The role of the extracellular matrix in primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm that arises from clonal proliferation of hematopoietic stem cells and leads to progressive bone marrow (BM) fibrosis. While cellular mutations involved in the development of PMF have been heavily investigated, noteworthy is the important role the extracellular matrix (ECM) plays in the progression of BM fibrosis. This review surveys ECM proteins contributors of PMF, and highlights how better understanding of the control of the ECM within the BM niche may lead to combined therapeutic options in PMF.

Restoration of MYC-repressed targets mediates the negative effects of GM-CSF on RUNX1-ETO leukemogenicity.

Granulocyte macrophage-colony-stimulating factor (GM-CSF) signaling regulates hematopoiesis and immune responses. CSF2RA, the gene encoding the α-subunit for GM-CSF, is significantly downregulated in t(8;21) (RUNX1-ETO or RE) leukemia patients, suggesting that it may serve as a tumor suppressor. We previously reported that GM-CSF signaling is inhibitory to RE leukemogenesis. Here we conducted gene expression profiling of primary RE hematopoietic stem/progenitor cells (HSPCs) treated with GM-CSF to elucidate the mechanisms mediating the negative effects of GM on RE leukemogenicity. We observed that GM treatment of RE HSPCs resulted in a unique gene expression profile that resembles primary human cells undergoing myelopoiesis, which was not observed in control HSPCs. Additionally, we discovered that GM-CSF signaling attenuates MYC-associated gene signatures in RE HSPCs. In agreement with this, a functional screen of a subset of GM-CSF-responsive genes demonstrated that a MYC inhibitor, MXI1 (Max interactor 1), reduced the leukemic potential of RE HSPCs and t(8;21) acute myeloid leukemia (AML) cells. Furthermore, MYC knockdown and treatment with the BET (bromodomain and extra terminal domain) inhibitor JQ1 reduced the leukemic potential of t(8;21) cell lines. Altogether, we discovered a novel molecular mechanism mediating the GM-CSF-induced reduction in leukemic potential of RE cells, and our findings support MYC inhibition as an effective strategy for reducing the leukemogenicity of t(8;21) AML.

Y-box binding protein-1 regulates cell proliferation and is associated with clinical outcomes of osteosarcoma.

BACKGROUND: Prognosis of osteosarcoma (OS) with distant metastasis and local recurrence is still poor. Y-box binding protein-1 (YB-1) is a multifunctional protein that can act as a regulator of transcription and translation and its high expression of YB-1 protein was observed in OS, however, the role of YB-1 in OS remains unclear. METHODS: Y-box binding protein-1 expression in OS cells was inhibited by specific small interfering RNAs to YB-1 (si-YB-1). The effects of si-YB-1 in cell proliferation and cell cycle transition in OS cells were analysed in vitro and in vivo. The association of nuclear expression of YB-1 and clinical prognosis was also investigated by immunohistochemistry. RESULTS: Proliferation of OS cell was suppressed by si-YB-1 in vivo and in vitro. The expression of cyclin D1 and cyclin A were also decreased by si-YB-1. In addition, si-YB-1 induced G1/S arrest with decreased cyclin D1 and cyclin A in OS cell lines. Direct binding of YB-1 in OS cell lines was also observed. Finally, the nuclear expression of YB-1 was significantly related to the poorer overall survival in OS patients. CONCLUSION: Y-box binding protein-1 would regulate cell cycle progression at G1/S and tumour growth in human OS cells in vitro and in vivo. Nuclear expression of YB-1 was closely associated with the prognosis of OS, thus, YB-1 simultaneously could be a potent molecular target and prognostic biomarker for OS.

Comparative examination of subcutaneous tissue reaction to high molecular materials in medical use.

