Tokuhiro Chano

Cytokine production in the infrapatellar fat pad: another source of cytokines in knee synovial fluids

Background: Recent studies have shown that adipose tissue is an endocrine organ that releases various cytokines. Objective: To investigate the production of growth factors and proinflammatory cytokines in infrapatellar fat pad specimens. Methods: Infrapatellar fat pad tissues were obtained from patients during knee surgery. Protein levels of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), tumour necrosis factor (TNF)α, and interleukin (IL)6 in homogenised tissues were measured by an enzyme immunoassay. Gene expressions for those cytokines were examined by reverse transcription-polymerase chain reaction (RT-PCR). Localisation of bFGF and VEGF was evaluated by immunohistochemistry and in situ hybridisation. Results: Infrapatellar fat pads were found to contain various protein levels of bFGF, VEGF, TNFα, and IL6. Further, gene expressions for these cytokines were detected by RT-PCR. Immunohistochemistry and in situ hybridisation showed that the expressions of both bFGF and VEGF were localised in immature adipocytes, interstitial undifferentiated mesenchymal cells, and vascular endothelial cells. Conclusion: The production of bFGF, VEGF, TNFα, and IL6 in the infrapatellar fat pad was demonstrated. Although synovial cells and articular chondrocytes are thought to be primary sources of cytokines found in knee synovial fluids, the results suggest that they may also originate from this fat pad.

MRI findings in intramuscular lipomas

Abstract Objective. To investigate the spectrum of magnetic resonance (MR) findings of intramuscular lipoma. Design and patients. A retrospective review of 17 consecutive cases of intramuscular lipoma examined with MR imaging was undertaken. Features assessed included the size and margin of the mass; the homogeneity of the contents, including the presence or absence of intermingled muscle fibers; whether the mass was uninodular or multinodular; and the presence of linear structures between and within the tumor nodules. Three well-differentiated liposarcomas and one dedifferentiated liposarcoma associated with lipoma-like components were also studied to allow a comparison of the benign and malignant lesions. Results. The diameter of the intramuscular lipomas varied from less than 3 cm to more than 10 cm. Ten of the intramuscular lipomas were homogeneous but the remaining seven were inhomogeneous with intermingled muscle fibers within the mass. The intramuscular lipomas were well defined in 12 cases, and infiltrative in five. In one case the margin of the lesion showed prominent infiltration of the surrounding muscle tissue. Of the 17 cases of intramuscular lipoma, 15 were composed of a single nodule, whereas three of four cases of liposarcoma were composed of multinodular masses. Conclusion. The MR findings of intramuscular lipoma varied from a small, single and homogeneous mass identical to ordinary (superficial) lipoma, to a large, inhomogeneous lesion with an infiltrative margin. The presence of infiltrative margins and intermingled muscle fibers in intramuscular lipoma indicates a benign lesion rather than malignancy. In addition, uninodularity of the mass is helpful in differentiating intramuscular lipoma from well-differentiated liposarcoma.

p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200

The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53K382R failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.

Redundancy of autocrine loops in human osteosarcoma cells.

With the aim of identifying innovative therapeutic strategies for osteosarcoma patients who are refractory to conventional chemotherapy, we analyzed the in vitro effects of the blockage of autocrine circuits. Since the insulin‐like growth factor‐I receptor (IGF‐IR)–mediated loop is relevant to the growth of osteosarcoma, we analyzed the activity of the IGF‐IR‐blocking antibody αIR3 in both sensitive and multidrug‐resistant osteosarcoma cell lines. Only limited effects, however, were observed, suggesting the simultaneous existence of other autocrine circuits. Indeed, in a representative panel of 12 human osteosarcoma cell lines, in addition to the IGF‐IR‐mediated circuit, we demonstrated also a loop mediated by epidermal growth factor receptor as well as the presence of nerve growth factor, low‐affinity nerve growth factor receptor as well as tyrosine receptor kinase A in the great majority of osteosarcomas. Therapies based on the inhibition of single circuits may have only limited effects in osteosarcoma, whereas the use of suramin, a drug which, besides other activities, non‐selectively interferes with the binding of growth factors to their receptors, appears as a promising alternative, in both sensitive and drug‐resistant osteosarcoma cells. Int. J. Cancer 80:581–588, 1999.

