Shear Stiffness of 4 Common Intracranial Tumors Measured Using MR Elastography: Comparison with Intraoperative Consistency Grading.

BACKGROUND AND PURPOSE: The stiffness of intracranial tumors affects the outcome of tumor removal. We evaluated the stiffness of 4 common intracranial tumors by using MR elastography and tested whether MR elastography had the potential to discriminate firm tumors preoperatively. MATERIALS AND METHODS: Thirty-four patients with meningiomas, pituitary adenomas, vestibular schwannomas, and gliomas scheduled for resection were recruited for MR elastography. On the elastogram, the mean and the maximum shear stiffnesses were measured by placing an ROI on the tumor. Blinded to the MR elastography findings, surgeons conducted qualitative intraoperative assessment of tumor consistency by using a 5-point scale. Histopathologic diagnosis was confirmed by using the resected specimens. The mean and maximum shear stiffnesses were compared with histopathologic subtypes, and the intraoperative tumor consistency was graded by the surgeons. RESULTS: The mean and maximum shear stiffnesses were the following: 1.9 ± 0.8 kPa and 3.4 ± 1.5 kPa for meningiomas, 1.2 ± 0.3 kPa and 1.8 ± 0.5 kPa for pituitary adenomas, 2.0 ± 0.4 kPa and 2.7 ± 0.8 kPa for vestibular schwannomas, and 1.5 ± 0.2 kPa and 2.7 ± 0.8 kPa for gliomas. The mean and maximum shear stiffnesses for meningiomas were higher than those of pituitary adenomas (P < .05). The mean and maximum shear stiffnesses were significantly correlated with the surgeon's qualitative assessment of tumor consistency (P < .05). The maximum shear stiffness for 5 firm tumors was higher than that of nonfirm tumors (P < .05). CONCLUSIONS: MR elastography could evaluate intracranial tumors on the basis of their physical property of shear stiffness. MR elastography may be useful in discriminating firm tumors preoperatively.

A second-generation profiling system for quantitative methylation analysis of multiple gene promoters: application to lung cancer.

Cancer-specific gene promoter methylation has been described in many types of cancers, and various semi-quantified results have shown their usefulness. Here, we show a more sensitive and specific second-generation system for profiling the DNA methylation status. This method is based on bisulfite reaction of DNA and real-time PCR using two TaqMan MGB probes labeled with different fluorescence, followed by clustering analysis. Primers were designed with CpG-less sequences, and TaqMan MGB probes were designed to contain three or four CpG sites and to be shorter than conventional TaqMan probes. We have added new criteria for primer and probe design for further specificity. We confirmed the reliability of this system and applied it to analysis of lung cancers. Using 10 promoters, 90 primary lung cancers were clustered into six groups consisting of cases having similar smoking status and pathological findings. EGFR mutation and p16 promoter DNA methylation were exclusive, as previously reported; however, DNA methylation in other genes was unrelated to EGFR mutation. This system was also useful to distinguish double primary lung cancers from a single cancer with intrapulmonary metastasis. As above, our system has widespread availability in clinical use and biological research.

Possible dominant-negative mutation of the SHIP gene in acute myeloid leukemia.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) is crucial in hematopoietic development. To evaluate the possible tumor suppressor role of the SHIP gene in myeloid leukemogenesis, we examined primary leukemia cells from 30 acute myeloid leukemia (AML) patients, together with eight myeloid leukemia cell lines. A somatic mutation at codon 684, replacing Val with Glu, was detected in one patient, lying within the signature motif 2, which is the phosphatase active site. The results of an in vitro inositol 5'-phosphatase assay revealed that the mutation reduced catalytic activity of SHIP. Leukemia cells with the mutation showed enhanced Akt phosphorylation following IL-3 stimulation. K562 cells transfected with the mutated SHIP-V684E cDNA showed a growth advantage even at lower serum concentrations and resistance to apoptosis induced by serum deprivation and exposure to etoposide. These results suggest a possible role of the mutated SHIP gene in the development of acute leukemia and chemotherapy resistance through the deregulation of the phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3)/Akt signaling pathway. This is the first report of a mutation in the SHIP gene in any given human cancer, and indicates the need for more attention to be paid to this gene with respect to cancer pathogenesis.

Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis.

