Pancreatic cancer cells overexpress gelsolin family-capping proteins, which contribute to their cell motility.

BACKGROUND: Previously, proteomic methods were applied to characterise differentially expressed proteins in microdissected pancreatic ductal adenocarcinoma cells. AIMS: To report that CapG and a related protein, gelsolin, which have established roles in cell motility, are overexpressed in metastatic pancreatic cancer; and to describe their pattern of expression in pancreatic cancer tissue and their effect on cell motility in pancreatic cancer cell lines. METHODS: CapG was identified by mass spectrometry and immunoblotting. CapG and gelsolin expression was assessed by immunohistochemical analysis on a pancreatic cancer tissue microarray and correlated with clinical and pathological parameters. CapG and gelsolin levels were reduced using RNA interface in Suit-2, Panc-1 and MiaPaCa-2 cells. Cell motility was assessed using modified Boyden chamber or wound-healing assays. RESULTS: Multiple isoforms of CapG were detected in pancreatic cancer tissue and cell lines. Immunohistochemical analysis of benign (n = 44 patients) and malignant (n = 69) pancreatic ductal cells showed significantly higher CapG staining intensity in nuclear (p<0.001) and cytoplasmic (p<0.001) compartments of malignant cells. Similarly, gelsolin immunostaining of benign (n = 24 patients) and malignant (n = 68 patients) pancreatic ductal cells showed higher expression in both compartments (both p<0.001). High nuclear CapG was associated with increased tumour size (p = 0.001). High nuclear gelsolin was associated with reduced survival (p = 0.01). Reduction of CapG or gelsolin expression in cell lines by RNAi was accompanied by significantly impaired motility. CONCLUSIONS: Up regulation of these actin-capping proteins in pancreatic cancer and their ability to modulate cell motility in vitro suggest their potentially important role in pancreatic cancer cell motility and consequently dissemination.

Upregulation of heat shock protein 27 in metaplastic and neoplastic lesions of the endocervix.

Heat shock proteins (hsps) are molecular chaperones that are known to play a pivotal role in regulating intracellular homeostasis. hsp27 may have diagnostic and prognostic values for different gynecological malignancies. A cross-sectional analytical study was conducted at the Department of Pathology, The University of Liverpool, Liverpool, UK. Included in the study were 80 cervical glandular lesions of various histologic types, representing tuboendometrial metaplasia/endometriosis (n = 19), cervical glandular intraepithelial neoplasia (n = 33), and invasive adenocarcinoma (n = 28). Paraffin-embedded sections were stained using a commercial mouse monoclonal anti-hsp27 antibody with prior pressure-cooking for antigen retrieval. Sections of 11 normal cervices were used as controls. The median percentage of cells expressing hsp27 in each group was calculated. Normal cervical glands showed minimal expression of hsp27 (median: 10%, interquartile ranges [IQ]: 5-15). Expression was significantly more widespread in tuboendometrial metaplasia/endometriosis (median: 35%, IQ: 15-80), cervical glandular intraepithelial neoplasia (median: 60%, IQ: 32-80), and invasive adenocarcinoma (median: 40%, IQ: 25-80) when compared with normal endocervix (P = 0.007, < 0.001, and 0.001, respectively). However, no significant difference in hsp27 protein expression was found between cervical glandular intraepithelial neoplasia and invasive adenocarcinoma. In invasive adenocarcinoma, hsp27 showed no correlation with tumor grade, lymph node involvement, and lymphovascular space invasion. Our data highlight early dysregulation of hsp27 expression in both metaplastic and neoplastic lesions of the cervix.

Correlation of mRNA for oestrogen receptor beta splice variants ERbeta1, ERbeta2/ERbetacx and ERbeta5 with outcome in endocrine-treated breast cancer.

This study has been performed to test the hypothesis that different oestrogen receptor beta (ERbeta) splice variants may be important determinants of clinical parameters, including outcome, in post-menopausal women with breast cancer receiving adjuvant endocrine treatment but no chemotherapy. Splice variants ERbeta1, ERbeta2 and ERbeta5 have been analysed by semi-quantitative RT-PCR in a cohort of 105 patients with primary breast cancer. Clinical correlates included age, grade, size, nodal status, ERalpha, progesterone receptor, Ki67, relapse-free survival (RFS) and overall survival (OS). Seventy per cent of cases were ERbeta1 positive, 69% ERbeta2 positive and 70% ERbeta5 positive. Within the cohort, 47% were positive for all three variants while 10% were negative for all three. ERbeta1 exhibited no discernible relationship with disease outcome. ERbeta2 and ERbeta5 expression was significantly associated with better RFS (P<0.005), and ERbeta2 with better OS (P=0.0002). In multivariate analysis, ERbeta2 (P=0.006), nodal status and the level of Ki67 expression were independent predictors for RFS while ERbeta2 (P=0.0008) and Ki67 status were independent predictors for OS. In the ERalpha-positive cases, or in the subset of those receiving adjuvant tamoxifen, ERbeta2 was significantly associated with good RFS (P<0.0005) and was the only independent marker of OS. We conclude that precise identification of splice variants of ERbeta are more important assessors than is ERbeta1 alone of the biological status of individual breast cancers, and hence in predicting their response to endocrine therapy.

