induced differential TOP2-mediated DNA cleavage in vitro using recombinant hTOP2α proteins and the formation of hTOP2α/ßcc in the cell culture system. Interestingly, d-aa-conjugated MACs often caused a lower level in hTOP2-mediated DNA breaks and cell-killing than the corresponding l-form ones indicating a steric-specific effect of MACs. Together, our results suggest that both enzyme- and DNA-drug interactions might contribute to TOP2-targeting by M/AACs. Furthermore, Met-MACs are poor substrates for the MDR1 transporter. Therefore, L/LMet-MAC represents a promising class of TOP2-targeting drugs with favorable drug resistance profiles.

TRPM7 regulates polarized cell movements.

TRPM7 (transient receptor potential melastatin 7) is a Ca²+- and Mg²+-permeant ion channel in possession of its own kinase domain. As a kinase, the protein has been linked to the control of actomyosin contractility, whereas the channel has been found to regulate cell adhesion as well as cellular Mg²+ homoeostasis. In the present study we show that depletion of TRPM7 by RNA interference in fibroblasts alters cell morphology, the cytoskeleton, and the ability of cells to form lamellipodia and to execute polarized cell movements. A pulldown-purification assay revealed that knockdown of TRPM7 prevents cells from activating Rac and Cdc42 (cell division cycle 42) when stimulated to migrate into a cellular wound. Re-expression of TRPM7 reverses these phenotypic changes, as does, unexpectedly, expression of a kinase-inactive mutant of TRPM7. Surprisingly, expression of the Mg²+ transporter SLC41A2 (solute carrier family 41 member 2) is also effective in restoring the change in cell morphology, disruption of the cytoskeleton and directional cell motility caused by depletion of the channel-kinase. The results of the present study uncover an essential role for Mg²+ in the control of TRPM7 over the cytoskeleton and its ability to regulate polarized cell movements.

Calcium-induced cleavage of DNA topoisomerase I involves the cytoplasmic-nuclear shuttling of calpain 2.

Important to the function of calpains is temporal and spatial regulation of their proteolytic activity. Here, we demonstrate that cytoplasm-resident calpain 2 cleaves human nuclear topoisomerase I (hTOP1) via Ca(2+)-activated proteolysis and nucleoplasmic shuttling of proteases. This proteolysis of hTOP1 was induced by either ionomycin-caused Ca(2+) influx or addition of Ca(2+) in cellular extracts. Ca(2+) failed to induce hTOP1 proteolysis in calpain 2-knockdown cells. Moreover, calpain 2 cleaved hTOP1 in vitro. Furthermore, calpain 2 entered the nucleus upon Ca(2+) influx, and calpastatin interfered with this process. Calpain 2 cleavage sites were mapped at K(158) and K(183) of hTOP1. Calpain 2-truncated hTOP1 exhibited greater relaxation activity but remained able to interact with nucleolin and to form cleavable complexes. Interestingly, calpain 2 appears to be involved in ionomycin-induced protection from camptothecin-induced cytotoxicity. Thus, our data suggest that nucleocytoplasmic shuttling may serve as a novel type of regulation for calpain 2-mediated nuclear proteolysis.

Cellular processing determinants for the activation of damage signals in response to topoisomerase I-linked DNA breakage.