Polypropylene (PP), Polyethylene (PE) and polytetrafluoroethylene (FE) are high molecular materials in medical use. They are also used as the negative control materials for ISO 10993-6 international standard biological evaluation of medical devices. We examined tissue reactions to these materials embedded subcutaneously in the dorsal area of male ddY mice. One week and 12 weeks after embedding, the tissue surrounding the embedding site was removed and then histopathological examination was performed. Our results demonstrate that the basic histopathological reaction is the formation of fibrous capsule consisting of granulation tissue around the embedded materials. Based on our results, we believe that the high molecular materials such as, PP, PE and FE, can be considered for medical use as a biomaterials within the body.

A novel tumor-derived SGOL1 variant causes abnormal mitosis and unstable chromatid cohesion.

Mitosis is the most conspicuous cell cycle phase, because it is the phase in which the dynamic physical distributions of cellular components into the two daughter cells occur. The separation of sister chromatids is especially important during mitosis, because of the extreme accuracy required for distribution to the next generation of cells. Shugoshin-like 1 (SGOL1) is a key protein in protecting sister chromatids from precocious separation. We have reported finding that chromosome instability is more likely in SGOL1-downregulated colorectal cancers, but it is still unknown whether there is an association between cancer and SGOL1 transcript variation. Here, we identified a novel SGOL1 variant, SGOL1-P1, in human colon cancer. The SGOL1-P1 transcript contains an exon-skip of exon 3 that results in a stop codon occurring within exon 4. Overexpression of SGOL1-P1 in HCT116 cells resulted in an increased number of cells with aberrant chromosome alignment, precociously separated chromatids and delayed mitotic progression, occasionally followed by inaccurate distribution of the chromosomes. These phenotypes, observed when SGOL1-P1 was present, were also observed very frequently in SGOL1-knockdown cells. Furthermore, the overexpression of SGOL1-P1 inhibited the localization of endogenous SGOL1 and cohesin subunit RAD21/SCC1 to the centromere. These results suggest that SGOL1-P1 may function as a negative factor to native SGOL1, and that abundant expression of SGOL1-P1 may be responsible for chromosomal instability.

BubR1 localizes to centrosomes and suppresses centrosome amplification via regulating Plk1 activity in interphase cells.

BubR1 is a critical component of the mitotic checkpoint that delays the onset of anaphase until all chromosomes have established bipolar attachment to the microtubules. We previously reported that mutations of the BUB1B gene (encoding BubR1) caused premature chromatid separation (PCS) syndrome, a condition characterized by constitutional aneuploidy and a high risk of childhood cancer. We here report that the cells from PCS syndrome patients have loss of regulation of the centrosome duplication machinery, resulting in centrosome amplification and multipolar mitosis. PCS syndrome cells show increased activity of Polo-like kinase 1 (Plk1), whose knockdown suppresses centrosome amplification. BubR1 localizes to centrosomes, physically interacts with Plk1 and inhibits Plk1 phosphorylation and its kinase activity during interphase. These results unravel a crucial role of BubR1 in preventing centrosome reduplication through negative regulation of Plk1 in interphase cells.

[Tracheostenosis caused by blunt thoracic trauma].

Blunt thoracic trauma rarely implicate retropharyngeal hematoma obstructing trachea. 85-year-old woman being struck on her cheek and anterior chest, visited our emergency room. She was nearly suffocated following stridor and dyspnea. Tracheal intubation relieved her dyspnea Chest computed tomography (CT) showed retropharyngeal hematoma obstructing trachea. 5 days conservative management reduced the hematoma and tracheal tube was extubated through an uneventful course.

NBS1 prevents chromatid-type aberrations through ATM-dependent interactions with SMC1.