Allelic imbalance at the DNA mismatch repair loci, hMSH2, hMLH1, hPMS1, hPMS2 and hMSH3, in squamous cell carcinoma of the head and neck.

BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) is one of the 10 most frequently occurring cancers in the world. Defective mismatch repair, as exhibited by the phenomenon of microsatellite instability, has been observed in SCCHN although no reports of mismatch repair gene mutations or altered protein expression have been published. In a variety of microsatellite instability (MSI) positive cancers where mutations in the mismatch repair (MMR) genes were not observed, allelic imbalance at the loci of the MMR genes was prevalent. OBJECTIVE: To investigate whether allelic imbalance at the MMR genetic loci contributes to the development of SCCHN. MATERIALS AND METHODS: 35 matched normal/tumour SCCHN pairs were studied using 29 microsatellite markers located within and adjacent to six known DNA mismatch repair genes. In addition, mutational analysis and protein expression of hMSH2 and hMLH1 were investigated. RESULTS AND CONCLUSIONS: We demonstrated that 36 and 17% of the analysed SCCHN specimens exhibited allele imbalance at the hMLH1 and hMSH3 genetic loci, respectively. Allelic instability at these two loci was found to be correlated with the MSI status of the SCCHN tumours. Allelic instability was found to be uncommon at the other MMR gene loci analysed. One mutation was found in hMSH2 and none in hMLH1 in this series of tumours. 23 of 24 (96%) of the examined SCCHN tumours showed reduced expression of either hMSH2 or hMCH1 genes. Allelic instability in the MMR genes, hMLH1 and hMSH3, is proposed to be involved in the aetiology of SCCHN tumours.

S100A4 (p9Ka) protein in colon carcinoma and liver metastases: association with carcinoma cells and T-lymphocytes.

The presence of the EF-hand-calcium-binding protein S100A4 in the carcinoma cells of the primary tumour is associated with a shorter survival time of a group of breast cancer patients. In colon cancer, primary tumours as well as metastases to the liver can be studied. Here we show, using quantitative PCR applied to RNA from 24 normal colon, four liver tissues, 24 colon carcinoma specimens, and 24 livers containing colonic carcinoma metastases, that the level of S100A4 mRNA was significantly higher in the carcinomas compared to normal specimens (Mann-Whitney U-test, P=0.05), and in liver metastases compared to carcinoma specimens (P=0.039). The latter comparison included seven liver metastases and their matched primary carcinomas (P<0.001) from the same patient. In situ hybridization and immunocytochemistry techniques have localized S100A4 to both carcinoma cells and lymphocytes in the malignant specimens. The percentage of specimens stained for S100A4 in the epithelial cells is significantly higher for those isolated from carcinomas and metastases than from the corresponding normal tissue, and from metastases than from corresponding carcinoma (Fisher Exact text, P<0.0016, P=0.04, respectively). In most specimens, S100A4 is present in clusters of T lymphocytes and this distribution is also found in the lymphoid, uninflamed appendix.

hMLH1 and hMSH2 expression correlates with allelic imbalance on chromosome 3p in non-small cell lung carcinomas.

DNA mismatch repair genes have been implicated in the pathogenesis and predisposition of certain malignancies through a mutator phenotype. In this study, we investigated, in 150 non-small cell lung carcinomas, the expression levels of hMLH1 and hMSH2 proteins in relation to loss of heterozygosity on chromosomes 3p and 2p, the mutational status of these genes' promoters and the hot spot exons. We have demonstrated that 88 of 150 (58.6%) tumor specimens had reduced expression levels of the hMLH1 protein, whereas 85 of 147 (57.8%) specimens had reduced expression levels of the hMSH2 protein. Reduced expression levels of both proteins were observed in 51 of 150 (34%) specimens. In adenocarcinomas, the reduction of hMSH2 expression was more frequently observed than that of hMLH1 (P<0.003), whereas in squamous cell carcinoma of the lung hMLH1 expression was more frequently reduced than hMSH2 (P<0.006). Reduced expression of hMLH1correlated with allelic imbalance on loci D3S1289 (P<0.0002) and D2S391 (P<0.05). It is of note that an inverse correlation was found between hMSH2 reduced expression and loss of heterozygosity at locus D3S1300 (P = 0.016). In addition, hMLH1 reduced expression was more frequently associated with heavy smokers, assessed by daily tobacco uptake (P = 0.018) and total smoking exposure (pack-years; P<0.05). In addition, a correlation between hMLH1 reduced expression and nodal metastasis in squamous cell carcinoma of the lung was observed (P = 0.015). No mutations were identified in the promoters or exons examined in these two genes. These findings indicate that hMLH1 and hMSH2 gene inactivation is a common event in the development of non-small cell lung carcinoma and allelic loss seems to be a major genetic event involved in hMLH1 silencing. In addition, we propose that a putative negative regulator of hMSH2 gene may be located at the locus 3p14.

