Estudo da cromatina nos sítios de fixação à matriz nuclear no domínio do gene TP53 e das modificações epigenéticas e no modelo de progressão tumoral mamária 21T / MARs chromatin study on TP53 gene domain and epigenetic modifications in a breast cancer progression model

Dentre os diversos tipos de câncer agressivos, o câncer de mama é o mais comum em mulheres. Mutações hereditárias e adquiridas, assim como alterações epigenéticas atuam em sinergia na carcinogênese mamária e na progressão tumoral. A proteína P53 é uma supressora de tumor e possui uma atuação fundamental na integridade genômica. Apesar do vasto conhecimento sobre o controle da P53 a nível de proteína, ainda pouco se sabe sobre o controle transcricional do gene TP53. A série 21T, uma série de 4 linhagens celulares originadas da mama da mesma paciente, representando diferentes estágios de progressão tumoral mamária, é um eficiente modelo para investigação das alterações epigenéticas e suas influências na expressão gênica ao longo da progressão do câncer de mama. Nós analisamos a organização do domínio do gene TP53 através da técnica de arranjo de DNA, em diversas linhagens celulares de câncer de mama e linhagens controle, e realizamos uma tentativa de caracterizar estes elementos de DNA nas linhagens controle não-tumorais HB2 e MCF10A e nas tumorais MCF-7, MDA-MB-231, T47D, através dos marcadores epigenéticos de eucromatina, H4Ac, e heterocromatina, H3K9me3. Ainda analisamos a ligação de proteínas à região associada à matriz nuclear (MAR), denominada MAR 2, e a possível ligação da proteína ligante à matriz nuclear (MARBP), PARP-1, através de ensaios de gel shift (EMSA). Detectamos que na linhagem controle epitelial mamária, HB2, o gene TP53 está posicionado num domínio de DNA relativamente pequeno, aproximadamente 50 kb, delimitado por dois sítios de fixação à matriz nuclear. Interessantemente, esta estrutura de domínio se apresentou radicalmente diferente nas linhagens de câncer de mama estudadas, MCF7, T47D, MDA-MB-231 e BT474, nos quais o tamanho do domínio estudado estava aumentado e a transcrição do TP53 diminuída...

Breast cancer is the most common aggressive cancer type in women. Inherited and acquired mutations as well as epigenetic alterations act together in breast carcinogenesis and tumor progression. P53 is a tumor suppressor protein critical for genome integrity. Although its control at the protein level is well known, the transcriptional regulation of the TP53 gene is still unclear. The 21T series, a series of 4 breast cell lines originating from the same patient and representative of the breast tumor progression stages, is a suitable model to investigate epigenetic alterations and their influences upon gene expression during breast tumor progression. We have analyzed the organization of the TP53 gene domain using DNA arrays in several breast cancer and control cell lines and we made an attempt to characterize these DNA elements in breast non-cancerous cell lines HB2 and MCF-10, and cancerous MCF-7, MDA-MB-231 and T47D, through the determination of epigenetic markers of euchromatin, H4Ac, and heterochromatin, H3K9me3. We further analyzed the matrix attachment region (MAR), named MAR 2, protein binding, and possible MAR 2 binding of the important MAR binding protein (MARBP), PARP-1, by Electrophoretic mobility Shift Assay (EMSA). We have found that in the control breast epithelial cell line, HB2, the TP53 gene is positioned within a relatively small DNA domain, encompassing 50 kb, delimited by two nuclear matrix attachment sites. Interestingly, this domain structure was found to be radically different in the studied breast cancer cell lines, MCF7, T47D, MDA-MB-231 and BT474, in which the domain size was increased and TP53 transcription was decreased...