Nijmegen breakage syndrome shares several common cellular features with ataxia telangiectasia, including chromosomal instability and aberrant S- and G2-phase checkpoint regulation. We show here that after irradiation, NBS1 interacts physically with both BRCA1 and SMC1, a component of the cohesin complex, and that their interactions are completely abolished in AT cells. It is noted that BRCA1 is required for the interaction of NBS1 with SMC1, whereas the reverse is not the case, since BRCA1 is able to bind to NBS1 in the absence of an NBS1/SMC1 interaction as observed in MRE11- or RAD50-deficient cells. This indicates that ATM and BRCA1 are upstream of the NBS1/SMC1 interaction. Furthermore, the interaction of NBS1 with SMC1 requires both conserved domains of NBS in the N-terminus and the C-terminus, since they are indispensable for binding of NBS1 to BRCA1 and to MRE11/ATM, respectively. The interaction of NBS1 with SMC1 and the resulting phosphorylation are compromised in the clones lacking either the N- or C-terminus of NBS1, and as a consequence, chromatid-type aberrations are enhanced after irradiation. Our results reveal that ATM plays a fundamental role in promoting the radiation-induced interaction of NBS1 with SMC1 in the presence of BRCA1, leading to the maintenance of chromosomal integrity.

Centrosome amplification induced by survivin suppression enhances both chromosome instability and radiosensitivity in glioma cells.

Glioblastoma is characterised by invasive growth and a high degree of radioresistance. Survivin, a regulator of chromosome segregation, is highly expressed and known to induce radioresistance in human gliomas. In this study, we examined the effect of survivin suppression on radiosensitivity in malignant glioma cells, while focusing on centrosome aberration and chromosome instability (CIN). We suppressed survivin by small interfering RNA transfection, and examined the radiosensitivity using a clonogenic assay and a trypan blue exclusion assay in U251MG (p53 mutant) and D54MG (p53 wild type) cells. To assess the CIN status, we determined the number of centrosomes using an immunofluorescence analysis, and the centromeric copy number by fluorescence in situ hybridisation. As a result, the radiosensitisation differed regarding the p53 status as U251MG cells quickly developed extreme centrosome amplification (=CIN) and enhanced the radiosensitivity, while centrosome amplification and radiosensitivity increased more gradually in D54MG cells. TUNEL assay showed that survivin inhibition did not lead to apoptosis after irradiation. This cell death was accompanied by an increased degree of aneuploidy, suggesting mitotic cell death. Therefore, survivin inhibition may be an attractive therapeutic target to overcome the radioresistance while, in addition, proper attention to CIN (centrosome number) is considered important for improving radiosensitivity in human glioma.

Homologous recombination repair is regulated by domains at the N- and C-terminus of NBS1 and is dissociated with ATM functions.

The proteins responsible for radiation sensitive disorders, NBS1, kinase ataxia-telangiectasia-(A-T)-mutated (ATM) and MRE11, interact through the C-terminus of NBS1 in response to the generation of DNA double-strand breaks (DSBs) and are all implicated in checkpoint regulation and DSB repair, such as homologous recombination (HR). We measured the ability of several NBS1 mutant clones and A-T cells to regulate HR repair using the DR-GFP or SCneo systems. ATM deficiency did not reduce the HR repair frequency of an induced DSB, and it was confirmed by findings that HR frequencies are only slightly affected by deletion of ATM-binding site at the extreme C-terminus of NBS1. In contrast, The HR-regulating ability is dramatically reduced by deletion of the MRE11-binding domain at the C-terminus of NBS1 and markedly inhibited by mutations in the FHA/BRCT domains at the N-terminus. This impaired capability in HR is consistent with a failure to observe MRE11 foci formation. Furthermore, normal HR using sister chromatid was completely inhibited by the absence of FHA/BRCT domains. These results suggested that the N- and C-terminal domains of NBS1 are the major regulatory domains for HR pathways, very likely through the recruitment and retention of the MRE11 nuclease to DSB sites in an ATM-independent fashion.

Nijmegen breakage syndrome and functions of the responsible protein, NBS1.

