Downregulation of TCF7 and LEF1 is a key determinant of tumor-infiltrating regulatory T-cell function.

Forkhead box P3 (Foxp3)-expressing regulatory T (Treg) cells play essential roles in immune homeostasis but also contribute to establish a favorable environment for tumor growth by suppressing anti-tumor immune responses. It is thus necessary to specifically target tumor-infiltrating Treg cells to minimize effects on immune homeostasis in cancer immunotherapy. However, molecular features that distinguish tumor-infiltrating Treg cells from those in secondary lymphoid organs remain unknown. Here we characterize distinct features of tumor-infiltrating Treg cells by global analyses of the transcriptome and chromatin landscape. They exhibited activated phenotypes with enhanced Foxp3-dependent transcriptional regulation, yet being distinct from activated Treg cells in secondary lymphoid organs. Such differences may be attributed to the extensive clonal expansion of tumor-infiltrating Treg cells. Moreover, we found that TCF7 and LEF1 were specifically downregulated in tumor-infiltrating Treg cells both in mice and humans. These factors and Foxp3 co-occupied Treg suppressive function-related gene loci in secondary lymphoid organ Treg cells, whereas the absence of TCF7 and LEF1 accompanied altered gene expression and chromatin status at these gene loci in tumor-infiltrating Treg cells. Functionally, overexpression of TCF7 and LEF1 in Treg cells inhibited the enhancement of Treg suppressive function upon activation. Our results thus show the downregulation of TCF7 and LEF1 as markers of highly suppressive Treg cells in tumors and suggest that their absence controls the augmentation of Treg suppressive function in tumors. These molecules may be potential targets for novel cancer immunotherapy with minimum effects on immune homeostasis.

Circulating extracellular vesicles carrying Firmicutes reflective of the local immune status may predict clinical response to pembrolizumab in urothelial carcinoma patients.

Bacterial flora has clinical significance for the host. The metabolic environment created by this flora influences immunotherapy in urothelial carcinoma. However, there are no reports on the clinical significance of bacterial flora in the host bloodstream. We aimed to clarify the correlation between extracellular vesicle (EV)-derived blood microflora information and tumor immunological status in urothelial carcinoma (UC) patients. Serum samples were collected from 20 healthy donors, 50 patients with localized UC, and 31 patients with metastatic UC (mUC) who had undergone pembrolizumab treatment. Bacterial DNA in EVs was extracted from each sample. Metagenomic sequencing was performed after amplification of the V1-V2 region of the bacterial 16S rRNA gene. Using the matched tumor tissue and serum samples, we revealed that the smaller amount of peripheral EVs carrying Firmicutes DNA was significantly correlated with the higher number of infiltrating T cells within tumor tissues (CD3; p = 0.015, CD4; p = 0.039, CD8; p = 0.0084) and the higher expression of activation markers on their surface (ICOS on both CD4; p = 0.0013 and CD8 T cells; p = 0.016 and 4-1BB on CD4 T cells; p = 0.016). In terms of circulating metabolic information, L-Ser and L-Pro levels, which play important roles in T cell expansion and proliferation, were significantly higher in the Firmicutes-low group (p = 0.010). All of the patients with higher Firmicutes abundance had disease progression without any clinical response (p = 0.026) and significantly inferior prognosis for pembrolizumab therapy (p = 0.035). This is the first study on the importance of peripheral bacterial EVs in cancer patients treated with cancer immunotherapy.

CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory.

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8- CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

S-540956, a CpG Oligonucleotide Annealed to a Complementary Strand With an Amphiphilic Chain Unit, Acts as a Potent Cancer Vaccine Adjuvant by Targeting Draining Lymph Nodes.