The Clinical Usefulness of Nuclear Matrix Protein-22 in Patients with Atypical Urine Cytology

PURPOSE: Difficulty exists in interpreting the significance of atypical urine cytology. This study was performed to assess the diagnostic utility of nuclear matrix protein-22 (NMP-22) testing when atypical cells are detected during urine cytology. MATERIALS AND METHODS: Among patients whose urine cytology was reported as atypical between January 2004 and December 2009, a total of 275 who also underwent NMP-22 testing were enrolled in the present study. These patients were further divided into the screening group (143 patients examined as outpatients for hematuria) and the follow-up group (132 patients followed up for previously diagnosed bladder cancer). The sensitivity, specificity, positive and negative predictive values, and accuracy were assessed for atypical cytology alone and in conjunction with NMP-22. RESULTS: Of the 275 patients exhibiting atypical urine cytology, cancer was confirmed in 85, yielding a positive predictive value of 30.9% (85/275). Of the 96 patients testing positive for NMP-22, 58 were diagnosed with bladder cancer. The positive predictive value in conjunction with NMP-22 was 60.4% (58/96). The sensitivity, specificity, negative predictive value, and accuracy were 68.2% (58/85), 80.0% (152/190), 84.9% (152/179), and 76.2% (210/275), respectively. Testing for NMP-22 in the screening and follow-up groups increased the positive predictive value from 30.0% (43/143) to 64.0% (32/50) and from 31.3% (42/132) to 56.5% (26/46), respectively; there was no significant difference between the screening and follow-up groups (p=0.106). CONCLUSIONS: When only cases with atypical urine cytology were examined, NMP-22 testing increased the detection rate of bladder cancer regardless of whether the test was used in screening hematuria or in following up patients.

Usefulness of the NMP22BladderChek Test for Screening and Follow-up of Bladder Cancer

PURPOSE: We evaluated the usefulness of the nuclear matrix protein 22 BladderChek (NMP22BC) test for the screening and follow-up of bladder cancer. MATERIALS AND METHODS: From February 2006 to September 2009, we enrolled 1,070 patients who had hematuria or who were being followed up for bladder cancer. We compared the sensitivity and specificity of the NMP22BC test with those of urine cytology. RESULTS: The sensitivity of the NMP22BC test (77.5%) was significantly higher than that of urine cytology (46.3%). The specificity of the NMP22BC test was 88.8%, compared with 97.9% for urine cytology. The sensitivity of the NMP22BC test (81.8%) in non-muscle-invasive bladder cancer was higher than that of cytology (36.4%). However, the sensitivity of the NMP22BC test and of urine cytology in invasive bladder cancer were 57.1% and 92.9%, respectively. The sensitivity of the NMP22BC test was higher for low-grade bladder cancer (83.9%) than for high-grade (62.5%), and the sensitivity of cytology was higher for high-grade bladder cancer (66.7%) than for low-grade (37.5%). Follow-up bladder cancer was detected in 262 patients. The sensitivity of the NMP22BC test in that group (72.7%) was decreased and the specificity (91.7%) was increased. The sensitivity of cytology (54.5%) in the follow-up group was increased and the specificity (95.6%) was decreased. The presence of pyuria was significantly associated with the lower specificity of the NMP22BC test. CONCLUSIONS: The greater sensitivity of the NMP22BC test may be more useful for the diagnosis of non-muscle-invasive bladder cancer and low-grade bladder cancer than for the diagnosis of invasive or high-grade bladder cancer. If the NMP22BC test is performed in the absence of pyuria, it may play a compensatory role for urine cytology.

Comparison of the Urinary Melanoma Antigen Gene Expression (MAGE) Test and Urinary Cytology for Bladder Cancer Screening

PURPOSE: In bladder cancer screening, many methods such as urinary cytology, bladder tumor antigen, and nuclear matrix protein-22 are well known. To learn the value of urinary melanoma antigen gene expression (MAGE) in bladder cancer screening, we compared the urinary MAGE test with irrigated urinary cytology. MATERIALS AND METHODS: From July 2000 to July 2007, a total of 142 patients were enrolled in this study. We divided these patients into 2 groups. Eighty-eight patients with bladder cancer were included in group I. Group II consisted of 54 patients who had been treated for bladder cancer and had no evidence of tumor by cystoscopy and irrigated urinary cytology. Urinary cytology, urinary MAGE test, and cystoscopy were performed in all patients. The urinary MAGE test was done by reverse transcriptase polymerase chain reaction (RT-PCR). Sensitivity and specificity were investigated according to cancer grade and stage. RESULTS: The overall sensitivity of the urinary MAGE test and urinary cytology was 69.3% (61/88) and 53.4% (47/88), respectively (p=0.03). The specificity of the urinary MAGE test and urinary cytology was 75.9% (41/54) and 83.3% (45/54), respectively (p=0.34). The sensitivity of each test in superficial tumors (Ta, T1) was 65.5% (38/58) and 46.6% (27/58), respectively (p=0.04). In advanced disease (> or =T2), the sensitivity of the tests was 76.7% (23/30) and 66.7% (20/30), respectively (p=0.39). The sensitivity of the urinary MAGE test in grade 1 tumors (60.5%, 23/38) was significantly higher (p=0.01) than that of urinary cytology (31.6%, 12/38). CONCLUSIONS: The urinary MAGE test was more sensitive than urinary cytology in bladder cancer screening. We consider the urinary MAGE test to possibly be a valuable test together with urinary cytology, especially for Grade 1 and Ta, T1 bladder cancer.