Aberrantly hypermethylated genes in human lung cancers were searched for by a genome scanning technique, methylation-sensitive-representational difference analysis (MS-RDA). A total of 59 DNA fragments were isolated as those methylated more heavily in either/both of two lung squamous cell carcinoma cell lines, EBC-1 and LK-2, than in a primary culture of normal human bronchial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation was confirmed in primary squamous cell carcinomas, were sequenced. By database searches, 17 of them were shown to be located within 2 kb of putative CpG islands, and five of the 17 DNA fragments had transcribed regions of known genes in their vicinities. By RT-PCR of the five genes in the carcinoma cell lines and NHBE, decreased expression of HTR1B (5-hydroxytryptamine receptor 1B) and EDN1 (endothelin-1) was observed. Sequencing after bisulfite modification showed that the CpG island in the promoter region of HTR1B was hypermethylated, while that of EDN1 was not. Demethylation and re-expression of HTR1B were observed after treatment of LK-2 cells with 5-aza-2'-deoxycytidine. In primary lung cancers, decreased mRNA expression of HTR1B was observed in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohistochemical analysis of endothelin-1 confirmed that its immunoreactivity was reduced in squamous cell carcinoma cells compared with that in normal bronchial epithelial cells. Considering that endothelin-1 induces apoptosis in melanoma cells and that silencing of endothelin receptor B is observed in prostate cancers, its reduced expression was speculated to confer a growth advantage to lung cancer cells. MS-RDA was shown to isolate DNA fragments that are hypermethylated and silenced, such as HTR1B, and those whose expressions are altered and the methylation statuses outside the promoter region are altered, such as EDN1.

Stable operation of a 300-m laser interferometer with sufficient sensitivity to detect gravitational-wave events within our galaxy.

TAMA300, an interferometric gravitational-wave detector with 300-m baseline length, has been developed and operated with sufficient sensitivity to detect gravitational-wave events within our galaxy and sufficient stability for observations; the interferometer was operated for over 10 hours stably and continuously. With a strain-equivalent noise level of h approximately 5x10(-21)/sqrt[Hz], a signal-to-noise ratio of 30 is expected for gravitational waves generated by a coalescence of 1.4M-1.4M binary neutron stars at 10 kpc distance. We evaluated the stability of the detector sensitivity with a 2-week data-taking run, collecting 160 hours of data to be analyzed in the search for gravitational waves.

Overestimated frequency of a possible emphysema-susceptibility allele when microsomal epoxide hydrolase is genotyped by the conventional polymerase chain reaction-based method.

A recent association study suggested that the His113 variant of microsomal epoxide hydrolase (mEPHX) may confer a risk for development of emphysema, presumably by increasing susceptibility to smoking injury. Before considering a possible role of this enzyme in pulmonary disease, we attempted to characterize the genetic polymorphism further. The Tyr/His113 polymorphism within exon 3 of mEPHX was initially examined in 62 healthy individuals by conventional methods involving polymerase chain reaction (PCR)-based determination of a restriction fragment length polymorphism (RFLP). Genomic nucleotide sequences, including the polymorphic site and the downstream primer sequence, were further analyzed in 95 unrelated, healthy Japanese volunteers by single-stranded conformation polymorphism (SSCP) analysis and direct sequencing. Genotyping by the first method (PCR-RFLP) revealed that the allelic distribution in our test population apparently deviated from Hardy-Weinberg equilibrium. Sequence analysis showed that a synonymous nucleotide substitution, AAG to AAA (Lys119), was located just within the published primer site. The AAA at codon 119 was present only in alleles with Tyr113, and its frequency reached 0.31 in our panel of 190 Japanese alleles. This substitution potentially hampered PCR amplification because of the nucleotide mismatch, with the result that the frequency of the Tyr113 variation was underestimated. The frequency of His113, a possible emphysema susceptibility allele of the mEPHX gene, was thus overestimated when human DNA samples were genotyped in the conventional way. Depending on the population(s) tested, this anomaly could represent a pitfall for PCR-based association studies.

Effects of interferon-beta in combination with 5-fluorouracil on the growth of esophageal cancer cells in vitro.

The possible antiproliferative potency of human recombinant interferon-beta (hIFN-beta) towards ten human esophageal cancer cell lines was examined in comparison with the activity of the factor towards human malignant melanoma cell lines. The cell growth of esophageal cancer cell lines was inhibited by hIFN-beta in a dose- and time- dependent manner. The 50% inhibitory concentrations (IC50) of hIFN-beta on nine cell lines out of ten ranged between 23 to 332 IU/ml of culture medium. The remaining cell line, T.Tn, was less sensitive to the interferon (IC50, 611 IU/ml). Under the same culture conditions, the melanoma cell lines tested differed markedly in their sensitivity to hIFN-beta. When the esophageal cancer cells were treated with 5-fluorouracil (5-FU) in the presence of a low concentration of hIFN-beta, the effectiveness of 5-FU was markedly enhanced. In particular, the rate of growth inhibition of T.Tn cells was more than the added potencies of 5-FU and hIFN-beta indicating that the interferon is an effective biomodulator of 5-FU. All these data suggest that combination therapy with hIFN-beta and the anticancer drug 5-FU would be beneficial for the treatment of carcinoma of the esophagus.