Nijmegen breakage syndrome (NBS) is a rare recessive genetic disorder, characterized by bird-like facial appearance, early growth retardation, congenital microcephaly, immunodeficiency and high frequency of malignancies. NBS belongs to the so-called chromosome instability syndromes; in fact, NBS cells display spontaneous chromosomal aberrations and are hypersensitive to DNA double-strand break-inducing agents, such as ionizing radiations. NBS1, the gene underlying the disease, is located on human chromosome 8q21. The disease appears to be prevalent in the Eastern and Central European population where more than 90% of patients are homozygous for the founder mutation 657del5 leading to a truncated variant of the protein. NBS1 forms a multimeric complex with MRE11/RAD50 nuclease at the C-terminus and retains or recruits them at the vicinity of sites of DNA damage by direct binding to histone H2AX, which is phosphorylated by PI3-kinase family, such as ATM, in response to DNA damage. Thereafter, the NBS1-complex proceeds to rejoin double-strand breaks predominantly by homologous recombination repair in vertebrates. NBS cells also show to be defective in the activation of intra-S phase checkpoint. We review here some cellular and molecular aspects of NBS, which might contribute to the clinical symptoms of the disease.

'Scirrhous' type hepatocellular carcinomas: a special reference to expression of cytokeratin 7 and hepatocyte paraffin 1.

AIMS: 'Scirrhous' hepatocellular carcinoma (scirrhous HCC) is extremely rare and its characteristics remain unclear. We investigated the clinicopathological and immunohistochemical features of scirrhous HCC, compared with those of ordinary hepatocellular carcinoma (ordinary HCC). METHODS AND RESULTS: We compared the clinicopathological and immunohistochemical features of 20 resected cases of scirrhous HCC with those of 69 resected cases of ordinary HCC. Scirrhous HCC was characterized by its gross and histological findings, such as a higher proportion of contiguous multinodular type tumours, the absence of a complete fibrous capsule around the tumour, the absence of tumour necrosis and highly preserved portal tracts in the tumour. The immunohistochemical results revealed a significantly higher expression of cytokeratin 7 and a significantly lower expression of hepatocyte paraffin 1 in scirrhous HCC than in ordinary HCC (P<0.0001, respectively). There were no significant differences in proliferative activity and survival curves between the patients with scirrhous HCC and those with ordinary HCC. CONCLUSION: Scirrhous HCC has several particular gross, histological and immunohistochemical features. In particular, we would like to emphasize the greater immunohistochemical expression of cytokeratin 7 and lower expression of hepatocyte paraffin 1 in scirrhous HCC than in ordinary HCC.

The influence of mycophenolate mofetil versus azathioprine and mycophenolic acid pharmacokinetics on the incidence of acute rejection and infectious complications after renal transplantation.

PURPOSE: The present retrospective study investigated the influence of mycophenolate mofetil (MMF) instead of azathioprine (AZA) as part of tacrolimus-based immunosuppression. Mycophenolic acid (MPA) pharmacokinetic (PK) parameters were used for associations with the incidence of acute rejection (AR) episodes and infectious complications after renal transplantation. METHODS: The 66 consecutive renal transplant recipients reported herein excluded ABO-incompatible transplants or cytomegalovirus (CMV)-seronegative recipients. The immunosuppressive regimen consisted of tacrolimus, steroids, and AZA 1-2 mg/kg/d in 22 patients (between February 1998 and December 2000) or MMF 2 g/d in 44 patients (since January 2001). CMV infection was defined as positive CMV-antigenemia. MPA PK was studied on day 28 after transplantation in 21 recipients. RESULTS: AR occurred in 13.6% of patients in the MMF group compared with 18.2% in the AZA group. The viral infection (CMV, varicella zoster virus, adenovirus hemorrhagic cystitis, and malignancy related to Epstein-Barr [EB] virus) rate was 22.7% in the MMF group and 0% in the AZA group (P = .015). There were no bacterial or fungal infections observed in the 2 groups. MMF dose per body weight was significantly lower among patients with AR than those without AR (25.1 vs 35.6 mg/kg; P = .026). There were no differences in MPA PK parameters between patients with and without viral infections. CONCLUSIONS: Patients treated with MMF required less treatment for AR, however, there were no significant differences. MMF dose per body weight may play an important role in the occurrence of AR. Although virus infections occurred in recipients treated with MMF, MPA PK did not influence the infectious complications after renal transplantation.