Recent studies have suggested an involvement of processing pathways for the initiation of cellular responses induced by topoisomerase-targeting drugs. Here, we showed that cellular exposure to camptothecin (CPT) induced formation of topoisomerase I cleavable complex (TOP1cc), degradation of TOP1 and activation of DNA damage responses (DDR). Transcription and proteasome-dependent proteolysis, but not replication, were involved in CPT-induced TOP1 degradation, while none of above three processing activities affected TOP1cc formation. Replication- and transcription-initiated processing (RIP and TIP) of TOP1cc were identified as two independent pathways, which contribute distinctly to various CPT-activated DDR. Specifically, in cycling cells, RIP-processed TOP1cc triggered the CPT-induced RPA phosphorylation. At higher CPT dosages, the TIP pathway is required for other DDR activation, including ATM, p53 and Chk1/2 phosphorylation. The TIP pathway was further demonstrated to be S-phase independent by using three nonreplicating cell models. Furthermore, the effect of proteasome inhibitors mimicked that of transcription inhibition on the CPT-induced activation of DDR, suggesting the involvement of proteasome in the TIP pathway. Interestingly, the TIP pathway was important for TOP1cc-activated, but not ionization radiation-activated ATM, p53 and Chk2 phosphorylation. We have also found that pharmacological interferences of TIP and RIP pathways distinctively modulated the CPT-induced cell killing with treatments at low and high dosages, respectively. Together, our results support that both RIP and TIP pathways of TOP1cc are required for the activation of CPT-induced DDR and cytotoxicity.

Cellular processing pathways contribute to the activation of etoposide-induced DNA damage responses.

Cytotoxic action (tumor cell killing) and carcinogenic side effect (therapy-related secondary leukemia) of etoposide are closely related to its ability in stabilizing topoisomerase II cleavable complex (TOP2cc), a unique form of protein-linked DNA break. How cells process and detect TOP2-concealed DNA damage for the activation of downstream cellular responses remains unclear. Here, we showed proteasomal degradation of both TOP2 isozymes in a transcription-dependent manner upon etoposide treatment. Downregulation of TOP2 was preferentially associated with proteasomal removal of TOP2 in TOP2cc rather than proteolysis of free TOP2. Interestingly, blockage of TOP2 downregulation in TOP2cc also caused reduction in etoposide-induced activation of DNA damage molecules, an observation suggesting that the processing pathways of TOP2cc are involved in activation of etoposide-induced cellular responses. In this regard, we observed two TOP2cc processing pathways, replication- and transcription-initiated processing (RIP and TIP) with proteasome involved in the latter. Importantly, two processing pathways contributed to differential activation of various DNA damage signaling and downstream cellular responses. Etoposide-induced phosphorylation of p53 relied mainly on RIP, whereas activation of Chk1, Chk2 depended largely on TIP. Both RIP and TIP played roles in activating non-homologous end joining pathway, while only RIP modulated etoposide-induced cell killing in a p53-dependent manner. Collectively, our results are consistent with the notion that protein-linked DNA breakage (e.g., TOP2cc) requires processing pathways for initiating downstream DNA damage detection, repair as well as cell death programs.

The prosurvival activity of p53 protects cells from UV-induced apoptosis by inhibiting c-Jun NH2-terminal kinase activity and mitochondrial death signaling.

The cytoprotective function of p53 recently has been exploited as a therapeutic advantage for cancer prevention; agents activating the prosurvival activity of p53 are shown to prevent UV-induced damages. To explore the mechanisms of p53-mediated protection from UV-induced apoptosis, we have established stable clones of H1299 lung carcinoma cells expressing a temperature-sensitive p53 mutant, tsp53(V143A). At the permissive temperature of 32 degrees C, the tsp53(V143A)-expressing cells were arrested in G(1) phase without the occurrence of apoptosis; consistent with this is the preferential induction of genes related to growth arrest and DNA damage repair. Previous expression of functional tsp53(V143A) for > or =18 hours inhibited the release of proapoptotic molecules from mitochondria and protected the cells from UV-induced apoptosis; moreover, it suppressed the activation of c-Jun NH(2)-terminal kinase (JNK) signaling and relieved the effect of UV on p53 target gene activation. p53 associated with JNK and inhibited its kinase activity. Using the p53-null H1299 cells, we showed that inhibition of JNK blocked the UV-elicited mitochondrial death signaling and caspase activation. Our results suggest that the ability of p53 to bind and inactivate JNK, together with the activation of the p53 target genes related to cell cycle arrest and DNA damage repair, is responsible for its protection of cells against UV-induced apoptosis.