Fine localization of a major disease-susceptibility locus for diffuse panbronchiolitis.

Diffuse panbronchiolitis affecting East Asians is strongly associated with the class I human leukocyte antigen (HLA) alleles. Recent observations suggest that a major disease-susceptibility gene may be located between the HLA-B and HLA-A loci in the class I region of the major histocompatibility complex on chromosome 6. To test this possibility, we analyzed 14 polymorphic markers in 92 Japanese patients and 93 healthy controls. Of these, seven marker alleles, including HLA-B54 and HLA-A11, were significantly associated with the disease. Maximum-likelihood haplotype analysis and subsequent direct determination of individual haplotypes identified a group of disease-associated haplotypes, one of which contained all seven disease-associated marker alleles. Another haplotype, containing HLA-B*5504, was also associated with the disease. All these haplotypes seem to have diverged from a common ancestral haplotype in East Asians and share a specific segment containing three consecutive markers between the S and TFIIH loci in the class I region. Furthermore, one of the markers within the candidate region showed the highest delta value, indicating the strongest association. Of 20 Korean patients with diffuse panbronchiolitis, 17 also shared the combination of the disease-associated marker alleles within the candidate region. These results indicate that an HLA-associated major susceptibility gene for diffuse panbronchiolitis is probably located within the 200 kb in the class I region 300 kb telomeric of the HLA-B locus on the chromosome 6p21.3.

Increased activity and intranuclear expression of phospholipase D2 in human renal cancer.

We examined the PLD activities of human renal cancers and found that the PLD2 activity was greatly elevated in almost all cases examined as compared with the adjacent normal region. Western blot analysis showed the increased levels of PLD2 protein, but the PLD1 was not discernible. The oleate-dependent PU) activity was very low but appeared to increase in most cases. Interestingly, the immunohistochemical observations indicated the high expression of PLD2 in the nuclei of clear carcinoma cells. This is the first demonstration which suggests the possible involvement of PLD2 in tumorigenesis of renal cancer.

Increased leukotriene A(4) hydrolase expression in the heart of angiotensin II-induced hypertensive rat.

Leukotriene A(4) (LTA(4)) hydrolase is essential for the conversion of LTA(4) to LTB(4), an inflammatory lipid mediator. We investigated whether LTA(4) hydrolase was regulated in the heart by angiotensin II (ang II) infusion. Continuous ang II infusion via an osmotic minipump for up to 7 days upregulated mRNA and protein levels of LTA(4) hydrolase ( approximately 3.5-fold of control) in the heart in a pressor-dependent manner. Immunohistochemistry demonstrated intense LTA(4) hydrolase staining in the myofibroblast as well as migrated monocytes/macrophages. These data suggest that the cardiac LTA(4) hydrolase-LTB(4) system plays a positive role in the promotion of cardiac inflammation in hypertension.

Loss of molecular interaction between cytochrome c and cardiolipin due to lipid peroxidation.

To explore the molecular mechanism underlying the translocation of cytochrome c from the mitochondrial inner membrane to the cytosol during apoptosis, we analyzed the molecular interaction between cytochrome c and cardiolipin (CL) by (1)H NMR spectroscopy. Bovine heart CL induced a drastic broadening of the linewidth of the downfield signals at 31.4 and 34.2 ppm assigned to the heme methyl group-3 and -8, respectively, of horse heart cytochrome c. In contrast, CL mono- and dihydroperoxides were less active in broadening the signals than CL, and CL trihydroperoxides induced almost no broadening of their linewidth. This finding suggests that the peroxidation of CL induces a release of cytochrome c from mitochondria into the cytosol, which release induces apoptosis in the cells.

Basic study on gene therapy of human malignant glioma by use of the cationic multilamellar liposome-entrapped human interferon beta gene.