Evaluation of telomerase activity in bronchial lavage as a potential diagnostic marker for malignant lung disease.

Telomerase is a ribonucleoprotein DNA polymerase that maintains the telomeric region of chromosomes lost during successive rounds of cell division. We used the telomeric repeat amplification protocol (TRAP) assay to examine telomerase activity in bronchial lavage (BL) samples from individuals undergoing diagnosis of lung cancer. Telomerase activity was detected in 17 (47%) of 36 samples examined. In particular, 16 (70%) of 23 BL specimens obtained from lung cancer patients showed detectable telomerase activity, while only 1 of 13 (8%) specimens obtained from patients without lung cancer demonstrated activity (P=0.00038). Moreover, 9 (90%) of 10 BL specimens, which were cytologically positive for lung cancer, were also positive for telomerase activity, while 7 (54%) of 13 cytologically negative BL specimens for lung cancer showed detectable telomerase activity. Detection of telomerase activity combined with cytology were able to identify 17 (74%) of 23 lung cancer cases whereas cytology alone identified 10 (43%) of 23 such cases (P=0.035). Our findings indicate that telomerase is a specific marker for malignant lung disease and a potential complementary tool to cytology in the diagnosis of certain lung cancer cases.

Peptide amidating activity in human bronchoalveolar lavage fluid: relationship to lung cancer, inflammation and infection.

The most common post-translational modification of peptide hormones, present in half of all neuroendocrine (NE) peptides, is alpha-amidation and this is necessary for the biological activity of the peptides. Peptides are alpha-amidated by the action of two enzymes: peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL). As the common pathway for the formation of amidated peptides, the enzymes may provide a better indication of the NE status of a tumour or tissue than the presence of any single amidated peptide. PHM and PAL enzyme activities were measured in 39 BL fluid specimens from patients undergoing bronchoscopy for diagnosis of lung cancer. The assays revealed that PHM levels were higher in a group of specimens from patients undergoing bronchoscopy for chest infection, inflammation, asthma, or pneumonia compared to a group of specimens in which malignant cells were seen. The presence of elevated levels of amidating enzymes in specimens with non-cancerous conditions may reflect events of promotional phase cancer biology occurring simultaneously with inflammatory and infectious processes.

Utilization of human tissue in breast cancer research.

The use of human tissue, and material derived from such tissue, for research purposes is currently the subject of much debate. This debate needs to address several issues, including: the principle of abandonment; the distinction between identified and unidentified specimens; general versus specific informed consent; and, with the improvement in biotechnology and medical informatics, the design and security of research databases. The outcome of this debate will shape the way in which research studies using human biological materials are designed and executed.

Telomerase activity in non-small cell lung carcinomas correlates with smoking status.

Human telomerase is a ribonucleoprotein DNA polymerase which maintains the telomeric region of human chromosomes and has been detected in all types of human cancer tested. We used the telomeric repeat amplification protocol (TRAP) assay to examine 71 non-small cell lung carcinomas (NSCLC) and their adjacent normal tissue. Telomerase activity was detected in 61 (86%) of the 71 NSCLC examined but not in any of the matched normal lung tissues. A significant correlation was found between the presence of telomerase activity and current smoking status at the time of diagnosis (p=0. 0076). In addition, a trend was found between telomerase activity and smoking exposure (p=0.06). Our findings demonstrate that telomerase activity is a common phenomenon in NSCLC cases but not in the normal lung. However, certain cases in former smokers may follow a telomerase independent pathway.

Genetic alterations in bronchial lavage as a potential marker for individuals with a high risk of developing lung cancer.

