Effect of atorvastatin on microRNA 221 / 222 expression in endothelial progenitor cells obtained from patients with coronary artery disease.

BACKGROUND: Endothelial progenitor cells (EPCs) play an important role in the maintenance of vascular integrity. Lipid lowering therapy (LLT) with statins may contribute to biologically relevant activities including the proliferation of endothelial cells. The physiological role of microRNA (miR)-221/222, a newly discovered class of small RNA, is closely linked to the proliferation of endothelial cells. We therefore investigated whether LLT with statins might affect miR-221/222 expression in EPCs obtained from patients with coronary artery disease (CAD). MATERIALS AND METHODS: This study included 44 patients with stable CAD and 22 subjects without CAD (non-CAD). Patients with CAD were randomized to 12 months of LLT with atorvastatin (10 mg day(-1)) or pravastatin (10 mg day(-1)). EPCs were obtained from peripheral blood at baseline and after 12 months of statin therapy. Levels of miR-221/222 in EPCs were measured by real-time RT-PCR. RESULTS: Levels of miR-221/222 were significantly higher in the CAD group than in the non-CAD group (P < 0.01). Levels of miR-221/222 were weakly negatively correlated with EPC number in the CAD group. After 12 months of therapy, changes in lipid profiles were greater in the atorvastatin group than in the pravastatin group. LLT with atorvastatin markedly increased EPC numbers and decreased miR-221/222 levels (all P < 0.05), whereas LLT with pravastatin did not change EPC numbers or miR-221/222 levels. CONCLUSIONS: This study demonstrates that LLT with atorvastatin increases EPC numbers and decreases miR-221/222 levels in patients with CAD, possibly contributing to the beneficial effects of LLT with atorvastatin in this disorder.

Aberrant maspin expression in gallbladder epithelium is associated with intestinal metaplasia in patients with cholelithiasis.

OBJECTIVE: Aberrant expression of maspin protein related to DNA hypomethylation in the promoter region is frequently observed in gallbladder carcinomas, whereas the non-tumorous gallbladder epithelium is maspin negative. We investigated maspin expression in non-tumorous gallbladder epithelium in patients with cholelithiasis. METHODS: An immunohistochemical study of maspin expression was performed in 69 patients with cholelithiasis and 30 patients with gastric cancer without cholelithiasis. RESULTS: Immunoreactivity for maspin was observed in focal and patchy regions of the gallbladder epithelium. Positive immunoreactivity for maspin was significantly associated with the presence of intestinal metaplasia in patients with cholelithiasis (p<0.05). CONCLUSION: The high incidence of aberrant maspin expression in both intestinal metaplasia and carcinoma of the gallbladder supports the assumption that intestinal metaplasia of the gallbladder may predispose to gallbladder carcinoma.

Frequent microsatellite alterations on chromosome 3p in esophageal squamous cell carcinoma.

By the microsatellite assay, two types of genetic alterations, loss of heterozygosity (LOH) and replication error (RER), were examined using 7 dinucleotide repeat markers [D3S1317 (3p26); CI3-1169 (3p25); CI3-946 (3p25); D3S1255 (3p24.2-25); CI3-771 (3p21.3); CI3-1413 (3p14.1-14.3); and CI3-373 (3p13)] on the short arm of chromosome 3p as well as 3 markers [D2S123 (2p15-16), IFNA (9p22), and D16S408 (16q12.1-13)] on other chromosomes in 35 patients with esophageal squamous cell carcinoma. On 3p, LOH was detected in 34% (12 of 35) and RER was detected in 60% (21 of 35) at single or multiple loci. RER occurred at a similar frequency in all stages and did not correlate with clinicopathological characteristics. On the other hand, LOH at the 3p25 locus was more frequently detected in carcinomas with lymph node metastasis than in those without it (P < 0.05). The incidences of microsatellite alterations were low on the chromosomes other than 3p, except at D2S123, where the incidence of RER was 20%. These findings suggest that RER on 3p is an early event and that a tumor suppressor gene which is involved in the progression of esophageal squamous cell carcinoma may exist near the 3p25 locus.

Transactivation of the multidrug resistance 1 gene by T-cell factor 4/beta-catenin complex in early colorectal carcinogenesis.

