Anti-DARPP32 antibody-immunopositive inclusions in the brain of patients with multiple system atrophy.

MSA is a neurodegenerative disease and GCIs are specific pathological hallmarks in the brain of MSA patients. Recently, Cdk5 immunopositive GCIs were reported, but the function of Cdk5 in the adult human brain is not clear. Cdk5 has several substrates such as neurofilament and tau proteins. Among these substrates, tau and MAP 1B are immunopositive in GCIs. DARPP32 has been identified as a target for dopamine and PKA in the striatum. DARPP32 has multiple phosphorylation sites, and Cdk5 can phosphorylate DARPP32 at Thr75. The phosphorylation ofThr75 converts DARPP32 into an inhibitor of PKA. DARPP32 is also one of the major substrates of Cdk5, and DARPP32 is widely expressed in both neurons and glial cells. In this study, we determined the immunohistochemical localization of DARPP32 in the brains of a normal control group and patients with MSA. An anti-DARPP32 antibody revealed immunopositive oligodendrocytes and astrocytes widely distributed in the brains of the normal control group and the brain of patients with MSA. Neurons in the caudate, globus pallidus, substantia nigra, hypothalamus, neocortex layers II and III, and cerebellar Purkinje cells were all immunopositive for DARPP32 in the normal control brains, and the immunostaining patterns were very similar to those observed in patients with MSA. We found that DARPP32 was immunopositive in GCIs, and the localization of DARPP32 and Cdk5 was very similar in GCIs. We suggest that Cdk5 and its substrate DARPP32 may be involved in the formation of GCIs through the phosphorylation of DARPP32 in the oligodendrocytes of brains with MSA.

[Usefulness of the thoracoscopic surgery under local anesthesia and irrigation for the patient with Bacillus cereus empyema; report of a case].

The case was 54-year-old male with some risks such as chronic heart failure, atrial fibrillation, and liver chirrhosis. He was admitted because of severe back pain and diagnosed as empyema by preoperative thoracentesis. By thoracoscopic procedures under local anesthesia, fibrinopurulent tissues were cleaned as much as possible and 3 of chest tubes were replaced. The final diagnosis was Bacillus cereus pyothorax by bacterial cultures of pleural effusion. Intrathoracic cavity was cleaned with physiological saline solution. The patient made favorable progress and recovered. Thoracoscopic surgery under local anesthesia with thoracic irrigation was so effective and safe methods to control the infection.

Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity.

Recent studies have shown that mutations at amino-acid 482 in the ABCG2 gene affect the substrate specificity of the protein. To delineate the effects of these mutations clearly, human embryonic kidney cells (HEK-293) were stably transfected with wild-type 482R or mutant 482G and 482T ABCG2. By flow cytometry, mitoxantrone, BODIPY-prazosin, and Hoechst 33342 were found to be substrates of all ABCG2 proteins, while rhodamine 123, daunorubicin, and LysoTracker Green were transported only by mutant ABCG2. In cytotoxicity assays, all ABCG2 proteins conferred high levels of resistance to mitoxantrone, SN-38, and topotecan, while mutant ABCG2 also exhibited a gain of function for mitoxantrone as they conferred a four-fold greater resistance compared to wild type. Cells transfected with mutant ABCG2 were 13- to 71- fold resistant to the P-glycoprotein substrates doxorubicin, daunorubicin, epirubicin, bisantrene, and rhodamine 123 compared to cells transfected with wild-type ABCG2, which were only three- to four-fold resistant to these compounds. ABCG2 did not confer appreciable resistance to etoposide, taxol or the histone deacetylase inhibitor depsipeptide. None of the transfected cell lines demonstrated resistance to flavopiridol despite our previous observation that ABCG2-overexpressing cell lines are cross-resistant to the drug. Recently reported inhibitors of ABCG2 were evaluated and 50 microM novobiocin was found to reverse wild-type ABCG2 completely, but only reverse mutant ABCG2 partially. The studies presented here serve to underscore the importance of amino-acid 482 in defining the substrate specificity of the ABCG2 protein and raise the possibility that amino-acid 482 mutations in human cancers could affect the clinical application of antagonists for ABCG2.