Using 12 microsatellite markers, we have studied DNAs from the bronchial lavage of 90 individuals who were referred to an early-lung-cancer clinic in the Northwest of England with suspected lung cancer. Genetic alterations were detected in 15 (35%) of 43 patients with lung cancer but also in 11 (23%) of 47 patients with no cytological or radiological evidence of bronchial neoplasia. No significant differences were found between the referring symptoms in any of the second group of individuals with and without genetic alterations. No correlation was found between smoking exposure and loss of heterozygosity (LOH)/microsatellite alterations (MAs) in the microsatellite markers. On comparing LOH with MAs based on cytology review, we found that the prevalent type of alteration in specimens with cytological evidence of malignancy was LOH; in contrast, the individuals with no cytological evidence of malignancy showed a preponderance of MAs (P = 0.01). Our results indicate that a substantial proportion of cells in the bronchial lavage from suspected lung cancer patients carry identifiable genetic alterations. However, the presence of genetic alterations in the bronchial lavage of individuals with no clinical evidence of lung cancer raises the question whether instability is a phenomenon solely associated with cancer or represents a feature of nonneoplastic diseases. Our results suggest that microsatellite PCR-based assays can be developed as tools for the earlier identification of genetic changes in cells exfoliating in the bronchus.

Heterogeneous nuclear ribonucleoprotein A2/B1 up-regulation in bronchial lavage specimens: a clinical marker of early lung cancer detection.

This study examines the use of a new epithelial marker in the detection of early lung cancer in bronchial lavage samples. The monoclonal antibody 703D4 recognizes the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, and its overexpression/up-regulation was assessed and compared with routine diagnostic cytology. One hundred and three individuals were recruited into a prospective study. These individuals were referred to a chest physician with a request to examine for possible lung cancer, and a full clinical work-up was undertaken, including bronchoscopy and radiological investigations. In this study, we analyzed hnRNP expression in individuals with metaplastic bronchial epithelial cells or tumor cells in the bronchial lavage specimens, in a blinded study. The results from 103 bronchial lavage specimens indicate that hnRNP overexpression was more accurate in detecting evidence of a neoplasia than routine cytological examination. Twenty-two of 23 specimens in which malignant cells were identified cytologically demonstrated overexpression of hnRNP A2/B1. However, in the 80 specimens that were reported as cytologically negative, 41 of 80 demonstrated hnRNP overexpression, and 29 of these individuals were shown to have a lung neoplasm based on radiological findings and/or the biopsy taken at the bronchoscopy. An additional 4 of these 41 patients were shown to have a lung neoplasm within 8 months of the initial bronchoscopy. In conclusion, detection of hnRNPA2/B1 in bronchial lavage specimens that contain metaplastic bronchial epithelial cells or cancer cells predicts the presence of a neoplasm with a sensitivity of 96%, 82%, specificity.

p53 gene aberrations in non-small-cell lung carcinomas from a smoking population.

We examined 46 non-small-cell lung carcinomas (NSCLCs) for the presence of p53 mutations in exons 4-9, positive p53 immunostaining and loss of heterozygosity (LOH) in the TP53 locus. p53 mutations were detected in 13 tumour samples (28.3%), whereas overexpression of the p53 protein was found in 30 of 45 (67%) samples. Allelic loss was found in 9 of 38 (23.6%) informative cases. The statistical analysis revealed no significant correlation between p53 mutations and clinicopathological data, although mutations appear to occur more frequently in squamous cell carcinomas (7 of 18) than in adenocarcinomas (2 of 15). All but three individuals in this study group smoked. In contrast to previous reports, we found a higher prevalence of GC-->AT transitions than of GC-->TA transversions, as expected in a smoking population. A trend was found between p53-positive immunostaining and a history of heavy smoking (76-126 pack-years) and was inversely correlated with allelic deletion (LOH) at the TP53 locus. Eight of the 12 NSCLCs containing p53 mutations also had concomitant p53 overexpression, and it is of specific note that three of the four tumours containing p53 'mutations' with no overexpression of the p53 protein had either insertions or deletions in the p53 gene. No correlation was found between p53 mutations and fractional allele loss or ras mutations. p53 mutations in this Merseyside population in the UK do not appear to be as common as in other reports for NSCLC and exhibit predominance of GC-->AT transitions preferentially at non-CpG sites, suggesting that other carcinogens in addition to those in tobacco smoke may be involved in NSCLC in the Merseyside area of the UK.

Open-circuit gas exchange analysis in the non-steady-state.

The serial measurement of oxygen uptake (VO2) of four subjects was calculated during the transition from rest to constant-load cycle ergometer work using an open-circuit gas exchange analysis system. In calculating VO2, the mixed expired gas concentrations were matched with the ventilatory volume by various delay factors. A delay factor equivalent to the passing of 20 L of expired ventilation through the mixing chamber yielded results which were most similar to the VO2 obtained by a computerized breath-by-breath analysis. Previous checks of the response of the system to changes in calibrating gas mixtures had indicated that it was necessary to pass approximately 20-25 L of gas through the system before a plateau response was observed. This volume remained relatively constant, independent of flow rate. It is proposed that an understanding of the response characteristics of an open circuit system will enable the accurate calculation of VO2 over short time intervals in the non-steady-state.