The mutational inactivation of a tumor suppressor gene, adenomatous polyposis coli (APC), results in the accumulation of cytoplasmic beta-catenin protein and the activation of T-cell factor (TCF)/lymphoid enhancer factor transcriptional factors. A colorectal carcinoma cell line, DLD-1, was engineered to suppress transactivation by the TCF4/beta-catenin complex in a dominant-negative manner under the strict control of the tetracycline regulatory system. A large-scale comparison of the expression profiles, using two-color fluorescence hybridization of cDNA microarray, led to the identification of MDR1 as a target gene of the TCF4/beta-catenin complex. Luciferase reporter and gel retardation assays revealed the TCF4/beta-catenin responsive elements in the promoter of the human MDR1 gene. Corresponding to the accumulation of beta-catenin, expression of the MDR1 gene product was steadily up-regulated in adenomas and adenocarcinomas of 10 patients with familial adenomatous polyposis. In combination with cell proliferative activities of c-myc and cyclin D1, MDR1 may initiate colorectal tumorigenesis by suppressing cell death pathways programmed in intestinal epithelial cells.

Mutations of the APC gene occur during early stages of gastric adenoma development.

Mutations of the adenomatous polyposis coli (APC) gene have recently been shown to play an important role in colorectal tumorigenesis. We investigated mutations of the APC gene in 30 gastric adenomas obtained endoscopically. Mutations of the APC gene were examined by polymerase chain reaction-single-strand conformation polymorphism analysis followed by sequencing of the polymerase chain reaction products. Mutations were detected in 20% (6 of 30) of gastric adenomas. In addition, deletion of the remaining allele that subsequently led to complete inactivation of the APC gene was confirmed in one-half (3 of 6) of the tumors with APC gene mutations. Sequencing analysis confirmed that the mutations resulted in truncation of the gene products or in an amino acid change. The incidences of mutations of the APC gene remained constant regardless of the size or degree of histological atypia. Our observations suggest that mutations of the APC gene, similarly to those in colorectal tumorigenesis, occur during the early stages of gastric adenoma development.

Microsatellite alterations in adenoma and differentiated adenocarcinoma of the stomach.

In order to elucidate the significance of the adenoma-carcinoma sequence in gastric carcinogenesis from a genetic point of view, we examined microsatellite alterations (replication error and loss of heterozygosity) on chromosomes 2p (D2S123), 3p (D3S1317), 5q (D5S409), 9p (IFNA), and 13q (D13S153) as well as p53 gene mutations in 13 adenomas and 23 differentiated adenocarcinomas including 8 early carcinomas of the stomach. Replication error was detected in only one of the adenomas (8%, 1/13) at the D5S409 locus and in none at the other loci, and loss of heterozygosity was also an infrequent event found in one adenoma (14%, 1/7 informative cases) at D5S409 and in none at the other loci. A p53 gene mutation was detected in one (8%, 1/13) of the adenomas. Thus, microsatellite alterations and p53 gene mutations are rare events in adenomas. In differentiated adenocarcinomas, replication error was detected in 4 (17%, 4/23) at single or multiple loci, and loss of heterozygosity was observed frequently at D3S1317 (25%, 3/12), D5S409 (67%, 6/9), and IFNA (26%, 5/19). Mutations in the p53 gene were detected in 9 (39%, 9/23) of the differentiated adenocarcinomas. Microsatellite alterations on several chromosomes and mutations in the p53 gene were frequent in differentiated adenocarcinomas, even those at an early stage. These results suggest that the adenoma-carcinoma sequence is relatively rare in gastric carcinogenesis, and that the majority of differentiated adenocarcinomas of the stomach may develop through a de novo pathway.

Inactivation of the CDKN2 gene by homozygous deletion and de novo methylation is associated with advanced stage esophageal squamous cell carcinoma.

We examined the genomic status of the CDKN2 gene including de novo methylation of 5' CpG islands in primary and metastatic tumor samples from 31 patients with esophageal squamous cell carcinoma. One somatic frame shift mutation (1 of 31; 3.2%) was identified by PCR-single strand conformational polymorphism analysis and DNA sequencing. Homozygous deletion and de novo methylation of the gene were confirmed in 5 (16%) and 6 (19%) of 31 patients, respectively. Homozygous deletion and de novo methylation were significantly associated with silencing of gene expression (P < 0.01). Aberrations of the CDKN2 gene were detected in tumors with lymph node metastasis and muscular invasion (12 of 22; 54%) and in none of stage I tumors (0 of 9.0%; P < 0.05). These results suggest that homozygous deletion and de novo methylation are predominant mechanisms of inactivation of the CDKN2 gene and may be associated with metastatic and invasive phenotypes of esophageal squamous cell carcinoma.

Two distinct regions of deletion on the long arm of chromosome 5 in differentiated adenocarcinomas of the stomach.