Acquired mutations in the MXR/BCRP/ABCP gene alter substrate specificity in MXR/BCRP/ABCP-overexpressing cells.

A disparity was noted in the transport of rhodamine 123 among nine MXR/BCRP/ABCP-overexpressing cells studied; all demonstrated mitoxantrone transport, whereas only two effluxed rhodamine 123. When the MXR/BCRP/ABCP gene was sequenced in the cell lines studied, differences were noted at amino acid 482, predicted to be at the start of the third transmembrane domain. Sequencing genomic DNA revealed wild-type MXR/BCRP/ABCP to have an arginine at position 482. Cells having a threonine or glycine at position 482 were able to efflux rhodamine 123, whereas cells having an arginine were not. A vaccinia virus expression system confirmed that rhodamine as well as doxorubicin efflux is observed with R482T or R482G but not with the wild-type R482; all three MXR/BCRP/ABCP forms transported mitoxantrone. Cross-resistance studies suggest that, compared with wild-type MXR/BCRP/ABCP, cells having an R482T mutation have higher anthracycline resistance, whereas an R482G mutation seems to confer relatively less resistance to SN-38 and topotecan. These results suggest that amino acid 482 has a crucial role in MXR/BCRP/ABCP function and that mutation of a single amino acid residue significantly changes substrate specificity, thus altering the drug resistance phenotype.

A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2).

The fluorescent compounds rhodamine 123, LysoTracker Green DMD-26, mitoxantrone, and BODIPY-prazosin were used with the antagonist fumitremorgin C (FTC) in order to develop functional assays for the half-transporter, MXR/BCRP/ABCP1. A measure of FTC-inhibitable efflux was generated for each compound in a series of MXR-overexpressing drug-selected cell lines and in ten unselected cell lines which were used to determine if the four fluorescent compounds were sensitive enough to detect the low MXR levels found in drug-sensitive cell lines. FTC-inhibitable efflux of mitoxantrone and prazosin was found in four of the ten cell lines, SF295, KM12, NCI-H460, and A549, and low but detectable levels of MXR mRNA were also observed by Northern analysis in these cells. FTC-inhibitable mitoxantrone and prazosin efflux in both selected and unselected cell lines was found to correlate well with MXR levels as determined by Northern blotting, r(2)=0.89 and r(2)=0.70 respectively. In contrast, rhodamine and LysoTracker were not able to reliably detect MXR. Cytotoxicity assays performed on two of the four unselected cell lines confirmed increased sensitivity to mitoxantrone in the presence of FTC. FTC was found to be a specific inhibitor of MXR, with half-maximal inhibition of MXR-associated ATPase activity at 1 microM FTC. Short term selections of the SF295, KM12, NCI-H460 and A549 cell lines in mitoxantrone resulted in a small but measurable increase in MXR by both Northern blot and functional assay. These studies show that flow cytometric measurement of FTC-inhibitable mitoxantrone or prazosin efflux is a sensitive and specific method for measuring the function of the MXR half-transporter in both selected and unselected cell lines.

Down-regulation of galectin-3 suppresses tumorigenicity of human breast carcinoma cells.

Galectin-3 is an endogenous beta-galactoside-binding protein with specificity for type I and II ABH blood group epitopes and poly-N-acetyllactosamine glycan-containing cell surface glycoproteins and is the major nonintegrin cellular laminin-binding protein. Galectin-3 is expressed at an elevated level in a wide range of neoplasms, and expression was shown to be associated in some tumor cell systems with metastases. Here we determined the functional consequence of blocking galectin-3 expression in highly malignant human breast carcinoma MDA-MB-435 cells. Inhibition of galectin-3 expression led to reversion of the transformed phenotype as determined by altered morphology, loss of serum-independent growth, acquisition of growth inhibition properties by cell contact, and abrogation of anchorage-independent growth. The blockage of galectin-3 expression led to a significant suppression of tumor growth in nude mice. These results provide direct evidence that galectin-3 expression is necessary for the maintenance of the transformed and tumorigenic phenotype of MDA-MB-435 breast carcinoma cells.

