Restoration of plakoglobin expression in bladder carcinoma cell lines suppresses cell migration and tumorigenic potential.

The reduction or loss of plakoglobin expression in late-stage bladder cancer has been correlated with poor survival where upregulation of this catenin member by histone deacetylase inhibitors has been shown to accompany tumour suppression in an in vivo model. In this study, we directly addressed the question of the role of plakoglobin in bladder tumorigenesis following restoration, or knockdown of expression in bladder carcinoma cell lines. Restoration of plakoglobin expression resulted in a reduction in migration and suppression of tumorigenic potential in vivo. Immunocytochemistry revealed cytoplasmic and membranous localisation of plakoglobin in transfectants with < 1% of cells displaying detectable nuclear localisation of plakoglobin. siRNA knockdown experiments targeting plakoglobin, revealed enhanced migration in all cell lines in the presence and absence of E-cadherin expression. In bladder cell lines expressing low levels of plakoglobin and desmoglein-2, elevated levels of desmoglein-2 were detected following restoration of plakoglobin expression in transfected cell lines. Analysis of wnt signalling revealed no activation event associated with plakoglobin expression in the bladder model. These results show that plakoglobin acts as a tumour suppressor gene in bladder carcinoma cells and the silencing of plakoglobin gene expression in late-stage bladder cancer is a primary event in tumour progression.

Loss of heterozygosity and microsatellite instability at chromosomal sites 1Q and 10Q in morphologically distinct regions of late stage prostate lesions.

PURPOSE: We investigated the incidence of loss of heterozygosity (LOH) and microsatellite instability in sporadic prostate cancer and surrounding tissue at loci encompassing the HPC1 and PTEN genes. MATERIALS AND METHODS: Surgical specimens from 63 patients with sporadic stage T3 or T4 prostatic adenocarcinoma were analyzed for LOH and microsatellite instability. Microdissected tissue included morphologically normal foci, benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma. LOH analysis was performed using 4 microsatellite markers that map in the region of the 1q24 to 25 locus of the putative prostate cancer susceptibility gene HPC1 and 4 that map in the region of the 10q23 locus of the PTEN gene. RESULTS: The incidence of LOH on 10q was consistent with that previously reported in prostatic tumors. LOH associated with the PTEN locus was recorded in morphologically normal foci, BPH and adenocarcinoma. Sequence analysis of PTEN in a limited number of lesions revealed mutations in nontumor and tumor tissue. Analysis of the DS10215 locus showed significant LOH in tumor but not in benign tissue, suggestive of a tumor suppressor gene in this region associated with prostatic neoplastic progression. In contrast, no significant LOH was observed in the same tissues at 4 loci on chromosome 1q. In this study we recorded elevated levels of microsatellite instability in benign prostatic tissue with an additional increase associated with prostatic adenocarcinoma. CONCLUSIONS: The low incidence of LOH in the region of the HPC1 locus in all prostate lesions studied suggests that this putative hereditary prostate cancer susceptibility locus does not appear to have a role in sporadic prostate cancer, at least not in the context of LOH. In contrast, analysis of the same tissues for LOH at chromosome 10q confirmed frequent alterations in this region linked to late stage prostate cancer. PTEN mutations in microdissected morphologically normal and BPH tissue showed alterations in nontumor tissue surrounding adenocarcinoma. Microsatellite instability was increased in adenocarcinomas over an elevated background recorded in surrounding tissues.

Disparate E-cadherin mutations in LCIS and associated invasive breast carcinomas.

AIMS: The relation between lobular carcinoma in situ (LCIS) and invasive breast cancer is unresolved. In an attempt to establish whether LCIS is a precursor of invasive cancer the mutational status and the expression of E-cadherin was analysed in LCIS and associated invasive breast carcinoma in 23 patients. METHODS: Foci of LCIS and associated invasive carcinoma were individually microdissected from tissue from 23 patients. Exons 4-16 of the E-cadherin gene were analysed using single strand conformation polymorphism (SSCP); protein expression and the localisation of E-cadherin and beta-catenin were assessed with the use of immunohistochemistry. RESULTS: Immunohistochemistry revealed a lack of expression of E-cadherin and beta-catenin in most LCIS samples and invasive foci. In all but four cases, the staining pattern was identical in the LCIS and associated invasive areas. When E-cadherin was absent, beta-catenin was also undetected, suggesting a lack of expression of alternative classic cadherin members in these lesions. Coincident E-cadherin mutations in LCIS and associated invasive carcinoma were not identified in this series of patients. However, mutational analysis of E-cadherin in multiple foci of carcinoma in situ surrounding an invasive lesion provided evidence to support ductal carcinoma in situ as a precursor of invasive ductal carcinoma. CONCLUSION: These data support the hypothesis that LCIS is not a precursor of invasive breast carcinoma but a marker of increased risk of developing invasive disease.

Expression of classic cadherins type I in urothelial neoplastic progression.