Frequent loss of heterozygosity (LOH) on the long arm of chromosome 5 (5q) has been reported in many types of human malignancies, including gastric carcinoma. One of the targets of 5q-LOH in colorectal carcinoma is certainly the adenomatous polyposis coli (APC) gene on 5q21. However, other evidence has suggested the presence of another tumor suppressor gene in this region which may be inactivated in gastric carcinoma. In the present study, to determine the location of the putative tumor suppressor gene on 5q, LOH at nine microsatellite loci on 5q were investigated at 38 differentiated adenocarcinomas of the stomach that probably did not carry APC mutations. LOH at any locus on 5q occurred in 37% (14 of 38) of the tumors. Although many tumors exhibited large interstitial deletions on 5q that included the APC locus (5q21), we have identified minimum regions of deletion as the D5S428 locus and the interferon regulatory factor-1 (IRF-1) locus. Thus, at least two putative tumor suppressor genes, which play a crucial role in the genesis of differentiated adenocarcinoma of the stomach and are distinct from the APC gene, lie on 5q.

Tumor necrosis factor-alpha-converting enzyme and tumor necrosis factor-alpha in human dilated cardiomyopathy.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) has been implicated in the pathogenesis of dilated cardiomyopathy (DCM). TNF-alpha-converting enzyme (TACE) has recently been purified and its complementary DNA cloned. The expression of TACE results in the production of a functional enzyme that has precursor TNF-alpha in the mature form. The aim of this study was to determine whether TACE is expressed with TNF-alpha in myocardium and whether levels of TACE and TNF-alpha are related to clinical severity of DCM. METHODS AND RESULTS: Endomyocardial tissues were obtained from 30 patients with DCM and 5 control subjects. TNF-alpha and TACE mRNA levels were measured by a novel real-time quantitative reverse transcriptase-polymerase chain reaction method. Expression of TNF-alpha and TACE proteins was determined by immunohistochemical analysis. TNF-alpha mRNA was expressed in DCM patients (TNF-alpha/GAPDH ratio 0.85+/-0.24) but not in control subjects. TACE mRNA expression was significantly greater in DCM patients than in control subjects (TACE/GAPDH ratio 2.52+/-0.59 vs 0.03+/-0.02, P<0.05). A positive correlation was found between TNF-alpha and TACE mRNA levels (r=0.779, P<0.001). TACE and TNF-alpha immunostaining was observed in myocytes in patients with DCM. When 2 subgroups of DCM were divided on the basis of left ventricular end-systolic diameter (LVESD) of 45 mm and left ventricular ejection fraction (LVEF) of 40%, the DCM subgroup with high LVESD (>/=45 mm) showed significantly greater expression of TACE (P=0.02) and TNF-alpha (P=0. 001) than did the low LVESD subgroup (<45 mm). In addition, the DCM subgroup with lower LVEF (<40%) showed higher expression of TACE (P=0.006) and TNF-alpha (P=0.01) than did the subgroup with high LVEF (>/=40%). CONCLUSIONS: This study has shown that increased myocardial TACE expression is associated with elevated myocardial TNF-alpha expression in both mRNA and protein levels in clinically advanced DCM.

Drug-sensitivity testing in patients with human oesophageal squamous cell carcinoma.

To evaluate the response to chemotherapeutic agents against human oesophageal cancer, 19 samples were tested by the human tumour clonogenic assay (HTCA), 21 samples by subrenal capsule assay (SRCA) and 33 samples by SRCA with immunosuppressant (IS-SRCA). The evaluability rate of was 21% for HTCA, 95% for SRCA and 91% for IS-SRCA. No active agent was detected by HTCA, however, 29% of the drugs tested by SRCA and 22% by IS-SRCA were considered to be active. Histological analysis revealed substantial inflammatory infiltrates and poor tumour cell preservation with SRCA; however, infiltrates were minimal and there was a high degree of tumour cell preservation with IS-SRCA. The response rates of IS-SRCA were comparable with those of prior clinical tests for each drug. These results suggested that IS-SRCA is the most useful drug sensitivity test for human oesophageal cancer.

Mutations in mitochondrial control region DNA in gastric tumours of Japanese patients.

The non-coding control region of mitochondrial DNA (mtDNA), containing the hypervariable regions HV1 and HV2 and the D-loop region, was screened for mutations in 45 gastric tumours (15 tumours each of adenoma, differentiated adenocarcinoma and undifferentiated carcinoma). We found mutations in two of the 45 tumours (4%); a 1 bp A deletion at nucleotide position 248 in a differentiated adenocarcinoma and a G to A transition at nucleotide position 16,129 in an adenoma. We also observed 10 polymorphisms, four of which were not previously recorded. Both mtDNA mutations were present in replication error negative (RER-) tumours. Short mono- or dinucleotide repeats in the control region, such as (C)7, (A)5 or (CA)5, were not altered regardless of nuclear genetic instability. In summary, mtDNA is mutated in a subset of benign and malignant gastric tumours, but, disruption of the mtDNA repair system appears not to be significantly involved in gastric tumours of Japanese patients.