Galectin-3 maintains the transformed phenotype of thyroid papillary carcinoma cells.

Galectin-3, a beta-galactoside-binding protein, is highly expressed in thyroid papillary carcinomas, while functional relevance of galectin-3 overexpression to the malignant phenotype remains elusive. In the present study we transfected galectin-3 antisense cDNA into the human thyroid papillary carcinoma cell line NPA which expresses an innately high level of galectin-3, and examined the effect of antisense inhibition of galectin-3 expression on the transformed phenotype. There was no difference in anchorage-dependent growth between the antisense clones and either the control or parental clones. In contrast, anchorage-independent growth and saturation density of the antisense clones were significantly suppressed compared to those of either the control or parental clones. These results demonstrate that overexpression of galectin-3 in thyroid papillary carcinoma cells is necessary for the maintenance of transformed phenotype, and suggest galectin-3 as a potential target for therapeutic interventions in the future.

Immunohistochemical localization of phosphoinositide 3-kinase in brains with multiple system atrophy.

Oligodendrocytes have been shown to display some morphological characteristics of apoptosis in MSA. The accumulated evidence shows that phosphoinositide 3-kinase (PI3K) is closely associated with the regulation of apoptosis. Thus, we examined immunohistochemically PI3K in the cerebellum and pons from autopsy samples with MSA. In control tissues, PI3K was immunostained in some neurons and a few oligodendrocytes. In MSA samples, the larger number of oligodendrocytes was observed in the pons and cerebellum. Furthermore, some neurons were strongly immunolabeled in MSA samples. The recent study has shown that PI3K phosphorylates PKB/Akt which phosphorylates BAD resulting in the cessation of apoptotic process. The present results suggest that PI3K is upregulated in oligodendrocytes and some neurons in MSA, possibly in response to the apoptotic signals to these cells.

Galectin-3 binding and metastasis.

Galectin-3 (gal-3) is a member of a growing family of carbohydrate-binding proteins. It consists of two functional domains: an amino-(N)-terminal domain, which is cleavable by collagenases and is responsible for dimerization as well as secretion of the protein, and a carboxy-(C)-terminal domain with affinity for carbohydrates containing N-acetylactosamine residues. Gal-3 is present in the nucleus, the cytoplasm, and also the extracellular matrix of many normal and neoplastic cell types. However, an array of reports show an up-regulation of this protein in transformed and metastatic cell lines (1,2). Moreover, in many human carcinomas, an increased expression of gal-3 correlates with progressive tumor stages (3,6).

Association of p53 expression with second primary tumour development in hypopharyngeal carcinoma.

Abnormalities of p53 tumour suppressor gene are detected in a diversity of malignancies and play an important role in their pathogenesis. Hypopharyngeal carcinoma is the most morbid among head and neck squamous cell carcinomas because of the high incidence of treatment failures and because a biological marker predictive of the treatment failures remains elusive. The expression of p53 protein in 46 hypopharyngeal squamous cell carcinomas was examined histochemically and p53 immunoreactivity was found in 19 of 46 cases (41.3 per cent). The rate of second primary tumour development was significantly higher in the p53-positive group than in the p53-negative group (p = 0.039), whereas that of tumour recurrence was not significantly different between the two. Moreover, there was no statistically significant difference in either overall or disease-free survival between the p53-positive and -negative groups. These results indicate that although p53 expression significantly correlates with second primary tumour development in patients with hypopharyngeal squamous cell carcinomas, it is not predictive of the clinical outcome.

Blockade of cadherin-6B activity perturbs the distribution of PSD-95 family proteins in retinal neurones.