Robust induction of cancer-antigen-specific CD8+ T cells is essential for the success of cancer peptide vaccines, which are composed of a peptide derived from a cancer-specific antigen and an immune-potentiating adjuvant, such as a Toll-like receptor (TLR) agonist. Efficient delivery of a vaccine antigen and an adjuvant to antigen-presenting cells in the draining lymph nodes (LNs) holds key to maximize vaccine efficacy. Here, we developed S-540956, a novel TLR9-agonistic adjuvant consisting of B-type CpG ODN2006 (also known as CpG7909), annealed to its complementary sequence oligodeoxynucleotide (ODN) conjugated to a lipid; it could target both a cancer peptide antigen and a CpG-adjuvant in the draining LNs. S-540956 accumulation in the draining LNs and activation of plasmacytoid dendritic cells (pDCs) were significantly higher than that of ODN2006. Mechanistic analysis revealed that S-540956 enhanced the induction of MHC class I peptide-specific CD8+ T cell responses via TLR9 in a CD4+ T cell-independent manner. In mice, the therapeutic effect of S-540956-adjuvanted with a human papillomavirus (HPV)-E7 peptide vaccine against HPV-E7-expressing TC-1 tumors was significantly better than that of an ODN2006-adjuvanted vaccine. Our findings demonstrate a novel adjuvant discovery with the complementary strand conjugated to a lipid, which enabled draining LN targeting and increased ODN2006 accumulation in draining LNs, thereby enhancing the adjuvant effect. Our findings imply that S-540956 is a promising adjuvant for cancer peptide vaccines and has a high potential for applications in various vaccines, including recombinant protein vaccines.

Tumour grade significantly correlates with total dysfunction of tumour tissue-infiltrating lymphocytes in renal cell carcinoma.

It is important to evaluate the clinical importance of both CD8 T cells and CD4 T cells expression simultaneously because they have crucial networks in tumour targeting immune responses. In 97 RCC patients, RNA sequencing and gene set enrichment analysis of both CD8 and CD4 T cells based on the expression levels of PD-1 and TIM-3 implied that the populations of PD-1+TIM-3+ CD8 T cells and PD-1lowTIM-3 + CD4 T cells were characterized as exhausted CD8 T cells and regulatory CD4 T cells, respectively. These populations of CD4 and CD8 T cells were significantly upregulated in the patients with RCC of higher WHO/ISUP grade (grades 3, 4) (P < 0.001). Moreover, the cytokine productivities of each population in both CD4 and CD8 T cells of the higher-grade patients were significantly lower than those of the lower-grade patients (P < 0.05). Multivariate analysis showed the prognosis of patients with metastatic RCC of higher WHO/ISUP grade treated by nivolumab to be significantly worse than that of patients with lower grade (P = 0.026). This study showed that tumour grade significantly correlated with dysfunction of both CD4+ and CD8+ TILs and the efficacy of nivolumab treatment.

Docetaxel Upregulates HMGB1 Levels in Non-small Cell Lung Cancer.

Immune checkpoint inhibitors (ICIs) exert beneficial effects in non-small cell lung cancer (NSCLC) patients. However, ICIs are only advantageous for a limited population of NSCLC patients. Therefore to enhance their effects, combination therapies with ICIs have been developed. To identify preferable chemotherapy to combine with ICIs against lung cancer, we examined immunological effects of docetaxel compared with epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). We found no difference in peripheral lymphocyte counts and ratio of their subpopulations in lung cancer patients before and after both treatments. On the other hand, plasma levels of high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, showed significant increase after docetaxel treatment. Furthermore, we investigated effects of HMGB1 on tumor-infiltrating immune cells obtained from surgically resected tumor tissue from NSCLC patients. When the tumor infiltrating cells were stimulated with HMGB1, CD11c+ cells showed increased expression of activation markers. These findings imply that docetaxel could be involved in anti-tumor immunity via HMGB1. Therefore docetaxel might be a candidate for combination treatment with ICIs.

Clinical importance of the expression of CD4+CD8+ T cells in renal cell carcinoma.