For gene therapy of human malignant glioma, we adopted positively charged multilamellar liposomes entrapping the human interferon beta (hIFN-beta) gene. One week after the transplantation of human malignant glioma U251-SP cells to produce glioma in nude mouse brain, the liposomes entrapping the gene (500 ng of DNA per 25 nmol of lipids per 2 microl) were injected into the same site of the cell transplantation once every second day for a total of five injections; and by this means the tumor completely disappeared. To confirm the antiproliferative effect of hIFN-beta, we performed an in vitro study using a plasmid containing a secretion signal sequence-deleted hIFN-beta gene and one containing the hIFN-beta gene inserted in reverse. In both cases, there was no hIFN-beta release into the medium and no growth inhibition effect. On addition of anti-hIFN-beta antibody to the medium, the growth inhibition effect was abolished. As this cell line expresses IFN-alpha/beta receptor, the hIFN-beta produced in the transfected cells could be released and acted in a paracrine manner. For 120 days the body weight change of normal mice treated by the same procedure as used in the curing experiment was not significant among the groups injected with empty liposomes, plasmid only, and liposomes entrapping the gene. In all of these three groups, death, abnormal behavior, and significant histological changes were not observed.

Association of diffuse panbronchiolitis with microsatellite polymorphism of the human interleukin 8 (IL-8) gene.

Diffuse panbronchiolitis (DPB) is a distinctive chronic inflammatory lung disease predominantly found in Asian populations. Although its etiology is unknown, DPB is considered to be a multifactorial disease of whose susceptibility is determined by genetic predisposition unique to Asians. We and others have previously reported that the B*5401 allele of the human leukocyte antigen (HLA)-B gene or a closely linked gene in the HLA region on 6p21.3 is one of the major genetic factors in susceptibility to this disease. However, the association with B*5401 is not absolute and the contribution of other genetic or environmental factors should also be considered. Here, four candidate genes that are postulated to play a role in the pathophysiology of DPB, namely, RON-kinase, CYP3A4, motilin, and interleukin (IL)-8, were chosen, and association studies between microsatellite markers at these loci and DPB were conducted. We demonstrated the presence of a specific allele at the IL-8 locus was associated with the disease (c2 = 9.13; P = 0.0025; corrected P [Pc] < 0.05). Although further studies are needed to examine whether neutrophil accumulation in the airways of patients with DPB is controlled by a possible genetic variation of IL-8 or other chemokine genes located in the region 4q12-q13, our data suggest that genes other than those of the HLA system may also contribute to a genetic predisposition to DPB.

Contribution of TAP genes to genetic predisposition for diffuse panbronchiolitis.

Diffuse panbronchiolitis is a chronic obstructive pulmonary disease found in Asian populations. Although diffuse panbronchiolitis is considered to be a multifactorial disease of unknown etiology, the disease susceptibility appears to be determined by a genetic predisposition unique to Asians. An earlier study showed that human leukocyte antigen (HLA)-B54 predominantly found in East Asians was strongly associated with the disease. A possible interpretation of this association is that the class I molecule or class I antigen presenting system is directly involved in its pathogenesis. Recent observations in which impaired expression of class I molecules causes a syndrome resembling diffuse panbronchiolitis further prompted us to test this possibility. Genes of the molecules implicated in the class I pathway, TAP1, TAP2 and LMP2, which are located in the HLA region of the sixth chromosome were analyzed in 76 patients with diffuse panbronchiolitis and 120 normal controls. The combination of Ala-665 and Gln-687 in exon 11 of the TAP2 gene was associated with the disease (P=0.0028, Pc<0.05). Although this positive association might be partly explained by linkage disequilibrium with HLA-B*5401, this TAP2 variation was associated with the disease even in the B*5401-negative subgroup. On the other hand, the His-60 substitution within the LMP2 gene exhibited a negative association with the disease. This negative association, however, could be explained by a strong linkage disequilibrium with HLA-B44 which showed a negative association with the disease in the previous study. These results support the notion that diffuse panbronchiolitis is influenced by genetic factors in the HLA region. Besides the class I gene itself, genes relevant to the class I antigen presenting system might contribute to its genetic predisposition.

Induction of apoptosis in cultured colon cancer cells by transfection with human interferon beta gene.

The growth of SW480 colon cancer cells following the transfection with the human interferon beta (hIFNbeta) gene entrapped in cationic multilamellar liposomes was effectively inhibited, but not that of the cells transfected with the gene from which the secretion signal sequence of hIFNbeta had been deleted. The amount of hIFNbeta secreted in the medium from SW480 cells transfected with hIFNbeta gradually increased and became maximum 3 days after the transfection, but no hIFNbeta was detected in the medium of the cells transfected with the secretion signal-deleted hIFNbeta. These findings indicate that the growth inhibition of SW480 cells after the transfection with hIFNbeta was caused by hIFNbeta secreted from the transfected cells. At that time, SW480 cells were induced to undergo apoptosis, which was identified by morphological aspects, viz., chromatin condensation, nuclear segmentation, and nucleosomal DNA fragmentation. The hIFNbeta-induced apoptosis was found to be linked to the activation of caspases 3 and 8 as evidenced by immunoblot, enzymological, and cell death inhibition analyses.