Truncating mutations of RB1CC1 in human breast cancer.

The protein RB1CC1 (retinoblastoma 1 (RB1)-inducible coiled-coil 1) has been identified as a key regulator of the tumor-suppressor gene RB1 (ref. 1). RB1CC1 is localized in the nucleus and has been proposed to be a transcription factor because of its nuclear localization signal, leucine zipper motif and coiled-coil structure. The gene RB1CC1 is localized to a region of chromosome 8q11 (ref. 2) containing several loci of putative tumor-suppressor genes; however, its role in human cancers remains to be determined. Here we report that 20% (7 of 35) of primary breast cancers examined contained mutations in RB1CC1, including nine large interstitial deletions predicted to yield markedly truncated RB1CC1 proteins. Wildtype RB1CC1 and RB1 were absent or significantly less abundant than normal in the seven cancers with mutations in RB1CC1, but were abundant in cancers without such mutations. In all seven cancers, both RB1CC1 alleles were inactivated; two showed compound heterozygous deletions. Thus, RB1CC1 is frequently mutated in breast cancer and shows characteristics of a classical tumor-suppressor gene.

Identification of RB1CC1, a novel human gene that can induce RB1 in various human cells.

Multidrug resistance to anti-cancer agents (MDR) is a major barrier to successful cancer treatment. Current knowledge about genes that contribute to MDR is limited, however, and its mechanisms remain unclear. To identify genes involved in MDR, we performed differential display analysis and isolated a novel human gene, RB1CC1 (RBI-inducible Coiled-Coil 1). The 6.6-kb RB1CC1 cDNA encodes a putative 1594-amino-acid protein that contains a nuclear localization signal, a leucine zipper motif and a coiled-coil structure. Western blot analysis and immunocytochemical staining with anti-RB1CC1 antibody showed that endogenously expressed RB1CC1 protein localized to the nucleus. In MDR variants of human osteosarcoma cells, RB1CC1 expression increased in response to doxorubicin-induced cytotoxic stress and remained elevated for the duration of drug treatment. RB1CC1 expression levels correlated closely with those of RB1 (retinoblastoma 1 ) in cancer cell lines as well as in various normal human tissues. Moreover, introduction of wild-type RB1CC1 significantly induced RB1 expression in human leukemic cells. These data suggest that RB1CC1 may be a key regulator of RB1 gene expression.

RB1CC1 insufficiency causes neuronal atrophy through mTOR signaling alteration and involved in the pathology of Alzheimer's diseases.

RB1-inducible Coiled-Coil 1 (RB1CC1) has been shown to be a novel tumor suppressor regulating RB1 expression. Neuronal abundance of RB1CC1 is reported to contribute to the non-proliferating enlarged cell phenotype through the maintenance of RB1 and mTOR. To clarify whether RB1CC1 insufficiency is involved in neuronal atrophy and Alzheimers pathology, we investigated modifications of RB1CC1 as a possible cause of atrophy or death through the disturbance of mTOR signaling in Neuro-2a neuroblastoma cells. We also evaluated the correlation between RB1CC1 and mTOR signaling in a series of Alzheimers brain tissues. Though RB1CC1 introduction enhanced neurite growth, RNAi-mediated knockdown of RB1CC1 or rapamycin treatment caused neurite atrophy and apoptosis due to mTOR signaling reduction in the differentiated Neuro-2a cells. Both TSC1 and RB1CC1 were equally functional and maintained mTOR signaling, indicated by phospho-S6 (Ser240/244) expression in 69% of Alzheimers (9/13 cases) and 100% of normal brains (6/6 cases). However, scanty RB1CC1 expression, less than TSC1, caused phospho-S6 disappearance in 31% of Alzheimers tissues (4/13 cases). These findings suggest that RB1CC1 insufficiency may result in mTOR signaling repression through unbalanced TSC1 abundance and may induce neuronal atrophy. These observations may have implications for the pathogenesis of Alzheimers disease.