BACKGROUND: Synaptic junctions have cadherin-catenin complexes, but their functions are poorly understood. Using retinal neurones, we investigated the role of this adhesion machinery in synaptic organization. RESULTS: In cultures of chicken retinal cells, cadherin-6B (cad6B) and cadherin-7 (cad7) are expressed by distinct neurones, each being distributed in a punctate pattern along their neurites as well as in the soma. Double-immunostaining for cad6B and PSD-95/SAP90 or other PSD-95 family members, known to localize in the postsynaptic density, showed that their distributions overlapped each other. To assess the role for cad6B, we incubated retinal cells with antibodies that could specifically block cad6B-mediated adhesion. In the antibody-treated neurones, the localization pattern of PSD-95 family proteins was altered, that is, their staining signals tended to be reduced or disarranged. We then examined whether cadherins interacted molecularly with PSD-95: Cadherin immunoprecipitates from brain lysates did not contain PSD-95; nevertheless, this protein was co-precipitated with alphaN- and beta-catenins. When PSD-95 proteins were ectopically expressed in epithelial cells, some of these molecules were concentrated in cell-cell junctions, co-localizing with E-cadherin, and this junctional localization of PSD-95 was abolished by blocking of E-cadherin activity. CONCLUSION: These results suggest that cadherins play a role in the subcellular organization of postsynaptic density components through some, perhaps indirect, molecular interactions.

Galectin-3 induces endothelial cell morphogenesis and angiogenesis.

Increasing evidence suggests that carbohydrate-binding proteins play an essential role in tumor growth and metastasis. However, conflicting results on their function in the regulation of cell proliferation and differentiation during angiogenesis have been reported. We have examined the role of galectin-3 in the regulation of human umbilical vein endothelial cell proliferation, differentiation, migration, and neovascularization. Galectin-3, a carbohydrate-binding protein, with specificity for type 1 and 11 ABH blood group epitopes and polylactosamine glycan containing cell surface glycoproteins, is the major nonintegrin cellular laminin-binding protein. Because galectin-3 expression was shown to be associated in some tumor systems with metastasis, we questioned whether it induces endothelial cell morphogenesis. Here we show that galectin-3 affects chemotaxis and morphology and stimulates capillary tube formation of HUV-EC-C in vitro and angiogenesis in vivo. Endothelial cell morphogenesis is a carbohydrate-dependent process, as it is neutralized by specific sugars and antibodies. These findings demonstrate that endothelial cell surface carbohydrate recognition event(s) can induce a signaling cascade leading to the differentiation and angiogenesis of endothelial cells.

Expression of cytoplasmic galectin-3 as a prognostic marker in tongue carcinoma.

Galectin-3 is a member of the beta-galactoside-binding mammalian lectin family with affinity to ABH group epitopes, cell surface and extracellular polylactosamine glycans. It has been shown to be involved in differentiation, morphogenesis, tumor progression, and metastasis. Here we questioned the possible involvement of galectin-3 in the neoplastic progression of the tongue epithelium and evaluated its prognostic value in tongue cancer patients. Galectin-3 expression was analyzed by the immunohistochemical method in 77 tongue specimens (54 squamous cell carcinomas and 23 specimens of distinct normal mucosa). Levels of nuclear expression of galectin-3 markedly decreased during the progression from normal to cancerous states (P < 0.0001), while cytoplasmic expression increased (P < 0.0001). Enhanced expression of galectin-3 in the cytoplasm was associated with a reduced disease-free survival of tongue cancer patients. Multivariate analysis identified enhanced expression of cytoplasmic galectin-3 as an independent predictor of disease recurrence (P = 0.0120). These results suggest that the observed translocation of galectin-3 from the nucleus to the cytoplasm during neoplastic progression may serve as a prognostic factor for tongue cancer patients.

Ki-67 positive fractions in benign and malignant thyroid tumours: application of flow cytometry.

We investigated the DNA ploidy pattern, cell cycle and the percentage of Ki-67 positive fractions in fresh surgical material from 17 benign and 33 malignant thyroid tumours using flow cytometry. DNA aneuploidy was not seen at all in benign tumours, but was seen in 3 out of 33 malignant tumours, suggesting that detection of DNA aneuploidy indicates malignancy, although the detection sensitivity was low. Regarding the cell cycle, there was no difference in the percentage of S-phase fractions (SPF) or G2 plus M phase fractions (G2M) between benign and malignant tumours. However, the percentage of Ki-67 positive fractions in malignant tumours (39.9 +/- 3.9%) was significantly higher than that in benign tumours (9.4 +/- 2.1%), indicating that malignant thyroid tumours contained a large population of G phase cells. When a cut-off value of 20%, was used for Ki-67 positive fractions, sensitivity was 82%, specificity was 88% and accuracy was 84% for the diagnosis of malignant tumours. Although this study was carried out on surgically derived materials, it is possible that flow cytometric analysis of fine needle aspiration-derived materials may have a place in preoperative histopathological assessment of thyroid tumours.

