Thoracic duct relationships to abnormal neurovascular structures in cervicothoracic regions: case study and clinical relevance.

The presence of variant intercostal and bronchial arteries and variable position of left recurrent laryngeal nerve (LRLN) along the course of thoracic duct (TD) may have clinical relevance in various cervicothoracic surgeries.

Fatty acid synthase expression associated with NAC1 is a potential therapeutic target in ovarian clear cell carcinomas.

BACKGROUND: This study examined the clinical significance of NAC1 and the expression level of its potential downstream target fatty acid synthase (FASN) in ovarian clear cell carcinomas (OCCCs), and evaluated the NAC1/FASN pathway as a potential therapeutic target. METHODS: NAC1 and FASN expression and NACC1 gene amplification were assessed in ovarian cancers by immunohistochemistry, fluorescence in situ hybridisation, and clinical data collected by a retrospective chart review. C75, a FASN inhibitor, was used to assess whether this pathway represented a therapeutic target in OCCC. RESULTS: High NAC1 expression was most frequent in clear cell tumours (40.0%:24/60). NACC1 gene amplification was identified in none of the 58 OCCCs. The frequency of NACC1 gene amplification was significantly higher in the high-grade serous histology than in the clear cell histology (P<0.01). NAC1 expression was significantly correlated with FASN expression in both OCCC samples and OCCC cell lines. Either high NAC1 expression or high FASN expression significantly correlated with shorter progression-free and overall survival (P=0.002 and 0.0048). NAC1 overexpression stimulated FASN expression, and NAC1 silencing using siRNA decreased FASN expression in OCCC cell lines. Profound growth inhibition was observed in C75-treated carcinoma cells with FASN overexpression when compared with the response in carcinoma cells without FASN expression. CONCLUSION: These findings indicate that NAC1/FASN overexpression is critical to the growth and survival of a subset of OCCC. The FASN silencing by the C75-induced phenotypes depends on the expression status of the targeted cell line. Therefore, NAC1/FASN pathway-targeted therapy may benefit selected OCCC patients.

EGFR gene amplification is related to adverse clinical outcomes in cervical squamous cell carcinoma, making the EGFR pathway a novel therapeutic target.

BACKGROUND: The aim of this study was to investigate the patterns of epidermal growth factor receptor (EGFR) overexpression, EGFR gene amplification, and the presence of activating mutations in the tyrosine kinase domain of this gene in squamous cell carcinomas and adenocarcinomas/adenosquamous carcinomas of the uterine cervix. METHODS: The EGFR expression, amplification, and mutation in cervical carcinomas were assessed by immunohistochemistry, fluorescence in situ hybridisation, and PCR-SSCP, respectively, and correlated with clinical data collected by a retrospective chart review. A functional assessment was performed by inactivating EGFR in cervical cancer cells with the potent inhibitor AG1478. RESULTS: Immunohistochemical analysis revealed that 6 out of 59 (10.2%) cervical squamous cell carcinomas showed significant amplification of the EGFR locus, whereas none of the 52 adeno/adenosquamous cell carcinomas had detectable EGFR amplification (P<0.05). The EGFR amplification significantly correlated with shorter overall survival (P=0.001) in cervical squamous cell carcinomas. Multivariate analysis showed that EGFR gene amplification was an independent prognostic factor for overall survival (P=0.011). None of the squamous cell carcinomas (0%: 0 out of 32) had detectable oncogenic mutations in EGFR exons 18 through 21. The frequencies of KRAS and BRAF mutations were very low in both squamous and adeno/adenosquamous cell carcinomas. Sensitivity of cervical cancer cells to AG1478 depended on the presence of EGFR overexpression. AG1478-induced EGFR inactivation in cell lines with EGFR overexpression significantly suppressed tumour development and progression in a mouse xenograft model. CONCLUSION: Our data suggest that EGFR signalling is important in a subset of cervical squamous cell carcinomas and that anti-EGFR therapy may benefit patients who carry the 7p11.2 amplicon in their tumours.

Anomalous branching pattern of external carotid artery: Clinical relevance to cervicofacial surgery.