Comparison of the Efficacy of Urine Cytology, Nuclear Matrix Protein 22 (NMP22), and Fluorescence in Situ Hybridization (FISH) for the Diagnosis of Bladder Cancer / 대한비뇨기과학회지

PURPOSE: We compared the efficacy of urine cytology, nuclear matrix protein 22 (NMP22), and fluorescence in situ hybridization (FISH) for the detection of bladder cancer. MATERIALS AND METHODS: Washing urine samples from 156 patients were evaluated for the detection of bladder cancer. Patients were divided into 3 groups. Group 1 was 106 patients with bladder cancer, group 2 was 30 patients with benign prostatic hyperplasia who underwent transurethral resection of the prostate without bladder cancer, and group 3 had gross hematuria without bladder cancer. The sensitivity and specificity of cytology, NMP22, and FISH were compared. NMP22 positivity was defined as > or =10U/ml. FISH was done with the UroVysion(R) system and FISH positivity was defined as > or =2 abnormal urothelial cells with an abnormal signal from any out of 4 probes. RESULTS: The overall sensitivity of urine cytology, NMP22, and FISH was 60.4%, 75.5%, and 84.9%, respectively (p<0.001). The overall specificity of cytology, NMP22, and FISH was 96.7%, 83.3%, and 93.3%, respectively (p=0.168). In group 3, the false-positive rates of cytology, NMP22, and FISH were 20.0%, 55.0%, and 10.0%, respectively. In these patients with gross hematuria, the false-positive rate with NMP22 was significantly higher than with cytology or FISH (p=0.004). The sensitivity of cytology, NMP22, and FISH in low-grade bladder cancer patients was 25.9%, 51.9%, and 77.8%, respectively, and that in pTa-1 bladder cancer patients was 40.6%, 65.6%, and 78.1%, respectively. In low-grade or in pTa-1 patients, the sensitivity of the three diagnostic tools was significantly different (low grade; p<0.001, pTa-1; p<0.001). CONCLUSIONS: FISH is more sensitive in low-grade bladder cancer than is urine cytology and can be used as a diagnostic tool for the detection of primary and recurrent bladder cancer. NMP22 was affected by gross hematuria and thus has limitations for screening of bladder cancer. However, it can be used to follow-up bladder cancer.

Urinary Survivin Test Compared to the Nuclear Matrix Protein (NMP)-22 Test and Urine Cytology for the Diagnosis of Bladder Cancer / 대한비뇨기과학회지

PURPOSE: The aim of this study was to compare the sensitivity and specificity of the urinary survivin test for the diagnosis of bladder cancer with the nuclear matrix protein (NMP)-22 test and the urine cytology, and we wanted to correlate survivin with the tumor stage and grade. MATERIALS AND METHODS: Between October 2002 and March 2004, voided urine samples were obtained from 41 patients with bladder cancer and also from 36 controls who had no evidence of bladder cancer. The urinary survivin and NMP-22 levels were measured using a DuoSet IC ELISA kit and an automated chemiluminescent assay system. The results were compared with the cytologic results and the pathologic findings. RESULTS: The comparative results showed the higher sensitivity for the urinary survivin test (78.0%) and the NMP-22 test (75.6%) than for the urine cytology (65.8%) for the detection of bladder cancer. The specificity of urinary survivin (86.1%) and urine cytology (97.2%) were higher than that for the NMP-22 test (66.6%). Measuring the urinary survivin level was a more accurate test than the urinary NMP-22 test and the urine cytology for the detection of lower grade and superficial bladder cancer. CONCLUSIONS: The urinary survivin test was superior to urine cytology for sensitivity and specificity, and these two parameters of the urinary survivin test were higher than those of the NMP-22 test. Especially, the urinary survivin test is an accurate diagnostic test for superficial and lower grade bladder tumor. Our results suggested that the urinary survivin test appears to be a reliable diagnostic test to identify patients with bladder cancer.