Expression of galectin-3 in fine-needle aspirates as a diagnostic marker differentiating benign from malignant thyroid neoplasms.

BACKGROUND: Galectin-3 is a beta-galactoside-binding protein that has been reported to be expressed preferentially in thyroid malignancies. The current study was designed to substantiate this finding further and to establish a presurgical diagnostic modality of differentiating between benign and malignant thyroid neoplasms by analyzing galectin-3 expression in fine-needle aspirates. METHODS: The expression of galectin-3 was examined immunohistochemically in total of 172 specimens: 45 primary and 20 metastatic papillary carcinomas, 8 primary and 2 metastatic follicular carcinomas, 5 primary and 3 metastatic anaplastic carcinomas, 3 primary medullary carcinomas, 25 follicular adenomas, 3 goiters, and 58 adjacent normal thyroid tissue. Alternatively, epithelial cells were isolated from the fine- needle aspirates of 14 thyroid nodules and subjected to immunoblotting analysis of galectin-3. RESULTS: Immunohistochemical analysis revealed that all thyroid malignancies of follicular cell origin (including papillary, follicular, and anaplastic carcinomas) showed high and diffuse expression of galectin-3, whereas one of the three medullary carcinomas of parafollicular cell origin displayed weaker and focal expression of galectin-3. In contrast, neither benign thyroid adenomas, goiters, nor normal thyroid tissues expressed galectin-3. Immunoblot analysis of the isolated epithelial cells detected galectin-3 in nine thyroid nodules that were proven histologically to be malignant ( eight papillary carcinomas and one follicular carcinoma) after surgical intervention, whereas galectin-3 was not detected in five nodules proven to be benign follicular adenomas. CONCLUSIONS: Galectin-3 serves as a marker of thyroid malignancy of follicular cell origin. Analysis of galectin-3 expression in fine-needle aspirates enhances the differential diagnostic accuracy between benign and malignant thyroid neoplasms.

The NH2 terminus of galectin-3 governs cellular compartmentalization and functions in cancer cells.

Galectin-3 is a member of the beta-galactoside-binding protein family shown to be involved in tumor progression and metastasis. It has a unique primary structure consisting of three domains: a 12-amino acid leader sequence containing a casein kinase I serine phosphorylation site, which is preceded by a collagenase-sensitive Pro-Gly-rich motif, and a COOH-terminal half encompassing the carbohydrate-binding site. To study the functional role of the unusual leader sequence of galectin-3, a mutant cDNA that causes an 11-amino acid deletion in the NH2-terminal region was generated and expressed in galectin-3-null BT-549 human breast carcinoma cells. Deletion of the NH2 terminus resulted in abolition of the secretion of truncated galectin-3, loss of nuclear localization, and reduced carbohydrate-mediated functions compared with the wild-type protein. When green fluorescent protein was fused to the galectin-3 leader sequence and transiently transfected into BT-549 cells, the uniform cellular distribution of native green fluorescent protein was changed mainly to a nuclear pattern. To further investigate whether the functional changes observed in a galectin-3 with the 11 NH2-terminal amino acids deleted were due to loss of phosphorylation at Ser6, two point mutations were created at this serine: Ser6-->Ala and Ser6-->Glu. No obvious difference was observed in cellular localization between wild-type and Ser6-mutated transfectants. These results suggest a structural role for the NH2 terminus leader motif of galectin-3 in determining its cellular targeting and biological functions independent of phosphorylation.

Pre-operative assessment of metastatic parotid tumors.