Loss or reduced expression of E-cadherin has been shown to be associated with poor survival in patients with bladder cancer. In numerous cases, loss of E-cadherin expression in bladder tumors has been accompanied by continued association of catenins with the membrane, suggestive of the expression of an alternative cadherin member. In this study we examined 75 bladder tumors using immunohistochemistry for the expression of E-, P-cadherin, and alpha-, beta-, and gamma-catenins. As reported previously, loss or reduced E-cadherin expression is a frequent event in late stage bladder cancer, accompanied by less frequent alterations associated with different catenin family members. Analysis of 51 tumors for expression of E-, P-, and N-cadherin showed P-cadherin localized to the basal cell layers of normal urothelium, with retention of expression in the majority of tumors. In low-grade tumors P-cadherin was found localized to an expanded basal cell compartment, contrasting with the more extensive staining observed in late stage tumors. Membranous P-cadherin staining was often found in the absence of E-cadherin staining. N-cadherin is not expressed in normal bladder mucosa, but detection of this cadherin member was recorded in 39% (20/51) of bladder tumors. Unlike P-cadherin, membranous N-cadherin was detected in focal regions within tumors, representing novel expression in urothelial neoplastic progression. Although focal N-cadherin staining was observed in 3 noninvasive lesions, the majority of tumors expressing N-cadherin were invasive (17/20). Coexpression of E-, P-, and N-cadherin was recorded in 5 grade 2 bladder tumors. Expression of P-cadherin is maintained throughout bladder tumorigenesis, accompanied by aberrant expression of N-cadherin. Clearly, neither P- nor N-cadherin act in an invasive-suppressor mode in bladder cancer, but whether they have a primary role to play in urothelial neoplastic progression has yet to be established.

Molecular analysis of PTEN and MXI1 in primary bladder carcinoma.

Loss of heterozygosity (LOH) on 10q is associated with late-stage events in urothelial neoplastic progression. The tumor suppressor gene PTEN, which is mutated or homozygously deleted in numerous cancers, maps to a region of 10q within the reported region of minimal loss in bladder tumors. In two recent studies alterations in the PTEN gene occur at a low frequency in bladder tumors displaying 10q LOH. We have screened 35 late-stage bladder tumors for mutations in PTEN and MXI1, both genes mapping to chromosome 10q. Using single-strand conformation polymorphism analysis, we identified 6 tumors harboring mutations in PTEN and 2 additional tumors displaying homozygous deletion at this locus. No MXI1 mutations were identified within the same tumor panel. Of 16 bladder tumor cell lines analyzed, 2 showed homozygous deletion of PTEN and 3 harbored point mutations resulting in an amino acid change. Two cell lines harbored missense mutations in MXI1. We report a significantly higher frequency of PTEN alterations in bladder carcinoma (23%) than was previously recorded, with no accompanying mutations in the MXI1 gene.

Somatic mutation of PTEN in vulvar cancer.

PTEN, a candidate tumor suppressor gene located at chromosome 10q23.3, has been shown to be mutated in approximately 40% of endometrial cancers. Such mutations have also been identified in endometrial hyperplasia, indicating that inactivation of the PTEN tumor suppressor gene is an early event in the genesis of some endometrial cancers. In this study, we have extended the analysis of PTEN in gynecological cancer to include adenocarcinoma of the cervix and vulvar carcinomas. Microdissected tissue (including normal tissues), preneoplastic, and neoplastic lesions were analyzed from 9 patients with cervical cancer and 10 patients with vulvar cancer. Only 1 cervical adenocarcinoma displayed a PTEN mutation. In contrast, five of eight vulvar carcinomas studied harbored PTEN mutations. Alterations were identified in carcinoma in situ as well as squamous cell carcinoma of the vulva. In two patients, PTEN mutations were identified in mucosal regions with mild or focal dysplasia. These results suggest that PTEN is frequently altered in vulvar carcinomas and can be found associated with early dysplastic changes in vulvar mucosa.

Overexpression of pp60c-src elicits invasive behavior in rat colon epithelial cells.

BACKGROUND & AIMS: Src activation is reported as an early event found in preneoplastic colonic adenomas and in 70% of colon carcinomas. The aim of this study was to identify the biological consequences of c-src overexpression in rat colon epithelial cells. METHODS: Introduction and overexpression of c-src in an immortalized rat colon epithelial cell line was achieved using lipofection. Transfectants were tested for changes in growth and cell behavior using different in vitro assay systems. RESULTS: Colon epithelial cells overexpressing c-src showed the ability to form microcolonies in soft agar without acquiring tumorigenic potential. In in vitro assays, c-src transfectants displayed a gain of invasive potential through Matrigel without an accompanying change in migrational ability. No discernible qualitative changes were observed in the phosphotyrosyl protein profile between c-src and v-src transfectants. Assessment of the cadherin/catenin status in these cells revealed an intact, functional complex with no detectable tyrosine phosphorylation of different components of the complex. CONCLUSIONS: Overexpression of c-src in an immortalized rat colon epithelial cell line does not elicit full neoplastic transformation but enhances anchorage-independent growth and confers invasion capability. Increased invasion through Matrigel was not linked to inactivation of the cadherin complex in c-src transfectants.