Birefringence of cells grown in vitro and of mitotic chromosomes after staining with picrosirius red

Fibroblasts and neuroblastoma cells kept in monolayer cultures, as well as surface spreads of mitotic chromosomes, were stained with picrosirius red. Red staining (in normal light) and optical anisotropy of the stained structures (in polarized light) were observed intracellularly and in the chromosomes. The intracellular and extranuclear birefringence induced by staining with sirius red could not be abolished by digestion with collagenase prior to staining, or by treatments used to disrupt microtubules (vinblastine, colcemid) or microfilaments (cytochalasin B). We therefore propose that the parallelly-arranged intermediate filaments are responsible for the optical anisotropy induced by sirius red staining in these cells. In addition, the spatially oriented scaffold of chromosomes can be detected by sirius red-induced birefringence. These data argue against the collagen-specificity of picrosirius red staining and of the birefringence induced by this technique. Our results also suggest that picrosirius red staining combined with polarized light microscopy can be used to study the spatial orientation pattern of the intermediate filaments and chromosome scaffold.

Isolation and functional analysis of tobacco MARs / 生物工程学报

Two new MAR segments (M14 and M17) were cloned from tobacco genome. Both of the sequences contained several typical consensus sequences of MARs, which were different from the original MAR sequence, such as 90%AT-box, A-box, T-box, the base unpairing regions (BUR), autonomously replicating sequences (ARS), the consensus sequence for topoisomerase II, MAR recognition sequence (MRS), origin of replication (ORI), curved DNA motifs and ATATTT et al. To investigate the effects of these two sequences on gene expression in transgenic plants, 3 plant expression vectors were constructed with uidA gene coding beta-glucuronidase (GUS) which were flanked on one side and on both sides by the MARs we obtained. These plant expression vectors with one or two MARs were transformed into tobaccos via Agrobacterium-mediated transformation method, with the plant expression vector pCAMBIA2301 without MAR and wild type tobacco as controls. GUS histochemical staining results showed that the uidA gene expressed stably in transgenic tobaccos. Quantitative detection of GUS activity showed that the MARs could increase GUS expression levels in vivo in contrast to the controls, wherever they were flanked on one side or both sides of uidA gene. The vector ligated with MARs in the same direction on both sides of uidA could increase the GUS expression level much better than both vectors which just ligated with single MARs on one side. The former one increased the average GUS activity for 3.14 folds, but 1.56 and 2.43 folds for the latter two vectors with single MARs respectively contrasting to the pCAMBIA2301 control. But the expression differences among individual transformants were still obvious. Therefore, it was concluded that the DNA sequences we obtained in this experiment were two novel MARs and could enhance gene expression in vivo. In the meanwhile, although the numbers of the MARs typical motifs in M14 were more than in M17, especially the 90% AT box which had been considered to be the highest correlative motif with binding strength in vitro, the enhancement of gene expression was lower yet, which implied no correlation between improvement of gene expression and binding strength between MARs and nuclear matrix in vitro.

The potential role of nuclear matrix attachment regions (MARs) in regulation of gene expression / 生物工程学报

Gene transfer technology is being used to enhance agronomic performance or improve quality traits in a wide variety of crop species. However, it is sometimes severely handicapped by difficulty in obtaining material in which transgene expression is predictable and stable over many generations. Because integration seemed to occur randomly in the plant genome, it was thought that some transgenes would be integrated in a relatively uncondensed, transcriptionally active chromatin environment, while others in a condensed, transcriptionally inert chromatin structure. Nuclear matrix attachment regions (MARs) are defined as DNA sequences that bind preferentially to the proteins of the nuclear matrix. They typically are localized at the borders of gene domains, implicating them in the formation of individual loops of higher order chromatin structure and transcription regulation. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable espression in transgenic plants, most likely by minimizing gene silencing. In this review, we focus mainly on novel findings and our observations concerning the function of MARs in transcription regulation. Our objective is not only to summarize the current data and present several possible models to explain MAR effects on the transcription regulation, but also to point out some open questions involving the utilization of MARs in constructing high efficient expression vectors.