DNMT3L Is a Novel Marker and Is Essential for the Growth of Human Embryonal Carcinoma

Purpose: Testicular germ cell tumors (TGCT) have a unique epigenetic profile distinct from that of other types of cancer. Elucidation of these properties has a potential to identify novel markers for TGCTs. Experimental Design: We conducted comprehensive analysis of DNA methyltransferase (DNMT) gene expression in TGCTs. Based on the expression profiles of DNMT genes in TGCTs, we generated a rabbit polyclonal anti-human DNMT3L antibody. We then studied the role of DNMT3L in TGCTs by the treatment of two embryonal carcinoma (EC) cell lines with a small interfering RNA system. Finally, we evaluated the immunohistochemical detection of DNMT3L in TGCT tissues. We also compared the patterns of DNMT3L immunohistochemistry with those of CD30 and SOX2. Results: Among the DNMT genes, we found that mRNA for DNMT3L was specifically expressed in TGCTs, but neither in normal testicular tissues nor in cancer cells of somatic tissue origin. DNMT3L protein was strongly expressed in two EC cell lines, but not in the cell lines of somatic tissue origin. Transfection of small interfering RNA for DNMT3L significantly reduced DNMT3L expression and resulted in growth suppression and apoptosis in EC cells. Immunohistochemical analysis showed that DNMT3L protein was present only in EC cells, but not in the other types of TGCT components and cancer cells of somatic tissue origin. DNMT3L staining was more prominent and specific than CD30 or SOX2 staining for detecting EC cells. Conclusion: DNMT3L is a novel marker and is essential for the growth of human embryonal carcinoma. Clin Cancer Res; 16(10); 2751–9. ©2010 AACR.

RECQL1 and WRN Proteins Are Potential Therapeutic Targets in Head and Neck Squamous Cell Carcinoma

RECQL1 and WRN proteins are RecQ DNA helicases that participate in suppression of DNA hyper-recombination and repair. In this study, we report evidence supporting their candidacy as cancer therapeutic targets. In hypopharyngeal carcinomas, which have the worst prognosis among head and neck squamous cell carcinomas (HNSCC) that are rapidly rising in incidence, we found that RECQL1 and WRN proteins are highly expressed and that siRNA-mediated silencing of either gene suppressed carcinoma cell growth in vitro. Similarly, siRNA administration in a murine xenograft model of hypopharyngeal carcinoma markedly inhibited tumor growth. Moreover, combining either siRNA with cis-platinum (II) diammine dichloride significantly augmented the in vivo anticancer effects of this drug that is used commonly in HNSCC treatment. Notably, we observed no recurrence of some tumors following siRNA treatment in this model. Our findings offer a preclinical proof of concept for RECQL1 and WRN proteins as novel therapeutic targets to treat aggressive HNSCC and perhaps other cancers.

Identification and characterization of the human long form of Sox5 (L-SOX5) gene.

The Sox (Sry-type HMG box) group of transcription factors, which is defined by a high-mobility group (HMG) DNA-binding domain, is categorized into six subfamilies. Sox5 and Sox6 belong to the group D subfamily, which is characterized by conserved N-terminal domains including a leucine-zipper, a coiled-coil domain and a Q-box. Group D Sox genes are expressed as long and short transcripts that exhibit differential expression patterns. In mouse, the long form of Sox5, L-Sox5, is co-expressed and interacts with Sox6; together, these two proteins appear to play a key role in chondrogenesis and myogenesis. In humans, however, only the short form of Sox5 has previously been identified. To gain insight into Sox5 function, we have identified and characterized human L-SOX5. The human L-SOX5 cDNA encodes a 763-amino-acid protein that is 416 residues longer than the short form and contains all of the characteristic motifs of group D Sox proteins. The predicted L-SOX5 protein shares 97% amino acid identity with its mouse counterpart and 59% identity with human SOX6. The L-SOX5 gene contains 18 exons and shows similar genomic structure to SOX6. We have identified two transcription start sites in L-SOX5 and multiple alternatively spliced mRNA variants that are distinct from the short form. Unlike the short form, which shows testis-specific expression, L-SOX5 is expressed in multiple tissues. Like SOX6, L-SOX5 shows strong expression in chondrocytes and striated muscles, indicating a likely role in human cartilage and muscle development.