Loss of DCC expression and glioma progression.

The deleted in colorectal cancer (DCC) gene, a candidate tumor suppressor gene on chromosome 18q21, encodes a neural cell adhesion molecule family protein that is most highly expressed in the nervous system. To address the hypothesis that DCC may play a role in glioma development and/or progression, we examined DCC expression by immunohistochemistry in 57 resected human astrocytic tumors. Overall, low-grade astrocytomas were predominantly DCC positive (15 of 16, or 94%), whereas high-grade tumors significantly less often expressed the DCC protein (27 of 41, or 66%; P = 0.03). We were able to directly assess the relationship between DCC expression and tumor progression in 15 patients who initially presented with a low-grade astrocytoma and subsequently recurred with a glioblastoma. Within this panel of paired lesions from the same patient, 14 of 15 (93%) low-grade tumors expressed the DCC protein, whereas only 7 of 15 (47%) corresponding glioblastomas were DCC positive. We also observed that secondary glioblastomas resulting from malignant progression of low-grade astrocytomas were more often DCC negative (8 of 15, or 53%) compared with primary or de novo glioblastomas (6 of 26, or 23%; P = 0.05). These findings implicate DCC inactivation in glioma progression and also demonstrate that DCC expression is preferentially, but not exclusively, lost in the genetic pathway to secondary glioblastoma multiforme.

Decrease of contralateral neglect by neck muscle vibration and spatial orientation of trunk midline.

Three patients with a right hemisphere lesion and marked left-sided neglect without visual field defects were asked to detect and identify stimuli which were tachistoscopically presented in the left or right visual half-field. Neglect of stimuli presented in the contralesional left visual field, which was observed when the patient's body was in a normal upright position with trunk, head and gaze oriented straight ahead to the middle of the projection screen, could be reduced by vibrating the left posterior neck muscles as well as by turning the trunk 15 degrees to the left. In contrast, unspecific stimulation on the left side of the patient's body, produced by vibrating the left hand muscles or the proprioceptive signal induced by turning the head 15 degrees to the left, had no compensatory effects. The results showed that the afferent information about real lengthening of the left posterior neck muscles (produced by turning the trunk) as apparent lengthening of these muscles (produced by their vibration), leads to a remission of contralateral neglect. Thus, the proprioceptive input from the neck muscles, i.e. the head-on-trunk signal, appears to influence the extension of the neglected part of space in patients with neglect. The signal seems to contribute substantially to the neural generation of the egocentric frame of reference that allows the determination of body position with respect to visual space. We hypothesize that the reduction of neglect by vibration of the contralateral posterior neck muscles is based on a shift of the subjective spatial localization of the sagittal midplane in the contralesional direction and a corresponding alteration of the egocentric coordinate system necessary for visuomotor coordination and exploration of space.

Treatment of neurogenic bladder dysfunction in multiple sclerosis by ultrasound-controlled bladder training.

Neurogenic bladder dysfunction, the main cause of chronic urinary tract infections in multiple sclerosis (MS), is efficiently treated by bladder training with ultrasound control of the residual urine. However, the beneficial effects of bladder training in the hospital are often lost within a short time when the patient returns to his home. Reexamination at home of 97 MS patients with increased residual urine and/or chronic urinary tract infections showed that the group which claimed to continue bladder training at home had significantly less residual urine at home than the group which did not continue bladder training at home. The residual urine decreased from 210 ml on average to almost normal while the patients did bladder training in the hospital, but the volume nearly doubled within a short time at home. Thus, more decentralized rehabilitation by family members, volunteer personnel or local nurses is necessary. Decentralized symptomatic therapy is the most efficient treatment of MS at present. Family members. volunteers, and local nurses, however, need training. Without these improvements in decentralized rehabilitation the hospital treatment of MS is of little benefit because urinary tract infection quickly recurs at home. For efficient bladder training the patient needs feedback regarding the residual urine; this can be provided with minimum risk by ultrasound sonocystography. In those rare cases in which bladder training does not work, intermittent catheterization must be carried out by the patients or their families, volunteer personnel or a local nurse. A continuous indwelling catheter should not be used. antibiotic treatment should be applied only on the basis of a precise bacteriological diagnosis.

[Analysis of action potentials of muscles by a small general purpose computer (author's transl)]. / Analyse von Muskelaktionspotentialen mit Hilfe eines Prozess-Rechners

To measure the duration of action potentials by a computer, the following steps are necessary: 1. automatic selection of suitable potentials from the EMG 2. identification of equal potentials (action potentials of the single motor unit) 3. measuring the durations of potentials, defined by the distance between first and last deviation from the base line. We noticed that the automatic evaluation of durations of single potentials is greatly deteriorated by base-line fluctuations. Therefore we average about 16 action potentials, stored in the computer, of the same single motor unit and measure the duration of the averaged potential.