Influence of transfected EGFR-cDNA on bcl-2 and Bax in glioblastoma cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the correlation of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U87 as well as the effect of EGFR-cDNA transfection on the expression of bcl-2 and Bax in U87 cells.</p><p><b>METHODS</b>The correlation of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U87 was studied by confocal microscopy and Western blot. The expression of bcl-2 and Bax in EGFR-cDNA transfected and untransfected glioblastoma cell lines was studied by Western blot.</p><p><b>RESULTS</b>Confocal microscopic images showed that bcl-2 protein was localized in the periphery of the nuclear matrix and Bax in the nuclear matrix. A 26 kDa bcl-2 band and a specific band of Bax at about 66 000 were detected in nuclear matrix proteins by western blot. The expression of bcl-2 was lower but that of Bax was higher in EGFR-cDNA transfected cells than the control.</p><p><b>CONCLUSION</b>Bcl-2 and Bax, being nuclear matrix associated proteins, are probably involved in the EGFR-cDNA induced malignant conversion of glioblastoma cells by introducing EGFR cDNA into the tumor cells.</p>

Nuclear matrix associated protein PML: an arsenic trioxide apoptosis therapeutic target protein in HepG2 cells / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate arsenic trioxide (As(2)O(3))-induced apoptosis and the effects on cell nuclear matrix related protein promyelocytic leukaemia (PML).</p><p><b>METHODS</b>HepG2 cells were cultured in MEM medium and treated with 0.5, 2, 5 and 10 micro mol/L As(2)O(3) for either 24 h or 96 h at each concentration. In situ terminal deoxynucleotidyl transferase (TdT) labeling (TUNEL) and DNA ladders were used to detect apoptosis. Confocal microscopy and Western blotting were used to observe the expression of PML.</p><p><b>RESULTS</b>The growth rates of HepG2 cells were slower in the As(2)O(3) treated than the untreated control group. DNA ladder and TUNEL positive apoptotic cells could be detected in As(2)O(3) treated groups. The expression of PML decreased in HepG2 cells with 2 micro mol/L As(2)O(3) treatment. Confocal images demonstrated that the expression of PML protein in HepG2 cell nuclei decreased after treatment with 2 micro mol/L As(2)O(3), and micropunctates characteristic of PML protein in HepG2 cell nuclei disappeared after treatment with 5 micro mol/L As(2)O(3).</p><p><b>CONCLUSIONS</b>Our results show that arsenic trioxide can significantly inhibit the growth of HepG2 cells in vitro. As(2)O(3) induces apoptosis in HepG2 tumor cells in a time and concentration dependent manner. As(2)O(3) may degrade the PML protein in HepG2 cell nuclei. The decreased expression of PML in As(2)O(3) treated tumor cells is most likely to be caused by apoptosis. Nuclear matrix associated protein PML could be the target of As(2)O(3) therapy.</p>

Nuclear matrix changes by the antisense inhibition of transglutaminase c in in vitro culture of snu-1 cells

It has been known that transglutaminase C (TGase C, TGase II) is directly participated in the DNA organization of chromosome, and affects the cellular processes such as proliferation, differentiation, and apoptosis of cells, but still not known what mechanism is working on. In this study, the cytogenetic and the immunohistochemical methods were used to observe the TGase C expression in the nuclear chromosome of the proliferating cells, especially in mitotic stage. The human gastric adenocarcinoma (SNU-1) cell line was used for immunohistochemistry and antisense inhibition study in vitro. The present study was also aimed to disclose the efficiency of antisense inhibition by using antisense oligonucleotide DNA labeled with fluorescence, and found that anti-TGase C probe was diffusely infiltrated into the cytoplasm and the nucleus of the cell. By the antisense inhibition the nuclei of SNU-1 cells became rough nuclear shape, as they were greatly reduced in TGase C immunoreactivity both for the normal and apoptotic SNU-1 cells. However, it is clearly presumed that the TGase C directly interacts with the chromosome of SNU-1 cells and it may play an important role in the division and organization of the chromosome during the mitotic stage.