OBJECTIVE: CD4+CD8+ T cells are expressed in some cancer patients including those with renal cell carcinoma (RCC). However, no reports have mentioned the clinical importance of this expression. We evaluated the expression of CD4+CD8+ T cells in patients with various cancer types to clarify clinical characteristics and prognostic importance significantly correlating with these T cells. METHODS: Expression of CD4+CD8+ T cells was evaluated using flowcytometry in tissue-infiltrating lymphocytes extracted from 260 cancer tissues including 104 RCC samples. RNA sequencing and characterization and regression (Citrus) was used to determine characteristics. The prognostic importance of CD4+CD8+ T cells was evaluated by Cox regression analysis. RESULTS: Among eight cancer types, expression of CD4+CD8+ T cells was significantly highest in RCC patients. According to the expression of CD4+CD8+ T cells in adjacent normal tissue-infiltrating lymphocytes, 24 patients (23.1%) were defined as being positive for CD4+CD8+ with an expression higher than 9.29% in RCC patients. Citrus showed CD8+PD-1+TIM-3+CD103- T cells to be a specific subpopulation of CD4+CD8+ T cells. RNA sequencing revealed that CD4+CD8+ T cells had significantly lower diversity than the other T cells and shared most T-cell receptor clones with CD8+ not CD4+ T cells. Expression of CD4+CD8+ T cells was identified as an independent predictor of overall survival (hazard ratio: 0.11, 95% confidence interval: 0.01-0.86, P = 0.035) in multivariate analysis. CONCLUSIONS: The expression of CD4+CD8+ T cells was significantly up-regulated in RCC patients and correlated significantly with prognostic importance in surgically treated RCC patients.

Phenotypic Analysis of Tumor Tissue-Infiltrating Lymphocytes in Tumor Microenvironment of Bladder Cancer and Upper Urinary Tract Carcinoma.

BACKGROUND: There are no previous reports directly evaluating immunologic conditions in tumor microenvironment including both bladder cancer (BCa) and upper urinary tract carcinoma (UTUC). In this study, we aimed to clarify the difference of immunity status and its clinical significance depending on the tumor site in urothelial carcinoma. PATIENTS AND METHODS: Tumor tissue-infiltrating lymphocytes were extracted from 70 urothelial cancer patients who underwent surgical resection (52 cases of BCa and 18 cases of UTUC). The immunologic classification was established by unsupervised clustering analysis according to the expression ratio of 9 extracellular surface markers measured by flow cytometry, and we examined the relationship between immunologic classification and clinical importance such as pathologic status and prognosis (progression-free survival and cancer-specific survival). RESULTS: The immunologic condition was classified into 2 groups. Group 1 (n = 41) comprised the CD4 T-cell-dominant group and group 2 (n = 29) the immunologically activated group. This immunologic classification was significantly correlated with tumor grade (P = .020) but not tumor location in multivariate analysis. In invasive BCa patients (n = 33), progression-free survival and cancer-specific survival of group 2 were significantly worse than those of group 1 (P = .021 and P = .022, respectively), while there was no significant difference between groups 1 and 2 in patients with invasive UTUC (n = 17). CONCLUSION: Although there was no difference in the local immunologic condition of urothelial carcinoma between BCa and UTUC, its significance as a prognostic predictor might vary depending on tumor site.

Immunological classification of renal cell carcinoma patients based on phenotypic analysis of immune check-point molecules.

OBJECTIVES: To clarify comprehensive immunological signature patterns of tumour tissue-infiltrating lymphocytes in patients with renal cell carcinoma and show its clinical significance. MATERIALS AND METHODS: We investigated the surface marker expressions of tumour tissue-infiltrating lymphocytes quantitatively and classified them based on their functional populations. We extracted 109 sets of tumour tissue-infiltrating lymphocytes from 80 patients who underwent surgical resection of renal cell carcinoma, of which 44 tumour tissue-infiltrating lymphocytes were multiply extracted from 15 patients. Each tumour tissue-infiltrating lymphocyte was characterised on the basis of functional T-cell populations using ten surface marker expressions measured by flow cytometry. RESULTS: All sets of the tumour tissue-infiltrating lymphocytes were classified into three groups, which correlated significantly with Fuhrman grade (OR 0.253, 95% CI 0.094-0.678, P = 0.006). Importantly, both overall metastasis-free survival (HR 0.449, 95% CI 0.243-0.832, P = 0.011) and recurrence-free survival (HR 0.475, 95% CI 0.238-0.948, P = 0.035) of the patients with the higher marker expressions were significantly inferior to those of the patients with the lower marker expressions by multivariate analysis. Six specific genes for this classification identified by microarray analysis verified our results using the TCGA KIRC data set. In addition, we discovered the presence of intra-tumoural diversity in the classification of 3 (20%) of the 15 patients. CONCLUSIONS: This study showed that the presence of classable diversity in the immunological signature of tumour tissue-infiltrating lymphocytes correlated with prognosis and tumour aggressiveness that was observed even within individual tumours in some patients with renal cell carcinoma.