Loss of heterozygosity at the DCC gene locus is not crucial for the acquisition of metastatic potential in oesophageal squamous cell carcinoma.

Tumour specimens from 111 patients with oesophageal squamous cell carcinoma were screened for loss of heterozygosity (LOH) at the deleted colorectal carcinoma (DCC) gene locus. DCC-LOH occurred in 10 of 61 informative cases (16%). No statistically significant correlation was observed between DCC-LOH and lymph node metastasis, histopathological grade or tumour stage. The survival of patients exhibiting DCC-LOH was not statistically different from that of patients without LOH. These results suggest that LOH at the DCC locus is not related to the acquisition of metastatic potential or the state of tumour cell differentiation in oesophageal squamous cell carcinoma.

Lack of mutations of the adenomatous polyposis coli gene in oesophageal and gastric carcinomas.

The adenomatous polyposis coli (APC) gene is the target of the loss of chromosome 5q heterozygosity observed frequently in gastrointestinal tract carcinomas and is inactivated in these carcinomas. We screened 94 gastrointestinal tract carcinomas for APC mutations, by polymerase chain reaction single-strand conformation polymorphism (SSCP) analysis. Mutations were detected in 8 of 21 (38%) colorectal carcinomas in the mutation cluster region of the APC gene whereas no mutation was detected in any of 49 oesophageal and 24 gastric carcinomas, even though SSCP analysis was extended to include the 5' half of the APC gene exon 15. Direct DNA sequencing revealed that six of eight (75%) mutations in colorectal carcinomas resulted in truncated gene products. These findings confirm the significance of APC gene mutations in colorectal, but not oesophageal or gastric carcinomas. Some other tumour suppressor genes near the APC gene may be the target of the frequent allelic loss of chromosome 5q in oesophageal and gastric carcinomas.

Analysis of genetic alterations in renal cell carcinoma using the polymerase chain reaction.

Very frequent loss of heterozygosity (LOH) on chromosome 3p has been found in human renal cell carcinoma (RCC). In the present study, we examined LOH at the retinoblastoma (RB), mutated in colorectal cancer (MCC) and adenomatous polyposis coli (APC) tumour suppressor genes loci, and mutations of the H-, K-, and N-ras oncogenes. We performed these studies using the polymerase chain reaction (PCR) method followed by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) analyses. LOH was detected in 2 of 11 (18.2%), and 2 of 14 (14.3%) informative cases at the MCC and APC loci, respectively, and in none of 15 informative cases at the RB locus in 25 RCCs. LOH at the MCC was accompanied by LOH at the APC locus in two RCCs. No mutation was detected in H-, K-, and N-ras genes in 39 RCCs. Thus, alterations of the known tumour suppressor genes and the ras oncogenes were infrequent events in RCC. The results suggest that the genetic pathway in the genesis of RCC differs considerably from that of other common human carcinomas.

Consensus primers for detecting monoclonal immunoglobulin heavy chain rearrangement in B cell lymphomas.

AIMS: To demonstrate the usefulness of polymerase chain reaction (PCR) methodology with both the FR2A/LJH/VLJH and the FR1c/LJH/VLJH primer sets for detecting monoclonal immunoglobulin heavy chain (IgH) gene rearrangement in B cell non-Hodgkin lymphomas (B-NHLs). METHODS: Eighty three patients with B-NHL were enrolled in this study. DNA was isolated from paraffin wax embedded sections and amplified by PCR to determine the sequences of the rearranged IgH gene. Each PCR product was subcloned. Cycle sequences and sequence analyses were done to determine the clone specific IgH variable region (VH) sequences. RESULTS: Clonal IgH gene rearrangements were detected in 45 cases with FR2a/JH/VLJH and in 14 of the remaining cases with FR1c/JH/VLJH. Most of the cases detectable with FR2a/JH/VLJH were derived from VH3 and VH4 families. Five of six cases in the VH1 family and one in the VH7 family were amplified with the FR1c/JH/VLJH primer set only. CONCLUSION: The detection rate of IgH rearrangement in B-NHLs can be increased by using both FR2A/LJH/VLJH and FR1c/LJH/VLJH, and these two primer sets are suitable for routine PCR methodology. Moreover, each primer set appears to be closely related to VH family specificity.

Development of immunoglobulin variable heavy chain gene consensus probes with conjugated 3' minor groove binder groups for monitoring minimal residual disease in childhood acute lymphoblastic leukaemia.