Anomalous branching pattern of the left external carotid artery (ECA) was detected in an old man. The ECA branched into high submental artery and large transverse facial artery ending as angular artery compensating for concurrent agenesis of ipsilateral facial artery. The lingual artery gave direct branch to the submandibular gland, whereas the superior thyroid artery arose directly from common carotid artery with high bifurcation level. This unreported branching pattern of the ECA may have important clinical relevance to cervicofacial surgery.

Persistent median artery: cadaveric study and review of the literature.

The persistent median artery (PMA) may compress the median nerve (MN) and may be a significant supply of blood to the hand. Two cases of unilateral PMA (4%) were detected during the dissection of 50 upper limbs. The first case was a 75-year-old, right-handed male who suffered from chronic pain in both upper limbs, especially the left side. A dissection of his left upper limb revealed a PMA piercing both the MN and the medial branch of the anterior interosseous nerve. This artery coursed distally, deep to the transverse carpal ligament (TCL), forming a median-ulnar pattern for the superficial palmar arch (SPA). The PMA was superficial to two nerves at the distal edge of the TCL; the extraligamentous recurrent thenar (RT) branch of the MN and the third common digital nerve (TCDN). The second case was from the left side of an 80-year-old female found to have a high origin of the radial artery with trifurcation of the latter into PMA, common interosseous, and ulnar arteries. The PMA passed deep to the TCL forming a radial-median-ulnar pattern of SPA. Both the transligamentous RT branch of the MN and the TCDN passed deep to the PMA inside the carpal tunnel, before the abnormal crossing of the latter nerve ventral to the SPA on its way to the digits. The relationships of the PMA to various MN branches may have important implications regarding the diagnosis and treatment of MN compressive neuropathies.

Inguinal lymph node metastases are recognized with high frequency in rectal adenocarcinoma invading the dentate line. The histological features at the invasive front may predict inguinal lymph node metastasis.

AIM: Inguinal lymph node (ILN) metastasis occurs with high frequency in some of the patients with lower rectal cancer. The aim of this study was to identify risk factors for ILN metastasis in patients with low rectal adenocarcinoma. METHOD: We retrospectively analysed 156 patients with lower rectal adenocarcinoma who underwent radical resection (R0) at a single institution. RESULTS: Twenty-five (16%) patients had a tumour that invaded the dentate line, seven of whom had ILN metastasis. Invasion of the dentate line was significantly associated with a high rate of ILN metastasis, worse prognosis and local recurrence than with a tumour not invading the dentate line (P = 0.03). A Cox proportional hazard regression analysis revealed the histological characteristics at the invading front (Hif) also to be a risk factor for ILN metastasis. CONCLUSION: Tumours which invade the dentate line have a high rate of ILN metastases and worse cancer specific end-points. The presence of poorly differentiated or mucinous adenocarcinoma components is an indication for bilateral groin irradiation.

Cytological criteria of endometrial lesions with emphasis on stromal and epithelial cell clusters: result of 8 years of experience with intrauterine sampling.

OBJECTIVE: There are a number of unresolved issues in endometrial cytology. They include the significance of nuclear atypia for the diagnosis of grade1 adenocarcinoma (G1AC) and atypical endometrial hyperplasia (AEH), cytological criteria of endometrial hyperplasia without atypia, and recognition of stromal cell cluster (SC) and its distinction from epithelial cell cluster (EC). METHODS: We examined nuclear atypia, SC and EC in typical cases of five categories: normal endometrium (NEM), simple endometrial hyperplasia without atypia (SEH), complex endometrial hyperplasia without atypia (CEH), G1AC and grade2 adenocarcinoma (G2AC). We classified EC into four types: simple EC (SPEC), large regular EC (LREC), large irregular EC (LIEC) and small irregular EC (SIEC). Based on the results, we developed criteria of endometrial cytology and have evaluated 13 639 cases over 8 years. RESULTS: Nuclear atypia was significantly more frequent in G2AC than in any of the other four categories (P < 0.001). SC was significantly more frequent in NEM and SEH than in the other three categories (P < 0.001). G1AC and G2AC showed significantly higher frequency of LIEC than the other three categories (P < 0.001). CEH exhibited significantly higher frequency of LREC than the four categories (P < 0.001). The sensitivity and the specificity was 88.8% and 99.0% respectively. CONCLUSIONS: We could diagnose G1AC, G2AC and CEH with high accuracy using the established criteria mainly based on SC and EC. We think that the criteria may facilitate an effective screening and an objective interpretation of endometrial samples.