NY-ESO-1 Protein Cancer Vaccine With Poly-ICLC and OK-432: Rapid and Strong Induction of NY-ESO-1-specific Immune Responses by Poly-ICLC.

We conducted a clinical trial of a cancer vaccine using NY-ESO-1 protein with polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) and/or OK-432 against solid tumors. A total of 15 patients were sequentially enrolled in 4 cohorts. Patients in cohort 1 received NY-ESO-1 protein; cohort 2a received NY-ESO-1 protein+OK-432; cohort 2b received NY-ESO-1 protein+poly-ICLC; cohort 3 received NY-ESO-1 protein+OK-432+poly-ICLC with Montanide ISA-51. The endpoints of this trial were safety, NY-ESO-1 immune responses, and clinical response. Vaccine-related adverse events observed were fever and injection-site reaction (grade 1). Two patients showed stable disease after vaccination. NY-ESO-1 antibodies were observed in 4 patients at the baseline (sero-positive) and augmented in all patients after vaccination. Eleven patients showed a conversion of negative antibody responses at baseline to positive after vaccination (seroconversion). The seroconversions were observed in all 11 sero-negative patients by the fourth immunization; in particular, it was observed by the second immunization in patients with poly-ICLC, and these induced antibody responses were stronger than those in patients immunized without poly-ICLC. The number of NY-ESO-1-specific interferon (IFN)γ-producing T cells was increased in patients immunized with poly-ICLC and/or OK-432, and furthermore, the increase of IFNγ-producing CD8 T cells in patients immunized with poly-ICLC was significantly higher than that in patients without poly-ICLC. Nonspecific activations of T-cell or antigen presenting cells were not observed. Our present study showed that poly-ICLC is a promising adjuvant for cancer vaccines.

A unique hinge binder of extremely selective aminopyridine-based Mps1 (TTK) kinase inhibitors with cellular activity.

Mps1, also known as TTK, is a dual-specificity kinase that regulates the spindle assembly check point. Increased expression levels of Mps1 are observed in cancer cells, and the expression levels correlate well with tumor grade. Such evidence points to selective inhibition of Mps1 as an attractive strategy for cancer therapeutics. Starting from an aminopyridine-based lead 3a that binds to a flipped-peptide conformation at the hinge region in Mps1, elaboration of the aminopyridine scaffold at the 2- and 6-positions led to the discovery of 19c that exhibited no significant inhibition for 287 kinases as well as improved cellular Mps1 and antiproliferative activities in A549 lung carcinoma cells (cellular Mps1 IC50=5.3 nM, A549 IC50=26 nM). A clear correlation between cellular Mps1 and antiproliferative IC50 values indicated that the antiproliferative activity observed in A549 cells would be responsible for the cellular inhibition of Mps1. The X-ray structure of 19c in complex with Mps1 revealed that this compound retains the ability to bind to the peptide flip conformation. Finally, comparative analysis of the X-ray structures of 19c, a deamino analogue 33, and a known Mps1 inhibitor bound to Mps1 provided insights into the unique binding mode at the hinge region.

Discovery of imidazo[1,2-b]pyridazine derivatives: selective and orally available Mps1 (TTK) kinase inhibitors exhibiting remarkable antiproliferative activity.