AIMS: To develop immunoglobulin heavy chain variable (VH) gene probes that are shorter and more flexible in position for monitoring minimal residual disease (MRD) in childhood leukaemia (ALL), using minor groove binder (MGB) technology. METHODS: All VH germline sequences registered in the database were aligned and the consensus regions were determined. The reliability of the MGB probes was compared with non-MGB probes in all 24 cases of ALL. RESULTS: Ten MGB probes (16 to 18 mers) were designed that enabled all the germline sequences on the database to be analysed, whereas the conventional non-MGB probes (21 to 27 mers) did not allow the analysis of four of the VH1 and five of the VH3 germline sequences. The sequencing results in five of the 24 cases of ALL were not matched to the non-MGB probes. CONCLUSIONS: MGB technology allows shorter probes to be designed, enabling MRD to be detected in childhood ALL. This would provide a considerable reduction in cost for a large MRD study.

Successful steroid therapy for cefdinir-induced acute tubulointerstitial nephritis with progressive renal failure.

A 58-year-old woman was admitted to our hospital because of renal dysfunction that continued to progress even after withdrawal of cefdinir, the presumed cause of acute renal failure. Renal histologic findings included interstitial fibrosis accompanied by moderate lymphocytic infiltration, and tubular atrophy with reduced numbers of epithelial cells. Mesangial cells and glomerular basement membranes were nearly normal. Scintigraphy with 67gallium disclosed diffuse abnormal accumulation in both kidneys. A lymphocyte stimulation test with cefdinir was positive. The patient was diagnosed with acute tubulointerstitial nephritis caused by cefdinir. Serum creatinine concentrations continued to rise after withdrawal of the drug, but steroid therapy was effective in normalizing renal function.

[SIRS in surgical stress].

It has been suggested that SIRS are triggered by superfluous pro-inflammatory cytokine production, and that organ injury is caused by uncontrolled inflammatory responses. However, the results of clinical studies, on the usefulness of specific cytokine antagonists and anti-TNF antibodies for the treatment of septic shock, have been unsatisfactory. The reason for this might have been that when uncontrolled inflammatory reactions progressed locally, anti-inflammatory reactions were elevated in the circulated blood by way of CARS, thus the timing of administration and pharmacokinetics did not match clinical course. Recent research has shown that SIRS is always accompanied by CARS, and since it seems to do the amplitude of SIRS and CARS to each other so that there may be a deep valley, if there is a high mountain. We introduce the recent knowledge which indicates that SIRS is a preliminary alert for not only organ dysfunction but also immunosuppression after severe injury or major surgery.

[Experimental study of biochemical modulation of 5-fluorouracil with l-leucovorin against human esophageal cancer].

Potentiation of cytotoxic effects of 5-fluorouracil (5-FU) were investigated by simultaneous or sequential combination of l-leucovorin (LV) against human esophageal cancer cell line (TE-1, TE-2) and 23 human clinical cancer samples in vitro. LV enhanced the cytotoxic effects of 5-FU on human esophageal cancer cell line as a dose dependent manner, and increased the cytotoxic effect of 5-FU about 1.5-fold with 10 microM and about 2-fold with 100 microM. The incubation time did not affect the effects. The potentiation with LV was also demonstrated against human clinical cancer samples, and the cytotoxic effects of 5-FU increased 7.6% in esophageal cancer, 20.9% in gastric cancer and 25.5% in colorectal cancer. As a result, the potentiating effects of LV against 5-FU seemed to be limited on human esophageal cancer.

[Preclinical evaluation of several cisplatinum analogs against human esophageal carcinoma by subrenal capsule assay].

The antitumor activities of CDDP analogs (CBD-CA, NK-121, 254-S) were evaluated preclinically by subrenal capsule assay (SRCA) with cyclophosphamide pretreatment. In the fundamental study, the antitumor activities against serially transplanted human esophageal cancer xenograft (IMEs-1) were compared with subcutaneous transplantation assay in nude mice and SRCA. The antitumor activities in SRCA were similar to those of in nude mice assay system (CBDCA greater than CDDP greater than 254-S greater than NK-121). Thus SRCA was considered to be useful for the evaluation of the activities of these agents. The activities were also tested against 10 human esophageal tumors obtained clinically. The sensitivity rate of these agents were 50% in CDDP, 30% in CBDCA, 30% in NK-121, and 30% in 254-S, respectively. These analogs seemed to be less effective than CDDP. However, in two cases, analogs were active though CDDP were inactive. The results suggest that these analogs are useful for the cases in which CDDP can not be given due to the toxicities and also for outpatient use.