Urinary NMP22 and BTA tests as screening markers for bladder transitional cell carcinoma / 대한임상병리학회지

BACKGROUND: Urinary bladder cancer has been diagnosed by urine cytology and cystoscopy with biopsy. Recently, in vitro noninvasive diagnostic tests, measuring urinary nuclear matrix protein22(NMP22) and bladder tumor antigen(BTA), were introduced. We analyzed the usefulness of the NMP22 and BTA tests for diagnosing bladder cancer and compared those with voided urine cytology. MATERIALS AND METHODS: Single voided urine specimens were obtained from 27 patients with bladder cancer and 23 healthy volunteers. The urine specimens were assayed by enzyme immunoassay(NMP22, Matrietech(R), Newton, MA.) and latex immunoassay(BTA, Bard, USA). Urine cytology was performed in patients with bladder cancer. RESULTS: Mean urinary NMP22 level of patients with bladder cancer(144.6 U/mL) was significantly higher than those of normal controls(2.9 U/mL, P<0.01). The sensitivities were 89% and 74% for NMP22 and BTA tests, respectively, compared with 41% for voided urine cytology. The sensitivities of NMP22 and BTA tests were 88%, 63% at grade 1(G1), 82%, 73% at G2, and 100%, 88% at G3, respectively(P<0.01; NMP22, P=0.580; BTA). According to tumor stage, the sensitivities of NMP22 and BTA tests were both 79% at superficial, and 100% and 69% at invasive cancer, respectively(P=0.110; NMP22, P=0.678; BTA). The sensitivities of urine NMP22 and BTA tests combined with urine cytology were both 96%. In following of transitional cell carcinoma patients, agreement between urine cytology and BTA test was 75%(24/32). Among the various urologic disease, false positive rate for BTA test was 17%(8/47). CONCLUSION: Urinary NMP22 and BTA tests were more sensitive than voided urine cytology regardless of tumor grade and stage, so these noninvasive and simple tests can be used as screening tests for urinary bladder transitional cell carcinoma.

Morphological and biochemical analysis of anti-nuclear matrix protein antibodies in human sera

Autoimmune sera have been used in the diagnosis of autoimmune diseases as well as the analysis of nuclear substructures. In an attempt to study the biological characteristics of the nuclear matrix, we screened human sera using immunofluorescent staining and immunoblot. We detected antibodies against nuclear matrix (NM), a remnant nonchromatin protein compartment after the treatment of detergent, salt and nuclease, in 212 out of 284 tested sera (74.6%) by immunoblot. Peptides with molecular weights of 70 kDa, 50 kDa and 25 kDa were detected in the order of frequency. Clinical informations of 198 out of 212 cases were available and went as follows: 38 cases were autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis; 132 non-autoimmune and non-neoplastic diseases; 16 neoplastic diseases and 12 cases unclassified. The immunofluorescent staining intensity by anti-nuclear matrix protein (NMP) antibodies decreased variably, but fibrillogranular, speckled and nucleolar immunolocalization patterns were retained after in situ fractionation. Ku70 and La protein were detected by anti-NMP antibodies. Immunolocalization by anti-NMP antibodies indicates that the NMPs constitute a variety of characteristic nuclear substructures and may serve as autoantigens in diverse human diseases. In addition, the presence of Ku70 and La protein as NMPs suggests that the NM can be functionally active in association with DNA or RNA.

Effect of Reperfusion after 20 min Ligation of the Left Coronary Artery in Open-chest Bovine Heart: An Ultrastructural Study / 대한흉부외과학회지