Monopolar spindle 1 (Mps1) is an attractive oncology target due to its high expression level in cancer cells as well as the correlation of its expression levels with histological grades of cancers. An imidazo[1,2-a]pyrazine 10a was identified during an HTS campaign. Although 10a exhibited good biochemical activity, its moderate cellular as well as antiproliferative activities needed to be improved. The cocrystal structure of an analogue of 10a guided our lead optimization to introduce substituents at the 6-position of the scaffold, giving the 6-aryl substituted 21b which had improved cellular activity but no oral bioavailability in rat. Property-based optimization at the 6-position and a scaffold change led to the discovery of the imidazo[1,2-b]pyridazine-based 27f, an extremely potent (cellular Mps1 IC50 = 0.70 nM, A549 IC50 = 6.0 nM), selective Mps1 inhibitor over 192 kinases, which could be orally administered and was active in vivo. This 27f demonstrated remarkable antiproliferative activity in the nanomolar range against various tissue cancer cell lines.

Indazole-based potent and cell-active Mps1 kinase inhibitors: rational design from pan-kinase inhibitor anthrapyrazolone (SP600125).

Monopolar spindle 1 (Mps1) is essential for centrosome duplication, the spindle assembly check point, and the maintenance of chromosomal instability. Mps1 is highly expressed in cancer cells, and its expression levels correlate with the histological grades of cancers. Thus, selective Mps1 inhibitors offer an attractive opportunity for the development of novel cancer therapies. To design novel Mps1 inhibitors, we utilized the pan-kinase inhibitor anthrapyrazolone (4, SP600125) and its crystal structure bound to JNK1. Our design efforts led to the identification of indazole-based lead 6 with an Mps1 IC50 value of 498 nM. Optimization of the 3- and 6-positions on the indazole core of 6 resulted in 23c with improved Mps1 activity (IC50 = 3.06 nM). Finally, application of structure-based design using the X-ray structure of 23d bound to Mps1 culminated in the discovery of 32a and 32b with improved potency for cellular Mps1 and A549 lung cancer cells. Moreover, 32a and 32b exhibited reasonable selectivities over 120 and 166 kinases, respectively.

Diaminopyridine-based potent and selective mps1 kinase inhibitors binding to an unusual flipped-Peptide conformation.

Monopolar spindle 1 (Mps1) is an attractive cancer drug target due to the important role that it plays in centrosome duplication, the spindle assembly checkpoint, and the maintenance of chromosomal stability. A design based on JNK inhibitors with an aminopyridine scaffold and subsequent modifications identified diaminopyridine 9 with an IC50 of 37 nM. The X-ray structure of 9 revealed that the Cys604 carbonyl group of the hinge region flips to form a hydrogen bond with the aniline NH group in 9. Further optimization of 9 led to 12 with improved cellular activity, suitable pharmacokinetic profiles, and good in vivo efficacy in the mouse A549 xenograft model. Moreover, 12 displayed excellent selectivity over 95 kinases, indicating the contribution of its unusual flipped-peptide conformation to its selectivity.

Sphingosylphosphorylcholine and lysosulfatide have inverse regulatory functions in monocytic cell differentiation into macrophages.

Sphingolipids act as signaling mediators that regulate a diverse range of cellular events. Although numerous sphingolipid functions have been studied, little is known about the effect of sphingolipids on monocyte differentiation into macrophages. Here, we report that two lysosphingolipids, sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF), inversely affect macrophagic differentiation of monocytic cell lines, U937 and THP-1. Molecular analyses revealed that SPC enhances, whereas LSF suppresses, phorbol ester-induced classical (M1-polarized) differentiation to macrophages. The expression of CD11b, a macrophage marker, was induced in accordance with the activation status of the Raf/MEK/ERK signaling pathway in which SPC and LSF had opposite effects. Pharmacological inhibition of this pathway aborted the differentiation, indicating that this signaling pathway is required. Consistently, SPC promoted, while LSF inhibited, monocyte adhesion to fibronectin, through the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. The effects of SPC on Raf/MEK/ERK and PI3K/Akt signaling were dependent on G(i/o), whereas the SPC-induced calcium influx was dependent on G(q). Thus SPC utilizes G-protein coupled receptor. In contrast, the effects of LSF were independent of G(i/o) and G(q). These results suggest that SPC enhances, whereas LSF suppresses, monocyte differentiation into macrophages through regulating the Raf/MEK/ERK and PI3K/Akt signaling pathways via distinct mechanisms.