Serum sex steroid hormone levels and polymorphisms of CYP17 and SRD5A2: implication for prostate cancer risk.

Polymorphism of the steroid hormone-related genes might affect life-long androgen exposure, thus altering a risk of prostate cancer incidence. To evaluate the effect of the polymorphisms of CYP17 and SRD5A2 on serum steroid hormone levels, the 164 male Japanese cohort were tested for serum hormone levels and the genotype of the polymorphisms of CYP17 (T-C base substitution in the promoter region) and SRD5A2 (V89L). The linear trends across the CYP17 genotypes in serum-free testosterone and androstenedione levels were found, suggesting the importance of the polymorphism of CYP17 in determining the circulating androgen levels.

Gluteal muscular and sciatic nerve metastases in advanced urinary bladder carcinoma: case report.

We present a case of gluteal muscular and sciatic nerve metastases from urinary bladder carcinoma. T2-weighted magnetic resonance images demonstrated diffuse swelling and an increase in the signal of the right gluteus maximus muscle without destruction of the original arrangement of muscular fibers. Further, remarkable thickening of the right sciatic nerve showing a relatively hypointense signal was detected. Postcontrast T1-weighted images showed strong enhancement of these structures. Fine-needle aspiration biopsy with ultrasonographic guidance confirmed metastatic carcinoma cells in the right gluteal muscle and the sciatic nerve. These radiologic findings may represent a rare pattern of metastasis from urinary bladder carcinoma.

Fat detection in gallbladder carcinoma with extensive xanthogranulomatous change demonstrated by chemical shift MR imaging.

We present a case of gallbladder carcinoma, in which fat was detected on dual-echo chemical shift magnetic resonance imaging (MRI). Histologic analysis showed poorly differentiated adenocarcinoma associated with massive xanthogranulomatous change. Sudan IV staining successfully confirmed the presence of fat within the interstitial histiocytes. Although rare, gallbladder carcinoma with xanthogranulomatous change should be included in the differential diagnosis of fatty tumor involving the region of the liver as observed on chemical shift MRI.

p16 Gene transfer increases cell killing with abnormal nucleation after ionising radiation in glioma cells.

It is well established that cells synchronised at the G1-S phase are highly radiosensitive. In this study, p16-null human glioma cell lines were induced into G1 cell cycle arrest by adenovirus-mediated p16 gene transfer, and examined for radiation-induced cell killing. Clonogenic analysis and trypan blue extraction test showed that the p16 gene transfer enhanced radiation-induced cell killing in p16-null glioma cell lines. TUNEL assays and pulse-field gel electrophoresis confirmed that the radiation-induced cell killing of p16-transfected cells could be caused by a nonapoptotic mechanism. Gimsa staining demonstrated that irradiation alone or Ax-mock infection plus irradiation results in a slight increase in the frequency of cells with abnormal nucleus, compared to unirradiated uninfected or Ax-mock infected cells. However, Ax-hp16 or Ax-hp21 infection alone modestly increased the frequency of cells with abnormal nucleus (especially bi- and multinucleation), and 4-Gy irradiation of Ax-hp16 or Ax-hp21 infected cells substantially enhanced this frequency. These results suggest that there exists some unknown interaction between radiation and p16 in cytoplasm/membranes, which decreases cytokinesis and promotes abnormal nucleation. Thus, p16 expression prevented radiation-induced apoptosis by promoting abnormal nucleation, thereby leading to another mode of cell death.

Gene expression of type I collagen in neoplastic chondrocytes.

In the present examination, we detected type I collagen mRNA in neoplastic chondrocytes in osteochondromas, typical benign bone neoplasms. We believe that the cells involved in >chondroid bone< appearing in osteochondromas temporally express cartilage phenotypes and then change directly into bone-forming cells that survive in the >chondroid bone< until the tissue is resorbed and remodelled into true bone tissue.