Electrogene therapy using endostatin, with or without suicide gene therapy, suppresses murine mammary tumor growth and metastasis.

Syngeneic inoculated metastatic mammary cancers received direct intratumoral injection of a plasmid vector containing either endostatin (pEndo) with or without a suicide gene (pHSVtk), pHSVtk alone or control vector once a week for 8 weeks. We applied electrogene transfer to the tumors after each injection and administered ganciclovir (GCV) to pHSVtk-transfected mice using an osmotic minipump. Anticancer efficacy was monitored using a variety of parameters, namely tumor volume, intratumoral microvessel density and DNA synthesis, number of mice with metastasis, and number of sites of metastasis per mouse. Tumor volume was significantly lower in all therapeutic groups, with the most effective growth suppression in the pEndo+pHSVtk/GCV group. Lymph node metastasis was significantly less frequent in all therapeutic groups, whereas the multiplicity of lung metastases was significantly lower only in the pEndo and pEndo+pHSVtk/GCV groups. All therapeutic groups showed significantly lower intratumor microvessel density and DNA synthesis. The pEndo and pEndo+pHSVtk/GCV groups also showed a significant reduction in the numbers of dilated lymphatic vessels containing intralumenal tumor cells. Our data suggest that endostatin electrogene therapy alone or in combination with pHSVtk/GCV suicide gene therapy is more beneficial than suicide gene therapy alone. The observed antimetastatic activity of endostatin may be of high clinical significance in the treatment of metastatic breast cancer.

Involvement of alpha-PAK-interacting exchange factor in the PAK1-c-Jun NH(2)-terminal kinase 1 activation and apoptosis induced by benzo[a]pyrene.

Benzo[a]pyrene [B(a)P], a potent procarcinogen found in combustion products such as diesel exhaust and cigarette smoke, has been recently shown to activate the c-Jun NH(2)-terminal kinase 1 (JNK1) and induce caspase-3-mediated apoptosis in Hepa1c1c7 cells. However, the molecules of the signaling pathway that control the mitogen-activated protein kinase cascades induced by B(a)P and the interaction between those and apoptosis by B(a)P have not been well defined. We report here that B(a)P promoted Cdc42/Rac1, p21-activated kinase 1 (PAK1), and JNK1 activities in 293T and HeLa cells. Moreover, alpha-PAK-interacting exchange factor (alpha PIX) mRNA and its protein expression were upregulated by B(a)P. While overexpression of an active mutant of alpha PIX (DeltaCH) facilitated B(a)P-induced activation of Cdc42/Rac1, PAK1, and JNK1, overexpression of mutated alphaPIX (L383R, L384S), which lacks guanine nucleotide exchange factor activity, SH3 domain-deleted alphaPIX (Delta SH3), which lacks the ability to bind PAK, kinase-negative PAK1 (K299R), and kinase-negative SEK1 (K220A, K224L) inhibited B(a)P-triggered JNK1 activation. Interestingly, overexpression of alphaPIX (Delta CH) and a catalytically active mutant PAK1 (T423E) accelerated B(a)P-induced apoptosis in HeLa cells, whereas alphaPIX (Delta SH3), PAK1 (K299R), and SEK 1 (K220A, K224L) inhibited B(a)P-initiated apoptosis. Finally, a preferential caspase inhibitor, Z-Asp-CH2-DCB, strongly blocked the alphaPIX (Delta CH)-enhanced apoptosis in cells treated with B(a)P but did not block PAK1/JNK1 activation. Taken together, these results indicate that alphaPIX plays a crucial role in B(a)P-induced apoptosis through activation of the JNK1 pathway kinases.

Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes.