The C. elegans Opa1 homologue EAT-3 is essential for resistance to free radicals.

The C. elegans eat-3 gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-3 locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-3 mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-3 mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-3 and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-3 mutants. Instead, the phenotypes of eat-3 mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-3 mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase sod-2. We conclude that free radicals contribute to the pathology of C. elegans eat-3 mutants.

Wheat germ agglutinin stains dispersed post-golgi vesicles after treatment with the cytokinesis inhibitor psychosine.

The galactosylsphingosine psychosine (Psy) is one of the sphingolipids and induce the formation of multinuclear cells in several cell lines by inhibiting cytokinesis. In the present report, we show that intracellular organelles, including wheat germ agglutinin (WGA)-positive vesicles and early endosomes, are selectively dispersed by Psy. WGA is a conventional Golgi marker and WGA-positive vesicles appeared to co-localize with the Golgi apparatus in untreated cells. Psy treatment induced the dispersal of WGA-positive vesicles without affecting the structure of the Golgi apparatus, resulting in discrimination of WGA-positive vesicles from the Golgi apparatus. In sharp contrast to this effect of Psy, WGA-positive vesicles were not affected by brefeldin A treatment, which induced the disappearance of the Golgi apparatus. Immunostaining with anti-TGN46 antibodies revealed that a large portion of the WGA-positive vesicles were derived from the trans-Golgi network. Notably, the dispersed WGA-positive vesicles did not stain with anti-syntaxin 6, another marker of the trans-Golgi network. During cytokinesis, WGA-positive vesicles in the cytoplasm decreased, and WGA staining accumulated at the cleavage furrow, which was apparently inhibited by the presence of Psy. These data suggest that the transport of WGA-positive vesicles to the cleavage furrow is associated with the progression of cytokinesis.

Regulation of the mitochondrial dynamin-like protein Opa1 by proteolytic cleavage.

The dynamin-related protein Opa1 is localized to the mitochondrial intermembrane space, where it facilitates fusion between mitochondria. Apoptosis causes Opa1 release into the cytosol and causes mitochondria to fragment. Loss of mitochondrial membrane potential also causes mitochondrial fragmentation but not Opa1 release into the cytosol. Both conditions induce the proteolytic cleavage of Opa1, suggesting that mitochondrial fragmentation is triggered by Opa1 inactivation. The opposite effect was observed with knockdown of the mitochondrial intermembrane space protease Yme1. Knockdown of Yme1 prevents the constitutive cleavage of a subset of Opa1 splice variants but does not affect carbonyl cyanide m-chlorophenyl hydrazone or apoptosis-induced cleavage. Knockdown of Yme1 also increases mitochondrial connectivity, but this effect is independent of Opa1 because it also occurs in Opa1 knockdown cells. We conclude that Yme1 constitutively regulates a subset of Opa1 isoforms and an unknown mitochondrial morphology protein, whereas the loss of membrane potential induces the further proteolysis of Opa1.

Sphingolipids involved in the induction of multinuclear cell formation.

In this review, we focus on sphingolipids as potential regulators of the induction of multinuclear cell formation through the inhibition of cytokinesis. A sphingolipid, psychosine (Psy) (galactosylsphingosine), was demonstrated to be a trigger lipid for the inhibition of cytokinesis and the induction of multinuclear giant cells associated with a sphingolipid metabolic disease, globoid cell leukodystrophy (GLD). Indeed, Psy is known to accumulate in the patients' brains. Interestingly, inhibition of sphingolipid biosynthesis also induced multinuclear cells. When cells were treated with a new immunosuppressant, ISP-1/myriocin, which inhibits serine palmitoyltransferase, the first step enzyme of sphingolipid biosynthesis, the cells underwent multinucleation and apoptosis. At present, a definitive model of the function of sphingolipids as to the induction of multinuclear cell formation is not available due to the rudimentary information but possible mechanisms are discussed.