BACKGROUND: It has been well documented that transient occlusion of the coronary artery causes myocardial ischemia and finally cell death when ischemia is sustained for more than 20 minutes. Extensive studies have revealed that ischemic myocardium cannot recover without reperfusion by adequate restoration of blood flow, however, reperfusion can cause long-lasting cardiac dysfunction and aggravation of structural damage. The author therefore attempted to examine the effect of postischemic reperfusion on myocardial ultrastructure and to determine the rationales for recanalization therapy to salvage ischemic myocardium. MATERIALS AND METHODS: Young Holstein-Friesian cows (130~140 Kg body weight; n=40) of both sexes, maintained with nutritionally balanced diet and under constant conditions, were used. The left anterior descending coronary artery (LAD) was occluded by ligation with 4-0 silk snare for 20 minutes and recanalized by release of the ligation under continuous intravenous drip anesthesia with sodium pentobarbital (0.15 mg/Kg/min). Drill biopsies of the risk area (antero-lateral wall) were performed at just on reperfusion (5 minutes), 1-, 2-, 3-, 6-, 12-hours after recanalization, and at 1-hour assist (only with mechanical respiration and fluid replacement) after 12-hour recanalization. The materials were subdivided into subepicardial and subendocardial tissues. Tissue samples were examined with a transmission electron microscope (Philips EM 300) at the accelerating voltage of 60 KeV. RESULTS: After a 20-minute ligation of the LAD, myocytes showed slight to moderate degree of ultrastructural changes including subsarcolemmal bleb formation, loss of nuclear matrix, clumping of chromatin and margination, mitochondrial destruction, and contracture of sarcomeres. However, microvascular structures were relatively well preserved. After 1-hour reperfusion, nuclear and mitochondrial matrices reappeared and intravascular plugging by polymorphonuclear leukocytes or platelets was observed. However, nucleoli and intramitochondrial granules reappeared within 3 hours of reperfusion and a large number of myocytes were recovered progressively within 6 hours of reperfusion. Recovery was apparent in the subepicardial myocytes and there were no distinct changes in the ultrastructure except narrowed lumen of the microvessels in the later period of reperfusion. CONCLUSIONS: It is likely that the ischemic myocardium could not be salvaged without adequate restoration of coronary flow and that the microvasculature is more resistant to reversible period of ischemia than subendocardium and subepicardium. Therefore, thrombolysis and/or angioplasty may be a rational method of therapy for coronarogenic myocardial ischemia. However, it may take a relatively longer period of time to recover from ischemic insult and reperfusion injury should be considered.

Significance of Urinary NMP22 in Patients with Bladder Tumor as a Diagnostic Test / 대한비뇨기과학회지

PURPOSE: The objective of this study was to evaluate the significance of urinary Nuclear Matrix Protein22(NMP22) in patients wish bladder tumor as a diagnostic test MATERIALS AND METHODS: A total persons were divided into three groups. First group were 26 patients with bladder tumor, second group were 31 persons of normal control, third group were 28 patients with UTI(urinary tract infection). NMP22 was assayed using a commercial test kit. RESULTS: Mean NMP22 in bladder tumor group(50.6units/ml) was significantly higher than mean NMP22 in normal control group(8.4units/ml). But, mean NMP22 in bladder tumor group was not significantly higher than mean NMP22 in UTI group(47.3units/ml). Mean NMP22 did not correlate with tumor stage or tumor grade in patients with bladder tumor(p> 0.05). Sensitivity, specificity, positive and negative predictive value of NMP22 in bladder tumor(cut-of value: 10units/ml) were 73.1%(19/26), 67.7%(21/31), 65.5%(19/29) and 75%(21/28), respectively. Sensitivity of urine cytology were 50%(13/26) in the bladder tumor groups. CONCLUSIONS: Our data suggest that NMP22 in patient with bladder tumor could be a good diagnostic test. High NMP22 level in patients with UTI, however, might be decrease the availability of MMP22 in diagnosis of bladder tumor.

Urinary Nuclear Matrix Protein ( NMP 22 ) in the Detection of Transitional Cell Carcinoma of the Bladder / 대한암학회지

PURPOSE: The detection of bladder cancers by noninvasive techniques remains an unsolved problem. We evaluate the availability of an immunoassay for urinary nuclear matrix protein, NMP 22, as an indicator for transitional cell carcinoma of the bladder. MATERIALS AND METHODS: Three groups of subjects participated in this trial of NMP 22: 22 patients with transitional cell carcinoma (group 1), 12 patients with urinary tract infection (group 2) and 31 healthy volunteers (group 3). NMP 22 was determined by ELISA using a commercial test kit (NMP 22 Test Kit, Matritech Inc., USA), We compared urinary NMP 22 levels to the grade, stage, cytology and DNA flowcytometry of transitional cell carcinoma of bladder. RESULTS: NMP 22 values in these 3 groups were significantly different (group 1, median 24.81 U/mL; group 2, median 8.41 U/mL; and group 3, median 5.12 U/mL; Mann-Whitney U test for differences between 3 medians, p < 0.05). The patients with transitional cell carcinoma had significantly greater urinary NMP 22 levels than those with no evidence of tumor (Mann-Whitney U test for differences between 2 medians, p<0.01). There was no zelationship between the urinary NMP 22 levels and tumor grade, stage, cytology or DNA flowcytometry. CONCLUSIONS: It is possible that urinary NMP 22 could improve the detection of bladder transitional cell carcinoma.