The use of biochemical fractionation, immunofluorescence laser-scanning confocal microscopy, and immunoelectron microscopy with mouse anti-human bcl-2 monoclonal antibody to analyze the subcellular localization of the bcl-2 gene product revealed the protein prominently in the nuclear envelope, endoplasmic reticulum membrane, and mitochondrial membranes. Electron microscopy at high magnification more precisely localized bcl-2 to the nuclear outer membrane as confirmed by the biochemical fractionation, as well as to mitochondrial outer and, to a lesser degree, inner membrane. This multisite membrane distribution of bcl-2 suggests an important role for this protein in several different membrane compartments.

Apoptosis and tumor angiogenesis in cervical cancer after preoperative chemotherapy.

The correlation between apoptosis and tumor angiogenesis in uterine cervical cancer treated by preoperative intraarterial infusion chemotherapy (IAC) was investigated. Cervical cancer samples surgically obtained from 12 patients (stages Ib-IIIb) receiving IAC and from 10 patients (stages Ib-IIb) receiving no chemotherapy and biopsy specimens from the 12 patients before IAC were examined. The apoptotic index (AI) was determined with an in situ end-labeling assay. Intratumoral microvessel density (IMVD) and thymidine phosphorylase (dThdPase) expression were evaluated immunohistochemically using anti-CD34 and anti-dThdPase antibodies. AIs were higher in the 8 patients with complete or partial response to IAC than they were in the 4 nonchemoresponsive patients and in the 10 patients who received no chemotherapy (P < 0.01) and were inversely related to IMVDs (r = 0.724; P < 0.01). AIs and IMVDs after IAC were higher and lower than those before IAC (P < 0.01), respectively. The expression of dThdPase, which has angiogenic activity, was markedly decreased after IAC. These results suggest that the antitumor effects of IAC are closely associated with apoptotic cell death, which may be influenced in part by the extent of tumor angiogenesis inhibition.

Induction of apoptosis as well as necrosis by hypoxia and predominant prevention of apoptosis by Bcl-2 and Bcl-XL.

The molecular mechanism of cell death due to hypoxia has not been elucidated. Our recent observations that overexpression of the anti-apoptotic proto-oncogene bcl-2 and a bcl-2-related gene, bcl-x, prevents hypoxic cell death suggest that hypoxia induces apoptosis. Using electron microscopy and confocal and nonconfocal fluorescence microscopy, we show here that hypoxia does, in fact, induce both necrosis and apoptosis, and that the proportion of these two modes is highly dependent on the cell type. Overexpression of Bcl-2 or Bcl-Xl blocks hypoxia-induced apoptosis in a dose-dependent manner.

alphaPIX nucleotide exchange factor is activated by interaction with phosphatidylinositol 3-kinase.

p21-activated kinase (PAK) is a common effector protein of the small GTPases Cdc42 and Rac, leading to the activation of downstream mitogen activated protein kinases. PAK also mediates polarized cytoskeletal changes induced by these GTPases. The recently identified PAK-interacting exchange factor (PIX) acts as a guanine nucleotide exchange factor on Rac, and colocalizes with PAK in a focal complex, but little is known about the associated signaling cascades, including upstream activators of PIX. In this study, we show that one of the isoforms of PIX, alphaPIX, is activated by signaling cascades from the platelet-derived growth factor (PDGF) receptor and EphB2 receptor, and from integrin-induced signaling through phosphatidylinositol 3-kinase (PI3-kinase). alphaPIX is activated by forming a complex with these receptors either via association with PAK and Nck, or direct association with the p85 regulatory subunit of PI3-kinase. Synthetic phosphoinositide and membrane targeted PI3-kinase augmented the alphaPIX activity in vivo. In Xenopus, aggregates of mesodermal cells derived from embryos microinjected with alphaPIX significantly increased the peripheral spreading on fibronectin substrate in response to PDGF through PI3-kinase. These results indicate that alphaPIX is activated by PI3-kinase, and is involved in the receptor mediated signaling leading to the activation of the kinase activity of PAK, and the migration of mesodermal cells on extracellular matrix.

Genetic polymorphism of the CYP2C subfamily and its effect on the pharmacokinetics of phenytoin in Japanese patients with epilepsy.