Ubiquitous localization of leukotriene A4 hydrolase in the rat nephron.

BACKGROUND: Leukotriene (LT) B4 is a well-known inflammatory mediator and is implied to play some roles in glomerulonephritis. Although LTA4 hydrolase, a final-step key enzyme to produce LTB4, is located in glomerular mesangial cells, as well as in leukocytes, platelets, and endothelial cells, its precise distribution in the kidney other than in mesangial cells remains unknown. Therefore, we have investigated the localization of mRNA, protein, and enzyme activity of LTA4 hydrolase in the rat kidney. METHODS: Microdissection reverse transcriptase-polymerase chain reaction was used for the determination of LTA4 hydrolase mRNA. The enzyme protein was detected by Western blot, and immunohistochemistry was performed. Finally, LTA4 hydrolase activity and LTB4 were assayed in kidney tissues. RESULTS: LTA4 hydrolase mRNA was detectable in all microdissected nephron segments of the cortex and outer medulla. The corresponding size of approximately 70 kDa protein was shown in descending order in the inner medullary > outer medullary >/= cortical homogenates. The immunohistochemical study demonstrated the ubiquitous presence of the enzyme in all nephron segments of cortex, outer medulla, and inner collecting tubules. LTA4 hydrolase activity was detected in the inner medullary >/= outer medullary >/= cortical tissue homogenates. LTB4 was demonstrated in the inner medullary > outer medullary >/= cortical tissues during the basal condition, and was time-dependently increased by stimulation with arachidonic acid and ionomycin in the cytosolic fraction from outer medulla and in the glomerular suspension. CONCLUSIONS: These results strongly suggest that renal tubular cells as well as glomerular cells have an LTB4-forming potency, which may participate in physiological and pathophysiological roles in the kidney.

Contribution of HLA genes to genetic predisposition in diffuse panbronchiolitis.

Diffuse panbronchiolitis (DPB) in East Asia is a distinctive chronic obstructive pulmonary disease of unknown etiology. We hypothesize that the disease susceptibility is due to genetic predisposition unique to Asians. Association between human leukocyte antigen (HLA)-Bw54 and the disease was previously reported. In the present study, using newly developed polymerase chain reaction (PCR)- based methods, we directly analyzed HLA class I and II alleles in 76 Japanese patients. HLA-A, -B, and -C antigens were screened by the conventional typing method, and then B22-group alleles including HLA-B54 were genotyped by single-strand conformation polymorphism analysis. Alleles of HLA-DRB1 gene were fully determined by the microtiter plate hybridization method. Thirty-seven percent of the patients possessed HLA-B*5401 allele conserved predominantly in East Asians, as compared with 15% of 110 healthy volunteers (chi2 = 12.4, p = 0.0004). In addition, 4% of the patients possessed B*5504 also unique to Asians but a rare allele which was not found in normal control subjects in this study. Typing of HLA-DRB1 class II gene did not demonstrate strong positive association with the disease. A33, B44, and DRB1*1302 showed negative association with the disease. We conclude that distinctive molecular structure of HLA-B alleles or a closely linked gene in the HLA region contributes to genetic predisposition in diffuse panbronchiolitis. This may partly explain why this disorder is found primarily in Asians.

Antisense oligodeoxyribonucleotide against the MLL-LTG19 chimeric transcript inhibits cell growth and induces apoptosis in cells of an infantile leukemia cell line carrying the t(11;19) chromosomal translocation.

To clarify the role of the multiple lineage leukemia gene-leukemia translocation gene of chromosome 19 (MLL-LTG19) protein in leukemogenesis, we synthesized antisense oligodeoxyribonucleotide (ODN) against the fused region of the MLL-LTG19 chimeric transcript and treated KOCL33 cells carrying the t(11;19) translocation with antisense ODN. The antisense ODN inhibited cell growth and induced apoptosis in KOCL33 cells but not in Daudi cells, which have no t(11;19). The levels of MLL-LTG19 mRNA and MLL-LTG19 protein in KOCL33 cells treated with antisense ODN were shown to decrease with time by reverse transcription-PCR and Western blot analysis. These results suggest that the MLL-LTG19 fusion protein contributes to cell proliferation and malignant transformation in infantile acute leukemia cells carrying the t(11;19) translocation.