The Significance of Urinary Nuclear Matrix Protein ( NMP22 ) Measurement in Patients with transitional Cell Carcinoma of the Bladder / 대한암학회지

PURPOSE: The objective of this study was to evaluate an immunoassay for urinary nuclear matrix protein (NMP22) as an indicator for transitional cell carcinoma of the bladder. MATERIALS AND METHODS: Three groups of subjects attended the trial of NMP22. First group was 27 patients with transitional cell carcinoma of the bladder, second group was 24 patients with other urinary cancer consisted of prostate cancer and renal cell carcinoma, and third group was 24 healthy volunteers. NMP22 was determined using a commercial test kit, which is based on an enzyme-linked immunosorbent assay. RESULTS: In normal healthy volunteers and other urinary cancer group median NMP22 levels were 2.24 and 3.27 U/ml, respectively. Median urinary NMP22 levels in patients with transitional cell carcinoma of the bladder were 54.30 U/ml. It was significantly greater than other two groups. Median NMP22 levels according to the tumor stage and the tumor grade did not show the significant difference statistically. CONCLUSIONS: Urinary NMP22 is a useful marker that is more specific for bladder cancer thsn for other urinary cancer. Further tests are required to clarify the influence of other spe- cific conditions, such as urinary tract infection, and intravesical drug instillation or procedure.

The Significance of Urinary Nuclear Matrix Protein ( NMP ) as a Marker for Transitional Cell Carcinoma of the Bladder / 대한비뇨기과학회지

NMP is a kind of protein relating to the internal structural framework of the nucleus, which is related to gene expression and regulation such as DNA replication and processing of RNA, and is made in tumor cell more than in normal cell. The object of this study is to evaluate the utility of NMP22 in urine as the possible marker of monitoring the transitional cell carcinoma of the bladder. Two groups attended the trial of NMP22; 1) 25 healthy volunteers 2) 25 patients with the transitional cell carcinoma of the bladder. The result is that the values of the mean NMP22 of the healthy volunteers and the patients with the transitional cell carcinoma of the bladder were 4.04+/-1.83 U/ml and 186.9+/-405.9 U/ml, respectively. The difference was statistically significant (p=0.028). The value of urinary mean NMP22 according to the tumor grade and the tumor stage didn`t show the significant difference statistically (grade I: 41.3+/-51.9 U/ml, grade II: 167.6+/-369.3 U/ml, grade HI: 362.7+/-605.5 U/ml, superficial TCC: 204.2+/-453.0 U/ml, invasive TCC:132.0+/-217.1 U/ml). In detecting the transitional cell carcinoma of the bladder, the sensitivity of urine cytology was 68% and the sensitivity of combining urinary NMP22 and urine cytology was 88%, when the value of the urinary NMP22 over 7.70 U/ml was considered as the positive. Urinary NMP22 is expected to increase the diagnosis and the detection of recurrence of the transitional cell carcinoma of the bladder if it is used together with the urine cytology as the urinary tumor marker of the transitional cell carcinoma of the bladder.

Nuclear matrix bound DNA polymerase-beta in mouse fibrosarcoma: effect of gamma-radiation.

Nuclear matrices isolated from the mouse fibrosarcoma tumour cells contain the eukaryotic replicative enzyme DNA polymerase-alpha and the presumptive repair enzyme DNA polymerase-beta. Exposure of tumors to various doses of gamma-radiation (1.95 to 6.5 Gy) causes a 2-fold increase in the levels of only DNA polymerase-beta in the nuclear matrix. The increase in the levels of this enzyme is not discernible if the matrices are isolated 24 hr after irradiation. The rise in the levels of the repair enzyme DNA polymerase-beta could be indicative of radiation stress response of the tumour cells and their repair ability.