OBJECTIVE: To examine the genetic polymorphism of CYP2C9 and CYP2C19 and its effect on the pharmacokinetics of phenytoin among 44 Japanese patients with epilepsy. METHODS: Polymerase chain reaction tests with leukocyte deoxyribonucleic acid were used to detect the mutations for the amino acid substitution (Arg144-->Cys and Ile359-->Leu) in CgammaP2C9 and for the defective allele (m1 and m2) in CgammaP2C19. The pharmacokinetic parameters of phenytoin in individual patients were estimated by means of empirical bayesian analysis, in which the prior information was the population parameters for Japanese patients with epilepsy. RESULTS: Of the 44 patients, none had the CgammaP2C9 mutation for the Cys144 allele, whereas six patients were heterozygous for the wild-type (wt) and Leu359 allele (wt/Leu359) in cgammaP2C9. The maximal elimination rate (Vmax) of phenytoin among patients with heterozygous wt/Leu359 in CgammaP2C9 was 33% lower than that among patients with normal CgammaP2C9. A total of 21 patients were heterozygous for the CgammaP2C19 mutation (wt/m1 or wt/m2), and five patients had the homozygous or heterozygous mutations in CgammaP2C19 (m1/m1 or m1/m2). The Vmax values of phenytoin were slightly decreased (up to 14%) among patients with CgammaP2C19 mutations compared with patients with normal CgammaP2C19. CONCLUSION: The findings indicated that the genetic polymorphisms of CYP2C isozymes play an important role in the pharmacokinetic variability of phenytoin and that the mutation in CYP2C9 proteins (Ile359-->Leu) is a determinant of impaired metabolism of the drug among Japanese persons.

Localization of apoptotic cells in the human epidermis by an in situ DNA nick end-labeling method using confocal reflectant laser microscopy.

We describe an immunohistochemical method that allows the detection of apoptotic cells in human epidermis by use of confocal laser reflectance and antibody-immunogold-silver complexes. For this purpose, the site of free 3'-OH DNA ends was detected by the reflectance from heavy metal products (anti-digoxigenin antibody-immunogold-silver complexes) instead of 3, 3'-diaminobenzidine (DAB) reaction products in the conventional in situ nick end-labeling of DNA strand breaks (ISEL) technique. Localization of double-stranded DNA was demonstrated by the autofluorescence of methyl green. The ISEL technique using confocal reflectant laser microscopy (CRLM) clearly showed the most intense reflectance in the nuclei of granular cells, in contrast to only a weaker reflectance in those of basal cells. On the other hand, the extent of autofluorescence of methyl green was significantly more intense in the nuclei of basal cells and showed a reciprocal relation to that of the reflectance. Therefore, granular cells were most prone to apoptosis and did not contain double-stranded DNA, as indicated by the lack of stainability with methyl green. In addition, this method demonstrating the simultaneous localization of both free 3'-OH DNA ends and double-stranded DNA proved to have a wide range of applications, including the study of other DNA autolytic processes.

Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies.

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.

Quantitative differentiation of both free 3'-OH and 5'-OH DNA ends between heat-induced apoptosis and necrosis.

Cell death is roughly categorized as either apoptosis or necrosis. For better understanding of the differences in DNA cleavage between them, we performed quantitative analysis of both the 3'-OH and the 5'-OH ends of DNA strand breaks via in situ nick-end labeling (ISEL) combined with transmission electron microscopy (TEM) of both heat-induced apoptosis and necrosis in mouse B-cells derived from a lymphoma cell line. To detect the 5'-OH ends, the 3'-P ends located on the opposite side holding the 5'-OH ends were dephosphorylated into 3'-OH ends with alkaline phosphatase. As assessed by statistical analysis of both the 3'-OH and the 5'-OH ends, their labeling densities were significantly higher in both the apoptotic and the necrotic cells in the early stage than in control cells. The labeling densities increased during the apoptotic and necrotic processes, except for a decrease in the density of the 3'-OH ends in necrotic cells in the late stages. Therefore, DNA degradation in both necrosis and apoptosis provides early evidence for these processes, and both apoptosis and necrosis may share at least the first steps of DNA degradation pathways.