Metastatic disease to the parotid gland is rare and its diagnosing procedures are not established. We assessed several parameters, including the prior history of malignancy, fine needle aspiration cytology (FNAC) and magnetic resonance (MR) imaging and determined the most useful combinations for diagnosing metastatic parotid tumors. The primary tumors were squamous cell carcinomas of the eyelid, larynx, tonsil and of unknown origin, malignant melanoma of the auricle and small cell lung carcinoma. In three of four patients with parotid lymph node metastasis who underwent MR imaging, rim enhancement of the tumor was observed on gadolinium (Gd)-enhanced fat-suppressed T1-weighted MR images (sensitivity 75%). In the histopathologic examinations, the tumor was encapsulated and massive infiltration of normal lymphocytes was found peripheral to the capsule. These were not seen in the patients with metastasis to the parotid parenchyma via hematogeneous dissemination or with advanced-stage parotid node metastasis. The FNAC diagnosis accorded with the tumor histology in five of six patients (sensitivity 83%). Three of the six patients had a prior history of malignancy in other sites (sensitivity 50%). The triad of FNAC, prior history of malignancy and enhanced MR images was identified as the combination most useful in the diagnosis of metastatic parotid tumors (sensitivity 100%).

Primary thyroid lymphoma associated with metastatic thyroid tumor: discrimination with US.

We surgically treated a 75-year-old man who suffered from metastatic adenocarcinoma from the colon associated with primary thyroid lymphoma measuring 1 cm maximum in diameter. Radiologic findings were correlated with histopathology of excised specimens. Ultrasonography could discriminate between these two tumors based on the margin characteristics and the lesion echogenicity: the metastatic tumor was shown as an ill-defined hypo to iso echoic mass, while the malignant lymphoma was detected as a well-defined markedly hypoechoic mass. In addition, we could confirm that early-stage primary thyroid lymphoma even as small as 1 cm shows the same radiologic appearance as that of a bulky lymphoma.

DNA ploidy, proliferative activities, and immunophenotype of malignant lymphoma: application of flow cytometry.

BACKGROUND: To explore the flow cytometric diagnosis of malignant lymphoma, we examined the deoxyribonucleic acid (DNA) ploidy, proliferative activities, and immunophenotype of surgical biopsy- and fine-needle aspiration (FNA)-derived materials. Our goal was to determine the possibility of making a diagnosis of malignant lymphoma by flow cytometric analysis of FNA-derived materials. METHODS: The DNA ploidy and proliferative indices including the percentage of S-phase fraction (SPF), G2 + M fraction (G2M), and Ki-67-positive fraction (Ki-67) were analyzed on the fresh materials from 84 consecutive patients with suspected malignant lymphoma. Flow cytometric analysis of surface antigens was simultaneously performed. Fourteen of the patients underwent FNA and subsequent surgical biopsy of the same lymph nodes for flow cytometric analysis. RESULTS: The proliferative indices of intermediate-grade non-Hodgkin's lymphomas (NHL) (n = 28) and high-grade NHL (n = 23) were significantly higher than those of the reactive hyperplasia (n = 25). The total for SPF + G2M of 6% was a satisfactory threshold for differentiating these NHL from reactive hyperplasia (sensitivity of 84%, specificity of 88%, and accuracy of 86%). However, low-grade NHL (n = 3) and Hodgkin's lymphoma (HL, n = 5) could not be discriminated by employing this parameter. DNA aneuploidy was seen in 13 of the 28 intermediate-grade NHL and 8 of the 23 high-grade NHL, whereas it was not seen in 25 reactive hyperplasia, 3 low-grade NHL, and 5 HL. The percentage of CD19-positive cells in B-cell NHL or CD3-positive cells in T-cell NHL was significantly higher compared with those for reactive hyperplasia. The percentage of CD16 + CD56-positive cells in natural killer (NK) cell NHL was extremely high, with a mean of 91.8%. Flow cytometric results for FNA-derived materials showed excellent correlation with those for surgical biopsy-derived specimens. CONCLUSIONS: Analyses of DNA ploidy, proliferative activities, and immunophenotype by flow cytometry (FCM) are useful for diagnosing intermediate- and high-grade NHL. Fine-needle aspiration is a less invasive approach than surgical biopsy, and, when combined with FCM, it may have a place in the diagnosis of NHL.